Rac1
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of dominant-inherited Parkinson's disease (PD), and yet the physiological functions of LRRK2 are not fully understood. Various components of the clathrin machinery have been recently found mutated in familial forms of PD. This study provides molecular insight into the association of LRRK2 with the clathrin machinery. Through its GTPase domain, LRRK2 binds directly to clathrin-light chains (CLCs). Using genome-edited HA-LRRK2 cells, LRRK2 was localized to endosomes on the degradative pathway, where it partially co-localizes with CLCs. Knockdown of CLCs and/or LRRK2 enhances the activation of the small GTPase Rac1, leading to alterations in cell morphology, including the disruption of neuronal dendritic spines. In Drosophila, a minimal rough eye phenotype caused by overexpression of Rac1, is dramatically enhanced by loss of function of CLC and LRRK2 homologues, confirming the importance of this pathway in vivo. These data identify a new pathway in which CLCs function with LRRK2 to control Rac1 activation on endosomes, providing a new link between the clathrin machinery, the cytoskeleton and PD (Schreij, 2014).
Recent studies established that the planar cell polarity (PCP) pathway is critical for various aspects of nervous system development and function,
including axonal guidance. Although it seems clear that PCP signaling
regulates actin dynamics, the mechanisms through which this occurs
remain elusive. This study established a functional link between the PCP
system and one specific actin regulator, the formin DAAM, which has previously been shown to be required
for embryonic axonal morphogenesis and filopodia formation in the growth
cone. DAAM also plays a pivotal role during axonal growth and guidance
in the adult Drosophila mushroom
body, a brain center for learning and memory. By using a combination
of genetic and biochemical assays, it was demonstrated that Wnt5 and the PCP signaling proteins
Frizzled, Strabismus, and Dishevelled act in concert with the
small GTPase Rac1 to activate the
actin assembly functions of DAAM essential for correct targeting of
mushroom body axons. Collectively, these data suggest that DAAM is used
as a major molecular effector of the PCP guidance pathway. By uncovering
a signaling system from the Wnt5 guidance cue to an actin assembly
factor, it is proposed that the Wnt5/PCP navigation system is linked by
DAAM to the regulation of the growth cone actin cytoskeleton, and
thereby growth cone behavior, in a direct way (Gombos, 2015).
This study has shown that DAAM plays an important role in the regulation of axonal growth and guidance of the Drosophila MB neurons. Several lines of evidence suggest that DAAM acts in concert with Wnt5 and the core PCP proteins to ensure correct targeting of the KC axons. DAAM functions downstream of Dsh and Rac1, and its ability to promote actin assembly is absolutely required for neural development in the MB. These data suggest a simple model in which axon guidance cues, such as Wnt5, signal through the PCP pathway to activate DAAM to control actin filament formation in the neuronal growth cone. Thus, PCP signaling appears to be linked to cytoskeleton regulation in a direct way, and these results provide compelling experimental evidence suggesting that, at least in neuronal cells, the major cellular target of PCP signaling is the actin cytoskeleton (Gombos, 2015).
Formins are highly potent actin assembly factors that are under tight regulation in vivo. The major mechanism of controlling the activity of the Diaphanous-related formin (DRF) subfamily involves an intramolecular autoinhibitory interaction between the N-terminal diaphanous inhibitory domain (DID) and the C-terminal Diaphanous autoinhibitory domain (DAD). This inhibition can be relieved upon binding of an activated Rho family GTPase that interacts with the GBD (GTP-ase binding domain)/DID region and also by proteins that bind to the DAD domain. Consistently, this study found that the Rac1 GTPase and the DAD domain binding Dsh protein both play role in DAAM activation in MB neurons. With this regard, it is notable that, despite that dsh1 is considered a PCP-null allele, the DAAMEx1, dsh1 double hemizygous mutants exhibit a stronger MB phenotype than dsh1 mutants alone, suggesting that DAAM must receive Dsh-independent regulatory inputs for which Rac1 is a prime candidate. Although previous work indicated that Rho GTPases might function downstream of Dsh in a linear pathway), the data suggest that Dsh and Rac1 act in parallel pathways in the MB. As the impairment of GTPase binding severely, but not completely, abolishes DAAM activity, it is concluded that Rac1 is likely to have a stronger contribution to DAAM activation in vivo; nonetheless, the simultaneous binding of Dsh appears to be required for full activation (Gombos, 2015).
Presumably, the most remarkable feature of the PCP system relies in its ability to create subcellular asymmetries. Therefore, it is a tempting idea that, upon guidance signaling, the PCP proteins are involved in the generation of molecular asymmetries within axonal growth cones, yet recent attempts failed to reveal such polarized distributions in MB neurons. Interestingly, however, it was shown that Fz and Vang display a differential requirement during development of the MBs, with Fz predominantly acting in the dorsal lobes and Vang predominantly acting in the medial lobes (MLs). This study found that, in contrast to Fz and Vang, DAAM plays a crucial role in both lobes of the MBs. Additionally, it was demonstrated that Fz promotes the formation of membrane-associated Dsh-DAAM complexes in S2 cells. This result, together with genetic data, suggests that DAAM acts as the downstream effector of a Fz/Dsh module, which is required for the correct growth and guidance of the dorsal MB axon branches (Gombos, 2015).
In addition to their potential connection to Fz signaling in the dorsal lobe, DAAM and Dsh were linked to Vang- and Wnt5-dependent ML development as well. Wnt5 and Vang have an identical effect on ML development when overexpressed, and this GOF phenotype can be suppressed by the same set of mutations (DAAM, dsh, Rac1). In particular, the putative PCP-null dsh1 allele and heterozygosity for Rac1 cause an almost equally strong, yet partial, suppression with regard to the ML fusion phenotype. This is best explained by assuming that Wnt5 and Vang signal both in a Dsh-dependent and in a Dsh-independent, but Rac-dependent, manner. With regard to DAAM, this study has shown that DAAM nearly completely suppresses the GOF of Wnt5 and Vang, and Dsh and Rac1 both contribute to DAAM activation. Collectively, these data suggest a model in which Wnt5 and Vang promote β lobe extension by signaling to Dsh and Rac1 that will activate DAAM in parallel to each other. The colocalization of Vang and DAAM, observed in S2 cells, indicates that they may bind each other directly, which would be in good accordance with genetic data suggesting a close functional link between DAAM and Vang during β lobe development. However, formins are not known to bind Vang proteins; therefore, an indirect interaction, mediated by Rac1, which has recently been shown to be bound and redistributed by Vangl2 in epithelial cell lines, appears a more likely possibility (Gombos, 2015).
As discussed above, and contrary to Vang, Fz does not appear to be required for ML development, or if anything, it might play an opposite role, as loss of fz leads to ML fusion in 16.1% of the lobes. This is a surprising observation at first glance as Wnt proteins are thought to activate members of the Fz receptor family, but former analysis of Wnt5 signaling during MB development also failed to reveal a Fz requirement in the β lobes. Instead, Wnt5 has been linked to other type of Wnt receptors, the Ryk/Derailed atypical tyrosine kinase receptors, which are known to be involved in axonal guidance in flies and vertebrates. In light of these results, it will be of future interest to analyze the Wnt5-Vang connection in the MB in more details and identify the Wnt5 receptor in this context (Gombos, 2015).
Consistent with the lack of lobe-specific requirement for dsh and DAAM, the current studies revealed that Dsh, DAAM, and Rac1 are used as common effector elements of a dorsal lobe-specific Fz-dependent signal and a Vang-dependent ML-specific signal. It follows that Dsh and DAAM are likely to take part in two types of PCP complexes. Although, in vitro, Dsh has the ability to interact with both Fz and Vang, the conclusion that Dsh functions downstream of Vang in the β lobes is markedly different from the classical PCP regulatory context in which the Fz/Dsh and Vang/Pk complexes have opposing effects. Thus, this result, together with the Wnt5-Vang data, substantiates the earlier findings that the PCP system operates at least partly differently in neurons than during tissue polarity signaling (Gombos, 2015).
During PCP signaling, the vertebrate DAAM orthologs control convergence and extension movements, polarized cell movements during vertebrate gastrulatio. In contrast, DAAM is dispensable for classical planar polarity establishment in flies, suggesting that the tissue polarity function of DAAM might be restricted only to vertebrates. Despite the lack of direct function in establishing tissue polarization, this study provides evidence that DAAM is linked to the PCP pathway in another important regulatory context, notably directed neuronal development in the adult brain. Consistent with the results, recent studies revealed that PCP signaling and DAAM regulate neural development in planarians and in Xenopus embryos. Given that the vertebrate PCP proteins are known to be involved in multiple aspects of CNS development, and the vertebrate DAAM orthologs are strongly expressed in the CNS, it is conceivable that the PCP/DAAM module represents a highly conserved regulatory system that is used to regulate various aspects of neuronal development throughout evolution (Gombos, 2015).
Neurons develop highly stereotyped receptive fields by coordinated growth of their dendrites. Although cell surface cues play a major role in this process, few dendrite specific signals have been identified to date. An in vivo RNAi screen in Drosophila class IV dendritic arborization (C4da) neurons identified the conserved Ret receptor (Ret oncogene), known to play a role in axon guidance, as an important regulator of dendrite development. The loss of Ret results in severe dendrite defects due to loss of extracellular matrix adhesion, thus impairing growth within a 2D plane. Evidence is provided that Ret interacts with integrins to regulate dendrite adhesion via rac1. In addition, Ret is required for dendrite stability and normal F-actin distribution suggesting it has an essential role in dendrite maintenance. Novel functions are proposed for Ret as a regulator in dendrite patterning and adhesion distinct from its role in axon guidance (Soba, 2015).
Accurate functional connectivity and sensory perception require proper development of the neuronal dendritic field, which ultimately determines the (sensory) input a specific neuron can receive and detect. Thus, coordinated dendrite growth and patterning is important for establishing the often complex, but highly stereotyped organization of receptive fields. Two of the organizing principles in dendrite development are self-avoidance and tiling. While self-avoidance describes the phenomenon of recognition and repulsion of isoneuronal dendritic branches, tiling refers to the complete yet non-redundant coverage of a receptive field by neighboring neurons of the same type. Both phenomena have been described in different systems across species including the mouse, zebrafish, medicinal leech, Caenorhabditis elegans, and Drosophila melanogaster (Soba, 2015).
Dendritic patterning by self-avoidance, tiling, and other mechanisms is thought to be mediated by cell surface receptors and cell adhesion molecules (CAMs), which play a pivotal role in integrating environmental and cellular cues into appropriate growth and adhesion responses. Many such receptors, prominently Robo and Ephrin receptors, have well understood roles in axon guidance. Although some of these axonal cues including Robo/Slit play a role in dendrite development as well, dendritic surface receptors and their functions are not fully characterized to date. Recent efforts have yielded some progress in this area. Down's syndrome cell adhesion molecule (Dscam) has been shown to regulate dendrite self-avoidance in Drosophila. Studies on protocadherins have revealed that they play an important role in dendrite self-avoidance in mammals. In C. elegans, sax-7/L1-CAM and menorin (mnr-1) form a defined pattern in the surrounding hypodermal tissue to guide PVD sensory neuron dendrite growth via the neuronal receptor dma-1. However, given the complexity and stereotypy of dendritic arbors within individual neuronal subtypes, it is important to search for additional signals for directing dendrite growth (Soba, 2015).
The Drosophila peripheral nervous system (PNS) has served as an excellent model which has helped to elucidate several molecular mechanisms regulating dendrite development. The larval PNS contains segmentally repeated dendritic arborization (da) neurons which have been classified as class I-IV according to their increasing dendritic complexity. All da neuron classes feature highly stereotyped sensory dendrite projections. Moreover, all da neurons exhibit self-avoidance behavior allowing them to develop their individual receptive fields without overlap. It has been demonstrated that all da neuron classes require Dscam for dendrite self-avoidance. In addition, the atypical cadherin flamingo and immunoglobulin super family (IgSF) member turtle might play a more restricted role in C4da neuron self-avoidance. Netrin and its receptor frazzled have also been shown to act in parallel to Dscam in class III da neurons ensuring their proper dendritic field size and location by providing an attractive growth cue which is counterbalanced by self-avoidance. For tiling, no surface receptor has been identified to date. However, the conserved hippo and tricornered kinases, and more recently the torc2 complex, have been implicated in C4da neuron tiling, as the loss of function of these genes results in iso- and hetero-neuronal crossing of dendrites (Soba, 2015).
Recent work has further shown that dendrite substrate adhesion plays an essential role in patterning. Da neuron dendrites are normally confined to a 2D space through interaction with the epithelial cell layer and the extracellular matrix (ECM) on the basal side of the epidermis. 2D growth of da neuron dendrites requires integrins, as loss of the α-integrin mew (multiple edomatous wing) or ß-integrin mys (myospheroid) results in dendrites being freed from the 2D confinement due to detachment from the ECM. Thus, they can avoid dendrites by growing into the epidermis leading to 3D crossing of iso- and hetero-neuronal branches . Integrins are therefore essential to ensure repulsion-mediated self-avoidance and tiling mechanisms, which restrict growth of dendrites competing for the same territory. How integrins are recruited to dendrite adhesion sites and whether they cooperate with other cell surface receptors is unknown (Soba, 2015).
To identify novel receptors required for generating complex, stereotypical dendritic fields, an in vivo RNAi screen was performed for cell surface molecules in C4da neurons. The Drosophila homolog of Ret (rearranged during transfection) was identified as a patterning receptor of C4da dendrites. Loss of Ret function in C4da neurons severely affects dendrite coverage, dynamics, growth, and adhesion. In particular, dendrite stability and 2D growth are impaired resulting in reduced dendritic field coverage and abnormal 3D dendrite crossing, respectively. These defects can be completely rescued by Ret expression in C4da neurons. It was further shown that Ret interaction with integrins is needed to mediate C4da dendrite-ECM adhesion, but not dendrite growth. These data suggest that Ret together with integrins acts through the small GTPase rac1, which is required for dendrite adhesion and 2D growth of C4da neuron dendrites as well. This study thus describes a novel role for the Ret receptor in dendrite development and adhesion by direct receptor crosstalk with integrins and its downstream signals (Soba, 2015).
This study provides evidence that Ret is a regulator of dendrite growth and patterning of C4da neurons. Ret is a conserved receptor tyrosine kinase (RTK) expressed in the nervous system of vertebrates and D. melanogaster , and has been shown to have a number of important functions in nervous system development and maintenance: it regulates motor neuron axon guidance (Kramer, 2006), dopaminergic neuron maintenance and regeneration, and mechanoreceptor differentiation and projection to the spinal cord and medulla. Ret signaling is activated by binding to glial cell line derived neurotrophic factor (GDNF) family ligands and their high affinity co-receptors, the GDNF family receptors (GFRα). Ret also plays an important role in human development and disease as loss of function mutations of Ret lead to Hirschprung's disease displaying colonic aganglionosis due to defective enteric nervous system development. Conversely, Ret gain of function mutations are causal for autosomal dominant MEN2 (multiple endocrine neoplasia type 2) type medullary thyroid carcinoma (Soba, 2015 and references).
Prior to this study, Ret has not been implicated in dendrite development. This study shows that Ret is required specifically for 2D growth of C4da neurons by regulating integrin dependent dendrite-ECM adhesion. Normally, C4da neuron dendrites are virtually always in contact with the ECM and the basal surface of the epithelium lining the larval cuticle, and thus tightly sandwiched between the two compartments. In both integrin and Ret mutants, dendrite-ECM adhesion is impaired. Ret and integrins can co-localize in dendrites and thus likely form a functional complex that could induce and maintain adhesion of dendrites to the ECM. Since Ret loss of function primarily leads to detached terminal dendrite branches, it is tempting to speculate that Ret might be required to recruit integrins to sites of growing dendrites to promote ECM interaction. This is supported by the colocalization of Ret and integrins on the dendrite surface. Their cooperative interaction could thus ensure proper adhesion of growing branches and, conversely, the fidelity of self-avoidance and tiling (Soba, 2015).
These results also highlight the importance of integrating different guidance and adhesion cues to achieve precise neuronal patterning. This has so far only been studied in axon guidance in vivo. Interestingly, vertebrate Ret has been shown to cooperate with Ephrins to ensure high fidelity axon guidance in motor neurons by mediating attractive EphrinA reverse signaling (Kramer, 2006; Bonanomi, 2012). Similar mechanisms may conceivably be employed for growing dendrites, which also encounter a multitude of attractive, repulsive, and adhesive cues that have to be properly integrated. Besides pathways acting independently or in a parallel fashion, an emerging view is that receptors exhibit direct crosstalk to integrate incoming signals. So far, only parallel receptor pathways like Dscam and Netrin-Frazzled signaling in class III da neurons or Dscam/integrins have been identified co-regulating dendrite morphogenesis. The current data show that the Ret receptor and integrins integrate dendrite adhesion and growth by collaborative interaction of the two cell surface receptors. The molecular and genetic link between Ret and integrins suggests that in this case direct receptor crosstalk plays a major role in their function. How exactly these cell surface receptors cooperate and interact remains to be elucidated. Integrins have been shown to display extensive crosstalk with other signaling receptors, including RTKs. Although integrins are involved in adhesion of virtually all cell types, the underlying signaling and recruitment of integrins to sites of adhesion in vivo is complex and not completely understood. It has been suggested that integrin and growth factor receptor crosstalk can occur by concomitant signaling, collaborative activation, or direct activation of associated signaling pathways. For example, matrix-bound VEGF can induce complex formation between VEGFR2 and β1-integrin with concomitant targeting of β1-integrin to focal adhesions in endothelial cells. The current findings of biochemical interaction and colocalization of Ret with the α/β-integrins mys and mew in C4da neuron dendrites argue in favor of direct receptor interaction and subsequent activation of a common signaling pathway (Soba, 2015).
Integrins and RTKs like Ret do share some of the same intracellular signaling components. These comprise, among others, the MAPK (mitogen-activated protein kinase) pathway, Pi3-Kinase (Pi3K), and Rho family GTPases including Rac1. Previous studies provide evidence for Ret-integrin-Rac1 interplay in vitro showing that Ret can enhance integrin mediated adhesion and induce Rac1 dependent lamellipodia formation in cell culture models. In primary chick motor neurons, Rac1 is involved in neurite outgrowth on the integrin substrates laminin and fibronectin. Interestingly, Rac1 has previously been shown to regulate dendrite branching in C4da neurons, however a role in dendrite adhesion in vivo has not been described before. This study shows that Rac1 is required for dendrite-ECM adhesion similarly to what has been described for integrins, and Ret and integrin dependent adhesion was genetically linked with Rac1 function. In Drosophila, MAPK, Src and PI3K can be activated by constitutively active Ret overexpression in the compound eye. Moreover, novel inhibitors of Ret signaling targeting Raf, Src, and S6-Kinase (S6K) prevent lethality induced by Ret over-activation in a Drosophila multiple endocrine neoplasia (MEN2) model. Interestingly, S6K has been shown to be involved in dendrite growth but not tiling in C4da neurons. It remains to be shown if these pathways play a direct role in Ret function in dendrite adhesion and growth (Soba, 2015).
Notwithstanding important commonalities, Ret function in C4da neurons cannot be fully explained by crosstalk with integrins and rac1. Reduced dendritic field coverage, likely due to the observed increase in dendrite turnover, is only evident in Ret but not in integrin or rac1 mutant C4da neurons. Moreover, increasing integrin expression in a Ret mutant background did not rescue dendrite coverage defects, albeit it prevented dendrite crossing. These findings indicate that Ret has additional functions in dendritic branch growth and stability that require as yet unknown extracellular and intracellular mediators. This is also supported by the aberrant F-actin localization in neurons lacking Ret. In this study, Ret dependent intracellular effectors are likely important for F-actin assembly to support directed dendrite growth and stabilization, and their localization and activity might be deregulated in the absence of Ret (Soba, 2015).
Drosophila Ret is a highly conserved molecule, its cognate vertebrate ligand GDNF, however is not (Airaksinen, 2006). In addition, Drosophila Ret can neither bind GDNF nor transduce GDNF signaling, although it has been shown to contain a functional tyrosine kinase domain. In mammals, the GFRα co-receptors are essential components of GDNF/Ret signaling. A Drosophila GFR-like homolog (dGFRL) has recently been characterized and was found to function and interact with the NCAM homolog FasII. Therefore, it appears that Ret's functional interaction partners in dendrite development differ significantly from the previously described co-factors in other systems. It is interesting to speculate that a yet undiscovered Ret ligand is involved in Ret mediated dendrite growth and branch stabilization, which might have implications for mammalian Ret function as well: due to its role in the maintenance of dopaminergic neurons and motor axon growth in mouse, adhesion related signaling via integrins could well be important during these processes. Moreover, the formation of a dorsal root ganglia derived mechanosensory neurons and their afferent and efferent fiber growth and innervation depends on Ret expression. It will be interesting to investigate the functional interplay of Ret and integrins in central and peripheral target innervation and neurite maintenance in these systems, given the interdependent function of Ret and integrins in sensory dendrite growth as shown in this study (Soba, 2015).
In summary, this study describes a novel role for the Ret receptor in dendrite branch growth and stability in Drosophila C4da neurons. This role involves cell-autonomous effects of Ret on ECM adhesion, and F-actin localization in these neurons. Moreover, dendritic adhesion defects attributable to Ret have been linked to integrin and rac1 function featuring a novel and possibly conserved mode of action for Ret in dendrite development (Soba, 2015).
puckered encodes a VH1-like phosphatase that acts directly as a Jun kinase
phosphatase to down-regulate Jun kinase (JNK) activity during dorsal closure
of the Drosophila embryo. puc plays a role in
follicle cell morphogenesis during oogenesis. The follicle cells (FCs) form an epithelial sheet around a cyst of germ cells (the oocyte and 15 nurse cells)
that then develops as an egg chamber unit. Late in oogenesis,
specific groups of follicle cells become distinct. During stage
9, most follicle cells migrate posteriorly to cover the oocyte
surface in a columnar epithelium. Approximately
50 cells -- the nurse cell FCs (NCFCs) -- flatten to form
a squamous domain over the nurse cells. Subsequently,
subgroups of the columnar FCs change shape and migrate. Beginning in stage 10B, the centripetally migrating FCs (CMFCs) move inward between the oocyte and nurse
cells. These cells will create anterior eggshell
structures such as the operculum. The main body FCs
(MBFCs) stretch during stage 11, as the oocyte rapidly enlarges with the transfer of the nurse cell contents, a process called nurse cell dumping. During stages 12
through 14, two dorsal anterior groups of FCs migrate anteriorly
to create the dorsal appendages. The posterior pole FCs (PPFCs) include cells that produce the aeropyle of the eggshell. The FCs that will create a specific eggshell
structure are fated earlier in oogenesis, as revealed by specific
patterns of gene expression (Dobens, 2001 and references therein).
The Rho family of small GTPases are important modulators of
cell shape and epithelial cell reorganization. Specific Rho
proteins can manifest specialized cellular functions. The cell-type
specific effects extend to their abilities to stimulate the
JNK pathway. The differing requirements for Puc in different regions of the
follicular epithelium suggest that these regions show
distinct sensitivities to the actions of Rho family GTPases.
Because of evidence that Rac1 and Cdc42 regulate
expression of A251-lacZ during embryogenesis, the ability of these small GTPases to regulate
A251-lacZ expression in the FC was tested. Two opposing
effects were found: activated Rac1 and dominant negative Cdc42
activate A251-lacZ expression in posterior cells; dominant
negative Rac1 and activated Cdc42 repress A251-lacZ
expression in anterior cells. Clones for activated Rac1 were
not recovered in the anterior nurse cell FCs, suggesting that
these cells are highly sensitive to increased Rac activity. These
data suggest that a balance between these two small GTPases
may define the level of A251-lacZ expression in the nurse cell
FCs and the posterior pole FCs. This may reflect the shared
origins of these distinct cell groups as terminal follicle cells (Dobens, 2001).
Neither Rac1 nor Cdc42 appeared to regulate A251-lacZ
expression in the main body FCs or the centripetally migrating
FCs. This latter observation is surprising, given the strong
requirement for Puc activity in these cells. Perhaps a distinct
Rho family GTPase is active in these cells, such as Rho1.
Consistent with this, mutants in PAK kinase, which acts in
parallel to JNK signaling during dorsal closure, have a variable
defect in centripetal migration (Dobens, 2001).
Activated GTPase expression in the follicle cells results in
additional phenotypes. Activated Rac1 has non-autonomous
phenotypes in the posterior pole FCs. Similarly,
activated Rac1 has non-autonomous effects on gene
expression in the dorsal ectoderm of the embryo. This suggests that in both tissues, a short-range
signal dependent on Rac1 signaling can upregulate
expression of these intron II enhancer traps in adjacent cells.
Dpp is a local signal to the dorsal ectoderm during dorsal
closure; however, the data suggest that Dpp is not the key
signal to induce A251-lacZ in the posterior FC. It is noted that loss of puc in marked clones never results in upregulation of A251-lacZ
expression in adjacent cells, indicating that whatever its
identity, the short range signal is not regulated by puc (Dobens, 2001).
Expression of activated Rac1 in the posterior pole FCs
causes domain-specific disorganization of the epithelium.
Dominant negative Cdc42 also causes this phenotype, similar
to genetic loss of Cdc42 activity. Similar phenotype are seen for activated RhoL. Thus the balance of Rho GTPases in the posterior follicular epithelium may be
important to maintain epithelial structure, similar to roles
ascribed to Rac in metastasis (Dobens, 2001).
Based on data presented here, it is proposed that puc functions
as a rheostat to modulate gene expression responses to Rho
family GTPases. This modulation is critical to
coordinate morphogenesis in distinct FC domains. Because Puc can act as a Jun kinase
phosphatase in the embryo, it has been speculated that Puc modulates
the activity of similar kinases that act downstream of the small
GTPases at the FC termini. It is likely that puc modulates Jun
kinase signal in the anterior FCs, since the Jun kinase kinase
encoded by hemipterous is required for anterior A251-lacZ expression. However, puc
is also required at the posterior, whereas late A251-lacZ
expression is not dependent on hep. Components of the
posterior pathway remain to be identified (Dobens, 2001).
The insensitivity of main body FCs to stimulation by Rac or
Cdc42 might suggest that these cells are incompetent to
respond to small GTPase activation during the late stages of
oogenesis. However, responses are suppressed by an independent mechanism. The dynamic
pattern of Jun and Fos and puc itself, suggests
that FCs may vary greatly, both in time and in space, in their
competence to respond to the JNK pathway. To complicate this
picture, the data indicates that puc regulates levels of Fos and
Jun, even in the main body FCs. Loss of puc leads to elevation
of Fos/Jun protein levels. Conversely overexpression of either
Puc or DN-Rac1 lowers Fos/Jun levels. Currently the direct mechanism for puc regulation of Fos/Jun levels is unknown, but these results recall observations that stability of Fos and Jun depends on phosphorylation. In cell culture, site-specific
mutations in Fos or Jun that block phosphorylation confer
instability; conversely, phosphate-mimetic mutations confer
greater stability. It is concluded that proper levels of Puc
phosphatase modulates Rho family GTPase signal output to
coordinate follicle cell morphogenesis. These results are
similar to the effect of both loss- and gain-of-function Puc in
blocking dorsal closure and a role for puc in disc epithelial morphogenesis. The precise regulation of Puc activity levels is critical to coordinate epithelial cell sheet spreading in at least three tissues (Dobens, 2001 and references therein).
Border cell migration in the Drosophila ovary is a relatively simple and genetically tractable model for studying the conversion of epithelial cells to migratory cells. Like many cell migrations, border cell migration is inhibited by a dominant-negative form of the GTPase Rac. To identify new genes that function in Rac-dependent cell motility, a screen was performed for genes that when overexpressed suppressed the migration defect caused by dominant-negative Rac. Overexpression of the Drosophila inhibitor of apoptosis 1 (DIAP1), which is encoded by the thread (th) gene, suppresses the migration defect. Moreover, loss-of-function mutations in th cause migration defects but, surprisingly, did not cause apoptosis. Mutations affecting the Dark protein, an activator of the upstream caspase Dronc, also rescues RacN17 migration defects. These results indicate an apoptosis-independent role for DIAP1-mediated Dronc inhibition in Rac-mediated cell motility (Geisbrecht, 2004).
The work reported here demonstrates a new function for DIAP1 in promoting cell migration. The strongest evidence for this is that border cells lacking DIAP1 fail to migrate. This finding is surprising since there has been no previous indication that IAP proteins contribute to cell motility. However, in most cells, it would be difficult or impossible to uncover a requirement for DIAP1 in cell migration because loss of the protein typically results in cell death (Geisbrecht, 2004).
The effect of DIAP1 on cell migration appears to be through the small GTPase Rac and its effects on the actin cytoskeleton based on genetic, biochemical, and cell culture experiments. First, overexpression of DIAP1 suppresses RacN17 migration defects specifically and does not rescue border cell migration defects that are due to other causes. Moreover, overexpression of either actin5C or profilin, both of which would be expected to increase the amount of polymerization competent G-actin in the cell, also rescue RacN17 border cell migration defects. The association of DIAP1 protein with Rac and profilin in S2 cells together with the finding that overexpression of DIAP1 can enhance activated Rac's effects on the actin cytoskeleton in cultured cells further support the conclusion that DIAP1 affects cell migration via Rac and the actin cytoskeleton. Additional support is provided by the finding that overexpression of Rac in border cells results in increased accumulation of DIAP1 protein and F-actin in vivo (Geisbrecht, 2004).
The effect of DIAP1 on border cell migration is clearly independent of its role in preventing apoptosis. The lack of apoptosis in th mutant follicle cell clones is striking since at other stages of Drosophila development, cells fail to survive in the absence of DIAP1. However, IAP proteins are thought to play a less critical role in survival of certain mammalian cells as well, where the current view is that the balance between proapoptotic and antiapoptotic BCL-2 family proteins is the deciding factor between life and death. The results presented here suggest that DIAP1 is not required for survival of every cell and tissue of the fly either. It may be that in both flies and mammals, different cell types have distinct requirements for particular classes of survival molecules (Geisbrecht, 2004).
Although the ability of DIAP1 to rescue RacN17 border cell migration defects is independent of its function in preventing cell death, and independent of its inhibition of effector caspases, the effects result from inhibition of the initiator caspase Dronc. The finding that inhibition of Dronc can rescue RacN17 border cell migration defects indicates that Dronc activity has a negative effect on migration. Since Dronc is a protease, the most parsimonious hypothesis would be that Dronc cleaves one or more proteins required for Rac-mediated cell motility. Previous studies have shown that Rac can be cleaved and inactivated by caspase 3 in lymphocytes. Therefore one possibility is that Rac itself is a Dronc substrate in border cells. A number of cytoskeleton-associated proteins are cleaved by caspases, including actin. Since reduced profilin levels was observed in th mutant follicle cells, it is also possible that profilin is a Dronc substrate, though no increased accumulation of actin or profilin was detected in cells overexpressing DIAP1. Further study will be required to pinpoint the physiologically relevant Dronc substrate in border cells (Geisbrecht, 2004).
The observation that two different dark mutant alleles cause mild border cell migration defects suggests that Dronc, which is thought to be constitutively active at a low level in most cells, contributes to normal migration. In fact, caspases have been shown to function in cell proliferation and differentiation in a variety of cell types, in addition to their better known role in promoting apoptosis. In some cases, caspase activity is required for terminal differentiation events that resemble incomplete apoptosis. For example, terminal differentiation of Drosophila sperm requires removal of much of the cytoplasm and requires caspase activity. Similarly, differentiation of mammalian lens cells and erythrocytes requires caspase activity. Other differentiation events, such as those of macrophages and skeletal muscle, do not overtly resemble apoptosis and yet require caspase activity. There must be some mechanism in such cells, and in border cells, to restrict the caspase activity to selected substrates so that apoptosis does not occur (Geisbrecht, 2004).
DIAP1 is a member of an evolutionarily conserved family of proteins that contain BIR domains. BIR domain-containing proteins are found in organisms from yeast to man and seem to have arisen in evolution prior to the apoptotic machinery. For example, in yeast, BIR1p is a protein required for proper chromosome segregation and cytokinesis. Yet the yeast genome does not encode an obvious caspase and yeast are not known to undergo apoptosis. In C. elegans, there is a BIR domain protein that does not suppress apoptosis when overexpressed. Reduction in the expression of this protein by RNA interference leads to defective cytokinesis and a phenotype that is very similar to loss of the worm formin protein. This is interesting since formin homology proteins can bind Rac, stimulate actin polymerization in concert with profilin, and promote cell migration. However it is not known if the C. elegans BIR domain protein interacts with formin or profilin, or whether it functions downstream of Rac. Taken together, these observations suggest that a primitive function of BIR domain proteins may have been regulation of cell division and the cytoskeleton (Geisbrecht, 2004).
It is well known that growth factors promote both survival and proliferation, as well as migration of specific cells. For example, Steel factor acting through the c-kit receptor tyrosine kinase regulates survival and proliferation of primordial germ cells and melanocytes in the mouse embryo. In addition, Steel factor and c-kit may contribute to guiding the embryonic migrations of these two cell populations. Conversely, overexpression of a factor that functions in repulsive guidance of Drosophila primordial germ cells causes excessive germ cell death. This intimate relationship between guidance and survival may exist to ensure that only those cells that migrate to the appropriate location survive and proliferate (Geisbrecht, 2004).
Rho family GTPases have also been demonstrated to affect both migration and survival. By activating gene expression through the JNK pathway, Rac1 protects COS 7 cells from apoptosis induced by ultraviolet light. Rac is also required for survival of cerebellar granule neurons. Another pathway required for both cell survival and Rac-mediated cell migration is the phosphatidylinositol 3-kinase pathway (PI3K). Activation of PI3K, and its downstream effector Akt, is capable of promoting neuronal survival in the absence of growth factors. Akt is also essential for Rac-mediated motility in mammalian fibroblasts. Akt is activated by Rac, and phosphorylated Akt colocalizes with Rac at the leading edge of fibroblasts. Therefore, there are several biochemical pathways that control, and possibly coordinate, cell survival and cell motility.
The mammalian formin homology protein FRL functions as a survival signal, in addition to its role in Rac-mediated regulation of the cytoskeleton. Overexpression of a truncated form of FRL, containing only the N-terminal Rac binding site, results in inhibition of cell growth and apoptosis in the macrophage cell line P388D1. These lines of evidence support the view that regulation of the cytoskeleton and cell survival are intertwined. The present study demonstrates that the inhibitor of apoptosis proteins, well known for their role in cell survival, can also promote cell migration, thus demonstrating a new and unexpected molecular link between survival and migration (Geisbrecht, 2004).
Paxillin is a prominent focal adhesion docking protein that regulates cell adhesion and migration. Although numerous paxillin-binding proteins have been identified and paxillin is required for normal embryogenesis, the precise mechanism by which paxillin functions in vivo has not yet been determined. An ortholog of mammalian paxillin in Drosophila (D-Pax) has been identified and a genetic analysis of paxillin function during development was undertaken. Overexpression of Dpax disrupts leg and wing development, suggesting a role for paxillin in imaginal disc morphogenesis. These defects may reflect a function for paxillin in regulation of Rho family GTPase signaling since paxillin interacts genetically with Rac and Rho in the developing eye. Moreover, a gain-of-function suppressor screen identified a genetic interaction between Dpax and center divider cdi in wing development. Cdi belongs to the cofilin kinase family, which includes the downstream Rho target, LIM kinase (LIMK). Significantly, strong genetic interactions were detected between Dpax and Dlimk, as well as downstream effectors of Dlimk. Supporting these genetic data, biochemical studies indicate that paxillin regulates Rac and Rho activity, positively regulating Rac and negatively regulating Rho. Taken together, these data indicate the importance of paxillin modulation of Rho family GTPases during development and identify the LIMK pathway as a critical target of paxillin-mediated Rho regulation (Chen, 2004).
Paxillin is a scaffolding protein found in focal adhesions. Targeted disruption of paxillin in mice results in an early embryonic lethal phenotype with defects in multiple mesodermally derived structures. The recent completion of the Drosophila genome revealed the evolutionary conservation of many of the key molecules found in focal adhesions, including integrins, paxillin, vinculin, FAK, p130CAS (see CAS/CSE1 segregation protein), and ILK. The Drosophila paxillin is predominantly expressed in embryos, pupae, and male adults. In situ analysis of staged embryos reveals a restricted expression pattern of Dpax. In particular, Dpax is highly expressed in tissues undergoing cell shape changes or cell migration. Overexpression of Dpax in late larval stages results in a pupal lethal phenotype with few escapers bearing malformed phenotypes, suggesting that Dpax also plays an important role during later stages of development (Chen, 2004).
A loss-of-function mutant of Drosophila paxillin has not yet been reported. Therefore, the UAS/GAL4 system was employed to investigate the function of Dpax in the later stages of development. As has been reported for Drosophila FAK, overexpressing Dpax results in a blistered-wing phenotype. In mammals, paxillin is a substrate of FAK in transducing signals from integrins. FAK regulates focal adhesion disassembly and has been shown to be involved in Drosophila Wnt4-mediated cell movement during ovarian morphogenesis and is also required for border cell migration during oogenesis. The function of Dpax in oogenesis is not clear; however, Dpax is also highly expressed in the border cells (Chen, 2004).
The blistered-wing phenotype is also found in integrin mutant flies. In the prepupal stage, the wing is a single epithelial sheet, and integrins have been suggested to play a regulatory role. As development progresses this sheet folds into a dorsal and ventral side, and the integrins play an adhesive role at these later stages. Using drivers that are expressed at different stages of development, the studies suggest that paxillin could be important for both the regulatory and adhesive functions of the integrins. Such functions would be consistent with studies of mammalian systems in which paxillin functions downstream of multiple integrins and can regulate both inside out and outside in signaling. In addition, both paxillin and FAK are important for focal adhesion turnover. Thus, too much paxillin or FAK may increase the turnover of focal complexes and perturb the stable adhesion between two epithelia, thereby resulting in the blistering phenotype (Chen, 2004).
Using a gain-of-function screen for modifiers that can rescue the Dpax-induced wing blistering, Cdi/TESK was identified. Like LIMK, Cdi/TESK phosphorylates the actin-depolymerizing factor cofilin and stabilizes F-actin. Cdi/TESK is highly homologous to LIMK in the kinase domain; however, a recent study has demonstrated that Cdi/TESK functions downstream of Rac1 during spermatogenesis (Raymond, 2004). Drosophila LIMK functions downstream of Rho1 in regulating disk morphogenesis (Chen, 2004). Dlimk and components in the Rho-LIMK pathway, including ssh, tsr, and bs/DSRF, also rescue the blistering phenotype. In addition, another regulator of SRF and actin, diaphanous, also shows genetic interactions with Dpax. Diaphanous is a direct effector of Rho which cooperates with LIMK to regulate SRF activation. All of these components play important roles in regulating F-actin synthesis. Taken together, these data indicate that it is possible that an increase in actin levels can prevent the increase in focal adhesion turnover caused by the excess level of paxillin, therefore suppressing the blistering phenotype. It is possible that simply overexpressing actin might be sufficient to rescue the blistering phenotype, although the results suggest that paxillin itself does not affect F-actin synthesis or actin organization. The ability of paxillin, however, to coimmunoprecipitate with LIMK and the increased cofilin phosphorylation in Pxl–/– MEFs suggests that paxillin can modulate LIMK function. These data, combined with the genetic and biochemical evidence that paxillin can regulate Rho, suggest that paxillin could act at multiple points to regulate the Rho pathway (Chen, 2004).
Interestingly, while modulation of some components downstream of Rho is able to suppress the blistering phenotype, overexpression of other components such as ROK does not alter this phenotype. While this could reflect insufficient expression levels or more complex regulation of ROK, the data suggest that paxillin's regulation of the Rho pathway may involve either modulation of only certain downstream components or a lack of function for these components in the paxillin-induced phenotypes (Chen, 2004).
Rho GTPases play an important role in regulating actin cytoskeleton organization. Genetic and biochemical analysis reveal that paxillin activates Rac signaling but inactivates Rho signaling. Previous binding and localization studies suggest that mammalinan paxillin may regulate Rac through its indirect association with at least two Rac exchange factors. Pix/Cool is linked to paxillin via PKL/Git2, the ARF-GAP, and overexpression studies with mutants of paxillin and other members of this complex have led to the suggestion that paxillin may be important for recruiting this complex to focal contacts. A second binding partner, Crk, can also link paxillin to Rac activation via a nontraditional exchange factor, Dock180. Mislocalization of one or both complexes in Pxl–/– mouse embryo fibroblasts (MEF)s could therefore lead to defects in Rac activation and subsequent defects in lamellipodium dynamics and migration. Both Pix/Cool and Crk localization were examined in rescued and Pxl–/– MEFs and only a minor decrease was detected in Cool and Crk positive peripheral adhesions in Pxl–/– cells. In MEFs, therefore, paxillin is not required for localization of these proteins to peripheral adhesions. This may be due to functional redundancy, as the paxillin family member Hic-5 can also bind the PKL-Pix complex and Crk can bind to other focal adhesion proteins, including p130Cas. In any case, mislocalization of these complexes is unlikely to account for the differences in Rac activation. In contrast, genetic studies of Drosophila have shown that deletion of a region encompassing the Drosophila homolog of Cool was able to suppress the Dpax-induced blistering. Thus, one potential mechanism by which paxillin may control Rac activation in Drosophila is through regulation of Pix/Cool. Since Rac and Rho have been shown to antagonize each other, it remains possible that in higher eukaryotes, paxillin could indirectly regulate Rac via regulation of Rho (Chen, 2004).
It is not clear how paxillin down-regulates Rho activity. Paxillin might be important for spatial regulation of Rho activity and/or controlling the activity or localization of a Rho GAP or GEF. Two Rho GAPs have been linked to mammalian paxillin. Graf is a Rho GAP that was originally identified as a Fak-binding partner, and a homolog of this protein has been identified in Drosophila studies. Since paxillin can interact with Fak, it is possible that loss of paxillin may somehow affect Graf localization or activation. While Fak localization to focal adhesions is less efficient in Pxl–/– MEFs, the effects are minimal and thus this is unlikely to account for the enhanced Rho activity. It is worth noting that it has recently been reported that mammalian paxillin binds to the p120 RasGAP and competes with p120 RasGAP for binding to p190 RhoGAP. It has been suggested that paxillin inhibits Rho by promoting the formation of free p190 RhoGAP. The Drosophila ortholog of p190 RhoGAP does not bind to the Drosophila p120 RasGAP. In addition, only minor changes in p190 localization to the leading edge were detected in Pxl–/– MEFs. Thus, paxillin may antagonize Rho function through multiple distinct regulatory mechanisms (Chen, 2004).
Taken together, these data suggest that while paxillin has the ability to interact with multiple proteins involved in diverse signaling pathways, a major function of this scaffolding protein in vivo is to regulate Rho family GTPases. Thus, misregulation of these GTPases is likely to account for the adhesion defects observed during development in mouse and Drosophila studies (Chen, 2004).
Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. This study shows glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. Evidence is provided for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. It is proposed that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury (Doherty, 2014: PubMed).
The etiology of autism is so complicated because it involves the
effects of variants of several hundred risk genes along with the
contribution of environmental factors. Therefore, it has been
challenging to identify the causal paths that lead to the core
autistic symptoms such as social deficit, repetitive behaviors,
and behavioral inflexibility. As an alternative approach,
extensive efforts have been devoted to identifying the convergence
of the targets and functions of the autism-risk genes to
facilitate mapping out causal paths. This study used a
reversal-learning task to measure behavioral flexibility in Drosophila
and determined the effects of loss-of-function mutations in
multiple autism-risk gene homologs in flies. Mutations of five
autism-risk genes with diversified molecular functions all lead to
a similar phenotype of behavioral inflexibility indicated by
impaired reversal-learning. These reversal-learning defects result
from the inability to forget or rather, specifically, to activate
Rac1 (Ras-related C3 botulinum toxin substrate 1)-dependent
forgetting. Thus, behavior-evoked activation of Rac1-dependent
forgetting has a converging function for autism-risk genes (Dong,
2016). This study investigates whether forgetting-dependent behavioral
inflexibility is a common phenotype for autism-risk genes and
whether behavior-dependent activation of Rac1 is the convergent
molecular target for these genes. To minimize genetic and
behavioral variations, flies for all genotypes had the same
isogenic background, and behavioral assays were performed using
the well-established balanced protocol. Aversive conditioning and
its associated reversal-learning task was used to investigate the
functions of five autism-susceptibility genes: Fmr1, Ube3a,
Nrx-1, Nlg4, and Tsc1. Four major
findings emerge from the current study. First, mutation and
RNAi-induced knockdown of these five autism-risk genes result in
strong reversal-learning defects independent of learning ability.
Second, these reversal-learning phenotypes are all caused by the
inability to forget the old memory. Third, all the autism-risk
gene mutants studied failed to trigger the behavior-dependent
activation of Rac1, a reported regulator of forgetting. Fourth,
the effects of these genes are confined within the mushroom body,
in which Rac1-dependent forgetting is involved. These results
suggest that the inability to activate Rac1-dependent forgetting
is a converging mechanism for multiple autism-risk genes (Dong,
2016). It is interesting that all five of the autism-risk genes with
diverse functions funnel to a deficit in Rac1 activation. These
five autism-risk genes have been reported to be involved in the
Rac1 signaling pathway. The cytoplasmic FMRP-interacting protein
(CYFIP) directly links Rac1 and FMRP to modulate cytoskeleton
remodeling; Tsc1 functionally regulates Rac1 activity; Ube3a
promotes Rho-GEF Pbl degradation via ubiquitination to affect Rac1
activation; and upon synaptic activation Rho-GEF Kal-7
disassembles from the Nrx-1/Nlg4/DISC1 complex to modulate the
Rac1 pathway. Several other autism-risk genes, such as Nlg1,
Nrx-4, P-Rex1, and Shank-3, have also
been reported to participate in the Rac1-signaling pathway. In
addition, when the geneenvironment interactions of 122 genes and
191 factors in the autistic context were analyzed by systems
biology, Rac1 was predicted to be a converging node that
genetically links to the neurobiology of autism. Taken together,
these findings indicate that Rac1 is a functional converging site
for autism-risk genes (Dong, 2016). Although autism is considered to be a developmental disorder,
emerging evidence points to the postdevelopmental effects of
autism-risk genes in adults. In this study, acute down-regulation
of these five autism-risk genes at the adult stage lead to
impaired behavioral flexibility with reduced reversal-learning and
resistant old memory. Thus, all these five autism-risk genes are
physiologically involved in regulating behavioral flexibility
(Dong, 2016). Synaptic vesicles (SVs) form distinct pools at synaptic terminals, and this well-regulated separation is necessary for normal neuro-transmission. However, how SV cluster in particular synaptic compartments to maintain normal neurotransmitter release remains a mystery. The presynaptic protein Neurexin (NRX) plays a significant role in synaptic architecture and function, and some evidences suggest that NRX is associated with neurological disorders, including autism spectrum disorders. However, the role of NRX in SV clustering is unclear. Using the neuromuscular junction at the 2-3 instar stages of Drosophila larvae as a model and biochemical, imaging, and electrophysiology techniques, this study demonstrate that Drosophila NRX (DNRX) plays critical roles in regulating synaptic terminal clustering and release of SVs. DNRX controls the terminal clustering and release of SVs by stimulating presynaptic F-actin. Furthermore, the results indicate that DNRX functions through the scaffold protein Scribble and the GEF protein Pixie to activate the small GTPase Rac1. A direct interaction was observed between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of Scribble and that Scribble bridges DNRX to DPix, forming a DNRX/Scribble/DPix complex that activates Rac1 and subsequently stimulates presynaptic F-actin assembly and SV clustering. Taken together, this work provides important insights into the function of DNRX in regulating SV clustering, which could help inform further research into pathological neurexin-mediated mechanisms in neurological disorders such as autism (Rui, 2017).
Neurons are the basic unit of the nervous system, and they communicate with each other through synapses. After being captured by sensory organs, neural signals pass between synapses in the form of neurotransmitters. As the vehicles of neurotransmitters, synaptic vesicles (SV) are essential for neurotransmission. SVs can be divided into three distinct pools according to their localization and function. The SVs adjacent to the active zone and ready to be released are referred to as the ready release pool. The second pool of vesicles is the exo/endo-cycling pool, which is also found close to release sites and supplies the ready release pool. Finally, the pool located away from the active zone, and that contains the majority of SVs, is referred to as the reserve pool and is considered to be a storage pool. Actin is a major part of the cytoskeleton that is required for maintaining the architecture of synapses as well as contributing to their function, and a significant amount of evidence has shown that the localization, translocation, and release of SVs can be altered by disturbing the polymerization of pre-synaptic actin (Rui, 2017).
The synapse is a highly specialized structure, and synaptogenesis is a highly complex process. Previous studies have shown that synaptic adhesive molecules play important roles in synaptogenesis and neurotransmission, and a number of synaptic cell adhesion molecules, including Neuroligins and Neurexins have been identified over the past few decades. Neurexin was first recognized as a receptor for α-latrotoxin, a black widow spider venom component that triggers massive neurotransmitter release. There are three neurexin genes in mammals, each of which has two promoters generating α-Neurexin and β-Neurexins, whereas there is only one neurexin-1 gene in Drosophila (dnrx). Recent studies both in Drosophila and mammals showed that Neurexin plays a significant role in synaptic architecture and function, and there is evidence suggesting that neurexin is associated with autism spectrum disorders (ASDs). The complexity and redundancy of the neurexin genes in mammals motivated the authors to focus on simpler model systems, such as Drosophila, to investigate the in vivo function of DNRX (Rui, 2017).
Neurexin has been shown to bind to several molecules, including the presynaptic scaffolding proteins Mint (Biederer, 2000), CASK (Sun, 2009), and LRRTM2 (de wit, 2009; Ko, 2009). Recently, DNRX also has been demonstrated to interact with the N-ethylmaleimide sensitive factor to regulate short-term synaptic depression and to interact with Spinophilin to maintain active zone architecture (Muhammad, 2015; Li, 2015). A typical trans-synaptic complex is formed by the heterophilic interaction of presynaptic Neurexins and postsynaptic Neuroligins, and these complexes have attracted much attention as scaffolding complexes that not only maintain the normal structure of the synapse but also function in passing signals across the synapses. It is thus clear that Neurexin is a multifunctional molecule. Despite the identification and characterization of these proteins that functionally associated with DNRX, understanding of the pathways including DNRX that control synaptic function are still incomplete, with other partners and mechanisms yet to be uncovered and analyzed (Rui, 2017).
This study has investigated the role of DNRX in the cluster and release of SVs at synaptic terminals. The effect of DNRX is mediated by presynaptic F-actin and there is a direct interaction between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of the tumor suppressor protein Scribble. Furthermore, Scribble bridges DNRX to DPix, forming a DNRX-Scribble-DPix complex to activate Rac1 and affect presynaptic F-actin assembly and SV clustering. Taken together, these studies provide novel insight into the mechanisms underlying the regulation of neurotransmitter release by DNRX (Rui, 2017).
Neurexin is a highly conserved cell adhesion molecule that is predominantly localized at the presynaptic terminal. Previous studies have shown that Neurexin plays a significant role in synaptic architecture and function. In addition, accumulating evidence has implicated Neurexin in Autism Spectrum Disorders (ASDs). ASDs are neurodevelopmental disorders characterized by deficits in communication and social interaction as well as restricted interests and repetitive and stereotypic patterns of behavior. However, the precise function and underlying molecular mechanisms of Neurexin in both normal physiology and ASDs remain unclear. Drosophila Scribble is a cytoplasmic scaffolding protein that was first recognized as a tumor suppressor that regulates epithelial cell adhesion and migration in mammals. Recently, it has been shown that Scribble is also localized in the nervous system both in invertebrate and vertebrate animals, and plays a role in synaptic plasticity and animal behavior, including learning, memory, social behavior, and olfactory behavior. However, how Scribble functions at the synapse remains unknown. In this study, this study revealed that DNRX interacts directly with the Scribble PDZ domains through the very C-terminal PDZ-binding motif to regulate presynaptic F-actin and SVs (Rui, 2017).
F-actin is highly enriched at synaptic terminals and is vital for SV traffic, localization, and release
For decades, F-actin emerged as the major cytoskeleton identified in presynaptic nerve terminals. Considering the especial location of F-actin at the internal space of presynaptic nerve terminals, it raised a hypothesis that F-actin regulates synaptic vesicle localization and release. Previous studies from several groups provided consistent evidence that after disrupting the polymerization of actin, pre-synapse affected SV traffic, localization, and neurotransmitter release. Moreover, cortical actin has been identified as a barrier for vesicles during the process of moving to the active zone in the presynaptic terminal. However, whether and how F-actin participates in the regulation of SV at synapse is still controversial. Therefore, further investigation will be necessary to determine the function of F-actin at synapse and especially for advances in understanding of the relationship with SV. By now the function of F-actin at synapse is still poorly understood. To better understand the effect of F-actin on SV this study assessed the cluster and release of SV after ablating the actin-associated genes and found the obvious defects of SV cluster and release. To further test this possibility, additional experiments are needed to elaborate the vital role of F-actin in SV regulation in future (Rui, 2017).
SV distribution and dynamics are essential for normal neural signal transmission and for synaptic plasticity both at peripheral and central synapses. Disruptions in SV function will lead to various forms of neurological disorders. The process of synaptic vesicle priming, docking, and fusion with the presynaptic membrane has been investigated extensively, and numerous molecules involved in this process have been identified. These include synaptotagmins, synapsins, synaptobrevins, and Munc18. However, how SV cluster in particular compartments and their role in regulating neurotransmitter release at the presynaptic terminal are unclear. The results from this study provide compelling evidences that DNRX plays an essential role in the distribution and release of SVs (Rui, 2017).
In this study, the data suggest the amount of F-actin is significantly reduced. Moreover, presynaptic Cortactin or the active form of DPak, key regulators of the actin cytoskeleton, are able to rescue the defects in SV distribution and spontaneous release frequency in the dnrx mutant. These results illustrate the essential role of presynaptic actin in regulating SVs localization and release. The results are consistent with the recent findings showing that Arp2/3 complex-mediated actin regulation is important for presynaptic neurotransmitter release (Rui, 2017).
Previous work has shown that Scribble is a scaffolding, tumor suppressor protein that through its PDZ domains interacts with a number of proteins, including β-catenin, βPix, and NOS1AP. Although NOS1AP can bind directly to the fourth Scribble PDZ domain, βPix has binding affinity to all four PDZ domains of Scribble. The present study used co-immunoprecipitation to show that DNRX can form a complex with Scribble in the Drosophila nervous system in vivo. DNRX and Scribble are co-localized in both central and peripheral nervous system during embryonic, larval, and adult stages. Importantly, these two proteins are highly expressed in the mushroom body of Drosophila, suggesting a key role in learning and memory. DNRX can directly bind to all four Scribble PDZ domains through its C-terminal PDZ-binding motif, similar to what is observed for βPix. These interaction results suggest that there may be competitions and cross-effect between DNRX and βPix. These interactions may also relate to the mutual effect on the protein level of Scribble and DNRX. Because the mRNA level of Scribble is not altered in the dnrx mutant, it is possible that when the DNRX or Scribble was absent, the complex becomes destabilized and subsequently degraded. Further experiments are needed to address this possibility (Rui, 2017).
What is the functional consequence of the Scribble and DNRX interaction? The present study provides evidence that Scribble may act as a bridge between DNRX and DPix to regulate the actin cytoskeleton. βPix is a GEF specific to Rac1/Cdc2, a key mediator of actin reorganization in response to various stimuli. In the mammalian system, Rac1 is locally activated in dendritic spines, and this spatial restricted activation is regulated by Pix (Zhang, 2005). The present study shows that the active form of Rac1 (Rac1-GTP) is reduced in the dnrx mutant, dnrx, and scribble knockdown flies compared with wild-type for the protein level of DPix in these lines were decreased, supporting the idea that the DNRX and Scribble interaction activates Rac1. Recent studies show that Rac1 plays a critical roles in animal behavior, particularly in the process of forgetting (36). In addition, Scribble has recently been reported to activate forgetting through Rac1 in Drosophila (35). Taken together, it is suggested that DNRX may also be involved in forgetting by regulating the Rac1 signaling pathway. Indeed, it has been demonstrated that Rac1 activation is defective in multiple autism-related gene mutations, including the dnrx gene, and that Rac1 has been proposed to be a converging node linked to ASD (36). Thus, the present study demonstrating that DNRX interacts with Scribble and DPix to regulate Rac1 provides direct mechanistic insight into not only the fundamental mechanisms underlying the roles of the neuroligin-neurexin complex in actin-mediated presynaptic regulation, but also the pathological mechanism of ASD (Rui, 2017).
The Drosophila homolog of the proto-oncogenic RAC protein kinase
(DRAC-PK) gives rise to two transcripts with the same coding potential,
generated by the use of two different polyadenylation signals. Because of the presence of a weaker initiator ACG codon upstream from the major
AUG, each transcript encodes two
polypeptides, such that the larger protein contains an N-terminal extension. Like the human isoforms,
DRAC-PKs possess a novel signaling region, the pleckstrin homology domain. DRAC-PK proteins
have a similar expression pattern, being regulated both maternally and zygotically, and are expressed
throughout Drosophila development. Antisera specific for recombinant DRAC-PK and for its C
terminus detect two polypeptides of 66 and 85 kDa in Drosophila extracts. The antirecombinant
antisera also recognize a polypeptide of 120 kDa from Drosophila, which apparently shared an epitope
related to DRAC-PK sequences. The role of p120 appears to be restricted compared with that of
DRAC-PK, since it is not detected in larvae or adult flies. There is no spatial restriction of
DRAC-PK expression during embryogenesis, suggesting that localized activation might be a regulatory
mechanism for its function. DRAC-PK possesses an intrinsic kinase activity that is approximately
8-fold higher in adult flies than in 0-3-h embryos undergoing rapid mitotic cycles (Andjelkovic, 1995).
A Drosophila homolog of the
serine/threonine kinase PAK (DPAK, now termed PAK-kinase) is a target of the Rho subfamily proteins Rac and Cdc42.
Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases.
DPAK binds to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities
in the distributions of DPAK, integrin, and phosphotyrosine suggest an association of DPAK with
focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK is
elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of
the embryo. The accumulation of cytoskeletal elements initiating cell
shape changes in these cells can be inhibited by expression of a dominant-negative DRacA
transgene. Leading-edge epidermal cells, which segment borders, express
particularly large amounts of DPAK. These cells undergo transient losses of cytoskeletal structures during dorsal
closure. DPAK may be regulating the cytoskeleton through its association with focal
adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase
signaling pathway recently demonstrated to be required for dorsal closure (Harden, 1996).
The rotund (rn) locus of Drosophila at cytogenetic position 84D3,4 has been isolated. Rotund protein 1.7 is similar to the product of the human n-chimaerin gene, which is
expressed in brain and testes. Recently, the GAP activity of n-chimaerin has been demonstrated and shown
to be specific for the Rac subfamily of the Ras oncoproteins. The Rac proteins have been implicated in
the regulation of secretory processes. In addition to being expressed in the imaginal discs, the m1.7
racGAP transcript is detected in developmentally specific germ line cells of the testes, the primary spermatocytes (Agnel, 1992).
The rotund gene in Drosophila melanogaster is associated with a gene coding for a RacGTPase-activating protein,
RnRacGAP. Cellular studies have shown that RacGAP proteins function as negative regulators of
substrate Rac proteins which, in turn, control the localization and polymerization state of actin within
the cell. Previous sequence analysis of rn RacGTPase genomic DNA and incomplete cDNA clones suggests that
at least two differentially spliced forms of the transcript exist: rnRacGAP(1) and rnRacGAP(2). Using
nested reverse transcription-polymerase chain reaction (RT-PCR) methods, the missing
exon and intron sequences have been cloned, and differences have been detected between rnRacGAP(1) and rnRacGAP(2)
involving 24 nucleotides (nt) of coding sequences and 119 nt of 3'UTR. This translates to a difference
of seven amino acids at the C-termini of the polypeptide products. In RT-PCR analysis, utilization of
form-specific primers provides a simple assay for the tissue specificity of expression of the two forms.
rnRacGAP(1) is the predominant species in the testes and is expressed at a low level in the ovary and
somatic tissues. rnRacGAP(2) is only very weakly expressed and is detectable solely in the testes (Hoemann, 1996).
RacGAP proteins have been shown to down-regulate members of the Rho/Rac subfamily, small
GTPases controlling actin network organization. Only one RacGAP protein, RnRacGAP, has been
identified in Drosophila. To examine RnRacGAP function, transgenic strains were generated expressing
RnRacGAP under the control of the heat-shock promoter hsp70. In cellularising embryos, ectopic
RnRacGAP induces lethality, associated with radical cell-shape changes, apical F-actin delocalization,
and inhibition of basal actin polymerization. Overexpression of RnRacGAP in pupae induces a number
of phenotypes with distinct critical periods of induction. These include wing shape and margin changes,
wing vein defects, disorientation of wing hairs and thoracic bristles, and abdominal segment fusion.
Thus, changes in cell shape/adhesion and reorganization of the actin network are sensitive to
overexpression of RnRacGAP throughout development in Drosophila (Guichard, 1997).
A Drosophila gene has been identified that has substantial sequence homology to a distinct class of
proto-oncogenes that includes DBL, VAV, Tiam-1, ost and ect-2. It has predicted Rho or Rac guanine
exchange factor (Rho/RacGEF) and pleckstin homology (PH) domains with the PH immediately
downstream of the Rho/RacGEF. Rho/RacGEFs catalyze the dissociation of GDP from the Rho/Rac
subfamily of Ras-like GTPases, thus activating the target Rho/Rac. Members of the Rho/Rac subfamily regulate organization of the actin
cytoskeleton, which controls the morphology, adhesion and motility of cells.
Message from this gene is found throughout oogenesis and embryogenesis. Of particular interest,
message is most abundant in furrows and folds of the embryo where cell shapes are changing and the
cytoskeleton is likely to be undergoing reorganization (Werner, 1997).
A motif-based search method was used to identify putative effector
proteins for Rac and Cdc42. A search of the GenBankTM data base for similarity with the minimum
Cdc42/Rac interactive binding (CRIB) region of a potential effector protein p65PAK has identified
over 25 proteins containing a similar motif from a range of different species. These candidate
Cdc42/Rac-binding proteins include family members of the mixed lineage kinases (MLK), a novel
tyrosine kinase from Drosophila melanogaster (DPR2), a human protein MSE55, and several novel
yeast and Caenorhabditis elegans proteins. Two murine p65PAK isoforms and a candidate protein
from C. elegans, F09F7.5, interact strongly in
a filter binding assay with the GTP form of both Cdc42 and Rac, but not with Rho. Three additional candidate proteins, DPR2, MSE55, and MLK3 showed binding
to the GTP form of Cdc42 and weaker binding with Rac, and again no interaction with Rho. These
results indicate that proteins containing the CRIB motif bind to Cdc42 and/or Rac in a GTP-dependent
manner, and they may, therefore, participate in downstream signaling (Burbelo, 1995).
Dorsal closure depends on the activities of a Jun amino (N)-terminal
kinase kinase (JNKK) encoded by the hemipterous gene, and of a JNK
encoded by basket. Hep is required for cell determination in the leading edge of
migrating epithelia, by controlling specific expression of the puckered gene in
these cells. puc encodes a protein related to vertebrate dual-specificity MAPK phosphatases of the CL100 family (E. Martin-Blanco, A. Gampel, and A. Martinez Arias, pers. comm. to Glise, 1997). During dorsal closure, decapentaplegic is expressed in the row of cells
making up the leading edge of the epithelia. The small GTPases
Dcdc42, Drac1, and the Hep JNKK control decapentaplegic expression in this migratory process. Activated Drac1 and Dcdc42 induce distinct, although partly overlapping, responses. Dcdc42 appears to be a good inducer of ectopic puc and dpp expression in ectodermal cells located more ventrally, whereas Drac1 seems more active in cells nearest to the leading edge.
Appropriate dpp and puc expression in the leading edge also depends on the inhibitory
function of the puc gene. puc acts as a repressor of dpp expression in the ectoderm, likely acting to inhibit Basket, the Jun N-terminal kinase. In puc mutants ectopic expression of puc and dpp is induced in the ectoderm, that is, outside the normal domain of puc expression in the leading edge. In addition, puc (but not dpp) is expressed ectopically in amnioserosa cells. These observations indicate a cell non-autonomous effect of puc mutations. The data suggest that the leading edge is the source
of a JNK autocrine signal, and exclude a role of Dpp as such a ligand. Dorsal closure
couples JNK and Dpp signaling pathways, a situation that may be conserved in
vertebrate development (Glise, 1997).
Rac activation is thought to be important for stimulating dorsal
closure because expression of dominant negative forms of Rac (DN Rac)
or Cdc42 inhibits dorsal closure in the Drosophila embryo. Since
activated Jra/Djun rescues the defect in dorsal closure induced by
expression of DN Rac, Rac probably functions upstream of
JNK activation to stimulate dorsal closure. To begin
to address the mechanism whereby Rac and Misshapen cooperate to
activate JNK, cultured cells were transfected with either Msn or NIK,
together with DN Rac and an epitope-tagged JNK, and kinase activity
assays were performed on JNK precipitates. Although overexpression of either
NIK or msn leads to a four- to five-fold increase in JNK
activation, coexpression of DN Rac markedly decreases JNK activation. Because PAK
family Ste20 kinases are activated by GTP-bound Cdc42 and Rac, it had been assumed that this family
of Ste20 kinases rather than an SPS1 Ste20 kinase family member would cooperate with Rac to
activate JNK. Thus, discovery of the role of Misshapen in conveying Rac signals to JNK has stimulated consideration of new paradigms for how
Rac functions to activate JNK. It is not thought that Rac activates Msn directly. Unlike PAK family
members, Msn does not contain a consensus Rac-binding motif and no binding of Msn to
activated Rac in vitro can be detected. Rather, it is hypothesized that Rac cooperates with Msn
to activate a downstream MKKK. MKKKs have been shown to bind GTP-bound Cdc42 or Rac. Thus, Rac may cooperate with Msn to regulate a downstream MKKK in a manner similar
to the way Ras cooperates with a yet to be defined kinase to activate RAF. In this model, binding of an
MKKK to activated Rac would facilitate interaction of this MKKK with Msn, thereby enabling its
activation by Msn. However, the possibility cannot be excluded that Rac and Msn activate
parallel pathways converging on JNK activation (Su, 1998).
A novel Drosophila gene, DRacGAP/RacGAP50C
has been identified that behaves as a negative
regulator of Rho-family GTPases Rac1 and Cdc42. Reduced function of DRacGAP
or increased expression of Rac1 in the wing imaginal disc cause similar effects
on vein and sensory organ development and cell proliferation. These effects
result from enhanced activity of the EGFR/Ras signalling pathway.
In the wing disc, Rac1 enhances EGFR/Ras-dependent activation of MAP Kinase in
the prospective veins. Interestingly, DRacGAP expression is negatively regulated
by the EGFR/Ras pathway in these regions. During vein formation, local DRacGAP
repression would ensure maximal activity of Rac and, in turn, of Ras pathways in
vein territories. Additionally, maximal expression of DRacGAP at the
vein/intervein boundaries would help to refine the width of the veins. Hence,
control of DRacGAP expression by the EGFR/Ras pathway is a previously
undescribed feedback mechanism modulating the intensity and/or duration of its
signalling during Drosophila development (Sotillos, 2000).
Evidence is presented for the
cooperation of Rac and Ras signaling pathways in the context
of a whole organism. A Drosophila gene,
DRacGAP, is described that encodes a putative GAP for Rac and Cdc42
GTPases. Both DRacGAP and DRac1 are
involved in the control of cell proliferation. Moreover, reduced
activity of DRacGAP or overexpression of DRac1 in the wing
imaginal disc cause similar defects: widening of veins,
development of extra sensory organs (SOs), apoptosis and the
appearance of enlarged cells that differentiate multiple hairs
with abnormal polarity. These phenotypes result
from DRac1 enhancement of epidermal growth factor receptor
(Egfr)/Ras signaling. The Egfr/Ras pathway pathway, which
operates through activation of the Ras/Raf/MEK/MAP kinase
cascade controls multiple developmental processes and is accurately regulated.
Indeed, this pathway controls the expression of its own
negative and positive regulators. Interestingly, expression of DRacGAP is repressed by
Egfr/Ras signaling in the prospective veins and accumulates
at the vein/intervein boundaries. These results suggest that
control of DRacGAP expression by the Egfr/Ras pathway
provides a new mechanism to modulate the intensity of this
pathway during Drosophila development (Sotillos, 2000).
DRacGAP gives rise to a unique transcript of 2.3 kb present
throughout development. The expression pattern of
DRacGAP is highly dynamic. It is ubiquitous during the initial
stages of embryogenesis and becomes restricted, after germ band
retraction, to the central and peripheral nervous system. In the early second instar wing imaginal disc, DRacGAP
expression occurs mostly in the presumptive wing region. Afterwards, DRacGAP mRNA accumulation is widespread
in all imaginal discs. At late third instar
DRacGAP is expressed in the eye-antenna disc in two stripes of
cells located at, and ahead of, the morphogenetic furrow and in
several rings of cells in the presumptive antenna. In
the wing disc, DRacGAP mRNA accumulates in the presumptive
interveins, where it persists in pupae, being stronger at
the vein/intervein boundaries (Sotillos, 2000).
Overexpression of DRacGAP in the wing disc interfers with
vein development. Expression of dominant negative (DN) DRacGap in the wing
disc results in loss of wing tissue (wing
notching), veins of increased width, fusion of adjacent veins
and appearance of vein material connecting neighbouring veins. In addition, wings have extra SOs along the vein L3
and in the proximal regions of veins L4 and L5. DRacGAP DN wings showed enlarged cells (indicated
by low trichome density) and alteration in the number
and polarity of the trichomes. The enlarged cells are
also visible in the imaginal disc (phalloidin staining, which
labels cortical actin) indicating the early onset of this defect (Sotillos, 2000).
By analogy with its closest homologs,
DRacGAP should regulate RacGTPase. Indeed, overexpression of wild-type Rac1 causes vein thickening,
development of extra SOs and wing enlargement. Coexpression of Rac1 and DN DRacGAP
has a synergistic effect,
suggesting that DRacGAP reduces Rac1 activity.
Consistently, the lethality of larvae overexpressing Rac1 is
reversed by coexpression of RacGAP and the
wing size reduction associated with a decreased activity of
Rac1 in DN Rac1 flies
is enhanced by coexpression of overexpression of DRacGAP (Sotillos, 2000).
Overexpression of DN DRacGAP or Rac1
causes vein enlargement and the appearance of extra
SOs, two structures requiring Egfr/Ras/Raf/MAPK activity. Since Rac and
Ras pathways cooperate in mammalian cells in the control of
cell proliferation, an investigation was carried out to see whether the
phenotypes associated with increased Rac signaling could be due
to overactivity of the Ras pathway. This appears to be the case,
since a reduction in Egfr signaling (by expression of DN Raf) reduces vein, wing
notching and large cell phenotypes. Similarly, when levels of the Egfr
activators rhomboid and vein (vn) are decreased, the wing notching associated with DN DRacGAP
expresson is substantially corrected. In contrast,
activation of Egfr signaling enhances the mutant phenotype
of DN DRacGAP flies. Thus, although flies heterozygous for
mutant argos, a repressor ligand of Egfr, are phenotypically wild type, its
combination with DN DRacGAP significatively increases the
number of campaniform sensilla of DN DRacGAP flies. These
results further suggest that the efficacy of the Egfr pathway
is enhanced by increased Rac activity. Rac signaling ultimately
activates MAPK since expression of DN DRacGAP widens
the domains of accumulation of dp-ERK in the presumptive
veins and spreads them into the interveins. This effect
is enhanced by coexpression of Rac1 (Sotillos, 2000).
Expression of DRacGAP appears to be decreased in the
domains of Egfr activation. This is most apparent in the
notum region of second instar wing disc, where the
Egfr activator vein is expressed, and in the
presumptive veins of third instar wing disc, territories
of maximal Egfr signaling. This observation suggests that the Egfr pathway may repress the expression of DRacGAP. In agreement with this notion,
expression of DRacGAP is either decreased or enhanced in
cells ectopically expressing Ras V12 or argos in which
the EFGR/Ras pathway is activated or repressed, respectively (Sotillos, 2000).
The enlarged cell size of wing cells overexpressing Rac
and the small size of wings with reduced Rac function indicate a role for
Rac in cell proliferation control. In mammalian cells, Ras and Rac pathways
cooperate in stimulating cell proliferation. Similarly, Drosophila Rac1 and Ras also appear to cooperate in this process since the reduced size of the
wings of DN Raf-expressing flies is largely normalized by reduction of
DRacGAP function or by overactivity of
Rac1. The induction of cell death by DN DRacGAP, where Rac activity is
upregulated, is in apparent contradiction with these results. However, note that in mammalian cells, quantitative variations in the level of Ras signaling can cause very different effects. Thus, instead of cell proliferation, high levels of
Ras signaling may induce cell cycle arrest
at G1 and apoptosis as part of a cell self
protective mechanism. In that situation, G1 arrest is caused by
Raf-dependent induction of p21 WAF1/CIP1
expression, which inhibits Cdk activity and
indirectly represses CycE transcription. The situation appears to be very similar in
Drosophila. Cell death of DN DRacGAP flies
could be attributed to their arrest at the G1 stage, since the phenotype is
rescued by expression of CycE. Moreover, the accumulation of
p21 causes apoptosis, enlargement of cell size and polarity
defects, phenotypes that are corrected by expression of CycE. Accordingly, it is
hypothesised that induction of cell cycle arrest and apoptosis by
overactivity of the Rac pathway could be a consequence of
increased Ras signaling. Rac would potentiate the reduced Raf
signaling occurring in DN Raf flies, allowing wing disc cells to
proliferate, but in the presence of wild-type Raf, overactivity
of Rac would enhance Raf signal to such a high level as to
induce p21 expression and ultimately, apoptosis. This interpretation is supported
by the partial rescue of the wing notching phenotype of
DN DRacGAP flies in a ve;vn heterozygous background and in
DN Raf flies where Egfr/Ras signaling is reduced (Sotillos, 2000).
Interestingly, in the Drosophila wing disc, DRacGAP
transcription is repressed by the Egfr/Ras pathway.
Activity of this pathway is finely tuned by its control of the
expression of its own inhibitors and activators. The results indicate that Ras signaling can self-stimulate through activation of the Rac pathway by
repression of DRacGAP. During vein formation, the
Egfr/Ras pathway, once it has attained a certain threshold,
should repress expression of DRacGAP in the prospective vein
regions, thus ensuring maximal activity of Rac and, in turn, of
Ras pathways in these territories of the imaginal wing disc,
which should trigger vein differentiation. In contrast, maximal
expression of DRacGAP at the vein/intervein boundaries
should locally decrease Rac and Ras signaling, and in
collaboration with Notch and Dpp pathways help to refine the final width of the veins. Hence, this regulatory loop is another feedback mechanism
modulating the activity of the Egfr/Ras pathway during
Drosophila development (Sotillos, 2000).
Among the putative Rho/Rac effector targets in mammals
are the protein kinase
N/protein kinase C-related
kinase (PKN/PRK) family of
serine/threonine kinases. PKN (also referred to as PRK1) and the closely related protein PRK2 together account for the vast majority of Rho-binding autokinase activity detected in most mammalian tissues. The carboxy-terminal catalytic domains of
these kinases are highly homologous to the PKC family kinases, but they
possess unique amino-terminal regulatory sequences, including three
leucine zipper-like repeats shown to be important for the interaction with the Rho GTPase. These proteins also interact detectably with the Rac GTPase, suggesting that they may be shared effector targets of the
Rho and Rac GTPases.
Despite the identification of closely related PKN
homologs in several organisms, the precise biological function of these putative Rho targets
remains unknown. The ability of the Rho GTPases to regulate cell morphology and motility
suggests that these proteins and their associated signaling pathway
components are likely to perform functions essential to the normal
morphogenesis of developing multicellular organisms. Dorsal closure (DC) does
not require new cell divisions, and appears to depend solely on
dramatic cell shape changes within a subset of epidermal cells. These
shape changes are initially restricted to two symmetric rows of
epidermal cells, known as the leading edge (LE) cells, and are followed
by the stretching of the more lateral epidermal cells, ultimately
resulting in the meeting of the two rows of LE cells at the dorsal midline.
Three classes of genes have been implicated in DC; namely, the Rho
family of GTPases; the c-Jun amino (N)-terminal kinase (JNK) cascade
components, including the decapentaplegic (dpp) signaling
pathway genes, and several membrane-associated proteins. The recently described Drosophila loss-of-function Rho1 mutant is defective for DC, and
homozygous mutant embryos exhibit an obvious hole in the
dorsal-anterior portion of the larval epidermis.
Although Rac1 loss-of-function mutants have yet to be
reported, overexpression of Rac1N17, a dominant-negative form
of Rac1, in developing Drosophila embryos, results in a DC defect. Similar results have also been reported
for Cdc42N17 (Lu, 1999 and references).
Four Drosophila
homologs of the mammalian JNK pathway genes [hemipterous (hep; JNKK), basket (JNK), Djun, and kayak (Dfos)] are all required for DC. Mutations in
any of those genes result in a dorsal-open phenotype very similar to
that seen in either the Rho1 mutants or embryos overexpressing
dominant-negative Rac1 or Cdc42. Furthermore, disruption of the JNK
pathway in hep mutants abolishes the expression of two
independent downstream target genes of Djun, dpp and
puckered (a MAP kinase phosphatase), which are also required for DC. The
hep mutation also blocks an increase in dpp
expression in the LE cells induced by expression of activated Rac1V12 in those cells. These results have led to a model for
DC in which Rac1 (and possibly Cdc42) signals through the JNK pathway
to activate the expression in LE cells of Dpp, a secreted ligand of the
TGF-beta receptor. In turn, Dpp relays an instructive signal to
initiate stretching of the more lateral epidermal cells. The signaling role of Rho1 or downstream effector targets
of Rho1 in this process is unknown. A Drosophila homolog of the mammalian PKN family kinases,
Drosophila Pkn is shown to bind specifically to both Rho1 and Rac1 GTPases in a GTP-dependent manner, and its kinase activity is promoted
by both interactions, suggesting that Rho1 and Rac1 GTPases can
utilize Pkn as a downstream effector target. Both Rho1 and
Rac1 bind Pkn through the amino-terminal region of the protein. Significantly, it appears that Rho1 and Rac1 interact with Pkn
through distinct binding sites. A loss-of-function mutation in the Drosophila Pkn gene leads specifically to a DC defect during embryogenesis. However, this Pkn-mediated DC pathway is
independent of the Rac-JNK-Dpp pathway; rather, the Pkn-mediated DC pathway appears to act
coordinately with the Rac-JNK-Dpp pathway to regulate epidermal cell shape changes
during morphogenesis of the Drosophila embryo (Lu, 1999 and references).
The predicted Drosophila Pkn protein sequence is closely
related to both human PKN and PRK2 (60% overall identity to both) and has all of the conserved features found in other PKN family members, including the amino-terminal negative
regulatory pseudosubstrate motif, the three
leucine zipper repeats (HR1a, HR1b, and HR1c) that mediate GTPase
binding, a central conserved
region of unknown function found in the PKC-eta kinases, and the carboxyl-terminal PKC-like kinase domain (Lu, 1999).
The expression pattern of the Drosophila Pkn gene is highly
dynamic during embryogenesis. An in situ hybridization analysis of wild-type embryos reveals that Pkn mRNA is
abundant at the blastodermal stage, suggesting that it is maternally
loaded. At stage 13, when DC is normally initiated, the most prominent expression is seen in the dorsal LE cells and in two pairs of discontinuous stripes on the epidermis of each segment. However, the
expression becomes more restricted in later stages and can only be
detected in the anterior and posterior spiracles, the pharynx, and the
mouth tip at stage 16 (Lu, 1999).
Although homozygous Pkn mutant embryos do not exhibit
obvious developmental defects prior to stage 13, ~10% of them die
as embryos with an obvious hole in the anterior region of the dorsal
epidermis. This dorsal-open phenotype closely resembles the
DC defects observed previously with loss of function of the
Rho1 gene and several components of the Rac-mediated
JNK cascade. To examine the requirement for maternally loaded Pkn mRNA,
germ-line clone (GLC) mutants of Pkn were generated:
~50% of the mutant embryos derived from these clones are found to
display the same DC defect. Phenotypic analysis of these
embryos with various histological markers reveals that they are
correctly patterned and do not exhibit any detectable defects of the
central or peripheral nervous system or the somatic musculature, suggesting that Pkn is not required prior to DC (Lu, 1999).
According to the current working model of DC, the Rac1 and Cdc42
GTPases activate the downstream JNK cascade kinases to induce the
expression of several genes in the LE cells, including dpp. Thus, DPP mRNA expression in the LE cells is a convenient assay of activation of the JNK cascade in
vivo. Because Pkn functions biochemically as a Rac1 effector target,
and mutations in Pkn cause a DC defect similar to that seen
with JNK pathway mutants, it was of interest to determine whether Pkn is
required for the previously established Rac-JNK-Dpp pathway. Therefore, the expression of DPP mRNA was examined in
Pkn mutant embryos. Mutations in the JNK cascade gene hep
result in embryos that fail to express detectable levels of
DPP mRNA in the LE cells. In contrast to the loss of dpp
expression seen in hep mutant embryos, expression of
dpp in the LE cells of Pkn mutants is not detectably
affected, indicating that Pkn is not
required for the previously reported Rac-JNK-Dpp pathway. This
conclusion was also verified by examination of the expression of beta-galactosidase from a puckered-lacZ enhancer trap, which
has been shown previously to be eliminated in a hep mutant
background. Pkn mutant embryos
that exhibit an obvious dorsal-open phenotype retain normal levels of
puckered-lacZ expression, confirming that the
Rac-JNK pathway leading to transcriptional activation is not
detectably affected by the absence of Pkn (Lu, 1999).
The shapes of epidermal cells of Pkn mutant
embryos were examined. All epidermal cells adopt an unstretched polygonal shape following an initial apparently normal LE
cell stretching, similar to that seen in the JNK pathway
mutants, such as hep. Thus, it appears that while
both Rac-JNK- and Pkn-mediated signals are required for the stretching
of epidermal cells required for DC, they are associated with distinct
pathways. Because the expression of Pkn mRNA is enriched specifically
in the LE cells prior to DC, the possibility that Pkn
expression is regulated by the JNK pathway was examined. However, Pkn
expression is unaffected in hep mutant embryos (Lu, 1999).
Accumulating evidence suggests that the Rac1 GTPase, as well as some
of the JNK pathway components, performs a function in the LE cells that
may be distinct from the regulation of gene expression, but is
necessary for the stretching of the LE cells. Because Pkn is a biochemical effector of the
Rac1 GTPase that mediates DC, but is not required for dpp or
puckered gene expression, the possibility was tested that the
Pkn mutant interacts genetically with the JNK pathway
component, basket. Removal of one copy of the
basket gene from a Pkn mutant GLC background
significantly increases the frequency of dorsal-open embryos, suggesting that both Pkn and JNK activities converge at some point to affect a related aspect of the DC process (Lu, 1999).
The Rho1 GTPase has also been implicated in DC.
Null alleles of Rho1 exhibit a DC defect that closely resembles that seen in Pkn mutants although the relevant Rho1-mediated pathway in this process has not been established. Because
the cell shape changes in the LE cells appear to initiate the DC
process, and Pkn expression is enriched in the LE cells just prior to
DC, the possibility was explored that a Rho1-Pkn signal
mediates shape changes in those cells. To start, the
requirement for Rho1 activity was examined specifically in the LE cells. A dominant-negative form of Rho1
(Rho1N19) is expressed in the LE cells of wild-type embryos. More than 60% of these
embryos display a dorsal-open phenotype very similar to that seen in
the Rho1, Pkn, and JNK pathway mutants. Similar to Rho1 and
Pkn mutants, but not to a hep mutant, embryos
expressing Rho1N19 in the LE cells exhibit normal levels of
DPP mRNA expression in the LE cells. Moreover, all
of the epidermal cells in these embryos ultimately adopt an unstretched
polygonal shape following a normal initial LE cell stretching at early
stage 13. This result demonstrates clearly that expression of the dominant-negative Rho1 in the LE cells does not cause a dorsal-open phenotype by nonspecifically blocking the Rac-JNK-Dpp pathway and
suggests that a Rho1-mediated second instructive signal is generated in
the LE cells, which together with Dpp, is required for the stretching
of the more lateral-ventral cells (Lu, 1999).
In light of the fact that Drosophila Pkn interacts equally
well with the activated Rac GTPase, it is possible that a Rac-Pkn interaction contributes to DC. However, the lack of a
Rac1 loss-of-function mutant in Drosophila makes it
difficult to examine the specific role of that interaction. Because the
JNK pathway mutants are also associated with a defect in stretching of
the LE cells, it has been suggested that components of the JNK pathway may mediate a Rac-dependent cell stretching signal that is unrelated to
transcriptional regulation. It is difficult to imagine how Pkn
could transduce a signal from Rac to this JNK-mediated cell shape
change pathway and yet not be required for the Rac-JNK transcriptional
pathway. However, it is possible that Pkn can transmit a Rac signal
independent of the JNK-Dpp pathway. Indeed, recent evidence suggests
that the Drosophila gene Myoblast city, which is
required for DC, encodes a Rac-specific activator that does not appear to regulate dpp expression
(Nolan, 1998). This observation suggests that Rac may perform
multiple functions in dorsal closure (Lu, 1999).
Significantly, there does seem to be some cross-talk between the
Pkn-mediated signaling pathway and the JNK pathway. Removal of one copy of
basket from a Pkn mutant GLC background significantly
increases the frequency of dorsal-open embryos. This result suggests
that some component of JNK cascade signaling is sensitive to the
activity of Pkn. Taken together with the fact that Rho1 generates a
JNK-Dpp independent signal in the LE cells that is required for DC, it is clear from these studies that distinct but coordinated signaling pathways mediated by the Rho and Rac GTPases within the LE cells are
essential for normal DC, and that Pkn is a strong candidate for an
effector that mediates signals downstream of both GTPases (Lu, 1999).
spire is a maternal effect locus that affects both the dorsal-ventral
and anterior-posterior axes of the Drosophila egg
and embryo. It is required for localization of determinants
within the developing oocyte to the posterior pole and to
the dorsal anterior corner. During mid-oogenesis, spire
mutants display premature microtubule-dependent
cytoplasmic streaming, a phenotype that can be mimicked
by pharmacological disruption of the actin cytoskeleton
with cytochalasin D. spire has been cloned by transposon
tagging and is related to posterior end mark-5, a gene from
sea squirts that encodes a posteriorly localized mRNA.
Spire mRNA is not, however, localized to the posterior
pole. Spire also contains two domains with similarity to
the actin monomer-binding WH2 domain, and Spire binds to actin in the interaction
trap system and in vitro. In addition, Spire interacts with
the rho family GTPases RhoA, Rac1 and Cdc42 in the
interaction trap system. This evidence supports the
model that Spire links rho family signaling to the actin
cytoskeleton (Wellington, 1999).
Previous work has shown that in spir mutants, the
microtubules bundle at the cortex prematurely, during stage
8, and this bundling of the microtubules is accompanied by
rapid, microtubule-dependent swirling of the cytoplasm. Both the bundling of
microtubules and cytoplasmic streaming are normally seen
later in stage 10 wild-type oocyte. A bi-directional signaling process occurs between the oocyte
and the posterior follicle cells to establish the posterior pole of
the egg.
Phenotypes indicative of a defect in this signaling process
include transformation of the posterior follicle cells into an
anterior follicle cell fate; misorganization of the microtubules
at stage 6; localization of Oskar mRNA to the center of the
oocyte; localization of Bicoid mRNA to the posterior pole, and
premature cytoplasmic streaming. The posterior follicle cell fates are established correctly in
spir. In contrast to a previous report, no central spot of Oskar mRNA
staining is observed in spir mutants. Finally, Bicoid mRNA localization
appears relatively normal in spir. These results
suggest that signaling between the posterior follicle cells and
the oocyte is not abnormal in spir mutants. In spir mutant oocytes, microtubules sometimes show abnormal
distributions during stage 6, but it is believed that
this probably reflects an earlier manifestation of the known spir
microtubule defect (Wellington, 1999 and references therein).
WH2 domains, like those found in Spir, have been found
in the Wiskott-Aldrich syndrome protein (WASP), verprolin,
Scar-1 (see Drosophila SCAR), and a number of other proteins of unknown function. The WH2 domains of N-WASP and of Scar1
have been shown to bind directly to G-actin in vitro. In
addition, Spir binds to unpolymerized
actin in vitro.
Although Spir is capable of binding to actin monomers
through its WH2 domain, no defects in the
actin cytoskeleton have been observed in spir mutants, suggesting a number of
possibilities. The defects may be in actin structures that are
difficult to observe, such as the cell cortex. In fact, spir phenotypes can be mimicked by treatment with
cytochalasin D, a drug that affects the polymerization state of
actin; defects in the actin
cytoskeleton in cytochalasin D-treated oocytes have not been observed. Alternatively,
spir may act downstream of the actin cytoskeleton and, thus,
not change it. Finally, in vitro experiments have shown that, in
the absence of the neighboring cofilin homology and acidic
domains, the two WH2 domains of N-WASP have either no
effect on or slowly depolymerize filamentous actin. Since Spir is lacking the cofilin homology
and acidic domains, Spir may have only minor or no
effect on filamentous actin (Wellington, 1999 and references therein).
It is becoming more apparent that a relationship exists
between the actin cytoskeleton and premature microtubule-dependent
cytoplasmic streaming. The premature cytoplasmic streaming phenotype of spir can be
mimicked by the addition of cytochalasin D,
a drug which depolymerizes actin filaments. Additional
evidence that the actin cytoskeleton is involved in repressing
microtubule bundling and streaming comes from analysis of
mutant phenotypes of genes linked to the actin cytoskeleton.
In addition to spir,
mutants in chickadee, which encodes profilin and capu, which
is thought to bind to profilin, also exhibit these microtubule
behaviors. While phenotypes for cdc42 and rac1 have been
described during oogenesis, their effects on patterning during
oogenesis are unknown. The
finding that Spir interacts with rho family GTPases suggests
that at least one of the rho family GTPases is functioning in
patterning. Further genetic and biochemical studies will be
required to determine the nature of Spir's interaction with rho
family GTPases in vivo.
The analysis of spir suggests that rho family GTPAses and
actin function with Spir in patterning the Drosophila oocyte.
Further studies on spir should elucidate the role of rho family
GTPases and the actin cytoskeleton in patterning during
oogenesis (Wellington, 1999).
Axon terminals change morphology with differentiation to become mature synapses. A molecule that might regulate this process has been identified by a screen of Drosophila mutants for abnormal motor activities. The still life (sif) gene encodes a protein homologous to guanine nucleotide
exchange factors, which convert Rho-like guanosine triphosphatases (GTPases) from a guanosine diphosphate-bound inactive state to a guanosine triphosphate-bound active state. The Sif proteins are found adjacent to the plasma membrane of synaptic terminals. Expression of a truncated Sif protein results in defects in neuronal morphology and induced membrane ruffling with altered actin localization in human KB cells. Thus, Sif proteins may regulate synaptic differentiation through the organization of the actin cytoskeleton by activating Rho-like GTPases (Sone, 1997).
The structure and localization of Sif suggest that Sif activates
Rho-family G proteins in the periactive zones. However, the
family comprises several members including Rac, Cdc42
and Rho, each playing a distinct role in a variety of cellular
events. It
is therefore important to estimate which member of the family
is activated by Sif in the periactive zone. The
GEF activity of Sif was assayed using Sif's catalytic ability to
dissociate [3H]GDP from mammalian RAC1, CDC42 and
RHOA, employing bacterially expressed a glutathione-S-transferase
(GST)-fusion protein carrying the DH domain and adjacent
Pleckstrin-homology (PH) domain of Sif. The
GST-SIF stimulates the dissociation of GDP from RAC1 both
in a time-dependent and dose-dependent manner, but does not show the activity for CDC42 and RHOA. Thus, these data demonstrate that Sif specifically
activates Rac1 in vitro, suggesting that Sif activates Rac in
the periactive zones (Sone, 2000).
A cell-adhesion molecule Fasciclin 2, which is required for synaptic growth, and Still life (Sif), an activator of Rac, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. betaPS integrin and Discs large, both involved in synaptic development, also decorate the zone. However, Shibire (Shi), the Drosophila dynamin that regulates endocytosis, is found in the distinct region. Mutant analyses show that sif genetically interacts with Fas2 in synaptic growth and that the proper localization of Sif requires Fas2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. It is proposed that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively (Sone, 2000).
To characterize the Sif localization pattern, particularly in reference to synaptic functional domains, the subcellular distribution of Sif in the boutons of larval neuromuscular junctions was examined by concomitant staining with anti-Pak antibody using laser-scanning confocal microscopy. Anti-Sif antibody labels the synaptic boutons in a network-like pattern, which is strikingly complementary with Pak staining in the boutons. The cross-section profile of the fluorescent intensity also shows that the staining patterns of Sif and Pak are mostly complementary to each other. These staining patterns demonstrate that the areas stained for Sif surround the active zones. Close examinations further reveal that anti-Sif and anti-Pak antibodies produce a number of concentric figures that are occasionally separated from each other. These data suggest that the active zone and the outer ring together form a structural unit that constitutes a synapse. The Sif-positive regions around the active zones are referred to as periactive zones (Sone, 2000).
To characterize the periactive zone, especially in identifying its functional significance, the distribution patterns of other molecules were examined with the aid of Pak staining. Monoclonal antibody, MAb1D4, against Fas2 labels the boutons in a complementary pattern with Pak staining. Fas2 staining surrounds the Pak-positive regions and forms concentric patterns as observed for Sif staining. The cross-section profile also shows similar patterns as Sif and Pak double staining. Sif and Fas2 are indeed co-localized in overlapping network-like patterns. Fas2 is involved in synaptic growth, stabilization and structural plasticity, possibly through its homophilic adhesion. These data suggest that Fas2 controls these synaptic events locally in the periactive zones. Thus, the periactive zone is characterized by the specific localization of two distinct types of molecules: a cell adhesion molecule (Fas2) that controls synaptic development and an intracellular molecule (Sif) that is a GEF to Rac (Sone, 2000).
In an attempt to understand the function of the periactive zone further, other molecular markers that stain the zone were sought. MAb6G11, the monoclonal antibody against betaPs integrin (Myospheroid) that is structurally similar to the vertebrate integrin beta1 subunit also shows staining that is complementary to Pak staining. Unlike Sif and Fas2, however, MAb6G11 staining is observed much more diffusely on the muscle surfaces surrounding the outside of the bouton, suggesting the staining in the postsynaptic specialization: the subsynaptic reticulum. In mutants of the mys gene the extent of the cell contact between nerve terminals and muscles is altered by either a primary or secondary effect of the mutation, and the growth of larval neuromuscular synapses is affected. These synaptic defects observed in the betaPs integrin mutants may represent its function in the periactive zones (Sone, 2000).
The polyclonal antibody against Dlg protein stains synaptic boutons in a way similar to MAb6G11. The Dlg staining also appears to be moderately diffused on the muscle surfaces surrounding the bouton. This pattern is complementary with the anti-Pak staining when the bouton is scanned at the surface level. In dlg mutants, the structural properties of synapses, including the formation of subsynaptic reticulum at the postsynapses and the number of active zones at the presynapses, are altered. Furthermore Dlg regulates the synaptic localization of Fas2 by binding directly to the cytoplasmic tail of Fas2. Therefore, one of the roles for Dlg in synaptic development is probably the localization of Fas2 to the periactive zone. These observations indicate that two additional molecules, betaPs integrin and Dlg, are present in synaptic areas including the periactive zones. They are both involved in the structural development of the neuromuscular synapses, and therefore appear to participate in the control of synaptic development in the periactive zones (Sone, 2000).
It has been suggested that a dynamin-rich domain in the presynaptic terminal functions as a site for the vesicular endocytosis. A recent study has also indicated that this domain is distinct from the active zone for exocytosis and instead surrounds the active zone. It is of interest to find out whether the periactive zone is involved in endocytosis and therefore the spatial relationships between the dynamin-rich domain and the periactive zone were examined. Synaptic boutons were co-stained with anti-Fas2 antibody and a polyclonal antibody against the Shi protein, the Drosophila homolog of dynamin. Shi is an essential factor for endocytosis, since endocytosis is completely blocked in the shi mutant. Anti-Shi antibody stains synaptic boutons in donut-like patterns but these patterns are found almost within the holes of the Fas2 rings. This Shi staining may partly overlap with Fas2 staining, but these staining patterns are distinct from each other. Thus, it is concluded that the periactive zone does not coincide with the dynamin-rich zone and therefore does not likely represent a functional domain for endocytosis (Sone, 2000).
Molecules involved in the synaptic development are present in the periactive zone. This implies that the periactive zone is the membrane domain that is specialized for the control of synaptic development. To test this possibility, the NMJ phenotype of the sif mutants was examined. Since the originally identified sif mutation is a hypomorphic allele, stronger sif mutant alleles induced with a chemical mutagen, ethyl methanesulfonic acid, were isolated. Among the alleles newly isolated in this study, one allele, sif ES11, is possibly functionally null, because the wild-type protein of more than 200 kDa has disappeared in the mutant flies, as revealed by a Western blot. Sequence analyses of the cDNAs indicate that the allele produces a truncated protein as a result of a frameshift mutation. The truncated protein lacks the Dbl-homology (DH) domain that catalyzes the GEF activity (Sone, 2000).
The mutant flies exhibit reduced locomotor activity, as observed for the original sif allele. In the mutant larvae, the synaptic bouton number of NMJ is slightly but significantly reduced when compared with the control larvae. Moreover, the reduced bouton number found in the mutant is rescued by expression of a sif minigene in neurons under the control of the elav promoter. These results suggest that sif functions in the regulation of synaptic growth. However, properties of basic synaptic transmission at the NMJs, including the frequency and amplitude of spontaneous MJPs, the amplitude of EJPs and the quantal content, are not significantly altered in the sif ES11 mutants. In addition, repetitive stimulation at 0.35 mM Ca2+ causes a similar decrease of EJPs in the mutants, as has been observed in the wild-type NMJs, and vesicle endocytosis is apparently unchanged, as estimated by the uptake of FM1-43. These data indicate that the lack of GEF activity of Sif results in no apparent effects on basic electrophysiological functions, which is consistent with the separate location of the active and periactive zones in the synaptic terminals (Sone, 2000).
Since Sif and Fas2 are co-localized in the periactive zone, a test was performed to see whether there is a genetic interaction between sif and Fas2 loci. The hypomorphic allele of Fas2, Fas2e76, shows a reduced number of boutons: would changing the dose of sif+ affect this bouton number phenotype? Strikingly, the double mutant of Fas2e76 and sif ES11 recovered the bouton number phenotype to the wild-type level, which suggests the presence of a suppressive genetic interaction between the two loci. To assess this genetic interaction further, the effect of Sif overexpression in the Fas2e76 background was examined. Sif overexpression does not clearly affect the synaptic bouton number in the wild-type background; rather, it causes a significant reduction in Fas2e76. Moreover, NMJs with extremely few synaptic boutons (less than 20) are observed in the Fas2e76 larvae overexpressing Sif, when compared with the NMJs in Fas2e76. Taken together, these data suggest that Sif and Fas2 are the components of related signaling pathways that control synaptic development, and Sif may, in an inhibitory manner, modulate the effect of Fas2 that regulates synaptic growth (Sone, 2000).
The possibility that the molecules in the periactive zone may affect each other in establishing their zonal localization was examined. Because Sif and Fas2 co-localize typically in the periactive zones and interact genetically, focus was placed on these proteins and an investigation was carried out to see if any perturbation occurs in the distribution of several molecular markers in the mutant background of Fas2 or sif. In the sif mutants, the localization of Fas2 is indistinguishable from the wild type. Conversely the Sif localization is normal in a hypomorphic allele of Fas2. However, in the boutons of a more severe hypomorphic allele of Fas2, Fas2e76, the Sif localization to the periactive zones is perturbed. The Fas2e76 mutation reduces Fas2 expression to 10% of the wild-type level. In most Fas2e76 boutons, the network pattern of Sif staining is still observed, but frequently in an irregular or diffused fashion. In more extreme cases, Sif is distributed almost evenly throughout the boutons or is largely concentrated on one side of the boutons so that Sif staining considerably overlaps Pak staining (Sone, 2000).
The mutant larvae could be distinguished from the wild type by the blind test for the Sif and Pak co-staining patterns. Similar results were obtained in the heterozygotes with Fas2e76 and a Fas2 null allele, suggesting that the alteration of Sif localization is not due to a second-site mutation on the Fas2e76 chromosome. To investigate the altered distribution of Sif further, the mutant boutons were examined under the electron microscope. A large number of Sif signals are occasionally present in the medial portions of the electron-dense regions in the Fas2e76 boutons and these signals are still associated with the plasma membrane, as are the signals observed in the wild type. It is therefore concluded that the reduction of Fas2 in the periactive zones results in the improper localization of Sif along the plasma membrane. Previous study has shown that the synaptic localization of Fas2 requires Dlg. Therefore, the localization of Sif was examined in the dlg mutant background, but no apparent alteration was found in the network pattern. A considerable amount of Fas2 is still present in the periactive zones of dlg mutant boutons, while faint or no staining is detected in the Fas2e76 boutons. This residual Fas2 seems to be sufficient to sustain the proper localization of Sif in the dlg mutants (Sone, 2000).
In mice, TIAM1 and STEF have been identified as GEFs
that specifically activate RAC1, and both are highly related to Sif in domain
organization and amino acid sequence in several domains,
indicating that these two proteins are likely to be the mouse
orthologs of Sif. Interestingly, both Tiam1 and Stef are
expressed in the brain, and Tiam1 expression is observed in the adult
hippocampus. Therefore, it would be important
to examine whether TIAM1 and STEF are localized
in the synaptic terminals (Sone, 2000 and references therein).
RAC is well known as a regulator of the actin-based
cytoskeleton and cell adhesion in various cells. RAC is also implicated in the
structural changes of nerve terminals, including growth cones and
dendrites. Therefore, in the periactive zones, activated
RAC may locally regulate the processes of the structural change
in the synaptic terminals, which include reorganization of the
actin-based cytoskeleton and cell adhesion (Sone, 2000 and references therein).
Previous studies have shown that RAC acts in the neurite
outgrowth of neuroblastoma cells that depend on the signal
from integrin on the cell surface. The
mammalian SIF homolog TIAM1, which functions as a RAC
GEF, recruits integrin to specific adhesive contacts at the cell
periphery. Moreover, expression of
TIAM1 increases cadherin-mediated cell adhesion in epithelial
MDCK cells. Therefore, there appear to
be signaling links between the RAC and cell-adhesion
molecules. Sif activates Rac; sif
genetically interacts with Fas2 in synaptic growth and the Sif
localization is perturbed in the Fas2 mutants. Taken together,
these data suggest that the Sif-Rac pathway is linked to the
cell-adhesion molecule Fas2 in close vicinity in the periactive
zone (Sone, 2000).
The periactive zone has been indicated as a region for the
control of synaptic development. The periactive zone
surrounds the active zone, which is the site for vesicle
exocytosis or neurotransmission. This concentric organization
suggests that the two zones specialize for the different cellular
functions and constitute an elemental unit for the presynaptic
structure. Investigation of how these zones are incorporated
into the synaptic bouton during development will be of interest.
The segregated distribution of the two zones suggests that
the mechanisms controlling synaptic development and
neurotransmission may be separable. This view is supported
by the mutant analyses for Fas2 and Sif; both mutations affect
structural properties of synapses without changing basic
electrophysiological functions. In the NMJs of Fas2 mutants,
the bouton number is decreased or increased depending on the
alleles but the total synaptic strength is maintained at the
normal level. Functional strength of the synapse is regulated only through
the activity of a transcription factor, cAMP-response-element-binding
protein (CREB), which functions independently of
Fas2. Also in sif mutants, the basic
electrophysiological properties of NMJs are normal. These
observations clearly contrast with the mutant phenotypes for
the proteins controlling vesicle exocytosis: Synaptotagmin,
Cysteine string protein, n-Synaptobrevin and Syntaxin 1A. Mutants in genes coding for all these proteins show impaired EJPs. Taken together, these results indicate that synaptic development and neurotransmission are genetically
separable phenomena and are regulated by independent
pathways. It is proposed that these genetically separable
phenomena are spatially segregated into the two zones on the
presynaptic plasma membrane, although the possibility that the two zones interact with each other cannot be excluded (Sone, 2000).
Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance. Using yeast two-hybrid screening and in vitro interaction assays, it has been shown that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induces similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. The findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration (Driessens, 2001).
Many previously identified Rac targets contain a distinctive Rac binding site, the CRIB motif, but sequence analysis does not reveal any obvious CRIB-like sequence in plexin-B1. To identify the region of plexin-B1 that contains the Rac interaction site, a series of truncations were expressed as GST fusion proteins in E. coli. These were used in a dot blot assay. Rac interacts with a region encompassing 180 residues (amino acids 1724-1903) of the receptor (Driessens, 2001).
Plexin-B1 is a member of a large family of transmembrane proteins, and based on sequence alignments, four classes of plexins (A, B, C, and D) have been described. To test whether Rac could interact directly with other members of the family, cDNAs were obtained for human plexin-A2 (kiaa0463), plexin-B2 (kiaa0315), and plexin-D1 (kiaa0620). A region corresponding to amino acids 1724-1903 of plexin-B1 was cloned into the pGEX vector; GST fusion proteins were analyzed in the dot blot assay, but under these conditions only plexin-B1 was found to interact (Driessens, 2001).
In Drosophila, two plexins have been identified: Drosophila plexin-A and Drosophila plexin-B. Recombinant Drosophila plexin-B protein (C-terminal 435 amino acids, similar to plexin-B1 two-hybrid clone) interacts strongly with in vitro translated Drosophila L61Rac1 and weakly with wild-type Drosophila Rac1 in a pull-down experiment. Drosophila plexin-A does not interact with Drosophila Rac1 under the same conditions. A Drosophila plexin-B fragment corresponding to amino acids 1724-1903 of human plexin-B1 interacts similarly with Drosophila Rac1, as does a shorter, 149 amino acid region. Partial binding was observed with a 54 amino acid domain (Driessens, 2001).
Two blocks of sequence similarity, of approximately 320 and 150 amino acids each, have been identified in plexin cytoplasmic domains. These two blocks of sequence similarity are separated by a variable linker. This linker region is most divergent between the plexin subfamilies. The minimal Rac binding region in Drosophila plexin-B consists of the last 149 amino acids of the first conserved block but does not contain the linker region. Alignment of this 149 amino acid region of Drosophila plexin-B with other human plexins reveals a sequence highly conserved among all plexin subfamilies (Driessens, 2001).
A mechanism is proposed for plexin-B signaling to the actin cytoskeleton. In this mechanism, clustering of B plexins induces a Rac-dependent activation of Rho. These results provide a framework for the further exploration of the complex mechanisms by which plexins affect the actin cytoskeleton in different cell types, including neurons (Driessens, 2001).
Plexins are neuronal receptors for the repulsive axon guidance molecule Semaphorins. Plexin B (PlexB) binds directly to the active, GTP-bound form of the Rac GTPase. A seven amino acid sequence in PlexB is required for RacGTP binding. The interaction of PlexB with RacGTP is necessary for Plexin-mediated axon guidance in vivo. A different region of PlexB binds to RhoA. Dosage-sensitive genetic interactions suggest that PlexB suppresses Rac activity and enhances RhoA activity. Biochemical evidence indicates that PlexB sequesters RacGTP from its downstream effector PAK. These results suggest a model whereby PlexB mediates repulsion by coordinately regulating two small GTPases in opposite directions: PlexB binds to RacGTP and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA (Hu, 2001).
Plexin B binds to the active form of Rac (RacGTP); the binding maps to a 147 amino acid region, PlexBDelta3 (amino acids 1617 through 1765). To identify the critical binding sequence in PlexBDelta3, small deletions and point mutations were introduced and a seven amino acid sequence NTLAHYG (1722 through 1728) toward the C terminus of PlexBDelta3 has been identified that, when deleted, abolishes Rac binding (PlexBDelta3d7). Deletions in neighboring regions 1743 through 1759 (PlexBDelta3d17) and 1707 through 1714 do not affect Rac binding (Hu, 2001).
The NTLAHYG sequence is highly conserved among Plexin family members. In particular, the tyrosine residue within the sequence is invariable. In human Plexin B1, a putative Cdc42/Rac interactive binding (CRIB)-like motif right after this conserved sequence has been described. Although the CRIB-like motif is not found in Drosophila PlexB, this may reflect a conservation of the binding mechanism at a higher structural level. The Psi blast program predicts two blocks of sequences in the Plexin cytoplasmic domain that share similarity with R-ras family GAP proteins. The sequence needed for RacGTP binding is located between these two GAP-like regions (Hu, 2001).
In the Drosophila genome, there are six Rho family small GTPases: Rac1 (referred to here as Rac), Rac2, Cdc42, RhoA, Mtl, and RhoL. To gain some insight into the specificity of the interaction, the binding of PlexBDelta3 with all six Drosophila Rho-like GTPases was examined. Only Rac and Rac2, which share the highest degree of sequence similarity (93% identity), show strong interactions with the BDelta3 region of PlexB (Hu, 2001).
Several lines of evidence suggest that RhoA is also involved in PlexiB signaling. Clustering of the vertebrate PlexB in Swiss 3T3 cells leads to stress fiber formation, indicative of Rho activation. The response can be blocked by inhibitors of Rho or of its downstream effector Rho kinase. Genetic data also indicate that RhoA mediates part of Plexin B signaling in embryonic axon guidance. It was of interest, then, to enquire whether RhoA may also directly associate with PlexB (Hu, 2001).
PlexBDelta, a larger piece of the PlexB cytoplasmic domain (1617 through 1827) binds to RhoA. In contrast to a preferential binding to GTPgammaS-bound Rac, PlexBDelta binds to the GTPgammaS and GDP-bound forms of RhoA equally well. The binding requires the last 40 amino acids of PlexBDelta. The seven amino acid internal deletion that eliminates PlexBDelta binding to Rac does not affect its binding to RhoA. Thus, two independent regions in PlexB cytoplasmic domain have been defined that are important for PlexB association with Rac and RhoA, respectively. Cdc42, another Rho family GTPase, does not bind to PlexBDelta (Hu, 2001).
Dosage-sensitive genetic interactions suggest Rac antagonizes Plexin B signaling. Since there is no mutant available for PlexB with which to examine genetic interactions, whether the gain of function phenotype of PlexB is sensitive to the level of expression of Rac or RhoA was examined. PlexB is endogenously expressed by CNS neurons. Overexpression of PlexB in all embryonic CNS neurons can be achieved with the UAS-GAL4 binary expression system. Flies containing the UAS-PlexB transgene reporter are crossed to flies carrying a neuron-specific transcriptional control driver, elav-GAL4. With two independent UAS-PlexB transgenic lines, a consistent, GAL4-dependent phenotype was observed in specific motor nerve branches. In particular, a striking defect was observed in the ability of the ISNb (intersegmental nerve b) motor axons to innervate the ventral longitudinal muscles 7, 6, 13, and 12. In wild-type embryos, the ISNb projects into the ventral longitudinal muscles. Particular motor axons innervate specific muscles; for example, the RP3 motor axon innervates muscles 7 and 6, while other ISNb axons innervate muscles 13 and 12. When PlexB is overexpressed in these neurons, two types of phenotypes are observed that are consistent with PlexB being a repulsive guidance receptor for muscle-expressed Semaphorins: (1) the RP3 axon frequently fails to defasciculate from the ISNb motor nerve branch, and as a result muscles 7 and 6 are uninnervated; (2) ISNb axons often fail to reach their distal-most target muscle 12 (scored as 'stall'). The copy number of UAS-PlexB transgene and elav-GAL4 driver was varied to generate embryos with a range of levels of expression of PlexB; 'RP3 missing' and 'stall' phenotypes are dose dependent. This dosage sensitivity suggests that the PlexB gain-of-function phenotype may provide a sensitive background for revealing genetic interactions with genes encoding downstream components involved in Plexin B signaling (Hu, 2001).
Does the binding of PlexB to Rac increase or decrease the output of Rac? It was reasoned that if increasing PlexB expression produces its effect by activating Rac, then genetically limiting Rac gene dose might suppress the PlexB overexpression phenotype. Alternatively, if PlexB signals by turning down Rac activity, then an enhancement of the plexB overexpression phenotypes might result when Rac is reduced. Indeed, the results support the second alternative: PlexB inactivates Rac. Rac protein level was reduced by 50% using a small deficiency line, Df(3L)Ar14-8 (61C04-62A08), in a moderate PlexB overexpression background (one copy transgene, one copy driver). This resulted in a distinct increase in the penetrance of PlexB gain of function phenotypes. A complementary effect results when Rac dosage is increased in the same neurons where PlexB is overexpressed with a UAS-Rac transgene. Under such conditions, a suppression on the PlexB gain of function phenotypes is observed (Hu, 2001).
Consistent with the idea that PlexB signals by downregulating Rac activity, the Plexin gain-of-function stall phenotype is reminiscent of the loss-of-function phenotype of a positive regulator of Rac, the Trio GEF. Trio has been shown to play a role in axon guidance in Drosophila and nematode and has provided additional evidence that, in this capacity, Trio interacts with Rac and regulates PAK activity. Similar to reducing Rac, reducing Trio enhances PlexB gain-of-function phenotype. However, the enhancement caused by reducing Trio is not as great as that caused by reducing Rac. This probably reflects that Trio is not directly coupled to PlexB and is not the only positive regulator (GEF) for Rac in motor axons. Rather, Trio is likely to be one of many positive regulators of Rac in these axons (Hu, 2001).
The role of RhoA in PlexB signaling was examined by reducing RhoA gene dosage with two different RhoA mutant alleles, Rhorev220 and RhoAl(2)k07236. Instead of enhancing the PlexB gain-of-function phenotypes as the Rac deficiency does, partially removing RhoA suppresses the PlexB gain-of-function phenotypes. This result suggests that RhoA acts antagonistically to Rac and, moreover, that RhoA partially mediates Plexin B signaling (Hu, 2001).
To further test the model that PlexB downregulates Rac output, the effect of increasing Plexin was examined in Rac dominant-negative embryos. No mutant for Drosophila Rac has yet been published, but a loss-of-function analysis for Rac, achieved by overexpressing a dominant-negative form of Drac (N17Rac) in neurons, has revealed dramatic defects in motor axon guidance. The same ISNb nerve branch that is affected by PlexB overexpression is also sensitive to overexpression of dominant-negative Rac (N17Rac). The predominant ISNb defect in N17Rac embryos occurs at an earlier target entry point, where the whole ISNb branch normally branches off from ISN nerve. In N17Rac embryos, the ISNb fails to enter the ventral muscles and instead follows the ISN distally toward dorsal muscles (scored as 'bypass'). The difference in the quality of the ISNb phenotype of N17Rac and Plexin B gain of function embryos may likely reflect the fact that Rac is downstream of multiple guidance receptors (Hu, 2001).
The penetrance of the N17Rac bypass phenotype is very sensitive to gene dosage. When the N17Rac transgene is expressed using drivers of different strengths, different frequencies of defects result. This suggests that N17Rac only partially knocks out the wild-type gene function and that expressing N17Rac with a driver of medium strength may provide a sensitized background for testing genes that regulate the remaining Rac activity. It was reasoned that if the Plexin and Rac interaction regulates Rac activity, then it might be possible to alter the penetrance of the N17Rac bypass phenotype by simultaneously increasing PlexB gene dose in the same neurons. Indeed, coexpressing PlexB and RacN17 results in a distinct enhancement of the ISNb bypass phenotypes. N17Rac embryos also show bypass defects in the SNa motor axons that project to lateral muscle targets. This SNa bypass phenotype has never been observed in any other mutant background, and it also turns out to be enhanced by simultaneously overexpressing PlexB in these neurons (Hu, 2001).
In a reciprocal experiment PlexB was reduced in Rac dominant-negative embryos to see if this had an opposite effect. This was done by injecting double-strand RNA of PlexB into N17Rac embryos. N17Rac embryos injected with Plexin B dsRNA show distinct reduction in bypass defects compared with N17Rac embryos injected with buffer. Thus, reducing PlexB and increasing PlexB in Rac dominant embryos produces opposite modulations, consistent with the model that PlexB downregulates Rac activity (Hu, 2001).
To test whether the PlexB gain-of-function and the genetic interactions depend on the direct association between PlexB and Rac, a mutant PlexB transgene, UAS-Plex Bd7, was constructed containing the seven amino acid NTLAHYG deletion in the Rac binding region of an otherwise wild-type PlexB. The same enhancement test on N17Rac embryos was performed with this mutant transgene, and no enhancement was observed. The PlexB gain-of-function phenotypes also seem to be dependent on this Rac binding region. In contrast to wild-type PlexB, when the d7 mutant transgene is overexpressed under the control of the same neuronal GAL4 driver elav, the frequency of ISNb phenotypes is significantly lower. This low-penetrance phenotype is not enhanced by removing one copy of Rac (Hu, 2001).
In vivo expression and targeting of PlexBd7 transgene could not be examined due to the lack of PlexB antibody. Nevertheless, the seven amino acid deletion does not affect protein expression and stability of PlexBDelta3 in in vitro experiments. Three independent lines of PlexBd7 transgenes show consistent behavior when tested for their phenotypes and interactions with Rac, arguing that the negative result is not caused by the insertion site (Hu, 2001).
In light of the genetic interactions between PlexB and Rac, the biochemical nature of this negative regulation was investigated. PlexB was found to compete with the Rac downstream effector PAK (p21-activated kinase) for binding to RacGTP. PAK is a serine/threonine kinase that mediates a major part of Rac signaling output to actin polymerization. Upon binding to RacGTP, PAK undergoes a conformational change that releases an autoinhibition on the kinase domain and becomes active. Since Rac binding is critical for PAK activation and also because PlexB and PAK bind to Rac in the same GTP-dependent manner, it was asked whether PlexB and PAK may bind to the same region of Rac and whether their binding to RacGTP is mutually exclusive (Hu, 2001).
An in vitro pull-down competition assay was used in which in vitro translated L61Rac was incubated with bead-bound GST-PAK1-141 in the presence or absence of soluble PlexB protein fragment: PlexBDelta2(1619-1753). (PlexBDelta2 is a 135 amino acid fragment of PlexB. It binds to RacGTP equally well as PlexBDelta3, but it can be expressed at a higher level.) When Plexin BDelta2 is present in the binding solution, the amount of RacL61 pulled down by PAK1-141 is greatly reduced. The extent of reduction is dependent on the amount of PlexBDelta2 used. At the 48:1 molar ratio of PlexBDelta2 to PAK1-141, the reduction is close to complete. PlexBDelta2d7, a deletion PlexB fragment that is incapable of binding to Rac, does not compete with PAK for the Rac binding. Conversely, the presence of PAK protein fragment PAK78-151 also reduces RacL61 binding to PlexBDelta3. This shows that the binding of the two proteins to RacGTP is indeed mutually exclusive (Hu, 2001).
Does this competition exist in vivo? If it does, then it may be expected that overexpressing PAK together with PlexB in embryos will cancel out PlexB gain-of-function effect. Indeed, overexpressing PAK in a PlexB gain-of-function background suppresses the phenotypes of the latter, demonstrating that PlexB signaling can be antagonized by the Rac effector PAK in vivo (Hu, 2001).
It is concluded that PlexB mediates repulsion in vivo in part by binding to active Rac (RacGTP) and downregulating its effector output and in part by binding to and activating RhoA. Biochemical analysis shows that PlexB binds to RacGTP. A seven amino acid sequence in the cytoplasmic domain of PlexB is required for this binding. Genetic analysis shows that PlexB downregulates the output of RacGTP. Removal of one copy of Rac enhances a PlexB gain-of-function phenotype, while overexpression of PlexB enhances a Rac dominant-negative phenotype in motor axon guidance. Overexpression of a mutant form of PlexB that lacks the seven amino acid sequence required for Rac binding does not generate its own gain-of-function phenotype, and it does not enhance a Rac dominant-negative phenotype. It is also shown that PlexB binds to RhoA through a different region of its cytoplasmic domain. Although the biochemical mechanism is not known, genetic analysis suggests that PlexB increases the output of RhoA (Hu, 2001).
The results presented here allow a confirmation and extension of a current model concerning the role of GTPases in axon guidance. This model suggests that attractive guidance cues locally activate Rac or Cdc42 in the growth cone while repulsive guidance cues locally activate RhoA. It is argued that what is important is the relative balance in the output of Rac versus RhoA. An example is provided in which the PlexiB receptor mediates repulsive axon guidance by downregulating RacGTP output and simultaneously upregulating RhoA output. A coordinate regulation of these two small GTPases may allow the receptor to have a finer control over actin regulatory machinery. Semaphorin signaling can be converted from repulsion to attraction by changes in cGMP level. It would be interesting to test whether and how the cGMP signaling can affect this Rac/Rho balance (Hu, 2001).
Drosophila has two Plexins: A and B. Both Plexin A and B are highly expressed in the central nervous system. The two proteins share high sequence similarity in their cytoplasmic domain, indicating a similar mode of signaling shared by the two. A direct physical association of RacGTP with PlexB but not with PlexA has been demonstrated. However, genetic interactions have been found between Rac and both Plexins. For example, increasing PlexA also enhances the Rac dominant-negative phenotype as does PlexB. In COS cell and DRG neurons, Rac shows coclustering with PlexA upon Sema3A ligand treatment. It is likely that ligand binding to PlexA causes Rac binding (and subsequent inactivation of Rac) just as with PlexB, but it may be that PlexA requires an unknown third protein to help mediate or facilitate this physical interaction. From a genetic perspective, they both appear to function in the same way, mediating repulsion at least in part by inactivating Rac (Hu, 2001).
The transmembrane protein OTK associates with Plexin A and contributes to the Sema 1a/Plexin A signaling pathway. Mammalian Plexin B1 also coimmunoprecipitates with OTK. In the future, it will be interesting to test whether PlexB also interacts with OTK in vivo and to what degree the Rac/Rho GTPases and OTK signaling pathways function together or in parallel downstream of Plexins (Hu, 2001).
Mammalian Vav signal transducer proteins couple receptor tyrosine kinase signals to the activation of the Rho/Rac GTPases, leading to cell differentiation and/or proliferation. The unique and complex structure of mammalian Vav proteins is preserved in the Drosophila homologue, Vav. Drosophila Vav functions as a guanine-nucleotide exchange factor (GEF) for DRac. Drosophila cells overexpressing wild-type (wt) Vav exhibit a normal morphology. However, overexpression of a truncated Vav mutant (that functions as an oncogene when expressed in NIH3T3 cells) results in striking changes in the actin cytoskeleton, resembling those usually visible following Rac activation. Dominant-negative Rac abrogates these morphological changes, suggesting that the effect of the truncated Vav mutant is mediated by activation of Rac. In Drosophila cells, stimulation of the Drosophila EGF receptor (DER) increases tyrosine phosphorylation of Vav, which in turn associates with tyrosine-phosphorylated DER. In addition, the following results imply that Vav participates in downstream DER signalling, such as ERK phosphorylation: (1) overexpression of Vav induces ERK phosphorylation; and (2) 'knockout' of Vav by RNA interference blocks ERK phosphorylation induced by DER stimulation. Unlike mammalian Vav proteins, Drosophila Vav was not found to induce Jnk phosphorylation under the experimental circumstances tested in fly cells. These results establish the role of Vav as a signal transducer that participates in receptor tyrosine kinase pathways and functions as a GEF for the small RhoGTPase, Rac (Hornstein, 2003).
The receptor tyrosine kinases (RTKs) play an important role in the control of most fundamental cellular processes including the cell cycle, cell migration, cell metabolism and survival, as well as cell proliferation and differentiation. RTK stimulation leads to the deployment of signalling proteins that relay the appropriate specific signals, resulting in the desired cell fate. Many of the signal transducing proteins, including RTKs, are conserved throughout evolution. In the past few years, genetic and biochemical studies in Drosophila have revealed the identity and function of many signalling cascade molecules that are also shared by mammals. However, there are still many signalling proteins whose roles are still unknown. One such signal transducer protein, the Drosophila melanogaster homologue of mammalian Vav proteins, has been isolated and partially characterized (Hornstein, 2003 and references therein).
Vav proteins represent a novel family of signal transducers that couple tyrosine kinase signals with the activation of the Rho/Rac GTPases and are likely to play an integral role in the regulation of cell differentiation in many tissues. The first member of the mammalian Vav family of cytoplasmic signal transducer proteins to be identified, Vav1, was isolated as an oncogene. Removal of its amino terminus activates Vav1 as a transforming protein. Likewise, the corresponding molecular lesions in Vav2 and Vav3, two other members of the mammalian Vav family, render these proteins transforming. Unlike Vav1, which is exclusively expressed in hematopoietic cells, Vav2 and Vav3 are expressed in both hematopoietic cells and in many cells of nonhematopoietic origin. Numerous biochemical and overexpression experiments revealed that tyrosine phosphorylation of Vav1 in response to activation by one of several cytokines, growth factors or antigen receptors regulates its activity as a GEF for the Rho/Rac family of GTPases, RhoA, Rac1 and RhoG. Activation of these GTPases leads to cytoskeletal reorganization and activation of stress-activated protein kinases (SAPK/JNKs) in T cells. Vav2 and Vav3 function in a similar but not identical fashion. While both Vav2 and Vav3 also act as GEFs, there are conflicting reports regarding which GTPases are activated by them, and whether these GTPases are distinct from those activated by Vav1. Knockout experiments revealed that in T cells, Vav1 integrates signals from lymphocyte antigen receptors and costimulatory receptors to control differentiation, proliferation and the response to activation. Thus, mice deficient in Vav1 exhibit defects in numerous responses to T-cell stimulation, including capping of the T-cell receptor (TCR) postactivation, recruitment of the actin cytoskeleton to the CD3 chain of the TCR, interleukin-2 (IL-2) production and proliferation, cell cycle progression, activity of NF-AT, phosphorylation of SLP-76 and increase in Ca2+ influx. Mice deficient in Vav2 display no obvious defects in T-cell development yet exhibited some defects in B-cell function. Mice lacking both Vav1 and Vav2 displayed major defects in B-cell function that are as dramatic as the defects in T-cell development and activation observed in Vav1-/- mice. Since there are no reports regarding Vav3-/- mice at the present time, the picture of the intricate signalling network induced by the Vav proteins is incomplete. However, it is obvious that the redundancy and complexity of the mammalian Vav proteins even in hematopoietic cells together with the possibility that they differ both in their protein-protein interactions and in their activation of various GTPases, makes it difficult to clearly interpret the results of knockout and other experiments in mammals (Hornstein, 2003 and references therein).
In Drosophila, only one Vav homologue is present (Dekel, 2000). The highly conserved and unique structure of Vav suggests that the vav genes probably evolved from one ancestral gene and that they are important regulatory molecules in flies as well as in mammals. Drosophila Vav encodes a protein whose similarity with hVav1 is 47% and with hVav2 and hVav3 is 45%. Like mammalian Vav proteins, Droosophila Vav encodes a 'calponin-homology' (CH) region, a dbl homology (DH) domain, a pleckstrin homology (PH) domain and both an Src Homology 2 (SH2) and an Src Homology 3 (SH3) domains. However, unlike mammalian Vav proteins, Drosophila Vav lacks an amino-SH3 region. Vav is the only known Drosophila Rho GEF that encodes in addition to a DH region, both SH2 and SH3 domains, attesting that it may be a uniquely versatile signal transducer. The fact that only one homologue of Vav is present in flies, combined with the unique and highly conserved structure of the protein, promises that any study of Vav in flies should be highly beneficial and instructive (Hornstein, 2003).
A hallmark of Vav signal transducer proteins is that their tyrosine residues become phosphorylated on tyrosine residues in response to EGF stimulation. Furthermore, Vav proteins are known to bind to the stimulated EGFR through their SH2 region. In mammalian cells, Drosophila Vav is tyrosine phosphorylated in response to EGFR induction; in vitro, the Drosophila Vav SH2 region is associated with tyrosine-phosphorylated EGFR (Dekel, 2000). These results combined with the encoded domain structure of Vav strongly suggest that Drosophila Vav may function as a signal transducer protein in the Drosophila EGF receptor (DER) signalling cascades. This study investigated the role of Vav in downstream signalling from the DER, its interaction with the Drosophila Rac pathway, and its ability to effect cytoskeletal changes in Drosophila cells (Hornstein, 2003).
This study demonstrates that Vav can activate Rac in vivo. Rac is involved in Drosophila in various cellular processes including cell shape, cell adhesion, gene transcription, protein trafficking and cell cycle progression, as well as numerous developmental processes. One of the best known characteristics of Rac is that it is involved in actin cytoskeletal organization. Indeed, the expression of a constitutively activated form of DRac (V12DRac) in cultured S2 cells causes marked changes in the morphology of the cells, leading to lamellipodia and microspikes. These results indicate that overexpression of oncVav (Drosophila Vav that lacks 214 residues of its amino-terminus), but not wild-type Vav, can induce a morphology similar to that obtained with V12DRac (constitutively active mutant Rac). The fact that N17DRac inhibits the changes in the morphology obtained with oncVav further substantiates the conclusion that the activation of Rac by Vav is responsible for the observed cytoskeletal reorganization. Correspondingly, an inactive hVav1 variant defective in its ability to activate Rac inhibited the ability to induce actin cytoskeletal organization, thus further supporting the tight association between Vav, Rac and actin organization. Notably, coexpression of oncVav and V12DRac leads to more profound changes in cytoskeleton organization compared to those observed in cells overexpressing each protein alone. This result could be explained by the fact that the sum of activation reached by both the endogenous Rac activated by oncVav as well as the constitutively activated Rac yields a more striking morphology. Consistent with these results with wild-type Vav, wt hVav1 does not cause any change in morphology of NIH3T3 cells and COS cells. Conversely, a remarkable change in actin organization has been observed following overexpression of hVav1 in T cells. These conflicting results obtained with mammalian wtVav1 could stem from the use of different experimental systems, including the strong possibility that different levels of endogenous Vav2 and Vav3 exist in these systems. The cytoskeletal changes in S2 cells transfected with oncVav are compatible with a previous study demonstrating that an amino-terminus-truncated hVav1 caused depolarization of fibroblasts and triggered the bundling of actin stress fibers in NIH3T3 cells. Taken together, these results support a pathway in which Drosophila Vav serves as a GEF for Rac, thereby triggering the reorganization of the actin cytoskeleton (Hornstein, 2003).
Drosophila Vav is tyrosine phosphorylated in response to stimulation of DER and it also associates with the stimulated receptor. This result is compatible with the known characteristics of mammalian Vav proteins. For example, when ectopically expressed in nonhematopoietic cells, Vav1 associates with the EGFR through its SH2 region and becomes tyrosine phosphorylated upon induction with EGF. A similar result was reported for the ubiquitously expressed Vav2 and Vav3 proteins. Although it is well established that the Vav proteins are tyrosine phosphorylated upon EGFR stimulation, the exact contribution of the various mammalian Vav proteins to the EGFR signalling pathway is not understood. These studies with Drosophila Vav shed some light on these events. Thus, this study demonstrates that overexpression of Vav in D2F cells leads to increased ERK phosphorylation. Moreover, its elimination by the use of dsRNAi blocks phosphorylation of ERK even following stimulation of DER. There are opposing results regarding the link between Vav and ERK activation in mammals. Overexpression of Vav1 in Jurkat T cells induced 3-4-fold activation of ERK activation. Vav1 activates ERK when ectopically expressed in NIH3T3 or CHO cells. Furthermore, Vav1 coimmunoprecipitates with ERK in a human myeloma cell line stimulated with interleukin-6. Finally, T cells deficient in Vav1 exhibit defects in ERK phosphorylation. Conversely, the activation of Ras and ERK following stimulation of the TCR in Vav1 null Jurkat T cells appears normal. Without the complication of multiple homologues, these studies strongly suggest a role for Drosophila Vav in the ERK pathway in S2 cells (Hornstein, 2003).
Despite the existence of several studies that point to a link between hVav1 and Ras, it is still unclear how Vav proteins can affect ERK. Drosophila Vav may affect the Ras/ERK pathway through its function as a GEF towards DRac. Cross talk between the Rac and Ras pathways has been shown to exist in mammals. Rho family small GTPases were found to play an important role in mediating the activation of Raf by Ras. Thus, a dominant-negative mutant of Rac can block Raf activation by Ras. Additionally, the effect of Rac can be substituted by the PAK kinase, which is a direct downstream target of Rac. Moreover, PAK directly associates with Raf-1 under both physiological and overexpressed conditions. The extent of interaction between PAK and Raf-1 is correlated with the ability of PAK to phosphorylate Raf and induce mitogen-activated protein kinase activation. These studies strongly suggest that cross talk between Rac and Ras exist and it is mediated through activation of downstream effectors of Rac, such as PAK. Furthermore, it was demonstrated that MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42. A novel Drosophila gene, DRacGAP, has been identified which behaves as a negative regulator of the GTPases, DRac1 and DCdc42. Reduced function of DRacGAP or increased expression of DRac1 in the wing imaginal disk causes effects on vein and sensory organ development and cell proliferation as a result of enhanced activity of the EGFR/Ras signalling pathway. Thus, DRac and DRas are involved in cross-talk mechanisms that modulate Drosophila development (Hornstein, 2003 and references therein).
Drosophila Vav might also activate ERK in a GEF-independent manner. For instance, Vav might stimulate the Ras/ERK pathway via PLC activation, just as Vav1 was shown to activate PLC. PLC contributes to the activation of Ras, probably by stimulating the activity of the diacylglycerol-dependent exchange factor, Ras GRP. It is highly conceivable that the activity of hVav1 towards PLC is mediated in a GEF-independent mode. Whether such a mechanism is also elicited in flies remains to be determined. Drosophila Vav contains several protein-binding domains (SH2, SH3) that might participate in various pathways that result in activation of the Ras/ERK pathway. For instance, Vav may bind to the adapter molecule DShc, that was shown to be associated with the Grb2/Drk proteins leading to DRas activation. Indeed, mammalian Shc binds mammalian Vav proteins. Collectively, the current results clearly illustrate that Vav influences both the DRac and ERK pathways. However, it is not clear yet whether it exerts its influence on ERK by an exclusive inducement of the DRac pathway and/or through a GEF-independent activity (Hornstein, 2003).
The involvement of mammalian Vav proteins, by functioning as GEFs towards Rac in the JNK signalling cascade, is well establishe. Moreover, Vav proteins mediate this response through their function as GEFs towards Rac. This pathway is highly conserved between mammals and flies. In Drosophila, it can transduce signals of a diversified nature, leading to changes in cell polarity and mediating immunity in the adult. It is also required for dorsal closure during embryonic development. Genetic studies focusing on these processes placed the Rho family small GTPases in the JNK signalling cascade. However, although this study demonstrated that Vav functions as a GEF towards DRac in Drosophila, Vav does not seem to be involved in the sorbitol-induced JNK activation. In accordance, no effect has been detected of Rho family small GTPases on sorbitol-induced JNK activation in S2 cells. It is therefore conceivable that in S2 cells, the sorbitol-induced activation of JNK is not mediated through activation of DRac, and therefore does not require Vav. The possibility that Vav is involved in JNK activation under other physiological pathways in Drosophila, such as dorsal closure, still exists; however, this question merits further investigation (Hornstein, 2003).
In summary, these results show that, in fly cells, Vav functions in various signalling cascades in which it can play a role as a GEF or participate as an adapter protein. A P-element insertion has recently been reported to inactivate Vav, leading to lethality of flies (Bourbon, 2000). Further genetic experiments will be required to better understand the physiological function of Vav in developmental systems (Hornstein, 2003).
The capacity of stem cells to self renew and the ability of stem cell daughters to differentiate into highly specialized cells depend on external cues provided by their cellular microenvironments. However, how microenvironments are shaped is poorly understood. In testes of Drosophila, germ cells are enclosed by somatic support cells. This physical interrelationship depends on signaling from germ cells to the Epidermal growth factor receptor (Egfr) on somatic support cells. Germ cells signal via the Egf class ligand Spitz (Spi), and evidence is provided that the Egfr associates with and acts through the guanine nucleotide exchange factor Vav to regulate activities of Rac1. Reducing activity of the Egfr, Vav, or Rac1 from somatic support cells enhanced the germ cell enclosure defects of a conditional spi allele. Conversely, reducing activity of Rho1 from somatic support cells suppresses the germ cell enclosure defects of the conditional spi allele. It is proposed that a differential in Rac and Rho activities across somatic support cells guides their growth around the germ cells. These novel findings reveal how signals from one cell type regulate cell-shape changes in another to establish a critical partnership required for proper differentiation of a stem cell lineage (Sarkar, 2007).
In the male gonad of Drosophila, germ cells are surrounded by somatic cells that define their cellular microenvironmen. Germline stem cells (GSCs) are attached to a cluster of nondividing cells at the apical tip, called hub cells, and associated with cyst progenitor cells (CPCs) that act as stem cells for the somatic support cell lineage. Two CPCs extend their cytoplasm around one GSC, toward the hub, and toward each other such that each GSC appears to be completely enclosed in its cellular microenvironment. GSCc and CPCs generate differentiating daughters, called gonialblasts and cyst cells, respectively. The gonialblasts undergo transit amplification divisions to produce 16 spermatogonia, which become spermatocytes, grow in size, undergo the meiotic divisions, and differentiate into sperm. Two cyst cells grow cytoplasmic extensions around one gonialblast to form the germ cell cellular microenvironment that controls various aspects of germ cell differentiation (Sarkar, 2007).
Germ cells associated with somatic cells mutant for the Map-Kinase Raf fail to differentiate and accumulated as early-stage germ cells instead. A similar accumulation of early-stage germ cells was observed in Egfrts mutant testes shifted to nonpermissive temperature, and in testes from animals mutant for Stem cell tumor (Stet; Rhomboid2), a protease that cleaves Egfr ligands. However, stet mutant germ cells in addition fail to associate with somatic support cells, suggesting that the Egfr pathway is required for setting up the critical cellular microenvironment (Sarkar, 2007).
Loss of spi results in a failure of germ cells to differentiate, similar to the effects of loss of stet or the Egfr. Wild-type testes are long (~2 mm) tubular structures that contain germ cells in a spatio-temporal order along the apical-to-basal axis. Early germ cells (GSCs, gonialblasts, and spermatogonia) are small and have small, densely packed nuclei in DAPI-stained preparations. Spermatocytes are located basal to the spermatogonia, and differentiating spermatids fill the distal part of the testis (Sarkar, 2007).
Animals carrying a temperature-sensitive allele of spi, spi77-20, die when raised at 29°C. However, spi77-20 animals raised at a slightly permissive temperature (27°C) survive and have tiny testes. Most of these testes (40 of 50) contain only small cells, as seen at the tip of wild-type testes, and do not have spermatocytes or differentiating spermatids. Staining with molecular markers revealed that the testes contains increased numbers of GSCs, gonialblasts, and spermatogonia compared to wild-type. The remaining testes (10 of 50) have high numbers of early germ cells and a few spermatocytes, but no differentiating spermatids (Sarkar, 2007).
Testes from spi77-20 animals raised at an intermediate permissive temperature (25°C) are longer than testes from animals raised at 27°C, but significantly shorter (500 μm-1.5 mm) than wild-type testes. A substantial part of the testes is occupied by tumor-like aggregates of early-stage germ cells. However, spermatocytes and differentiating spermatids are also present (Sarkar, 2007).
spi activity is both sufficient and required within the germ cells. Expression of a cleaved version of Spi (sSpi) in germ cells but not in somatic support cells of spi77-20 testes restores the wild-type phenotype, and germ cell clones mutant for spi accumulate at early stages based on phase-contrast microscopy and DAPI-stained preparations) (Sarkar, 2007).
spi was also required for somatic support cells to associate with and enclose the germ cells. Germ cell clones mutant for the conditional spi77-20 allele from animals raised at 27°C either do not associate with somatic support cells or associated with only one somatic support cell (4 of 20 clones), based on staining with soma-specific antibodies, such as the transcription factor Traffic Jam (Tj). Tj labels the nuclei of somatic support cells that are normally associated with early-stage germ cells (Sarkar, 2007).
Germ cell enclosure can be investigated by labeling testes with molecular markers such as antibodies against the membrane-bound β-catenin Armadillo (Arm) that labels the cell membranes of somatic support cells as they surround the germ cells. In wild-type testes, each GSC, gonialblast, and cluster of developing germ cells is associated with and surrounded by two somatic support cells. In testes from spi77-20 animals raised at 27°C, Tj-positive cells did not form cytoplasmic extensions around the germ cells. Similar results were obtained with other markers, including a cytoplasmic UAS-Green Fluorescent Protein (UAS-GFP) expressed in somatic support cells under control of a soma-specific Gal4-driver. In control testes, GFP is detected in the cell bodies of the somatic cells surrounding the germ cells. In contrast, in spi77-20 testes from animals grown at 27°C, GFP is detected in balls, most likely small round cell bodies of somatic support cells. Occasionally, cytoplasmic extensions emerged from somatic support cells, but they remained short and did not enclose the germ cells (Sarkar, 2007).
The lack of cytoplasmic extensions from Tj-positive cells in spi77-20 mutant testes is similar to the phenotype observed in stet mutants. This suggests that the Egf class ligand Spi, expressed in germ cells and processed by Stet, stimulates the Egfr on somatic support cells, inducing them to send out cytoplasmic extensions to enclose the neighboring germ cells (Sarkar, 2007).
Association of germ cells with somatic support cells is sensitive to the level of Spi. Germ cell clones from spi77-20 animals raised at 25°C and germ cell clones from animals mutant for the spi2 allele often associated with more than two somatic support cells (Sarkar, 2007).
The growth of cytoplasmic extensions around the germ cells is also sensitive to the level of Spi. When spi77-20 animals are raised at 25°C, many Tj-positive cells form cytoplasmic extensions directed toward and/or around the germ cells. However, not every germ cell cluster appear to be associated with and/or surrounded by somatic support cells. Furthermore, many of the Tj-positive cells forme cytoplasmic extensions toward each other, suggesting that multiple somatic support cells may surround one tumor-like aggregate of germ cells. Similar abnormal associations of somatic support cells with germ cells are also seen in Egfrts mutants shifted to nonpermissive temperature. One possible explanation for the different phenotypes of loss compared to reduction of Egfr signaling is that different levels of Egfr stimulation may affect different cellular properties of somatic support cells, such as cell adhesiveness and/or growth (Sarkar, 2007).
To identify novel players in germ cell enclosure, the sensitized background of the spi77-20 allele was used to search for genetic modifiers. It was found that impaired activity of the small monomeric GTPase (small GTPase) Rac1 enhances the spi77-20 testes phenotype. Activity of Rac1 was impaired by two strategies-either by removing one copy of the rac1 gene or by expressing a dominant-negative version of Rac1 (dnRac1) in somatic support cells of testes from spi77-20 animals raised at 25°C. In either case, the enhanced testes are shorter than testes from spi77-20 animals raised at 25°C. In 12 of 20 enhanced testes, Tj-positive cells do not enclose the germ cells, and early-stage germ cells accumulate (Sarkar, 2007).
Reducing activity of Vav, a guanine nucleotide exchange factor for Rac-type small GTPases, from somatic support cells by antisense expression also enhances the spi77-20 testes phenotype from animals raised at 25°C. 11 of 20 enhanced testes were tiny and contained mostly early-stage germ cells that were not surrounded by somatic support cells. The enhanced phenotypes caused by impairing Rac or Vav raises the possibility that Rac1 and Vav act downstream of the Egfr in somatic support cells and that Vav plays a role in regulating somatic support cell-shape changes associated with germ cell enclosure (Sarkar, 2007).
In mammalian cells, autophosphorylation of specific Vav-binding motifs within the cytoplamic tail of the Egfr allows for binding and phosphorylation of mammalian Vav2. Phosphorylation of Drosophila Vav has been shown to depend on Egfr stimulation in both mammalian and Drosophila cultured cells, and Drosophila Vav bound to mammalian Egfr (Sarkar, 2007 and references therein).
Consistent with a role for Drosophila Vav in Egfr signaling in testes, Vav protein immunoprecipitates from testis extracts with an antibody against the Egfr. Vav does not immunoprecipitate from testis extracts that had been pretreated with phosphatase, suggesting that the interaction between Vav and the Egfr is phosphorylation dependent. The immunoprecipitated Vav band comigrates with a band detected by antibodies against phospho-tyrosine, suggesting that Vav is phosphorylated when in a complex with the Egfr (Sarkar, 2007).
In the classical view of the Drosophila Egfr pathway, only one docking protein-Downstream receptor kinase (Drk)-binds to the stimulated Egfr and activates a MAP-Kinase cascade for transcription of target genes. However, genetic and biochemical data suggest that the Egfr pathway is branched at the level of docking proteins and that the adaptor protein Vav binds to the Egfr to activate the small GTPase Rac1. These data suggest that Rac regulates cell-shape changes associated with germ cell enclosure, and studies on Raf suggested that it regulates the transcription of target genes. However, the possibility of crosstalk between the two branches cannot be excluded: Vav may contribute to transcriptional regulation and Map-Kinases may contribute to germ cell enclosure. A possible crosstalk is consistent with findings that in cultured Drosophila cells (Hornstein, 2003), Vav can contribute to Erk phosphorylation (Sarkar, 2007).
Surprisingly, impairing activity of the Rho-type small GTPase Rho1 has the opposite effect to impairing Rac1. Testes from spi77-20 animals raised at 27°C that express dominant-negative Rho1 (dnRho1) in somatic support cells are long and appear almost wild-type. In contrast to somatic support cells in spi77-20 testes from animals raised at 27°C without dnRho1 expression, the somatic support cells expressing dnRho1 enclose the germ cells. The same dominant suppression is observed in spi77-20, rho1/+ testes, indicating that expression of dnRho1 reflects loss of Rho1 activity (Sarkar, 2007).
These data raise the possibility that Rac and Rho have antagonistic effects on germ cell enclosure. Rac appears to be required for somatic support cells to grow cytoplasmic extensions around the germ cells, and Rho appears to suppress this growth. Antagonistic roles for Rac and Rho have also been reported in cultured mammalian cells, where Rac and Rho regulate cell-shape changes and growth via different effects on the actin cytoskeleton. Prominent readouts for small GTPase activities on the actin cytoskeleton are the appearances of ruffles and lamellipodia in the cell membranes (Sarkar, 2007).
To address a potential role of Rac and Rho in shape changes of somatic support cells, dominant-negative Rac or Rho were expressed in somatic support cells of otherwise wild-type testes, and transmission electron microscopy (TEM) was used to investigate changes in the membranes of somatic support cells surrounding single germ cells and spermatogonia at the apical tip of the testes. Germ cells and somatic support cells can be identified based on their different shapes and density of staining in TEM. In wild-type, the somatic support cells surrounding single germ cells and spermatogonia exhibit wavy plasma membranes, possibly analogous to membrane ruffles accompanying cellular growth and rearrangements of the actin cytoskeleton in cultured cells (Sarkar, 2007).
Somatic support cells expressing dnRac1 have much smoother plasma membranes than do wild-type somatic support cells. Conversely, somatic support cells expressing dnRho1 have lamellipodia-like extensions in their membranes. Lamellipodia-like extensions were not detected in somatic support cell membranes in serial sections of wild-type testes or in testes expressing dnRac1. In mammalian cells, formation of lamellipodia depends on Rac-type small GTPases. The presence of lamellipodia-like extensions in somatic support cells expressing dnRho1 suggests that Rac may become hyperactive in the absence of Rho. Based on these TEM data, it is hypothesized that, just as their mammalian counterparts do in cultured cells, Drosophila small GTPases may act on the cytoskeleton of somatic support cells to mediate cell-shape changes and growth of cellular extensions and that the effects of Rac and Rho are antagonistic (Sarkar, 2007).
This model predicts that expression of a constitutively active Egfr ligand in somatic support cells might compromise the differential in smGTPase activities. Indeed, forced expression of cleaved ligand in somatic support cells, but not in germ cells, closely mimics the effect of dnRho expression: the somatic support cells formed lamellipodia-like structures in their membranes (Sarkar, 2007).
This research on the Drosophila gonad provides a striking example how one cell type in tissue communicates with another cell type to induce and direct the formation of a proper cellular microenvironment: a signal from one cell induces subcellular changes throughout the body of another cells. This mechanism underlying the formation of a cellular microenvironment may be conserved across species (Sarkar, 2007).
Neuronal plasticity requires actin cytoskeleton remodeling and local protein translation in response to extracellular signals. Rho GTPase pathways control actin reorganization, while the fragile X mental retardation protein (FMRP) regulates the synthesis of specific proteins. Mutations affecting either pathway produce neuronal connectivity defects in model organisms and mental retardation in humans. CYFIP, the fly ortholog of vertebrate FMRP interactors CYFIP1 and CYFIP2, is specifically expressed in the nervous system. CYFIP mutations affect axons and synapses, much like mutations in Drosophila Fmr1 and in Drosophila Rho GTPase Rac1. CYFIP interacts biochemically and genetically with Fmr1 and Rac1. Finally, CYFIP acts as a Rac1 effector that antagonizes Fmr1 function, providing a bridge between signal-dependent cytoskeleton remodeling and translation (Schenck, 2003).
Drosophila Roundabout is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Evidence is presented that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance (Fan, 2003).
Biochemical data suggests that the interaction between Dock and Robo is an SH3-dependent interaction and that the first two SH3 domains of Dock are most important for mediating Robo binding. Based on the observations that a three-protein interaction can be detected between Robo, Dock, and Pak and that Pak has been shown to interact with the SH3-2 domain of Dock, it is believed that the SH3-1 domain is the most important for Robo and Dock binding. Furthermore, Slit stimulation enhances Dock's ability to bind to Robo, suggesting a ligand-regulated SH3 domain interaction. This represents a different kind of adaptor interaction to many that have been observed previously, where Nck appears to interact with a number of tyrosine-kinase receptors through an SH2 domain/phosphotyrosine interaction. In the latter case, how ligand binding to the receptor regulates the Nck SH2 domain interaction is quite well understood. The observation that the Robo receptor shows a ligand-regulated SH3 domain interaction with Dock/Nck suggests that somehow ligand binding results in an increased availability of the SH3 binding sites in the receptor (Fan, 2003).
The regions of Robo that appear to be most important for the interaction are the proline-rich regions CC2 and CC3. Individual mutations in these motifs strongly reduce the amount of Dock that coimmunoprecipitates with Robo in cell culture, while removing both of these motifs completely abolishes binding. Furthermore, expression of Robo receptors that lack the CC2 and CC3 motifs in transgenic Drosophila disrupt the in vivo function of the receptor. It is important to stress that the CC2 and CC3 sequences are not only involved in Dock binding, but also bind Ena, Abl, and potentially other proteins as well. In addition, CC2 and CC3 are also required for the observed upregulation of Rac activity. The fact that many proteins bind Robo at these sites prevents clear conclusions about why the ΔCC2ΔCC3 mutant receptor is nonfunctional. In the future, more precisely defining the binding requirements of the many proteins that interact with Robo may allow forms of Robo to be created that specifically disrupt the binding of some partners and not others, which in turn should provide insight into the relative roles of different Robo signaling outputs (Fan, 2003).
The implication of Rac in Robo repulsion (dominant negative Rac1 shows a strong enhancement of slit;robo/+ defects) was unexpected in view of the well-established role of Rac as a positive regulator of axon outgrowth. On the surface, this finding appears quite contradictory to the function of Rac to promote actin polymerization at the leading edge of motile cells and axons. One possible explanation of this finding is that perhaps Rac can have different or even opposite effects on the actin cytoskeleton, depending on the molecular context in which it is activated and its overall level of activity. For example, depending on the coordinate local function of other small GTPases and actin regulatory proteins, the consequences of Rac function could be different. It is interesting to note that in addition to a role for Rac, genetic analysis and previously published data also support an important role for Rho in midline repulsion. Furthermore, in addition to strongly stimulating Rac activity, Slit has been shown have a modest stimulatory effect on Rho activity. The implication of both Rac and Rho in mediating repulsive responses has also been suggested to explain the output of the Plexin receptor. It will be interesting in the future to determine the interrelationship between Rac and Rho outputs in the context of Robo repulsion as well as in signaling downstream of other attractive and repulsive axon guidance receptors (Fan, 2003).
As an alternative to the context- and level-dependent explanation of the role of Rac in Robo repulsion, the observed axon steering defects in embryos where both Rac and Slit function are reduced, or in embryos deficient for multiple rac genes, could be explained as a secondary consequence of defects in the rate of axon extension. In this scenario, Rac's role in repulsive axon guidance would be intimately coupled with its role in axon outgrowth. That is to say, that appropriate steering decisions go hand and hand with the appropriate regulation of the rate of axon outgrowth (e.g., you are more likely to miss your exit if you are driving too fast). In this regard, it is important to emphasize that even repulsive cues can have stimulatory effects on axon extension. For example, in addition to repelling Xenopus spinal neurons, Slit also has a stimulatory effect on the rate of axon extension (Fan, 2003 and references therein).
Perhaps the most difficult observation to explain is how reciprocal shifts in Pak levels can lead to similar consequences for Robo repulsion. Since the enhancing effects of Pak overexpression in partial loss-of-function robo backgrounds are more dramatic with the membrane-tethered form of Pak, it is tempting to speculate that in order to signal properly, turning Pak activity on and off needs to be tightly controlled. Little is known about how Pak signaling is terminated and it seems quite possible that the membrane-tethered version of pak is not as effectively regulated as the wild-type form of pak. Interestingly, in genetic backgrounds where robo signaling is specifically compromised in its output, through reduction of rac, introducing the UASPakMyr transgene can partially suppress the midline crossing defects. Given the clear ability of alterations in pak expression to modulate midline repulsion and the observation that Slit can promote the formation of a Robo, Dock, and Pak protein complex, it is somewhat surprising that complete removal of zygotic pak does not have major consequences for embryonic axon guidance. Indeed, in the absence of clear loss-of-function phenotypes in pak mutants, it is difficult to argue unequivocally for a critical role of endogenous pak in robo function. There are a number of potential explanations for these observations including, but not limited to, maternal pak contribution and the potential redundant function of a second pak-like gene. Future experiments should address these possibilities in order to link pak more firmly to robo (Fan, 2003).
The implication of Dock/Nck and Rac in both DCC-mediated attraction and Robo-mediated repulsion raises the obvious question of how the specificity of attraction and repulsion is controlled and argues against a committed role of either of these signaling molecules to either one or the other type of responses. This is perhaps not too surprising, given the fact that Robo and DCC receptors themselves are intimately connected through their ability to form a heteromeric receptor complex with potentially unique signaling properties. Although it remains possible that signaling molecules or adaptors will be identified that can account for the specificity, an alternative possibility is that it is the coordinate regulation, relative activity levels, and combinatorial action of a core group of common signaling molecules that makes the difference in attraction versus repulsion (Fan, 2003).
Biochemical data support the idea that Slit stimulation of Robo can regulate the recruitment of Dock and Pak to the Robo receptor and also trigger an increase in Rac activity. Both of these events are dependent on the CC2 and CC3 sequences in Robo's cytoplasmic domain. Thus, the observations are consistent with either a Dock-dependent or a Dock-independent recruitment of Rac to Robo. Based on the known physical interactions between Dock and Pak and between Pak and Rac, it is likely that the recruitment of Rac is dependent on Dock. Alternatively, another protein interacting through CC2 and/or CC3 could function to recruit Rac in a Dock-independent fashion (Fan, 2003).
Regardless of whether the recruitment of Rac to Robo is dependent on Dock and Pak or is an independent event, the data cannot explain how Slit stimulation of Robo results in increased Rac activity. Two obvious types of molecules that are missing from the model and the protein complex are the upstream regulators of Rac, the GEF and GAP proteins. Intriguingly, in the course of a genome-wide analysis of all RhoGEFs and RhoGAPs in Drosophila, one Rac-specific GAP has been identified that when overexpressed results in phenotypes reminiscent of robo loss of function (H. Hu et al., submitted, reported in Fan, 2003). There are a number of candidate GEFs that could explain how Rac activity is upregulated by Slit activation of Robo, most notably Sos, rtGEF (pix), and Trio. It will be interesting to determine which if any of these molecules could play such a role in Robo signaling (Fan, 2003).
Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. vilse is a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the conserved intracellular domains CC0, CC1, and CC2 of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse (Lundström, 2004).
Database searches with the sequence from genomic DNA flanking the vilse lacZ P-element showed that it was inserted in the 5'-untranslated region of the predicted gene CG3421 (RhoGap93B in GadFly). The search also identified several cDNAs deriving from this gene, and the longest available clone, LD10379 (BDGP), was sequenced. The predicted Vilse protein contains a number of conserved domains: two N-terminal WW domains (residues 6-36 and 45-75), a more C-terminal myosin tail homology 4 (MyTH4) domain (amino acids 997-1124), a RhoGAP domain (amino acids 1154-1303), and a Pfam-B 53745 domain (amino acids 1304-1330). A predicted human protein, KIAA1688 has an identical domain structure and overall 29% identity and 51% similarity to Drosophila Vilse, which is in turn the closest match to KIAA1688 in Drosophila. KIAA1688 is therefore referred to as the human Vilse protein. In addition, the human genome encodes a second Vilse homolog with an additional extensin-2 domain (48% similarity, 26% identity, GenBank accession no. gi|37574693|r). Vilse homologs can also be found in the mosquito (66% similarity, 29% identity for gi|30176853|) and mouse (50% similarity, 28% identity, gi|28380066|) genomes. No protein with the same modular structure as Vilse was identified in the Caenorhabditis elegans genome, the closest relative in worms (33% similarity, 13% identity) is encoded by C38D4.5 CE and contains a WW, a PH, and a RhoGAP domain (Lundström, 2004).
In situ hybridization revealed that vilse transcript is ubiquitous during the first stages of development, suggesting a robust maternal contribution. Zygotic transcripts were prominent at stage 15 in the tip cells of all tracheal branches, the muscles, and midline cells of the VNC. This pattern was the same as the ß-gal marker expression in the enhancer trap strain, and was absent in vilse1 mutants, indicating that vilse1 is a strong loss-of-function mutant in RhoGap93B. To analyze the expression of Vilse protein, anti-Vilse antisera were raised. Immunostainings of whole-mount wild-type embryos detected Vilse protein expression in a pattern that mirrored the pattern of the vilse transcript and that of ß-gal expression in the vilse LacZ enhancer trap. In addition, Vilse antisera stained the epidermis, the peripheral nervous system (PNS) segmental and intersegmental nerves, and the CNS longitudinal connectives and commissures. This expression was not detected in the enhancer trap or by in situ hybridization, and may in part reflect the maternal protein. The antiserum is specific for Vilse; the staining was much reduced in vilse1 mutants. Vilse staining showed a subcellular distribution consistent with a cytoplasmic localization of the protein (Lundström, 2004).
Vilse promotes the hydrolysis of RacGTP and to a lesser extent that of Cdc42GTP. It is thus expected to antagonize the activity of these GTPases on their known effectors. Increasing experimental evidence indicates that Rac and Cdc42 regulate a multitude of cellular responses ranging from establishment of epithelial polarity and integrity to membrane trafficking and the control of planar polarity, in addition to their well known function in modulating the actin cytoskeleton. The tracheal phenotypes of vilse embryos are remarkably specific; all branches form, fuse, and grow toward their targets without any apparent defects on epithelial polarity, integrity, or shape. In addition, the rest of the terminal cells that target other internal organs concurrently with GB1 migrate correctly and associate with their targets. It is therefore concluded that the primary function of Vilse in the trachea is in the guidance of GB1 migration (Lundström, 2004).
How then does Vilse fulfill its role in cell navigation? Both of its target GTPases are key regulators of the actin cytoskeleton in several cell types. In fibroblasts, GTP-bound Cdc42 generates actin bundles and characteristic filopodial extensions, possibly through its association with the WASP protein and subsequent stimulation of the actin polymerizing activity of the Arp2/3 complex. RacGTP, in contrast, generates distinct cytoskeletal attributes, membrane ruffling, and lamellipodial protrusions. Rac controls actin polymerization through the intermediary protein IRSp53, which associates to the SCAR/WAVE regulator of Arp2/3 activity. Cdc42 also binds to the IRSp53 adaptor, suggesting that both GTPases regulate actin polymerization through SCAR/WAVE (Lundström, 2004).
An additional regulatory role for Rac and Cdc42 in cytoskeletal dynamics is exerted through their activating role on PAK (p21 activated kinase). PAK in turn activates the LIM kinase, which can phosphorylate the actin depolymerization factor (ADF/cofilin). Cofilin mediates depolymerization of actin filaments and can also function as a filament-severing factor. Its phosphorylation by LIM-kinase down-regulates its activity and inhibits F-actin depolymerization. Thus, the two GTPases in their active form promote the growth of actin filaments by both enhancing polymerization through the Arp2/3 complex and inhibiting severing and depolymerization at the minus end of the filaments. The phenotypic analysis of vilse and GTPase mutants leads to a proposal that Vilse antagonizes the function of Rac in promoting actin polymerization locally at the migrating tip of GB1 (Lundström, 2004).
Slit-mediated repulsive responses involve the regulation of cytoskeletal organization in the growth cone. In Drosophila, the Abelson kinase (Abl) binds to CC3 and phosphorylates a tyrosine in CC1, thereby modulating Robo activity. In contrast, the Abl substrate Enabled (Ena), a member of the profilin-binding family of proteins, associates with CC2 and mediates the repulsive role of Robo through an unknown mechanism that may involve control of cytoskeletal organization. More recently, Abl was found to collaborate with the cyclase-associated protein CAP, this time to mediate Robo repulsion. srGAPs bind to the CC3 domain of Robo in response to Slit and aid Cdc42GTP hydrolysis to directly mediate the repulsive response to Slit in cultured anterior subventricular rat neurons. This Cdc42GTP hydrolysis at the site of Robo activation would then result in actin filament depolymerization and severing, thus promoting the turn of the growth cone to the opposite direction. The functional analysis of Vilse identifies a direct transducer of the Slit signal to the inactivation of Rac. vilse, robo, and slit mutants show qualitatively the same phenotypes of midline crossings of tracheal cells and axons. The effect of vilse overexpression on the robo tracheal phenotypes and the dose-dependent interaction between slit, robo, and vilse, combined with the biochemical analysis indicate that Vilse acts downstream of Robo. Hence, Vilse may play an analogous role to srGAP in locally down-regulating actin polymerization through the hydrolysis of RacGTP and facilitating turning away from the midline (Lundström, 2004).
Paradoxically, both activation and inactivation of Rac appear to interfere with midline crossing and Slit signaling. Expression of constitutively activated Rac causes longitudinal axons to cross the midline, and reduction of Robo signaling enhances this phenotype. In contrast, rac mutants show strong phenotypes in axonal growth and guidance, including midline crosses, and the latter phenotype becomes more prominent by reduction of Slit. One possible explanation is that Rac might be involved in multiple cellular processes affecting different aspects of the Slit/Robo pathway. For example Rac might mediate Slit secretion by midline cells or intracellular trafficking of Robo in the axons, in addition to its effect on cytoskeletal dynamics downstream of Robo. The protein adaptor Dock has also been implicated in midline repulsion downstream of Robo. In response to Slit, Dock's binding to the intracellular domain of Robo is enhanced, leading to the recruitment of the Rac effector kinase Pak. This chain of events has been proposed to bring activated Rac to Robo in response to Slit. Yet, it is not clear how Rac becomes activated in response to Slit, or how the recruitment of active Rac and Pak might translate in the cellular events that lead to repulsion from Slit. The contradicting models of the function of rac downstream of robo may be reconciled by considering a sequential interaction of the effectors with the receptor. For example, Vilse may be required initially for severing of actin filaments at the cell extensions that first encounter Slit. The inducible recruitment of Pak to Robo might occur subsequently, perhaps in response to higher concentrations of Slit, promoting cytoskeletal reorganizations that lead to a sustained turning response. This involves a new function of Rac in the context of repulsion from the signal source. The genetic analysis of midline repulsion reveals that Slit signaling relies on the dynamic and spatially coordinated control of Rac activity. Vilse provides both the first direct link from Robo to the inactivation of Rac, and a molecular handle to address the complex interactions that control repulsion during cell migration (Lundström, 2004).
Rho GTPases are essential regulators of cytoskeletal reorganization, but how they do so during neuronal morphogenesis in vivo is poorly understood. The actin-depolymerizing and actin-severing protein factor cofilin, encoded by twinstar, is essential for axon growth in Drosophila neurons. Cofilin function in axon growth is inhibited by LIM kinase and activated by Slingshot phosphatase. Dephosphorylating cofilin appears to be the major function of Slingshot in regulating axon growth in vivo. Genetic data provide evidence that Rho or Rac/Cdc42, via effector kinases Rho-associated kinase (Rok, also named Rho kinase or ROCK), or p21-activated kinase (Pak), respectively, activate LIM kinase to inhibit axon growth. Importantly, Rac also activates a Pak-independent pathway that promotes axon growth, and different RacGEFs regulate these distinct pathways. These genetic analyses reveal convergent and divergent pathways from Rho GTPases to the cytoskeleton during axon growth in vivo and suggest that different developmental outcomes could be achieved by biases in pathway selection (Ng, 2004).
Biochemical studies have shown that LIM kinase is activated by Pak, a downstream effector kinase for Rac and Cdc42. Whether Pak activation could affect the LIMK pathway in axon growth was tested. In similar genetic interaction experiments, it was found that introducing three independently generated Drosophila Pak mutant alleles suppresses the LIMK1 overexpression phenotype. In addition, Pak overexpression also results in axon growth and guidance defects similar to those seen with LIMK1 overexpression, consistent with the model in which Pak activates LIMK1, leading to cofilin hyperphosphorylation. As predicted from this model, coexpression of active versions of cofilin with Pak results in a partial suppression of the growth and guidance defects (Ng, 2004).
Next it was determined which Rho GTPase pathway (Cdc42, Rac1/Rac2, or Rho1) regulates Drosophila Pak. It was found that loss of one copy of Cdc42 results in a strong suppression of the Pak overexpression phenotypes. Similar reduction of Rac1 and Rac2 (Rac1J10, Rac2Δ) results in weaker suppression. In contrast, reducing Rho1 results in a slight enhancement of the Pak overexpression phenotype. This suggests that the Pak overexpression phenotype specifically reflects the positive signaling input from Cdc42 and Rac in vivo (Ng, 2004).
Next it was tested whether mutations in the Cdc42 or Rac genes (Rac1, Rac2, Mtl, and the different combinations) would modify the LIMK1 overexpression phenotype. Reducing the Cdc42 activity leads to the suppression of the LIMK1 phenotype, suggesting that Cdc42 acts through Pak to activate LIMK1. Loss of one copy of both Rac2 and Mtl does not alter the LIMK1 phenotype. However, the LIMK1 phenotype is very sensitive to the levels of endogenous Rac1, since introducing one copy of a strong hypomorphic allele of Rac1 (Rac1J10) significantly suppresses the LIMK1 phenotype. This suppression is further enhanced when one copy of a null allele of Rac2 is introduced. These results suggest that Rac signaling (in particular, Rac1 and Rac2) also acts to activate LIMK1. This suggestion was also supported by the overexpression experiment. Overexpression of wild-type Rac1 (UAS-Rac1 WT) results in a weak LIMK1-like phenotype. However, when Rac1 and LIMK1 are coexpressed, axon growth defects were enhanced. Together with previous biochemical studies, these results suggest that Cdc42 and Rac act via Pak to activate LIMK1 (Ng, 2004).
To verify these genetic interactions with endogenous components, whether reducing either Cdc42 or Rac activity would modify the ssh-/- phenotype was examined. As with Rho1, reducing Cdc42 or Rac activity also partially suppresses the ssh-/- defects. Thus, these data indicate that Rho1, Cdc42, and Rac can all act through distinct downstream kinases to activate LIMK1 (Ng, 2004).
Surprisingly, reducing Rac GTPase activity can also result in enhancing the LIMK1 overexpression phenotype. For instance, the suppression effect of Rac1J10 Rac2Δ/+ was reverted by heterozygosity of Mtl (Rac1J10 Rac2Δ MtlΔ/+). This enhancement is more evident when one copy of the strongest allele of Rac1 was introduced into the LIMK1 overexpression background (Rac1J11/+). This effect is not due to dominant effects of this Rac1 allele, since in the same genetic background Rac1J11/+ animals did not display LIMK1-like growth defects without the LIMK1 expression transgene. Introduction of another hypomorphic allele of Rac1 (Rac1J6) together with Rac2 (Rac1J6 Rac2Δ/+) also strongly enhances the LIMK1 overexpression phenotype (Ng, 2004).
One interpretation of these results is that, while Rac can activate LIMK1 (via Pak), there is an alternative pathway downstream of Rac, acting antagonistically to LIMK1, that promotes axon growth. This is consistent with the finding that loss of Rac GTPase activity leads to axon growth defects in MB neurons (Ng, 2002). This pathway is likely to be Pak independent, since the Rac axon growth-promoting activity does not require direct binding to Pak (Ng, 2002), and Pak activation leads to axon growth inhibition. To further verify the existence of a Pak-independent pathway in regulating axon growth, use was made of a well-described Rac1 effector domain mutant. In mammalian fibroblasts, Rac1 activates Pak1 and, through an independent downstream pathway, promotes lamellipodia formation. A Y40C mutation in the effector binding domain of Rac1 results in the loss of Pak1 activation, but the lamellipodia promoting activity is maintained. Transgenic overexpression of Rac1 Y40C alone at levels comparable to those of wild-type Rac did not result in gross axon phenotypes. However, in contrast to wild-type Rac1 that strongly enhances LIMK1, coexpression of Rac1 Y40C strongly suppresses the LIMK1 overexpression phenotypes. These results suggest that Rac1 activates a Pak-independent pathway to counteract the effects of LIMK1 activity on axon growth (Ng, 2004).
Whether upstream activators of Rac (RacGEFs), could regulate these distinct axon growth pathways was tested. Trio encodes a RacGEF essential for axon guidance in MB neurons. Introducing one mutant copy of trio significantly suppressed the LIMK1 overexpression phenotype, suggesting that Trio acts to activate LIMK1. This was further verified by the overexpression experiments in which overexpression of wild-type Trio (UAS-trio) alone results in a mild LIMK1-like phenotype, while coexpression with LIMK1 results in a strong enhancement of the axon growth defects. Trio has two GEF domains—GEF1 is specific for Rac1, Rac2, and Mtl in vitro and in vivo, and GEF2 can activate Rho1/RhoA in vitro. Overexpression of the isolated Trio GEF1 domain (UAS-trio GEF1) in MB neurons results in severe axon growth defects, whereas overexpression of the isolated Trio GEF2 domain (UAS-trio GEF2) does not result in any gross defects. These results support the model that, in MB neurons, Trio, via its GEF1 domain, acts through Rac and Pak to activate LIMK1. These findings are also consistent with those of previous studies in which Trio acted via Rac/Pak to regulate Drosophila photoreceptor axon guidance (Ng, 2004).
In contrast to Trio, loss of one copy of still life (sif), encoding a different RacGEF, markedly enhances the LIMK1 phenotype. In overexpression experiments, UAS-sif alone does not result in gross axon defects. However, coexpression of Sif resulted in a strong suppression of the LIMK1 phenotype. These experiments suggest that Sif activates the pathway that acts antagonistically to LIMK1 (Ng, 2004).
The data suggest that Rac promotes axon growth via a pathway antagonistic to Pak and LIMK1. How does this pathway act to promote axon growth? One strong possibility is that Rac stimulates actin polymerization to promote axon growth. Therefore a number of candidate genes known to promote actin polymerization were tested. The following genetic criteria were establised by which these candidate pathways should work: (1) like Rac, loss of the candidate gene should result in axon growth defects; (2) genetic interactions with LIMK1, either through loss- or gain-of-function analyses, should show that they act antagonistically to LIMK1 (Ng, 2004).
The role of the actin nucleation factor SCAR-Arp2/3 complex was tested. Rac has been shown to promote de novo actin polymerization through interactions via SCAR and the Arp2/3 complex. Activation of the SCAR-Arp2/3 complex is required to establish cell protrusions during lamellipodia and filopodia formation, making it a good candidate pathway for promoting axon growth. This hypothesis was tested by making MARCM clones in MB neurons using null alleles of SCAR, WASp (a protein related to SCAR, also called Wsp), double mutants for SCAR and WASp, or Arpc1 (Flybase-Sop2), an essential component of the Arp2/3 complex. No axon growth defects were detected in single-cell or neuroblast clones. In addition, reduction of SCAR or Sop2 levels did not modify the LIMK1 overexpression phenotype. Furthermore, overexpression of SCAR did not suppress, but mildly enhanced, the LIMK1 overexpression phenotype. Taken together, these results suggest that the SCAR/WASp-Arp2/3 pathway does not play an essential role in axon growth of MB neurons, and it is unlikely that this pathway contributes to the Rac pathway that promotes axon growth (Ng, 2004).
Several other known regulators of actin polymerization were tested for their contribution to MB axon growth. The actin polymerization stimulators profilin (encoded by chickadee or chic) and Enabled (Ena) have been shown to be essential for axon growth and guidance in Drosophila. In addition, genetic interaction studies suggest that these proteins may be involved in Rac GTPase signaling. When assayed for MB axon growth, both ena-/- and chic-/- single-cell and neuroblast clones exhibited drastic axon growth defects. Interestingly, when examined at higher resolutions, neither ena nor chic axons displayed filopodia-like and lamellipodia-like protrusions at the axon termini characteristic of tsr-/- neurons. These genetic interaction experiments found no evidence that ena or chic act antagonistically to LIMK1. In fact, Ena overexpression strongly enhances the LIMK1 overexpression phenotype. Thus, although both Ena and profilin are essential for MB axon growth, they do not appear to constitute the Rac-mediated axon growth-promoting pathway (Ng, 2004).
Finally, the Formin-class protein Diaphanous (Dia) was tested, since it has been implicated in regulating actin polymerization downstream of Rho GTPases. dia-/- MB neurons do not exhibit axon growth defects in single-cell clones or in neuroblast clones, which exhibit strong cell proliferation defects. Interestingly, reducing Dia activity appears to suppress the LIMK1 overexpression phenotype, suggesting that Dia can act in a pathway that enhances, but does not antagonize, LIMK1 (Ng, 2004).
The Drosophila Ndr serine/threonine kinase Tricornered (Trc) is required for the normal morphogenesis of epidermal hairs, bristles, laterals, and dendrites.
Mutations in trc and its interacting factor furry (fry) cause cells to form ectopic wing hairs, implying that these genes function temporally before hair initiation (Geng, 2000; Cong, 2001).
In vivo evidence has been obtained that Trc function is regulated by phosphorylation and that mutations in key regulatory sites result in dominant negative alleles. Wild-type, but not mutant Trc, is found in growing hairs, and no Trc has been detected in pupal wing nuclei, implying that in this developmental context Trc functions in the cytoplasm. The furry gene and its homologues in yeast and Caenorhabditis elegans have been implicated as being essential for the function of the Ndr kinase family. Drosophila furry is also found in growing hairs, its subcellular localization is dependent on Trc function, and it can be coimmunoprecipitated with Trc. These data suggest a feedback mechanism involving Trc activity that regulates the accumulation of Fry in developing hairs (He, 2005).
Tricornered is involved in a repulsion mechanism regulating the distribution of dendrites in dendritic fields. To cover neuronal receptive fields completely but without redundancy, neurons of certain functional groups exhibit tiling of their dendrites via dendritic repulsion. Two evolutionarily conserved proteins, the Tricornered and Furry, are essential for tiling and branching control of Drosophila sensory neuron dendrites. Dendrites of fry and trc mutants display excessive terminal branching and fail to avoid homologous dendritic branches, resulting in significant overlap of the dendritic fields. Trc control of dendritic branching involves regulation of RacGTPase, a pathway distinct from the action of Trc in tiling. Time-lapse analysis further reveals a specific loss of the ability of growing dendrites to turn away from nearby dendritic branches in fry mutants, suggestive of a defect in like-repels-like avoidance. Thus, the Trc/Fry signaling pathway plays a key role in patterning dendritic fields by promoting avoidance between homologous dendrites as well as by limiting dendritic branching (Emoto, 2004).
The RhoGTPase family, including Rho, Rac, and Cdc42, plays a crucial role in neuronal morphogenesis. In particular, proper activation of Rac in developing neurons is essential for establishing and maintaining their unique dendritic branching pattern. To test whether Trc signaling involves Rac regulation, it was first asked whether overexpression of wild-type and mutant Rac1 affects dendritic morphology, and then the effects of coexpressing wild-type or mutant Trc and Rac1 were examined. Overexpression of wild-type Rac1 (RacWT) in class IV neurons results in overbranching of dendrites but does not produce any obvious tiling phenotype. This overbranching phenotype is partially suppressed by coexpression of wild-type Trc. Importantly, whereas expressing the dominant-negative Rac1 (RacN17) alone does not cause a detectable dendritic phenotype, RacN17 significantly suppresses the overbranching phenotype but not the tiling phenotypes in neurons expressing the dominant-negative Trc(K112A) mutant. The involvement of Rac in Trc signaling appears specific; coexpression of the dominant-negative RhoL (RhoN25) did not result in a significant change of dendritic branching and tiling phenotypes in neurons expressing the K112A mutant. These results suggest that Trc/Fry may negatively regulate Rac signaling to control dendritic branching (Emoto, 2004).
To further test this possibility, coimmunoprecipitation experiments were carried out and Trc was found in a complex with Rac1 but not with Cdc42 in Drosophila S2 cells. Moreover, using a pull-down assay in which Rac-GTP (the activated form of Rac) but not Rac-GDP is isolated via the Rac-GTP binding domain of PAK conjugated to GST, it was found that overexpression of wild-type Trc in stably transfected cell lines causes a significant reduction of the amount of Rac1-GTP compared to control cells, whereas expression of the dominant-negative Trc(K112A) mutant increases Rac1-GTP level. Taken together, these findings suggest that the Trc/Fry signaling negatively regulates Rac activity to control dendritic branching whereas another, distinct pathway mediates the action of Trc in tiling (Emoto, 2004).
Members of the Rho family of small GTPases are required for many of the
morphogenetic processes required to shape the animal body. The activity of
this family is regulated in part by a class of proteins known as RhoGTPase
Activating Proteins (RhoGAPs) that catalyse the conversion of RhoGTPases to
their inactive state. In a search for genes that regulate
Drosophila morphogenesis, several lethal alleles have been isolated of
crossveinless-c (cv-c). Molecular characterisation reveals
that cv-c encodes the RhoGAP protein RhoGAP88C. During embryonic
development, cv-c is expressed in t RhoGAPs normally function to stimulate the intrinsic GTP hydrolysing
capacity of the GTPases, thereby converting them to the inactive GDP-bound
form. Thus, the phenotypes observed in the absence of Cv-c function are
likely to be caused by elevated activities of the cognate GTPase substrate(s)
of Cv-c. Such relationships can be tested by genetic interactions; a reduction
in the activity of a substrate GTPase would rescue the cv-c
phenotype, while an increase in GTPase activity would enhance it. To identify
the substrate(s) of Cv-c, genetic interactions between
cv-c and candidate GTPases were
analysed for which mutants are available. Embryos
homozygous for cv-cM62 and homozygous or hemizygous for
mutations in the GTPases Rho1, Rac1, Rac2, Mtl and Cdc42
were analysed for both the Malpighian tubule and embryonic cuticle phenotypes (Denholm, 2005).
Removal of any of the Rac candidates, Rac1, Rac2 or Mtl,
and reduction or removal of Cdc42 failed to modify the
cv-cM62 phenotype in the Malpighian tubules. By contrast, 50% of
cv-cM62 mutant embryos additionally homozygous for
Rho172R and 20% of
cv-cM62 mutant embryos heterozygous for
Rho172R had a phenotype significantly less
severe than that of the cv-cM62
homozygote alone. The Malpighian tubules
of doubly mutant embryos undergo convergent extension movements to some extent,
such that they resemble weaker alleles of
cv-c. These data strongly suggest that Rho1 is a
substrate for Cv-c in the Malpighian tubules (Denholm, 2005).
Genetic interations were examined in the embryonic epidermis. Dorsal
closure defects occur in 82% of embryos doubly mutant for Rac1 and
Rac2. However, if Rac1,Rac2 embryos are additionally mutant for
cv-cM62, 37% of these embryos are rescued, indicating that Rac
GTPases are substrates for Cv-c during dorsal closure (Denholm, 2005).
The posterior spiracle phenotype in cv-c embryos was not
suppressed by any of the RhoGTPase mutants, possibly because maternal
contribution of the substrate GTPase is sufficient to provide full activity in
this tissue. However, it was unexpectedly found that embryos mutant for both
Rac1 and Rac2 do not decrease, but enhance the
cv-cM62 posterior spiracle phenotype. One possible
explanation for this interaction comes from observations in cell culture,
where it has been shown that Rac activity downregulates Rho activation. If Rac
normally functions to inhibit Rho in the posterior spiracle, then loss of Rac
and Cv-c together, would lead to hyperactivity in Rho1 and this would lead to
the enhancement of the cv-c phenotype. In support of this,
cv-c-like posterior spiracle phenotypes were seen with low penetrance in
Rac1 Rac2 embryos (Denholm, 2005).
During myoblast fusion, cell-cell recognition along with cell
migration and adhesion are essential biological processes. The factors involved
in these processes include members of the immunoglobulin superfamily like Sticks
and stones (Sns), Dumbfounded (Duf) and Hibris (Hbs), SH3 domain-containing
adaptor molecules like Myoblast city (Mbc) and multidomain proteins like Rolling
pebbles (Rols). For rolling pebbles, two differentially expressed
transcripts have been defined (rols7 and rols6). However, to date,
only a muscle fusion phenotype has been described and assigned to the lack of
the mesoderm-specific expressed rols7 transcript. This study shows that a
loss of the second rolling pebbles transcript, rols6, which is
expressed from the early bud to later embryonic stages during Malpighian tubule
(MpT) development, leads to an abnormal MpT morphology that is not due to
defects in cell determination or proliferation but to aberrant morphogenesis. In
addition, when Myoblast city or Rac are knocked out, a similar phenotype is
observed. Myoblast city and Rac are essentially involved in the development of
the somatic muscles and are proposed to be interaction partners of Rols7.
Because of the predicted structural similarities of the Rols7 and Rols6
proteins, it is argued that genetic interaction of rols6, mbc and
rac might lead to proper MpT morphology. It is also proposed that these
interactions result in stable cell connections due to rearrangement of the
cytoskeleton (Putz, 2005).
The Malpighian tubules (MpTs) of Drosophila arise as four buds from
the hindgut anlage close to its boundary with the posterior midgut primordium.
The cells of the four buds are characterised by the expression of the
transcription factor Cut (Ct) at stage 10 of embryogenesis.
During germ band extension at stage 11, the cells of the four tubule primordia
undergo cell proliferation, and the tubules begin to bud out. By stage 13,
proliferation is complete and short tubules have formed. From stage 13
onwards, cells from the caudal mesoderm join the MpT primordia and later the
stellate cells (SCs). From the end of germ band retraction, the tubules begin to
elongate due to cell rearrangement. In stage 15 and 16 embryos, the
characteristic stereotypic course of the four renal tubules through the
embryonic body is clearly visible. The paired posterior tubules span the
posterior abdominal and terminal segments of the embryo. The anterior tubules
extend forwards into abdominal segments 2/3 where the tubule
loops back on itself so that the tips of both anterior tubules lie more
posteriorly within the abdomen (Putz, 2005).
Since Rols6 is expressed in the Malpighian tubules (MpTs) throughout their
development, the role of Rols6 in the generation of this tissue was investigated.
For this purpose, a rols6-specific mutant was generated, in which the
majority of the putative promoter region of rols6 was deleted, and thereby
rols6 transcription was knocked out, while rols7 expression persisted as in the wild type. In this rols6-specific mutant, the early phase of
organogenesis is the same as in wild type, i.e. the MpTs consist of two cell
types, the principal cells (PCs) and the SCs. As the SCs originate from the mesoderm, one might
expect that they would be affected in rols mutants. However, in the
specific rols6 mutation generated, the SCs are able to migrate and
integrate between the PCs as observed in the wild type. However, the PCs and SCs
do not arrange correctly, and therefore, the typical MpT arrangement as found in
wild-type embryo is not observed for stage 15 embryos onwards. The anterior
tubules often show abnormal curves and lasso-like structures and fail to extend
through the abdominal cavity. These navigation defects might well result from
incorrect cell rearrangements, indicated by thickened regions of the tubules,
whereas other parts seem to have a typical wild-type organisation (Putz, 2005).
Evidence is presented that correct cell rearrangement is dependent
on Rols6 and proteins such as Mbc and Rac. These factors have been proposed to
act with Rols7 in a common signalling cascade during myoblast fusion. An
additional defect is the disorientation of MpTs in the body cavity, which again
is characteristic for rols6, mbc and rac mutants (Putz, 2005).
Homozygous rols6 mutants are viable, indicating that the physiological functions of principal cells and stellate cells are largely unaffected. Loss of rols6 expression only moderately affects embryonic viability. Furthermore, homozygous EP(3)3330*5a flies do not die prematurely in contrast to those lacking another gene essential for MpT formation, hibris. The strongest hibris allelic combination die early as adults. Also, in contrast to rols6 mutants, in hibris mutants, the number of SCs is strongly reduced. This might cause defects in excretory function of the tubules, and thus leads to the observed lethality (Putz, 2005).
rols6-specific mutants show no distortion in rols7
transcription and in muscle development indicating that rols6 is specific
for MpT development, while rols7 is essential for myogenesis. This is
consistent with the observatio that Rols6 is not able to rescue the myogenic defect in rols mutants (Putz, 2005).
myoblast city mutants, rolling pebbles mutants and rac1/rac2 double mutants show late defects in Malpighian tubule differentiation. mbc mutants exhibit a MpT phenotype and it is proposed that this might be due to a failure to complete cell rearrangement; a phenomenon which is more apparent in mbc mutants than for rolling pebbles ones. Mbc, the homologue of vertebrate DOCK180 in Drosophila, associates with the adapter protein Crk. This interaction regulates cell migration and cytoskeleton organisation in a Rac-dependent manner. This agrees with the finding that rac1/rac2 double mutants exhibit the characteristic MpT defects as rols6 and mbc mutants do. Rols7 and Duf have been shown to interact in myogenesis. The strong similarity between the Rols proteins and their proposed functions leads to the hypothesis that Rols6 interacts with a so far unknown partner in the PCs. It is proposed that Rols6 initiates a signalling cascade via Mbc and Rac that leads to the correct rearrangement of cells, presumedly by rearranging the cytoskeleton, as has been proposed for Rols7 in the myogenic precursor cells. In the development of the
somatic musculature, rearrangement of cytoskeleton is mediated by Blown fuse
(Blow) and Kette in the second fusion wave (Putz, 2005).
Individual factors and protein complexes involved in cell migration and cytoskeleton arrangement have been described from many model organisms as well as from cell culture experiments. DOCK180/CED-5, the homologues of Drosophila Myoblast city (Mbc) in vertebrates and in C. elegans, form a complex with
ELMO1/CED-12 that functions as a guanine nucleotide exchange factor (GEF). This
functional GEF promotes Rac activation, and thus facilitates cell migration and
rearrangement of the cytoskeleton. In vertebrates, additional protein
complexes are built via DOCK180/p130Cas/Crk interaction and regulate
cell migration and cytoskeletal organisation in a Rac-dependent manner. From kidney
cells of human and mouse, the signalling molecule NEPHRIN is known to be of
major importance in the podocyte for slit-diaphragm formation. Mutations in the
nephrin gene are the major cause of congenital nephrotic syndrome in
humans. In Drosophila, the homologue of vertebrate Nephrin, Hibris (hbs), is expressed during
MpT development specifically in SCs. Therefore, it is likely that during MpT
differentiation, Hibris mediates cell adhesion and arrangement between the PCs
and the SCs, a mechanism comparable to myogenesis. In vertebrates, CMS/CD2AP has
been identified as an interaction partner for Nephrin. The CMS/CD2AP homologue
in Drosophila can be detected in silico as CG11316. CD2AP knock-out mice die due to kidney failure. Moreover, the Nephrin/CD2AP complex is able to bind to actin and to p130Cas (corresponding to CG1212). In Drosophila,
homologues have been identified for all the above-mentioned factors involved in
these protein complexes. However, little is known about their role in the
developmental processes taking place during MpT development (Putz, 2005).
In Drosophila, a group of immunoglobulin-like proteins act in
cell-cell recognition and attraction during myogenesis. These processes
are also of importance in MpT development. Rolling pebbles is a multidomain and adapter-like protein. It is proposed that Rols6 interacts in Malpighian
tubule development with proteins also involved in myogenesis such as Mbc and
Rac. It is assumed that Rolling pebbles interacts with Mbc, and thus activates Rac.
This hypothesis is supported by the observations that mbc and rac
mutants exhibit defects in MpT development which might be linked to cell
organisation in this tissue (Putz, 2005).
The mechanisms underlying the stereotypic course
of the MpTs through the body cavity are still unclear. However, studies of
phenotypes of early determination mutants like numb show that the tip
cell and its sibling might both play a critical role in controlling the spatial
arrangement of the growing tubules. This is indicated by the MpT phenotypes of
numb mutants and UAS-numb embryos, where numb is
overexpressed. These embryos lack either the tip cell or the sibling cell but
form elongated MpTs with normally rearranged PCs. Although the PCs rearrange normally in these numb alleles, the MpTs are misrouted through the body cavity, as has been observed for rols and rac mutants. This raises the question whether determination of the tip cells is affected in rols
mutants (Putz, 2005).
Essential transcription factors for Tip cell determination and PC
cell proliferation are the A-SC, Krüppel and Seven up. These factors could be required for rols6 expression in the MpTs. However, since rols6 is expressed in the rudimentary primordia of MpTs in Krüppel mutants and in seven up mutants, this is unlikely. It is
assumed, therefore, that Rolling pebbles is not a signalling molecule involved in
cell specification through direct regulation of early genes, but rather that it
plays a role as an adapter molecule in a protein complex connecting the cells in
the tissue as Rols7 does in myogenesis. Since rols6, mbc and rac
mutant embryos exhibit the described MpT phenotype, it is likely that they
belong to a group of genes that can be helpful in discovering the mechanisms in
MpT development that lead to the typical thin tubule morphology through cell
rearrangement (Putz, 2005).
Myoblast fusion is an intricate process that is initiated by cell recognition and adhesion, and culminates in cell membrane breakdown and formation of multinucleate syncytia. In the Drosophila embryo, this process occurs asymmetrically between founder cells that pattern the musculature and fusion-competent myoblasts (FCMs) that account for the bulk of the myoblasts. The present studies clarify and amplify current models of myoblast fusion in several important ways. They demonstrate that the non-conventional guanine nucleotide exchange factor (GEF) Mbc plays a fundamental role in the FCMs, where it functions to activate Rac1, but is not required in the founder cells for fusion. Mbc, active Rac1 and F-actin foci are highly enriched in the FCMs, where they localize to the Sns:Kirre junction. Furthermore, Mbc is crucial for the integrity of the F-actin foci and the FCM cytoskeleton, presumably via its activation of Rac1 in these cells. Finally, the local asymmetric distribution of these proteins at adhesion sites is reminiscent of invasive podosomes and, consistent with this model, they are enriched at sites of membrane deformation, where the FCM protrudes into the founder cell/myotube. These data are consistent with models promoting actin polymerization as the driving force for myoblast fusion (Haralalka, 2011).
Recent consideration of myoblast fusion have included a common model in which Mbc and Rac1 function downstream of Kirre in the founder cells to direct actin polymerization, and is based on studies showing that Mbc interacts with the Kirre-associated Rols/Ants protein. However, since founder cell-specific expression of Mbc is inadequate to rescue its loss of function fusion phenotype, this study reasoned that it must be required in the FCMs. It has also been suggested that F-actin foci are present symmetrically at points of contact between founder cells and FCMs. This symmetric F-actin-associated adhesive structure has been termed the FuRMAS (fusion-restricted myogenic-adhesive structure), and appears to be a ring of Sns and Kirre surrounding a central core of F-actin. The current results amplify these models in several important areas. First, the data reveal that Mbc is explicitly required in the FCMs and is not needed in the founder cells for fusion to occur. Thus, the essential function of Rols/Ants in fusion cannot be to direct recruitment of Mbc to Kirre in the founder cells. Second, high-resolution imaging has revealed that Mbc, active Rac1 and F-actin are concentrated in FCMs near Sns at the point of contact with founder cells, and are therefore localized asymmetrically in the fusion partners. Moreover, FCM-associated structures project into the founder cell and myotube at the Sns:Kirre adhesion site prior to fusion. Consistent with these observations, other approaches have recently reported the enrichment of actin foci in FCMs and the presence of invadopodia visible in EM that extend from the FCM into the founder cell/myotube. Finally, it was also found that Mbc is important for the integrity of the F-actin focus and for the overall integrity of the actin cytoskeleton in FCMs (Haralalka, 2011).
Although very limited fusion occurs in embryos lacking Mbc, more than 80% of the founder cells do not undergo even a single fusion event. By contrast, FCM-directed expression of Mbc rescues an almost wild-type pattern of muscle fibers. Thus, Mbc expression in the FCMs is both necessary and sufficient for their fusion with founder cells. Robust fusion was also observed when activated Rac1 is expressed in the FCMs of mbcD11.2 embryos, indicating that the primary role of Mbc is to activate Rac1. The data do not support a mechanism in which Rols/Ants functions in the founder cells to recruit Mbc to the cytodomain of Kirre, a mechanism that is also inconsistent with the ability of Kirre to direct precursor formation in the absence of its cytodomain. Rather, these data support a mechanism in which Rols/Ants stabilizes Kirre, thereby ensuring that Kirre continues to be present on the myotube surface for fusion with FCMs. Unfortunately, it not possible to determine in the embryo whether Mbc or activated Rac1 is sufficient in the FCMs for later Mbc-dependent fusion between syncitia and FCMs, as the contents of the FCMs become incorporated into the syncitia following fusion. In primary cultures, however, wild-type founder cells as well as founder cells and binucleate precursors lacking Mbc all fuse with wild-type FCMs and at similar rates. Thus, the asymmetric distribution of Mbc in the FCMs is also sufficient for later fusion events, at least in cultured myoblasts (Haralalka, 2011).
It was not possible to address whether Rac1 and Rac2 are specifically required in the FCMs as the founder cells of rac1J11, rac2? embryos are already syncytial owing to the perdurance of maternally provided gene product. Although localization of active Rac1 to points of cell contact in the FCMs may be an indication that, like Mbc, Rac1 is essential only in the FCMs, it is noted that Rac1 is required in both fusion partners in vertebrates. In either case, it can be concluded that any requirement for Rac1 in the founder cells must involve a GEF other than Mbc/Elmo (Haralalka, 2011).
The data localize Mbc, active Rac1, and F-actin to FCMs at the Sns:Kirre adhesion site with either founder cells or developing myotubes. This asymmetric distribution is independent of whether the FCMs are contacting founder cells or myotubes and, in combination with features of fusion in primary myoblasts, suggests that the first fusion event does not differ from subsequent events with respect to these proteins. These data support a model in which both early and later stages of fusion are highly asymmetric, and that this asymmetry extends beyond recognition and adhesion at the cell surface to cytoplasmic events associated with polymerization of actin. Mbc, which is present but not localized in founder cells/myotubes, may serve a different purpose in these cells such as activating Rac1-dependent myotube guidance or attachment. In addition, though no large F-actin foci were observed on the founder cell/myotube side of the adhesion site, a strong layer of cortical actin was observed along the surface of the founder/developing myotube (Haralalka, 2011).
The local accumulation of F-actin in FCMs is reminiscent of dynamic actin foci found at sites of fusion and associated with the WAVE/SCAR, Vrp/WASp and the Arp2/3 complex. F-actin is also present in the core of the muscle-specific fusion-restricted myogenic-adhesive structure (FuRMAS). Interestingly, similarities have been noted between the FuRMAS and the immunological synapse (IS), podosome and invadopodia. The data provides important new information in support of this analogy, as invasive podosomes, the IS and invadopodia are actually all associated with asymmetic F-actin. As noted earlier, strong support for this analogy has recently been demonstrated by the presence of invadopodia-like invasive structures at the level of EM, that extend multiple finger-like projections into the founder cell/myotube. Interestingly, the IS, invadopodia and muscle-specific FuRMAS have common F-actin regulators that include WASp, HEM/Kette, SCAR/WAVE and Rho-family GTPases. Thus, the FCM appears to provide the primary F-actin associated force for myoblast fusion (Haralalka, 2011).
Previous studies have reported that F-actin foci in mbc mutants are enlarged and increased in number. However, careful 3D reconstruction of F-actin in embryos and in primary cells suggests that the tight actin foci found in wild-type FCMs are less organized and more dispersed in mbc mutants. The current analysis also revealed that the cytoskeletal network at the periphery of the FCM has collapsed in the absence of mbc. Thus, it appears that Mbc positively regulates organization of the actin cytoskeleton and actin polymerization at the adhesion site (Haralalka, 2011).
Although the mechanism(s) by which Mbc-activated Rac1 accomplishes this role have yet to be elucidated, Rac1 is known to interact with components of the WAVE/SCAR pathway in Drosophila and mammalian cells. In mammals, SCAR exists as part of a multiprotein complex composed of HEM/Kette, Abi, Sra1 and HSPC300. These subunits control SCAR stability and localization at the membrane. Moreover, the pentameric SCAR complex can be activated by GTP-bound Rac to promote actin polymerization by Arp2/3. HEM/Kette and SCAR play crucial roles in Drosophila myoblast fusion, and Rac1 has been shown to synergize with SCAR in the myoblast/myotube. Moreover, SCAR is absent from sites of fusion in
rac1 mutant embryos. Notably, recent studies have shown that SCAR is required in both cell types, though it remains to be determined whether it plays similar roles in each. In summary, however, the current studies support a mechanism in which Mbc/Elmo mediates the cell-type specific activation of Rac1 and, in turn, activation of WAVE/SCAR to promote an invasive actin-associated structure in the FCMs (Haralalka, 2011).
Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion (Yang, 2006).
Sos was identified in Drosophila as a GEF for Ras in the sevenless signaling pathway during the development of the Drosophila compound eye, where it activates the Ras signaling cascade to determine R7 photoreceptor specification. Studies in mammalian cell culture demonstrated that Sos functions as a GEF for both Ras and Rac in the growth factor-induced receptor tyrosine kinase (RTK) signaling cascade. Upon RTK activation, the SH3/SH2 adaptor protein Grb2/Drk recruits Sos to autophosphorylated receptors at the plasma membrane, where Sos activates membrane-bound Ras. In a later event downstream of RTK activation, Sos is thought to be targeted to submembrane actin filaments by interaction with another SH3 adaptor, E3b1(Abi-1), where Sos activates Rac . Whether the activation of Rac by Sos is strictly dependent on prior activation of Ras remains controversial, nor is it clear how Sos coordinates the activity of its two GEF domains in vivo (Yang, 2006 and references therein).
Evidence is provided that Sos functions as a Rac-specific GEF during Drosophila midline guidance. Sos is enriched in developing axons, and sos exhibits dosage-sensitive genetic interactions with slit and robo. Strikingly, genetic rescue experiments show that the Dbl homology (DH) RhoGEF domain of Sos, but not its RasGEF domain, is required for its midline guidance function. Biochemical experiments show that Sos physically associates with the Robo receptor through Dock in both mammalian cells and Drosophila embryos. Furthermore, Slit stimulation of cultured cells results in the rapid recruitment of Sos to membrane Robo receptors. These results provide a molecular link between the Robo receptor and Rac activation, reveal an independent in vivo axon guidance function of the DH RhoGEF domain of Sos, and support the model that Slit stimulation recruits Sos to the membrane Robo receptor via Dock to activate Rac-dependent cytoskeletal changes within the growth cone during axon repulsion (Yang, 2006).
These data support the idea that Sos provides a direct molecular link between the Robo receptor and the activation of Rac during Drosophila midline guidance. Genetic interactions between sos, robo, dock, crGAP/vilse, and the Rho family of small GTPases strongly suggest that Sos functions in vivo to regulate Rac activity during Robo signaling. Genetic rescue experiments indicate that sos is required specifically in neurons to mediate its axon guidance function. Furthermore, genetic data establish that, in the context of midline axon guidance, the Ras-GEF and Rac-GEF activities of Sos can be functionally uncoupled. Biochemical experiments in cultured cells and Drosophila embryos show that Sos is recruited into a multiprotein complex consisting of the Robo receptor, the SH3-SH2 adaptor protein Dock, and Sos, in which Dock bridges the physical association between Robo and Sos. Finally, experiments in cultured cells support the idea that Slit activation of Robo can recruit Sos to the submembrane actin cytoskeleton to regulate cell morphology. Together, these results suggest a model in which Slit stimulation recruits Sos to the Robo receptor via Dock to regulate Rac-dependent cytoskeletal changes within the growth cone during axon repulsion (Yang, 2006).
Based on previous work implicating rac in Robo repulsion, as well as in vitro studies demonstrating that Sos exhibits GEF activity for Rac, but not Rho or Cdc42, Rac seemed the most likely Sos substrate. However, rho has also been implicated in mediating Robo repulsion, and genetic interactions between sos and dominant-negative Rho have been interpreted to suggest that Sos could act as a GEF for Rho. This question was investigated further, and two types of genetic evidence have been presented that suggest that indeed Rac is the favored substrate of Sos. First, ectopic expression experiments in the eye reveal interactions exclusively between sos and rac. Second, genetic interaction experiments using loss of function mutations in rac and rho (rather than the more problematic dominant-negative forms of the GTPases) reveal strong dose-dependent interactions between sos and rac, but not sos and rho during midline axon guidance. Together, these observations argue in favor of Rac as the primary in vivo Sos substrate. Nevertheless, the possibilities that Sos also contributes to Rho activation and that the combined activation of Rac and Rho is instrumental in mediating the Robo response cannot be excluded (Yang, 2006).
Previous studies have demonstrated that Slit stimulation of the Robo receptor leads to a rapid increase in Rac activity in cultured cells. However, the mechanism by which Rac is activated downstream of Robo was not clear. This study provides direct genetic and biochemical evidence that Sos is coupled to the Robo receptor through the Dock/Nck SH3-SH2 adaptor, where it can regulate local Rac activation. Studies in cultured mammalian cells have highlighted the importance of distinct Sos/adaptor protein complexes in controlling the subcellular localization and substrate specificity of Sos. In the context of Rac activation, the E3b1 (Abi-1) adaptor has been shown to play a critical and rate-limiting role in Sos-dependent Rac activation and subsequent formation of membrane ruffles (Innocenti, 2002). Could Sos regulation of Rac activity during Robo repulsion be similarly limited by the availability of specific adaptor proteins? It is interesting to note in this context that overexpression of dock does not lead to ectopic axon repulsion, suggesting that Dock may not be limiting for Robo signaling. However, although dock mutants do have phenotypes indicative of reduced Robo repulsion, their phenotype is considerably milder than that seen in robo mutants, raising the possibility that there may be additional links between Robo and Sos (Yang, 2006).
A number of studies in cultured mammalian cells have suggested that Rac activation induced by activated growth factor receptors requires the prior activation of Ras. For example, PDGF-induced membrane ruffling can be promoted or inhibited by expression of constitutively active or dominant-negative Ras, respectively. However, other studies have suggested that in Swiss 3T3 cell lines RTK activation of Rac is Ras independent. In addition, the observation that Ras activation and Rac activation display very different kinetics, with Rac activation persisting long after Ras activity has returned to basal levels, has been used to argue against an obligate role for Ras in Rac activation. In this study, using a genetic rescue approach, whether the ability of Sos to activate Rac during axon guidance in an intact organism requires its Ras-GEF function was directly tested. Genetic data indicate that the RasGEF domain of Sos is dispensable for axon guidance, while the DH RhoGEF domain is strictly required. This observation argues strongly in favor of the model that in vivo Sos activation of Rac does not strictly require Sos activation of Ras (Yang, 2006).
It is clear that subcellular localization plays a major role in regulating Sos activity and that different protein complexes containing Sos exist in different locations in the cell. This study has shown that activation of the Robo receptor by Slit triggers the recruitment of Sos to Robo receptors at the plasma membrane. Biochemical data argue that the adaptor Dock/Nck is instrumental in bridging this interaction, and given the diverse interactions between Dock/Nck and guidance receptors, it seems likely that Dock/Nck could fulfill this role in many guidance receptor contexts. This bridging function of Dock/Nck and guidance receptors is analogous to the role of Grb2 for growth factor receptors only insomuch as it brings signaling molecules to the receptor—the mechanism of interaction is distinct, since it is mediated through SH3 domain contacts rather than SH2/phosphotyrosine interactions. These observations suggest that there may be an additional pool of Sos that can function in a distinct adaptor protein/guidance receptor complex to regulate cell morphology in response to extracellular guidance cues (Yang, 2006).
Is regulating subcellular localization the only mechanism by which Sos activity is controlled? This seems unlikely. Indeed, a recent study has implicated tyrosine phosphorylation of Sos by Abl as an additional mechanism to activate the Rac-specific GEF activity of Sos in vertebrate cell culture models. This raises the intriguing possibility that Abl may fulfill a similar role for Robo signaling. This is a particularly appealing idea given the well-documented genetic and physical interactions between Robo and Abl. Indeed, sos and abl exhibit dose-dependent genetic interactions during midline axon guidance. A clear genetic test of whether Abl activates the Rac-GEF activity of Sos downstream of Robo may be complicated by the fact that Abl appears to play a dual role in Robo repulsion: both increasing and decreasing abl function lead to disruptions in Robo function. Nevertheless, it should be possible in the future to generate mutant versions of Sos that are refractory to Abl activation and to test whether these alterations disrupt the Sos guidance function. It will also be of great interest to determine whether the redistribution of Sos can also be observed in response to guidance receptor signaling in navigating growth cones, and if so, then what changes in actin dynamics and growth cone behavior are elicited (Yang, 2006).
Members of the CDM (CED-5, Dock180, Myoblast city) superfamily of guanine nucleotide exchange factors function as RAC activators in diverse processes that include cell migration and myoblast fusion. The SH3, DHR1 and DHR2 domains of Myoblast city (MBC) are essential for it to direct myoblast fusion in the Drosophila embryo, while the conserved DCrk-binding proline rich region is expendable. This study describes the isolation of Drosophila ELMO/CED-12, an ~82 kDa protein with a pleckstrin homology (PH) and proline-rich domain, by interaction with the MBC SH3 domain. ELMO has been shown to modulate the Rac activation by Dock180 by means of at least three distinct mechanisms: helping Dock180 stabilize Rac in its nucleotide-free transition state; relieving a self-inhibition of Dock180; and targeting Dock180 to the plasma membrane to gain access to Rac. Thus, Dock180 and ELMO function together as a bipartite GEF to optimally activate Rac, upon upstream stimulation, to mediate the engulfment of apoptotic cells and cell migration (Geisbrecht, 2008; Lu, 2006).
Mass spectrometry confirms the presence of an MBC/ELMO complex within the embryonic musculature at the time of myoblast fusion and embryos maternally and/or zygotically mutant for elmo exhibit defects in myoblast fusion. Overexpression of MBC and ELMO in the embryonic mesoderm causes defects in myoblast fusion reminiscent of those seen with constitutively-activated Rac1, supporting the previous finding that both the absence of and an excess of Rac activity are deleterious to myoblast fusion. Overexpression of MBC and ELMO/CED-12 in the eye causes perturbations in ommatidial organization that are suppressed by mutations in Rac1 and Rac2, demonstrating genetically that MBC and ELMO/CED-12 cooperate to activate these small GTPases in Drosophila (Geisbrecht, 2008).
Drosophila myoblast city (MBC), Caenorhabditis elegans CED-5, and vertebrate DOCK180, are closely related members of the evolutionarily conserved CDM family of proteins. They serve as key players in a signaling complex that includes the SH2- SH3 domain-containing adaptor protein CrkII/CED-2 and the PH domain containing protein ELMO/CED-12 (reviewed in Meller, 2005). This complex then acts at the membrane to relay signals to the small GTPase Rac1/CED-10. MBC/DOCK180/CED-5 function as non-conventional Guanine nucleotide Exchange Factors (GEFs) for Rac. Conventional GEFs bind to nucleotide-free Rac via a Dbl-homology (DH) domain, thereby facilitating exchange of GDP for GTP. DOCK180/CED-5, which lack DH domains, associate with nucleotide-free Rac through a conserved Dock-Homology Region (DHR2) (Brugnera, 2002; Cote, 2002). Deletion of this domain results in loss of Rac binding and the inability to direct formation of GTP-bound Rac (Brugnera, 2002; Geisbrecht, 2008 and references therein).
In addition to DHR2, CDM proteins have in common an N-terminal SH3 domain, a second Dock Homology Region (DHR1), and a C-terminal proline rich region. The C-terminal region directs interaction with the SH3 domain of CrkII/CED-2. The CrkII SH2 domain can then direct interaction with upstream proteins that are phosphorylated on tyrosine, such as transmembrane receptors and components of focal adhesions. In addition to membrane recruitment through Crk-related interactions, the C-terminal PH domain of ELMO/CED-12 can also mediate DOCK180 membrane localization (deBakker, 2004; Grimsley, 2004). The DHR1 region of DOCK180, which binds to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], is also required for its membrane localization (Cote, 2005; Kobayashi, 2001). The N-terminal SH3 domains of DOCK180 and CED-5 mediate interaction with the C-terminal proline-rich region of ELMO and CED-12, respectively (Lu, 2004; Lu, 2005; Wu, 2001). In vitro studies demonstrate that DOCK180 binding to Rac can be sufficient for its activation (Cote, 2005), but that this activation can be significantly enhanced by DOCK180 bound to ELMO (Brugnera, 2002; Katoh, 2003; Lu, 2004). Thus, the DOCK180/ELMO complex is a key component in CDM signaling to Rac (Geisbrecht, 2008).
In addition to extensive homology and conserved biochemical interactions between the DOCK180/CED-5 and ELMO/CED-12 protein families, complexes of these proteins perform similar biological functions. For example, genetic studies have shown that C. elegans CED-10 acts with CED-2/Crk, CED-5/DOCK180 and CED-12/ELMO to promote cell migration of the distal tip cells during development of the somatic gonad and engulfment of cell corpses following apoptosis. Membrane targeted DOCK180 increases cell spreading, and overexpression of wild-type DOCK180 in mammalian cells enhances cell migration and phagocytosis of apoptotic cells. Lastly, a reduction in wild-type DOCK180 or overexpression of mutant forms of DOCK180 decrease activated Rac and cause defects in cell spreading and cell migration (Geisbrecht, 2008).
As in C. elegans and vertebrates, Drosophila MBC interacts genetically with other molecules required for CDM pathway function. For example, mutations in mbc delay border cell migration and influence PVR mediated F-actin accumulation in the follicle cells during ovary development, reflecting its proposed role in the PVR-Rac pathway (Duchek, 2001 To address the involvement of Drosophila ELMO in signaling from MBC, its role in myoblast fusion, border cell migration and ommatidial organization has been isolated and characterized. ELMO is expressed in the ovary, where it interacts genetically with MBC and plays a role in border cell migration. Multiple loss-of-function alleles were generate and analyzed to demonstrate the importance of maternal and zygotic ELMO in myoblast fusion. It was confirmed, through a targeted mass spectrometry approach, that these proteins form a stable complex in the embryonic musculature. ELMO and MBC act cooperatively in the mesoderm, such that an excess of both causes serious defects in myoblast fusion reminiscent of those seen with excess Rac activity. Finally, it was established genetically that ELMO and MBC can cooperate to activate Rac GTPases in the adult eye (Geisbrecht, 2008).
Thus, the conserved PH-domain that is normally present within conventional GEFs is actually provided for CDM proteins by a separate protein family represented by ELMO/CED-12 (Brugnera, 2002; Lu, 2004). One member of each of these two protein families can combine to form an unconventional bipartite GEF. While the small adaptor protein Crk often forms a critical component of this complex, recent studies have suggested that both DOCK180 and MBC can function in its absence (Balagopalan, 2006; Tosello-Trampont, 2007]). Moreover, Crk has been shown to function in pathways that are totally independent of this bipartite GEF. Reminiscent of this diversity of interactions, recent studies have suggested that CDM and ELMO/CED-12 family proteins may also function through independent interactions. For example, DOCK180 is capable of activating Rac on its own and has positive effects on cell migration and phagocytosis, albeit enhanced by binding of ELMO (Brugnera; Katoh, 2003]; Lu, 2004), and ELMO interacts with radixin independent of its interaction with DOCK180 (Grimsley, 2005). Drosophila MBC functions in a wide variety of processes that include border cell migration in the ovary and myoblast fusion in the embryo. This study has demonstrated, through its biochemical and genetic analysis, that Drosophila elmo functions in concert with MBC in these processes. Like MBC, decreased levels of ELMO impair border cell migration and myoblast fusion (Bianco, 2007). Moreover, MBC interacts stoichiometrically in the mesoderm with ELMO. Coincident over-expression of this complex impairs myoblast fusion, reinforcing the model from constitutively-active Rac1 that excess active Rac1 also interferes with myoblast fusion. Co-expression of MBC and ELMO also impacts development of the adult eye, resulting in a rough eye phenotype that is suppressed by decreasing endogenous levels of Rac. These data indicate that MBC and ELMO function together in a complex, and as a RacGEF (Geisbrecht, 2008).
CDM family proteins are required for a diverse array of biological processes. If ELMO functions with MBC in these processes, one would expect it to be expressed in a temporal and spatial pattern coincident with that of MBC. Consistent with this expectation, RT-PCR throughout the fly life cycle, embryonic in situ hybridizations and antibody stainings at multiple stages of Drosophila development, reveal that elmo is broadly expressed. Interestingly, however, the MBC/ELMO complex may serve distinct roles in each of the tissues in which it is expressed. MBC and ELMO are required for migration of the border cells in the ovary (Bianco, 2007; Duchek, 2001); however, myoblast migration appears to occur normally in mbc mutant embryos, as the fusion competent cells in mbc mutants can be found clustered and aligned with the founder cells (Geisbrecht, 2008).
The CDM/ELMO(Ced-12) complexes in both vertebrate and C. elegans function upstream of Rac as unconventional bipartite GEFs to promote exchange of GDP for GTP in activation of monomeric GTPases (Meller, 2005). These studies support a similar role for Drosophila MBC/ELMO. First, during ommatidial development, the rough eye phenotype resulting from co-expression of MBC and ELMO can be suppressed by removing half the gene dosage contributed by Rac1 and Rac2. Also, overexpression of the MBC/ELMO complex is sufficient to provide enough GEF activity to overcome the effect of its sequestration by RacN17 in the eye. In both the musculature and eye, expression of neither MBC nor ELMO alone has a phenotypic consequence, yet co-expression of MBC and ELMO phenocopies activated Rac (Geisbrecht, 2008).
Notably, embryos that are completely lacking both maternal and zygotic elmo die before muscle development occurs, possibly reflecting an earlier role for the protein. Interestingly, however, the development of mutant embryos that lack both maternally and zygotically provided mbc continues until myogenesis (Balagopalan, 2006). These data suggest that, like its vertebrate counterparts, Drosophila ELMO has multiple binding partners. In addition to DOCK180, vertebrate ELMOs bind to three of the five additional CDM family members, suggesting that ELMO binding is a general feature of these proteins (Grimsley, 2004; Sanui, 2003). Based upon primary sequence homology, the fly genome contains at least four potential CDM superfamily members in addition to MBC. The predicted transcripts of two of these are most closely related to vertebrate DOCK9/11 and DOCK 7/8. The Drosophila protein most closely related to vertebrate DOCK4 has been reported as the protein product of the sponge locus, but has not been studied extensively. It remains to be seen whether Drosophila ELMO is capable of binding to these other CDM-like molecules, and functions in concert with them in other tissues. One such place to examine in this regard is the embryonic CNS, where ELMO expression is quite strong but MBC is strikingly low. Thus, alternative CDM/ELMO-like complexes may be present and required in different tissues throughout Drosophila development, or in the same tissues to regulate different GTPases (Geisbrecht, 2008).
The above studies, combined with recent reports of ELMO binding to non-CDM family members, may reflect a role for ELMO proteins in integrating signals from different pathways. Interaction of the N-terminal region of ELMO with RhoG is capable of translocating the ELMO/DOCK180 complex to the membrane to regulate neurite outgrowth and cell migration (Katoh, 2003). Simultaneously, the N-terminal region of ELMO can bind to both the inactive and active forms of the ERM protein radixin (Grimsley, 2005). Interestingly, this ELMO/radixin interaction does not affect the ability of the ELMO/DOCK180 complex to promote Rac activation. Hence, ELMO may be functioning at the membrane to regulate the actin cytoskeleton via Rac, while recruiting radixin and ERM family members to perform their recognized roles in cross-linking the actin cytoskeleton to the plasma membrane (Geisbrecht, 2008).
In addition to Rac activation via the CDM/ELMO proteins, the ARF (ADP-ribosylation factor) family of GTPases has been shown to signal through Rac. Both DOCK180 and ELMO colocalize with ARNO (an ARF-GEF) and overexpression of mutant forms of DOCK180 and ELMO mutants block ARNO-induced Rac activation (Santy, 2005). However, RhoG signaling does not seem to be required for the ARNO-ELMO activation of Rac. This suggests Rac activation is differentially regulated in cell or tissue-specific manners or that within a cell there are localized mechanisms defined by crosstalk between signaling pathways that are responsible for Rac membrane localization. Intriguingly, in the Drosophila musculature, expression of a dominant-negative ARF6 results in myoblast fusion defects while the corresponding ARF-GEF Loner/Schizo is required for membrane localization of Rac. More work is required to see if ARF6, Loner/Schizo, and the ELMO/MBC proteins function in a signaling pathway in Drosophila to activate Rac (Geisbrecht, 2008).
Numerous studies have established a crucial role for Rac activation in myoblast fusion. More than a decade ago, it was demonstrated that a dominant-negative form of Rac1 interferes with myoblast fusion. A genetic analysis of the small Rac GTPases proved that the fusion defects observed with dominant-negative Rac1 reflect the Rac1, Rac2 loss-of-function phenotype. The need for activated Rac in myoblast fusion was further supported by the phenotype of embryos mutant for mbc and elmo. Rac, in turn, likely functions to direct rearrangement of the actin cytoskeleton through regulation of the Arp2/3 complex via Kette and WAVE. Surprisingly, however, constitutively-active forms of Rac negatively impact this process in a manner that, on the surface, have the same phenotypic consequence as the absence of active Rac. The specific relevance of excess activated Rac1 to normal myoblast fusion remains unclear, and it cannot be ruled out that excess Rac interferes with other signaling pathways that do not normally impact myoblast fusion. However, perturbation of this phenotype also has the potential to uncover key components and regulators that contribute to the normal process (Geisbrecht, 2008).
These data establish, for example, that monomeric Rac GTPases are in excess, and are therefore available to be activated when the level of the MBC/ELMO GEF complex is increased. Under normal circumstances, then, the ability of the cell to activate this endogenous Rac remains low. Potential mechanisms for this include the direct regulation of MBC and/or ELMO levels through synthesis or turnover. Though little is known about the synthesis of either MBC or ELMO or their levels in the cell, it is intriguing that DOCK180 is ubiquitinated and this modification ensures its rapid turnover (Makino, 2006). Alternatively, GAPs may be present to ensure that Rac does not remain active. Finally, the MBC/ELMO pathway may be integrating with the loner-ARF6 associated pathway, in such a way that perturbation of MBC and ELMO is impacting Rac1 activity through components of the ARF6 pathway. Either way, the fact that this myoblast fusion phenotype is occurring in response to perturbation of the endogenous pathway by wild-type proteins in a stoichiometric manner suggests that it may be possible to modulate it in ways that provide insights into the myoblast fusion process (Geisbrecht, 2008).
The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. This study shows that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. It is further demonstrated that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms (Palamidessi, 2008).
Crosstalk among small GTPases is critical in the regulation of numerous cellular functions, and the cell has adopted several strategies to regulate it. In some cases, crosstalk is obtained through simple hierarchical cascades whereby two small GTPases are directly and orderly linked in a positive or negative amplification loop. This is, for instance, the case with Ras-to-Rac signaling or signaling among Rho-like GTPases (Palamidessi, 2008).
In the case described in this study, Rab5 regulates Rac not via a direct biochemical link but, rather, by activating an entire process, endocytosis, that provides the enabling conditions for the activation of Rac and the execution of its function. The data, therefore, add an extra layer to the complex strategies adopted by the cell to use endocytosis as a device to provide spatial and temporal dimensions to signaling. The regulation of Rac activity by endocytosis is reminiscent of the endocytic-dependent regulation of Ras signaling. Ras and/or MAPK signaling has been observed on different endomembranes, and it depends on functional endocytosis. Additionally, H-Ras is dynamically recruited, in both its active form and a Rab5 and Rab11 manner, to recycling endosomes (Palamidessi, 2008).
There are important differences, however, in the finalities of endocytic regulation of Ras and Rac. In the former case, the organelle location of Ras has invariably been proposed to serve as an intracellular platform, conferring signal specificity and diversity possibly by extending and segregating the repertoire of regulatory molecules and/or effectors. In the case of Rac, endocytosis, localization to endosomes, and recycling to the plasma membrane should be viewed primarily as a means to resolve and direct signals in space, thus preventing them from becoming uniformly distributed and, as such, uninformative (Palamidessi, 2008).
The Drosophila guanine nucleotide exchange factor Pebble (Pbl) is essential for cytokinesis and cell migration during gastrulation. In dividing cells, Pbl promotes Rho1 activation at the cell cortex, leading to formation of the contractile actin-myosin ring. The role of Pbl in fibroblast growth factor-triggered mesoderm spreading during gastrulation is less well understood and its targets and subcellular localization are unknown. To address these issues, a domain-function study in the embryo was performed. Pbl is shown to be localized to the nucleus and the cell cortex in migrating mesoderm cells and it was found that, in addition to the PH domain, the conserved C-terminal tail of the protein is crucial for cortical localization. Moreover, the Rac pathway plays an essential role during mesoderm migration. Genetic and biochemical interactions indicate that during mesoderm migration, Pbl functions by activating a Rac-dependent pathway. Furthermore, gain-of-function and rescue experiments suggest an important regulatory role of the C-terminal tail of Pbl for the selective activation of Rho1-versus Rac-dependent pathways (van Impel, 2009).
The Rho GEF Pbl provides one of the few molecular links between the proximal FGF receptor signalling events and the regulation of cell shape changes. Loss-of-function phenotype of pbl mutants have shown that Pbl acts in a pathway downstream or in parallel to Htl-dependent MAP kinase activation. This study used genetics and biochemistry to determine the regulation of Pbl and its downstream Rho GTPase pathways in migrating cells. The data demonstrate that Pbl partially localizes to the cell cortex of mesoderm cells and functionally interacts with Rac GTPases in this process (van Impel, 2009).
The tandem DH-PH domain of Pbl are shown to be essential for cell migration and employs not only Rho1, but also the Rac pathway. Several lines of evidence strongly suggest that Pbl acts through Rac GTPases during mesoderm migration. The dominant rough eye phenotype induced by PblDH-PH is sensitive to gene doses of Rac GTPases. Expression of constitutively active or dominant-negative Rac1 but not Rho1 enhances the mesoderm phenotype in the hypomorphic pbl11D allele. Moreover, co-expression of Rac1, but not of Rho1, promotes the suppression of mesoderm migration defects by PblδBRCT in pbl-null mutants. In addition, biochemical data is provided that strongly suggest the Rac pathway as a direct target of Pbl (van Impel, 2009).
Pbl has previously been reported to localize to the nucleus in interphase
cells. Nuclear localization was interpreted as a means of storing the protein until rapid release at mitosis. In cultured cells and C. elegans zygotes, homologues of Pbl localize at the cell cortex, e.g. cell junctions or the anterior cortex in the nematode zygote. This study detected functional Pbl-HA in the nucleus and the cytoplasm, including
membrane protrusions. These data are consistent with the model that Pbl
activates Rac GTPases at the cell cortex during cell migration (van Impel, 2009).
This study identified two domains, the conserved C-terminal tail and the PH domain, as candidates to mediate the association of Pbl with the cell cortex in interphase cells. The use of N-terminally deleted constructs facilitated these studies, because the respective proteins were excluded from the nucleus as they lack the NLS. Either domain alone is sufficient to localize to the cell cortex, and deletion studies suggest that both domains are crucial for cortical localization. It is proposed that the PH domain and the C-terminal tail might cooperate in localizing Pbl to the cell cortex. DH domain associated PH domains are essential for GEF function and are known to promote binding to specific membrane subdomains enriched in phosphoinositides. An attractive model therefore is that the PH domain provides specificity by targeting Pbl to membrane domains enriched for particular phospholipids, whereas the C-terminal tail functions in anchoring Pbl to the cell cortex. In addition, binding to phospholipids might promote the specific exchange activity of the tandem DH-PH domain, as described for other Dbl family GEFs (van Impel, 2009).
It is difficult to address the issue of whether cortical localization is important for the function of Pbl in mesoderm migration. The reduced rescuing capability of PblδC-term is consistent with a correlation of cortical localization through the C-terminal domain and the function of Pbl in cell migration. A more stringent experiment would involve the generation of a construct that lacks the PH and C-terminal domains for membrane association. However, as PH domains are essential for DH domain function in vivo, deletion of the PH domain will abolish activity in any case, as has been shown for the constitutively active DH-PH construct. Such an analysis would require a way to uncouple the activities of the PH domain that promote the exchange activity and membrane-phospholipid binding. It will therefore remain important to determine whether the function of the PH domain involves its interaction with lipid substrates or directly promotes the activity of the DH domain in migrating cells (van Impel, 2009).
The inhibition of invagination and cytokinesis by PblδNterm is probably caused by disruption of the local activation of Rho1 at the cell cortex. During invagination and cytokinesis, the Rho1 pathway is activated locally: either 1) in the apical domain of the mesoderm cells to trigger apical constriction or 2) at the cell equator of the dividing cell to promote assembly of the contractile ring. Since PblδNterm strongly accumulates at the cortex in a non-polarized fashion, it might activate Rho1 ectopically throughout the cell cortex and thereby overriding any polarizing cues for local activation (van Impel, 2009).
The dramatic differences in the overexpression phenotypes of PblDH-PH or PblδNterm suggest an important function of the C-terminal tail in controlling the biochemical activities of the tandem DH-PH domain. Strikingly, PblδNterm genetically interacts with Rho1, but not with Rac GTPases, supporting the idea that the C-terminus promotes the exchange activity towards Rho1. It is proposed that in the mesoderm cells this activity of the C-terminal domain is antagonized to activate the Rac rather than to the Rho1 pathway. In the presence of the NLS and PEST motifs, the cytoplasmic levels of Pbl are low and allow for this regulation to occur, whereas the oncogenic forms lacking these motifs are present in the cytoplasm at high levels and might escape regulation. Thus, constructs that lack the C-terminal tail promote interaction with Rac and rescue Rac-dependent mesoderm migration. This model is also supported by the observation that the C-terminal domain is essential for Rho1 activation, but not for Pbl localization in dividing cells. The same construct, PblδC-term, is still able to rescue Rac-dependent migration
defects. Thus, deletion of the C-terminal tail uncouples activation of Rho1-dependent from Rac-dependent processes and suggests that in the absence of the negative interaction with the C-terminal tail, the tandem DH-PH domain promotes activation of Rac (van Impel, 2009).
Although many receptor tyrosine kinases signal through Rho GTPases, only
few FGF receptors have been reported to regulate Rho GEFs. One
attractive model is that FGF signalling mediates post-translational
modification of the C-terminal tail to trigger the switch in the differential
interaction with Rho1 and Rac GTPases. The sequence of the C-terminal tail
contains several conserved putative phosphorylation sites that might represent
targets for FGF signalling. Interestingly, the exchange factor specificity of
oncogenic ect2 for GTPase substrates depends on the C-terminal tail
of the protein. Identification of proteins that interact with the
C-terminal domain might shed light on its role in controlling selectivity for
distinct GTPase pathways. Such studies will be important to advance our
understanding of the mechanism of the transforming potential of Pbl, as well
as its mechanism of action in cell polarity and cell migration (van Impel, 2009).
Epigenetic mechanisms regulate genome activation in diverse events, including normal development and cancerous transformation. Centromeres are epigenetically designated chromosomal regions that maintain genomic stability by directing chromosome segregation during cell division. The histone H3 variant CENP-A resides specifically at centromeres, is fundamental to centromere function and is thought to act as the epigenetic mark defining centromere loci. Mechanisms directing assembly of CENP-A nucleosomes have recently emerged, but how CENP-A is maintained after assembly is unknown. This study shows that a small GTPase switch functions to maintain newly assembled CENP-A nucleosomes. Using functional proteomics, it was found that MgcRacGAP (a Rho family GTPase activating protein) interacts with the CENP-A licensing factor HsKNL2. High-resolution live-cell imaging assays, designed in this study, demonstrated that MgcRacGAP, the Rho family guanine nucleotide exchange factor (GEF) Ect2, and the small GTPases Cdc42 and Rac, are required for stability of newly incorporated CENP-A at centromeres. Thus, a small GTPase switch ensures epigenetic centromere maintenance after loading of new CENP-A (Lagana, 2010).
Epigenetic regulation of genome activity is critical during development and stem cell maintenance, and increasing amounts of evidence highlight its importance in cancers. However, mechanisms controlling epigenetic regulation during a single cell cycle are generally less well understood, compared with those involved in transcriptional programmes. Centromere specification is an epigenetic regulatory event that controls genome activity at singular chromosomal loci and occurs each cell cycle. Nucleosomes that contain CENP-A are thought to epigenetically define centromeres. During DNA replication, centromere identity is maintained by segregating CENP-A equally to the two daughter chromosomes. Before the subsequent S-phase, additional CENP-A must be incorporated at centromeres, thus propagating the centromere epigenetic mark. Critical to this cycle is maintenance of the proper amount of CENP-A; too little or too much CENP-A incorporation could result in either loss of centromere identity or errors in chromosome segregation. This study describes a mechanism to ensure maintenance of the proper CENP-A levels during the cell cycle regulated by a Rho family small GTPase molecular switch (Lagana, 2010).
Proteomics and quantitative imaging assays were used to identify a previously unknown step in centromere maintenance. MgcRacGAP, together with the GEF ECT2, and their cognate small GTPase Cdc42 (or possibly Rac) specifically maintain CENP-A at centromeres. MgcRacGAP localization to centromeres at the end of G1 is incongruous with a role in CENP-A loading and strongly suggests that MgcRacGAP acts in maintenance and not licensing or loading of CENP-A. Pulse-chase analysis revealed that MgcRacGAP is required specifically for maintenance of newly incorporated CENP-A as old CENP-A from the previous cell cycle was present at normal levels at centromeres. Reciprocal immunoprecipitation of MgcRacGAP did not isolate HsKNL2, probably because of a large excess of MgcRacGAP bound to other known interacting proteins in the cytoplasm (data not shown). These results support the conclusion that a minor subset of MgcRacGAP is bound to HsKNL2 for a brief period each cell cycle and imply that non-overlapping MgcRacGAP-containing protein complexes function in cells. Overall, this work defines a new event in epigenetic centromere regulation and reveals its control by a small GTPase molecular switch (Lagana, 2010).
A model is proposed wherein the HsKNL2–Mis18 complex licenses centromeres for loading of new CENP-A by the combined activities of HJURP and CAF1. After loading (approximately 8–12 h after anaphase onset), HsKNL2–Mis18 recruits Cdc42. The activity of Cdc42 is required for preservation of newly incorporated CENP-A and thus finalizes centromere repopulation. Cdc42 activity requires GTPase cycling facilitated by MgcRacGAP and the GEF ECT2. The results predict that newly incorporated CENP-A is distinct from CENP-A remaining from the previous cell cycle and can be recognized and removed. It is proposed that Cdc42 activity modifies (by either adding or removing a mark on) newly incorporated CENP-A, rendering it identical to old CENP-A. The manifestation of this mark could be any distinguishing modification, including but not limited to, recruitment of an additional protein, conformational change of the CENP-A nucleosome, or any of a range of post-translational modifications. New CENP-A that is not modified would be recognized as erroneously incorporated and removed from chromatin during a late-G1 surveillance step, or during DNA replication (Lagana, 2010).
In budding yeast, excess CENP-A (CSE-4) mislocalized to the chromosome arms is removed and selectively degraded through a proteasome-based mechanism. If this mechanism is conserved in human cells, it is expected to be less stringent, as overexpressed CENP-A localizes diffusely to chromosome arms without causing obvious defects in cell division. Alternatively or additionally, centromere maintenance could involve the chromatin remodelling protein RSF-1, which is required for CENP-A nucleosome stability. However, because RSF-1 is proposed to function in mid-G1 before MgcRacGAP and Cdc42 localize to centromeres, it is unlikely to be the downstream target of small GTPase activity at centromeres (Perpelescu, 2009). Regardless of the removal mechanism, it is proposed that a GTPase switch is spatially and temporally restricted through regulated localization to centromeres precisely after CENP-A doubling to promote the removal of spurious CENP-A (either excess at centromeres, or outside true centromere loci). By restricting centromere size, this 'quality control' mechanism helps to ensure proper centromere function and kinetochore assembly, thus preventing aneuploidy. Furthermore, it is possible that this mechanistic theme will apply to other epigenetic events that contribute to genomic regulation (Lagana, 2010).
Abl tyrosine kinase and its effectors among the Rho family of GTPases each act to control dendritic morphogenesis in Drosophila. It has not been established, however, which of the many GTPase regulators in the cell link these signaling molecules in the dendrite. In axons, the bifunctional guanine exchange factor, Trio, is an essential link between the Abl tyrosine kinase signaling pathway and Rho GTPases, particularly Rac, allowing these systems to act coordinately to control actin organization. In dendritic morphogenesis, however, Abl and Rac have contrary rather than reinforcing effects, raising the question of whether Trio is involved, and if so, whether it acts through Rac, Rho or both. This study shows that Trio is expressed in sensory neurons of the Drosophila embryo and regulates their dendritic arborization. trio mutants display a reduction in dendritic branching and increase in average branch length, whereas over-expression of trio has the opposite effect. It is further shown that it is the Rac GEF domain of Trio, and not its Rho GEF domain that is primarily responsible for the dendritic function of Trio. Thus, Trio shapes the complexity of dendritic arbors and does so in a way that mimics the effects of its target, Rac (Shivalkar, 2012).
Trio has been associated with both Rho family GTPases and the Abl tyrosine kinase. Both these pathways control dendritic arborization in Drosophila, but they do so in different ways, with Rac, for example, promoting dendritic branching and Abl limiting it. This made it important to determine whether Trio plays a role in dendrogenesis, and if so, whether it was functioning in association with Rac or with Rho, and how its effects compared with those of Abl. This study shows that Trio also shapes dendritic structure in the fly. In both simple Class I sensory neurons and complex Class IV sensory neurons, Trio promotes formation of dendritic branches: over-expression of trio produces more elaborately branched dendritic trees whereas loss of trio reduces the number of dendritic branches. In both cases, the effect of Trio is concentrated on higher-order branches, which others have shown to be actin-dominated and more dynamic, and not in the primary branches, which tend to be microtubule-dominated and more stable (Shivalkar, 2012).
Trio not only affects dendritic branching but also dendritic length. In most assays, Trio limits the average length of some or all orders of dendritic branches to a degree that roughly offsets the increase in branch number, leading to a modest net change or no change in total dendritic length. The compensation is not exact, however. For example, in trio mutants, while average dendritic length is unchanged in Class I neurons, an increase in average branch length is seen in Class IV neurons but it is not enough to counteract the decrease in branch number, leading to an overall decrease in total length. Conversely, in trio over-expression, both Class I neurons and Class IV neurons show no net change in total length in spite of an increase in the average length of dendrites. This variability may suggest that total dendritic length is not strictly invariant for a given sensory neuron, with a fixed length parceled among a variable number of branches, but rather that Trio may have separate, and opposite, effects on branch length and number. Further experiments will be necessary, however, to test this idea (Shivalkar, 2012).
Expression of constructs bearing mutations in each of its GEF domains suggests that Trio acts primarily through its Rac GEF domain, and not its Rho GEF domain, to affect dendritic morphogenesis of the PNS sensory neurons. Thus, a Trio derivative lacking Rac GEF activity does not alter dendritic structure whereas a derivative lacking Rho GEF activity produces effects that are indistinguishable from those of the wild type protein. This is consistent with the similarity between the phenotype observed for gain and loss of trio function and that reported for gain and loss of Rac, and also with data from axonal development, both in embryonic motor neurons and adult photoreceptors showing that the Rac-specific GEF1 domain is the key effector domain of Trio in axons. It is in contrast, however, to results from the adult Drosophila mushroom body, in which trio mutant clones showed overextension of neurites similar to that in RhoA mutant clones in the dendritic portion of the structure (the calyx). Perhaps Trio pairs with different GTPases in different developmental settings, as has been observed for C. elegans Trio. The results also indicate that the dendritic phenotypes seen upon over-expression of trio are not due to changes in expression of the important neuronal class specific transcription factors, Abrupt and Knot, thus arguing against the idea that changes of cell fate are responsible for changes in dendritic morphology in these experiments (Shivalkar, 2012).
In contrast to the concordance between the effects of Trio and Rac, the phenotypes produced by altering Trio activity are opposite to those from manipulation of the Abl tyrosine kinase pathway. This was surprising in light of prior work showing that the effects of Trio mimic those of Abl in axonal development, and that led to the suggestion that Trio is a core component of the Abl pathway. Two hypotheses could account for this discrepancy. First, it could be that the Trio-Rac module should be thought of as an adjunct to the Abl signaling network, with a variable and context-dependent relationship to Abl, rather than as itself being a core element of that pathway. Such a relationship would allow the Trio-Abl interaction to produce different morphological outcomes in different developmental settings. Alternatively, the possibility cannot be ruled out that the relationship of Trio to Abl at the molecular level is the same in dendrites as in axons, but it manifests in opposite morphological consequences due to the complexities of the relationship between signaling, cytoskeletal dynamics and morphology. Indeed, there are many examples of a cytoplasmic signaling protein producing seemingly opposite effects in different developmental contexts. In the current setting, however, this interpretation is not favored since such non-linear effects of signaling proteins in other systems typically lead to observation of contradictory phenotypes upon manipulating the activity of a gene across a wide dynamic range. In the case of Trio, in contrast, all of the gain- and loss-of function manipulations give a consistent set of effects on dendritic branching. Additional experiments will be required, however, to distinguish fully between these hypotheses (Shivalkar, 2012).
The data reported in this study show that Trio, like its effector Rac, regulates dendritic arborization in Drosophila sensory neurons. The data also suggest that the relationship of Trio to the Abl tyrosine kinase signaling network may be more nuanced than was previously appreciated. It seems likely that the interplay of these signaling modules channels the molecular machinery of morphogenesis in a variety of ways to help produce the vast range of neuronal shapes (Shivalkar, 2012).
As the primary sites of synaptic or sensory input in the nervous system, dendrites play an essential role in processing neuronal and sensory information. Moreover, the specification of class specific dendrite arborization is critically important in establishing neural connectivity and the formation of functional networks. Cytoskeletal modulation provides a key mechanism for establishing, as well as reorganizing, dendritic morphology among distinct neuronal subtypes. While previous studies have established differential roles for the small GTPases Rac and Rho in mediating dendrite morphogenesis, little is known regarding the direct regulators of these genes in mediating distinct dendritic architectures. This study demonstrates that the RhoGEF Trio is required for the specification of class specific dendritic morphology in dendritic arborization (da) sensory neurons of the Drosophila peripheral nervous system (PNS). Trio is expressed in all da neuron subclasses and loss-of-function analyses indicate that Trio functions cell-autonomously in promoting dendritic branching, field coverage, and refining dendritic outgrowth in various da neuron subtypes. Moreover, overexpression studies demonstrate that Trio acts to promote higher order dendritic branching, including the formation of dendritic filopodia, through Trio GEF1-dependent interactions with Rac1, whereas Trio GEF-2-dependent interactions with Rho1 serve to restrict dendritic extension and higher order branching in da neurons. Finally, it was shown that de novo dendritic branching, induced by the homeodomain transcription factor Cut, requires Trio activity suggesting these molecules may act in a pathway to mediate dendrite morphogenesis. Collectively, these analyses implicate Trio as an important regulator of class specific da neuron dendrite morphogenesis via interactions with Rac1 and Rho1 and indicate that Trio is required as downstream effector in Cut-mediated regulation of dendrite branching and filopodia formation (Iyer, 2012).
This analysis demonstrates that Trio functions in promoting and refining class specific dendritic arborization patterns via GEF1- and GEF2-dependent interactions with Rac1 and Rho1, respectively. It was also demonstrated that Trio is required in mediating Cut induced effects on dendritic branching and filopodia formation suggesting that these molecules may operate in a common pathway to direct dendritic morphogenesis. Giniger and colleagues (NINDS/NIH) have likewise been investigating Trio function in da neurons via a non-overlapping, complementary experimental approach, and that they arrived at conclusions regarding Trio function largely consistent with those reported in this study (Iyer, 2012).
Previous studies have demonstrated that Trio functions via its GEF1 domain in mediating the regulation of axon morphogenesis by modulating Rac1 activity, however much less is known regarding the potential in vivo functional role(s) of the Trio GEF2 domain. Intriguingly, a previous study demonstrated that trio mutant neuroblast clones display a neurite overextension phenotype from the dendritic calyx region of the mushroom body which strongly resembled the dendrite-specific overextension phenotype observed in RhoA mutant mushroom body clones suggesting that RhoA/Rho1 activation may be required for restricting dendritic extension. In Drosophila da neurons, trio loss-of-function analyses reveal a reduction in dendritic branching in three distinct da neuron subclasses (class I, III, and IV), indicating a functional role for Trio in promoting dendritic branching. However, class specific differences are observed with Trio gain-of-function studies in which Trio overexpression in class I neurons increases dendritic branching, whereas in class III neurons there is no change in overall dendritic branching, but rather a redistribution of branches, and in class IV there is a reduction in overall dendritic branching. The basis for these differences appear to lie in the observation that refinement of dendritic branching in da neurons is subject to the opposing roles of Rac1 and Rho1 activation via Trio-GEF1 and Trio-GEF2, respectively, where Trio-GEF1 activity promotes higher order dendritic branching, whereas Trio-GEF2 activity restricts higher order branching and also limits overall dendritic length/extension (Iyer, 2012).
One of the key distinctions between class I versus class III and IV neurons relates to inherent differences in normal dendritic branching complexity and the relative roles of dynamic actin cytoskeletal based processes in these neurons which are known to mediate higher order branching including the dendritic filopodia of class III neurons and fine terminal branching in class IV neurons, whereas the class I neurons do not normally exhibit this degree of higher order branching and are predominantly populated by stable, microtubule-based primary and secondary branches. As such, Trio overexpression in these distinct subclasses may yield different effects on overall dendritic branching morphology based upon the normal distribution of actin cytoskeleton within these subclasses leading to unique effects on class specific dendritic architecture. Both loss-of-function and gain-of-function results support this hypothesis as the predominant effects are restricted to actin-rich higher order branching, whereas the primary branches populated by microtubles are relatively unaffected. This is further supported by the demonstration that trio knockdown suppresses Cut induced formation of actin-rich dendritic filopodia. Moreover, phenotypic analyses revealed that co-expression of Cut and Trio-GEF1 synergistically enhance dendritic branching in class I neurons likely due to increased activation of Rac1, whereas co-expression of Cut and Trio-GEF2 lead primarily to increased dendritic extension likely due to increased activation of Rho1. Thus, Trio mediated regulation of Rac1 and/or Rho1 signaling has the potential for sculpting dendritic branching and outgrowth/extension depending upon the combinatorial and opposing effects of Rac1 and Rho1 (Iyer, 2012).
In contrast to Cut, which has been shown to be differentially expressed in da neuron subclasses and exert distinct effects on class specific dendritic arborization, this study has demonstrated that Trio is expressed in all da neuron subclasses and can exert distinct effects on class specific dendritic branching. For example, in all subclasses examined, loss-of-function analyses indicate Trio is required to promote dendritic branching and yet individual subclasses exhibit strikingly distinct dendritic morphologies. These results suggest that Trio is generally required in each of these subclasses to regulate branching, however alone is insufficient to drive these class specific morphologies solely via activation of Rac1 and/or Rho1 signaling. One logical hypothesis is that differential expression of RhoGAP family members in distinct da neuron subclasses may work in concert with Trio to refine class specific morphologies. The potential for combinatorial activity between Trio and various RhoGAPs is significant given that 20 RhoGAPs have been defined in the Drosophila genome. For example, given that class I da neurons exhibit a simple branching morphology which becomes more complex when Trio or Trio-GEF1 domains are overexpressed, perhaps there is higher expression of Rac-inactivating GAPs in class I neurons that function in limiting dendritic branching, whereas in the more complex class III or IV da neurons, there may be lower expression of RacGAPs. Since overexpression of Trio-GEF2 reduces dendritic branching complexity in all three da neuron subclasses analyzed, it might be predicted that Rho1 activation limits dendritic branching and that therefore the expression of RhoGAPs may be modulated to facilitate branching in class III and IV neurons relative to class I neurons. In concert, differential expression of RacGAPs and RhoGAPs together with the uniform expression of Trio in all da neuron subclasses could potentially account for differential levels of activation/inactivation of Rac1 and/or Rho1 in individual subclasses and thereby influence overall class specific dendritic architecture (Iyer, 2012).
In support of this hypothesis, class-specific microarray analyses conducted in class I, III, and IV da neurons indeed reveal differential gene expression levels for most of the 20 known RhoGAP family members at a class-specific level. These expression analyses reveal one trend whereby select RhoGAP encoding genes are upregulated in the more complex class III and IV da neurons relative to the simple class I da neurons, whereas select RacGAP encoding genes are downregulated in complex neurons relative to simple neurons. Moreover, it is known that individual RhoGAPs display differential specificities for Rac, Rho and Cdc42 in vivo, such that a given RhoGAP may function in activating one or more of these small G proteins thereby increasing the potential for fine-tuning activation levels of a particular G protein at a class specific level. Furthermore recent studies provide direct evidence of the importance of RhoGAP family members in regulating da neuron dendritic morphogenesis. Analyses of the tumbleweed (tum) gene, which encodes the GTPase activating protein RacGAP50C, demonstrate that tum mutants display excessive da neuron dendritic branching. The dendritic phenotype observed in tum mutant da neurons is strikingly similar to that observed with Trio-GEF1 overexpression which also leads to excessive dendritic branching. Together these data suggest that Trio-GEF1 functions in activating Rac1 to promote dendritic branching whereas Tum/RacGAP50C function in inactivating Rac1 via its GTPase activity and thereby limit dendritic branching. In contrast, mutant analyses of the RhoGAP encoding gene, crossveinless-c, whose target in da neurons is the Rho1 small G protein, reveal defects in directional growth of da neuron dendrites. These results indicate that Crossveinless-C is required to inactivate Rho1 in order to promote directional dendritic growth and further suggest that a failure to inactivate Rho1 leads to restricted dendritic growth consistent with the phenotypes observed with Trio-GEF2 overexpression in all da neuron subclasses examined. These results, together with those presented herein, suggest that potential combinatorial activity of Trio and RhoGAP family proteins may converge in shaping the class specific dendritic architecture. Ultimately, future functional studies will be required to validate this hypothesis (Iyer, 2012).
While previous studies have revealed Trio acts in concert with Abl and Ena in coordinately regulating axon guidance, the same regulatory relationship does not appear to operate in da neuron dendrites as Abl has been shown to function in limiting dendritic branching and the formation of dendritic filopoda, whereas both Ena functions in promoting dendritic branching. This study demonstrates that Trio functions in promoting dendritic branching, consistent with Ena activity, but in da neuron dendrites works in an opposite direction to Abl. These findings suggest that, at least in da neuron dendrites, Trio may operate in either an Abl-independent pathway or that Trio and Abl may exhibit a context dependent regulatory interaction that is distinctly different in dendrites versus axons (Iyer, 2012).
Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. This study reports that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd's Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo (Law, 2013).
This study reveals that Lpd colocalizes with the Scar/WAVE complex at the very edge of lamellipodia and directly interacts with this complex by binding to the Abi-SH3 domain. Active Rac directly binds Lpd, thereby regulating the interaction between Lpd and the Scar/WAVE complex. It is therefore postulated that Lpd acts as a platform to link active Rac and the Scar/WAVE complex at the leading edge of cells to regulate Scar/WAVE-Arp2/3 activity and thereby lamellipodium formation and cell migration (Law, 2013).
Knockdown of Lpd expression or KO of Lpd highly impaired lamellipodium formation, phenocopying the effect of Scar/WAVE complex knockdown on lamellipodium formation. Conversely, it was observed that overexpression of Lpd increased lamellipodia size in Xenopus NC cells, and this was dependent on the interaction with Abi, linking it to the Scar/WAVE complex. Overexpression of Pico, the Lpd fly orthologue, aberrantly increased the number and frequency of cellular protrusions at the rear of border cell clusters in a Scar-dependent manner, which suggests that the regulation of Scar/WAVE by Lpd is evolutionary conserved. Collectively, these data suggest that Lpd functions to generate lamellipodia via the Scar/WAVE complex (Law, 2013).
Lpd or Pico knockdown or Lpd KO impaired cell migration in vitro and in vivo in Drosophila, Xenopus, and mice. Lpd KO or knockdown cells were unable to migrate via lamellipodia but instead migrated very slowly by extending filopodia. The same residual migration mode had been observed for Arp2/3 knockdown cells. Arp2/3 is activated by the Scar/WAVE complex to regulate cell migration. It was also observed that both Lpd and Abi knockdown impaired NC migration in vivo. Consistently, it was found that Lpd and Abi-Scar/WAVE are in the same pathway regulating cell migration. This is consistent with recent studies suggesting that the Lpd orthologue in C. elegans, mig-10, genetically interacts with abi-1 to regulate axon guidance, synaptic vesicle clustering, and excretory canal outgrowth in C. elegans (Stavoe, 2012; Xu, 2012; McShea, 2013). Collectively, these results suggest that Lpd functions in cell migration via the Scar/WAVE complex in mammalian cells, Xenopus NC cells, and Drosophila border cells (Law, 2013).
Lpd not only interacts with the Scar/WAVE complex but also directly binds to Ena/VASP proteins. Ena/VASP proteins regulate actin filament length by temporarily preventing capping of barbed ends and by recruiting profilin-actin to the growing end of actin filaments. In contrast, the Scar/WAVE-Arp2/3 complexes increase branching of actin filaments. Lamellipodia with a highly branched actin network protrude more slowly but are more persistent, whereas lamellipodia with longer, less branched actin filaments protrude faster but are less stable and quickly turn into ruffles. It was observed that Lpd overexpression increases cell migration in a Scar/WAVE- and not Ena/VASP-dependent manner. This is consistent with a predominant function of Scar/WAVE downstream of Lpd to regulate a highly branched actin network supporting persistent lamellipodia protrusion and cell migration. Other actin-dependent cell protrusions such as axon extension or dorsal ruffles of fibroblasts require Lpd-Ena/VASP-mediated F-actin structures (Law, 2013).
Collective cell migration describes a group of cells that moves together and affect each other, and various types of collective cell migration exists during development and cancer invasion. Xenopus NC cells migrate as loose streams, whereas Drosophila border cells migrate as a cluster of cells with close cell-cell contacts. This study found that Rac regulates Lpd and Scar/WAVE interaction and that both are required for Xenopus NC migration, which is consistent with previous work in which Rac activity mediates this type of migration. Similarly, NC-derived melanoblast migration in the mouse depends on Rac-Scar/WAVE-Arp2/3, and it was found that Lpd functions in this process as well (Law, 2013).
Drosophila border cell clusters migrate through the fly egg chamber in two phases: an early part characterized by large and persistent front extensions, which are regulated predominantly by PVR (the fly PDGF receptor); and a late part characterized by dynamic collective 'tumbling' behavior. Surprisingly, Pico overexpression resulted in the appearance of a higher proportion of rear facing extensions, a phenotype previously observed with dominant-negative PVR, causing premature tumbling of the border cell cluster. This suggests that Pico function is normally tightly controlled to stabilize specific extensions and functions also in guidance of collective cell migration. Because Lpd-Scar/WAVE control single cell migration as well as collective cell migration, this suggests that they function as general regulators of cell migration (Law, 2013).
Collectively, this study has identified a novel pathway in which Lpd functions as an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo (Law, 2013).
Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Using a multidisciplinary approach, this study identified the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). Elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. These results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements (Fernandez-Espartero, 2013).
Directed cell migration plays a crucial role in many normal and pathological processes such as embryo development, immune response, wound healing and tumor metastasis. During development, cells migrate to their final position in response to extracellular stimuli in the microenvironment. To migrate towards or away from a stimulus, individual cells or groups of cells must first achieve direction of migration through the establishment of cell polarity. Guidance cues, such as growth factors, control cell polarization through the regulated recruitment and activation of receptor tyrosine kinases (RTKs) to the leading edge. A key event downstream of RTK signalling in cell migration is the localization of activated Rac at the leading edge. However, little is known about the mechanisms by which external cues regulate Rac activity during cell migration. Rac is activated by GTP exchange factors (GEFs), which facilitate the transition of these GTPases from their inactive (GDP-bound) to their active (GTP-bound) states. Thus, GEFs appear as excellent candidates to regulate the cellular response to extracellular cues during cell migration (Fernandez-Espartero, 2013).
Among the different Rac GEF families characterized so far, the Vav proteins are the only ones known to combine in the same molecule the canonical Dbl (DH) and pleckstrin homology (PH) domains of Rac GEFs and the structural hallmark of tyrosine phosphorylation pathways, the SH2 domain. In addition, Vav activity is regulated by tyrosine phosphorylation in response to stimulation by transmembrane receptors with intrinsic or associated tyrosine kinase activity. These features make Vav proteins ideal candidates to act as signalling transducer molecules coupling growth factor receptors to Rac GTPase activation during cell migration. In fact, a number of cell culture experiments have suggested a role for the Vav proteins in cell migration downstream of growth factor signalling. Thus, the ubiquitously expressed mammalian Vav2 is tyrosine phosphorylated in response to different growth factors, including epidermal (EGF) and platelet-derived (PDGF) growth factors, and its phosphorylation correlates with enhanced Rac activity and migration in some cell types. However, the biological relevance for many of these interactions and the cellular mechanisms by which Vav regulates in vivo cell migration remains to be determined (Fernandez-Espartero, 2013).
The Vav proteins are present in all animal metazoans but not in unicellular organisms. There is a single representative in multicellular invertebrates and urochordata species (such as C. elegans, Drosophila melanogaster and Ciona intestinalis) and usually three representatives in vertebrates. The single Drosophila vav ortholog possesses the same catalytic and regulatory properties as its mammalian counterparts. In addition, the Drosophila Vav is tyrosine phosphorylated in response to EGF stimulation in S2 cells. Furthermore, a yeast two hybrid analysis has shown that the SH2-SH3 region of Vav can bind the epidermal growth factor receptor (EGFR) and the intracellular domain of PVR, PVRi, but not a kinase-dead version of PVRi, suggesting that Vav SH2-SH3-HA::PVRi interactions depend on PVR autophosphorylation. Altogether, these results suggest that the role of mammalian Vavs as transducer proteins coupling signalling from growth factors to Rho GTPase activation has been conserved in Drosophila. Thus, this study took advantage of Drosophila to analyse vav contribution to growth factor-induced cell migration in the physiological setting of a multicellular organism (Fernandez-Espartero, 2013).
The migration of the border cells (BCs) in the Drosophila egg chamber represents an excellent model system to study guided cell migration downstream of PVR/EGFR signalling in vivo. Each egg chamber contains one oocyte and 15 nurse cells surrounded by a monolayer of follicle cells (FCs), known as follicular epithelium (FE). The BC cluster is determined at the anterior pole of the FE and it comprises 6-8 outer cells and two anterior polar cells in a central position. BCs delaminate from the anterior FE and migrate posteriorly between the nurse cells until they contact the anterior membrane of the oocyte. BCs use the PVR and the EGFR to read guidance cues, the PDGF-related Pvf1 and the TGFβ-related Gurken, secreted by the oocyte. The Rho GTPase Rac is required for BC migration. The current model proposes that higher levels of Rac activity present in the leading cell determine the direction of migration and that this asymmetric distribution of Rac activity requires guidance receptor input. The unconventional Rac GEF Myoblast city, Mbc, is the only identified downstream signalling effector in this context. However, although genetic analysis have led to propose that the unconventional GEF for Rac, Mbc/DOCK 180, could activate Rac downstream of PVR during BC migration, this has not been formally proven. In addition, Mbc is unlikely to be the only Rac GEF actin downstream of guidance receptors in BCs as the migration phenotype due to complete removal of mbc is not as severe as the loss of both Pvr and Egfr. Thus, other effectors are likely to contribute to the complicated task of guiding BC migration. Many candidate molecules have been tested for their requirement in BC migration, MAPK pathway, PI3K, PLC-gamma, as well as RTK adaptors, such as DOCK, Trio, and Pak, but none of these is individually required (Fernandez-Espartero, 2013).
Vav proteins were initially involved in lymphocyte ontology. Only recently, cell culture experiments have implicated these proteins in cell migration events downstream of guidance factors. Interestingly, Vav proteins can either promote or inhibit cell migration. In macrophages, Vav is required for macrophage colony-stimulating factor-induced chemotaxis. In human peripheral blood lymphocytes, Vav is involved in the migratory response to the chemokine stromal cell-derived factor-1. Conversely, in Schwann cells, Vav2 is required to inhibit cell migration downstream of the brain-derived neurotrophic factor and ephrinA5. In spite of the knowledge gained from cell culture experiments, the biological relevance for many of the above interactions has remained elusive. In recent years, Vav proteins have started to emerge as critical Rho GEFs acting downstream of RTKs in diverse biological processes. Analysis of Vav2-/- Vav3-/- mice revealed retinogeniculate axonal projection defects and impaired ephrin-A1-induced migration during angiogenesis, suggesting a role for Vav in axonal targeting and angiogenesis downstream of Eph receptors in vivo. This study has shown that Vav can act downstream of growth factors receptors to promote BC migration in the developing Drosophila ovary, supporting a role of this family of GEFs in transducing signals from RTKs to regulate cell migration during development (Fernandez-Espartero, 2013).
Analysis of the cellular mechanisms by which Vav regulates cell migration in vertebrates is hampered by the inaccessibility of the cells and the difficulty of visualizing them in their natural environment within the embryo. Thus, it is not yet clear how Vav proteins regulate cell migration downstream of RTKs during development. In this study, by analysing cell movement in their physiological environment, it has been possible to show that Vav is required to control the length, stabilization and life of front cellular protrusions. In addition, disruption of Vav function in vivo was found to result in a decrease in Rac activity at the leading edge. Defective signalling downstream of EGFR/PVR results in defects in the dynamics of cellular protrusion and Rac activation, which are very similar to those observed in vav-/- BCs. In addition, this study found that ectopic activation of Vav in BCs, as it is the case for PVR/EGFR and Rac, causes non-polarized massive F-actin accumulation. Thus, it is suggested that one of the roles of Vav in directed cell migration downstream of EGF/PVF signals is to remodel the actin cytoskeleton via Rac activation, hence promoting the formation and stabilization of cellular protrusions in the direction of migration. Studies in cultured neurons, have shown that the main role for mouse Vav2 during axonal repulsion is to mediate a Rac-dependent endocytosis of ephrin-Eph. Although endocytosis has been normally shown to be involved in attenuation of RTKs signalling, in BCs it has been proposed to ensure RTKs recycling to regions of higher signalling, thus promoting directed BC movement. This is based on the fact that elimination in BCs of the ubiquitin ligase Cbl, which has been shown to regulate RTK endocytosis, leads to delocalized RTK signal and migration defects. In this context, another possible role for Vav downstream of EGFR/PVR could be to mediate RTK endocytosis, as it is the case during axonal repulsion. Further analysis will be needed to fully explore the molecular and cellular mechanisms by which Vav proteins regulate cell migration in vivo in other developmental contexts (Fernandez-Espartero, 2013).
BC migration is a complex event and activation of EGF/PDGF receptors will most likely engage different GEFs to affect the distinct cytoskeletal changes necessary to accomplish it. In fact, the migration phenotype of BCs mutant for
vav is less severe than that of BCs double mutant for both EGFR and PVR. In addition, although reducing Vav function decreases the asymmetry in Rac activity between front and back present in wild-type clusters, it does not eliminate it, as it happens when the function of both guidance receptors is compromised. All these results suggest that there are other GEFs besides Vav that could act downstream of EGFR and PVR to activate Rac. Previous analysis have implicated the Rac exchange factor Mbc/DOCK180 and its cofactor ELMO on BC migration. In this context, Vav and the Mbc/ELMO complex could act synergistically as GEFs to mediate Rac activation to a precise level and/or to a precise location. This awaits the validation of the Mbc/ELMO complex as a GEF for Rac in BCs. In the future, it will be important to determine how the different GEFs contribute to Rac activation, which specific downstream effectors of Rac they activate, and ultimately what cellular aspects of the migration process they control (Fernandez-Espartero, 2013).
In summary, this work demonstrates that Vav functions downstream of RTKs to control directed cell migration during development. Furthermore, this study has unravelled the cellular and molecular mechanism by which Vav regulates cell migration in the developing Drosophila egg chamber: binding of PDGF/EGF to their receptors would induce Vav activation through tyrosine phosphorylation and its association with the activated receptors. This would lead to an increase in Rac activity at the leading edge of migrating cells, which promotes the stabilization and growth of the cellular front extensions, thus controlling directed cell migration (Fernandez-Espartero, 2013).
Regulation of Vav signalling downstream of RTKs can participate not only in development or normal physiology but also in tumorigenesis. Vav1 is mis-expressed in a high percentage of pancreatic ductular adenocarcinomas and lung cancer patients. Thus, understanding the mechanisms by which Vav controls cellular processes downstream of RTKs is likely to be relevant for both developmental and tumor biology (Fernandez-Espartero, 2013).
The dedicator of cytokinesis (DOCK) family proteins that are conserved in a wide variety of species are known as DOCK1-DOCK11 in mammals. Sponge (Spg) is a Drosophila counterpart to the mammalian DOCK3. Specific knockdown of spg by pannier-GAL4 or apterous-GAL4 driver in wing discs induced split thorax phenotype in adults. Reduction of the Drosophila c-Jun N-terminal kinase (JNK), basket (bsk) gene dose enhanced the spg knockdown-induced phenotype. Conversely, overexpression of bsk suppressed the split thorax phenotype. Monitoring JNK activity in the wing imaginal discs by immunostaining with anti-phosphorylated JNK (anti-pJNK) antibody together with examination of lacZ expression in a puckered-lacZ enhancer trap line revealed the strong reduction of the JNK activity in the spg knockdown clones. This was further confirmed by Western immunoblot analysis of extracts from wing discs of spg knockdown fly with anti-pJNK antibody. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rac1 in the wing discs. Taken together, these results indicate Spg positively regulates JNK pathway that is required for thorax development and the regulation is mediated by interaction with Rac1 (Morishita, 2014PubMed).
Microtubules are regulated by a diverse set of proteins that localize to
microtubule plus ends (+TIPs) where they regulate dynamic instability
and mediate interactions with the cell cortex, actin filaments, and
organelles. Although individual +TIPs have been studied in depth and
their basic contributions to microtubule dynamics are understood, there
is a growing body of evidence that these proteins exhibit cross-talk and
likely function to collectively integrate microtubule behavior and
upstream signaling pathways. This study have identified a novel
protein-protein interaction between the XMAP215 homologue in Drosophila,
Mini spindles (Msps),
and the CLASP homologue, Orbit.
These proteins have been shown to promote and suppress microtubule
dynamics, respectively. Microtubule dynamics are regionally controlled
in cells by Rac acting to suppress GSK3β in the peripheral
lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers
a relocalization of Msps from the microtubule plus end to the lattice.
Mutation of the Msps-Orbit binding site revealed that this interaction
is required for regulating microtubule dynamic instability in the cell
periphery. Based on these findings, it is proposed that Msps is a novel
Rac effector that acts, in partnership with Orbit, to regionally
regulate microtubule dynamics (Trogden, 2015).
Microtubules interact with the small GTPase Rac in a complex pattern of cross-talk at the leading edge of motile cells. Growing microtubules induce cortical Rac activation by locally activating a guanine exchange factor (GEF) to induce protrusion and directional migration. In what is thought to be a positive feedback loop, active Rac promotes persistent microtubule growth in the lamellae and lamellipodia by locally regulating the activity of microtubule-associated proteins (MAPs). At least three MAPs have been implicated in regulation of microtubule dynamics downstream of Rac. The first is CLIP-170, a microtubule plus end interacting protein (+TIP) that interacts with the Rac effector IQGAP1 to capture microtubule plus ends at the plasma membrane. The second is Stathmin/OP18, a microtubule destabilizing factor that is locally inhibited at the leading edge due to phosphorylation by the Rac effector kinase Pak. The third is CLASP, another +TIP that suppresses dynamics, leading to increased stabilization of microtubules at the leading edge of polarized fibroblasts. CLASP binds directly to microtubules through a central lattice-binding domain and localizes to growing plus ends through an interaction with EB1. In the cell cortex, CLASP is phosphorylated by GSK3β, which blocks its ability to bind to the microtubule lattice, thus targeting it to growing plus ends. At the leading edge, GSK3β is locally inhibited by Rac and dephosphorylated CLASP binds along the microtubule lattice. Although local inhibition of Stathmin and activation of CLASP seem to be necessary for persistent microtubule growth at the leading edge, neither factor is sufficient, suggesting that other regulatory mechanisms remain to be discovered (Trogden, 2015 and references therein).
The present study identified the XMAP215 homolog, Msps, as a downstream effector of the Rac pathway and describes a novel regulatory mechanism for Msps through a protein-protein interaction with the microtubule stabilizer Orbit and the scaffolding protein Sentin. In S2 cells, the Drosophila CLASP homolog Orbit localizes to microtubule plus ends, but binds to the microtubule lattice upon expression of active Rac1 or depletion of GSK3β. These observations are similar to the dynamics of CLASP in mammalian cells and suggest that this mode of regulation is conserved. Activation of Rac or depletion of GSK3β promotes Msps binding to the microtubule lattice and this localization requires Orbit. The data suggests that, like CLASP, Orbit is directly phosphorylated by GSK3β which prevents it from interacting with and recruiting Msps to the microtubule lattice. The Orbit-Msps interaction further requires another +TIP, Sentin. As the localization of Sentin at the microtubule plus ends is not regulated by Rac-GSK3β, it is likely serving a scaffolding function to promote interactions between Msps and Orbit. This study mapped the protein-protein interaction sites on Msps and Orbit to their C-termini and found that mutations that block their interaction severely perturb microtubule dynamics. Both a non-phosophorylatable Orbit mutant and a mutant that prevented the Msps-Orbit interaction lead to more persistent growth, with the non-phosphorylatable Orbit mutant also causing an increase in microtubule pause. This may indicate that this interaction is important for persistent microtubule growth downstream of Rac-GSK3β (Trogden, 2015).
A growing body of evidence indicates that +TIPs exhibit cross-talk with one another to regulate microtubule dynamics in response to upstream regulatory cues. The current results indicate that Msps and Orbit function together during interphase to regulate dynamic instability in response to Rac and GSK3β activity. It is well established that members of the XMAP215 family promote microtubule dynamics by catalyzing microtubule polymerization and depolymerization. These activities are conserved in Drosophila Msps; microtubules in S2 cells lacking Msps are less dynamic, spending most of their lifetime in a pause state. In contrast, Orbit acts to suppress microtubule dynamics and promotes their stability. Thus, Msps and Orbit would be seem to regulate microtubule dynamics antagonistically, a functional relationship supported by recent genetic studies. However, the current results indicate that the two proteins share a more complex interaction (Trogden, 2015).
Msps exhibits two distinct localization patterns on microtubules in S2 cells- at the plus ends of microtubules and along the distal microtubule lattice in the periphery. The data support the model that these different modes of microtubule association represent functionally distinct pools of Msps. First, the Msps-Orbit interaction sites were identified, they were mutated to ablate the interaction, and these mutants were used to rescue cells depleted of either endogenous Msps or Orbit. Expression of either Msps-GFP 3A or 3K3A was able to rescue microtubule dynamics as compared to Msps-depleted cells. However, microtubules in these cells exhibited abnormally high frequencies of rescue and low frequencies of catastrophe, spending more time in growth and less in shrinkage compared to control cells. Cells expressing GFP-Orbit with the GSK3β phospho-acceptor sites mutated to alanine (5S->A) suppressed Orbit RNAi-induced increases in dynamic instability, but microtubules in these cells also exhibited higher frequencies of rescue, lower frequencies of catastrophe, and more time in the pause state compared to control cells. These results indicate that Msps must interact with Orbit in order to properly regulate microtubule dynamics in the cell periphery. Second, when the growth rates of microtubules were examined by tracking EB1-GFP, it was noted that EB1 comets in the cell periphery exhibit slower velocities than those in the cell cortex. These differences likely reflect interactions between growing microtubules and lamellipodial actin undergoing retrograde centripetal flow in the cell periphery. Recent work has also shown that EB1 comets are structurally different in the cortex versus the periphery, so the differences may be explained by changes in the microtubule as well. However, when EB1 comets on microtubules were compared with Msps at the plus end to those that had Msps localized along the distal lattice, it was discovered that the latter exhibited a significantly slower rate of growth. Collectively, the results indicate that the Msps-Orbit interaction 'tunes' microtubule dynamics in response to Rac activation in the cell periphery. It is suggested that Msps could be shunted onto the lattice to act as a localized 'sink' that attenuates its activity as a microtubule polymerase. This inactive pool may serve as a mechanism to partially suppress Msps activity so that microtubules grow at specific rates upon reaching the edge of the cell. The mechanism of how Msps regionally governs microtubule dynamics presents an intriguing problem; future studies employing biochemical reconstitution of microtubule dynamics with recombinant +TIPs and their regulators will likely be required to address these models (Trogden, 2015).
One puzzling aspect of this study is that, despite the biochemical and functional evidence for the Msps-Orbit interaction, colocalization of Msps and Orbit on the microtubule lattice in the cell periphery was not detected under unperturbed conditions. It is speculated that this protein-protein interaction is transient, occurring at the plus end, but is required for some conformational change in Msps that unmasks its microtubule lattice-binding activity. Two lattice-binding sites have been detected in the inter-TOG linker regions that seem to be inactive while the protein is localized to plus ends. This results were also confirmed using in vitro reconstitution assays. It is possible, however, that Orbit does localize to the lattice in the cell periphery, but at levels so low it was not possible to detect in living cells using the available probes. A third possibility is that Orbit is able to alter the structure of the microtubule lattice proximal to the plus end in order to promote lattice binding of Msps. This alteration could represent a change in the local nucleotide state of the polymer as a recent study indicated that mammalian CLASPs are able to promote GTP hydrolysis by polymerized tubulin. Alternatively, it has been shown that EB1 family members promote structural transitions within the microtubule lattice that favor GTP hydrolysis and compaction of the lattice itself. Perhaps similar localized changes in microtubule structure signal to Msps to transition from tip-association to lattice binding. Further work will be required to understand how these proteins interact to regulate their respective functions and it is expected that in vitro reconstitution assays will prove valuable to advance understanding of this protein-protein interaction (Trogden, 2015).
It is interesting to note that the localization patterns for Msps and Orbit observed in Drosophila cells seem to exhibit the converse relationships to those described for XMAP215/CH-TOG and CLASP in mammalian cells. Msps also differs from XMAP215 and ch-TOG through its lack of ability to either bind directly to EB1 or independently recognize growing microtubule ends. It is possible that the interaction with Orbit developed to increase Msps' ability to target the microtubule plus end. This interaction may also be present in mammalian cells, where it may serve to modulate growth rates of microtubules. Although Msps and XMAP215/CH-TOG exhibit high degrees of identity overall, it will be interesting to compare their relative activities in living cells using Msps to replace CH-TOG, and vice-versa, using heterologous systems (Trogden, 2015).
The data point to an outstanding question about how this localized regulation of dynamic instability impact behavior at the level of the cell. Dynamic microtubules exhibit a complex, bidirectional cross-talk with the Rho family of small G proteins. It is suggested that Msps and other XMAP215 family members are critical components of these pathways. In migrating cells, for example, Rac activity promotes processive microtubule growth while microtubule dynamics also promote Rac activation. It is predicted that Msps/XMAP215 family members are likely to participate in this positive feedback look and are, therefore, likely to play crucial roles in cell motility. Microtubules are also essential for directed membrane traffic to the leading edge. Msps-induced microtubule growth may also contribute to this polarized delivery of cargo to the front of motile cells. In order to address these fascinating questions, the Msps-Orbit interaction will have to be addressed in the context of migratory cell lines or, better still, within the developing embryo (Trogden, 2015).
In the current model in the cortex of the cell, Rac activity is low and therefore GSK3β is active, leading to phosphorylation of Orbit on 5 serine residues. Both Orbit and Msps are at the plus end, but cannot interact with each other. Msps is localized to the plus end through its interaction with Sentin. Orbit can bind either Sentin or EB1 to target the plus end. In the periphery of an S2 cell (or the leading edge of a migrating cell), Rac is active, which leads to the local inactivation of GSK3β and dephosphorylation of Orbit. Orbit is still on the plus end, but is now able to interact with Msps, allowing Msps to bind to the lattice. How this interaction allows Msps to bind the lattice with Orbit remaining on the plus end remains to be determined. It is hypothesized that when Msps is at the plus end it is in a closed conformation where the C-terminus covers the Linker4-TOG5 region that can bind the microtubule lattice. When Msps and Orbit bind to one another, this causes Msps to adopt an open conformation, exposing the lattice binding region which allows Msps to diffuse along to lattice (Trogden, 2015).
Wiskott-Aldrich syndrome proteins (WASP) are nucleation promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR/WAVE, WASP and WASH, have been analyzed so far. This study characterizes WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, WHAMY specifically interacts with activated Rac1 through its two CRIB domains that are sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promotes exceptionally fast actin filament elongation, while it does not activate the Arp2/3 complex. Loss- and gain-of function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further imply synergistic functions between WHAMY and WASP during morphogenesis. Double mutants are late-embryonic lethal and show severe defects in myoblast fusion. Trans-heterozygous mutant animals show strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution (Brinkmann, 2015).
The actin cytoskeleton plays a central role in a number of different cellular functions, such as cell shape changes, cell motility and membrane trafficking. Members of the Wiskott–Aldrich syndrome protein (WASP) family are conserved nucleation-promoting factors (NPF) that activate the Arp2/3 complex, a major actin nucleator in eukaryotic cells. In mammals, the WASP protein family consists of eight different members: the two Wiskott-Aldrich syndrome proteins WASP and N-WASP (also known as WAS and WASL, respectively), the related WASP family Verprolin homologous proteins WAVE1–WAVE3 (also known as SCAR1–SCAR3 and WASF1–WASF3, the Wiskott–Aldrich syndrome protein and SCAR homolog WASH (also known as WASH1), and the WHAMM and JMY proteins. WASP proteins share a conserved C-terminal Arp2/3-complex-activating WCA module. This module consists of either one or multiple actin-monomer-binding WH2 (W) domains, a central domain (C) and an acidic (A) domain, which mediate Arp2/3 binding. Apart from the catalytic WCA module, WASP proteins often share a proline-rich region and a basic region, which bind SH3-domain containing proteins and acidic phosphoinositides, respectively. WASP proteins are regulated by similar molecular principles. Under resting conditions NPFs are primarily inactive and become activated upon binding of the Rho GTPases Cdc42 and Rac1. Additionally, a variety of factors further modulate proper activation and recruitment of WASP proteins (Brinkmann, 2015).
In Drosophila, only three WASP subfamily members have been described, namely WAVE, WASP and WASH (also known as CG13176). Insects like Drosophila have subsequently lost a WHAMM/JMY gene, although the common ancestor first arose in invertebrates. Genetic studies indicate that WAVE and WASP are the central activators of the Arp2/3 complex, differentially regulating most aspects of Arp2/3 function in Drosophila. These studies highlight distinct, but also overlapping cellular requirements of WAVE and WASP during development. WAVE function is in particular essential for cell shape and morphogenetic cell movements during development. By contrast, WASP function is needed for cell fate specification of sensory organ precursors (SOPs) and spermatid Both, WASP and WAVE are required for myoblast fusion (Brinkmann, 2015).
Loss of maternal and zygotic WASP results in late-embryonic lethality due to strong defects in cell fate decisions of neuronal cell lineages and myoblast fusion defects. Remarkably, animals lacking zygotic WASP function survive until early adulthood. Thus, maternally provided WASP protein is sufficient for proper embryonic and larval development. Mutant wasp flies show no strong morphological defects except a partial loss of sensory bristles. Loss of zygotic Arp2/3 function results in a similar, albeit stronger, neurogenic phenotype suggesting an involvement of additional factors in Arp2/3-dependent SOP development (Rajan et al., 2009). The loss of sensory bristles in wasp and arp2/3 mutants phenocopies Notch loss-of-function and is caused by a pIIa-to-pIIb cell fate transformation. This results in an excess of neurons at the expense of bristle sheath, shaft and socket cells. Recent work further suggests that the WASP–Arp2/3 pathway rather plays an important role in the trafficking of Delta-positive vesicles from the basal area to the apical cortex of the signal-sending pIIb cell (Brinkmann, 2015).
Remarkably, rescue experiments have implied that established activators of WASP, such as Cdc42 or phosphatidylinositol 4,5-bisphosphate (PIP2), are not required for WASP function, neither for the myoblast fusion process nor for SOP development. The identity of an independent activator that might act cooperatively to control Arp2/3 function in these contexts is unknown. This study presents a functional analysis of WHAMY, a new WASP-like protein that regulates cell motility of Drosophila blood cells but also synergizes with WASP during embryonic muscle formation and cell fate specification of adult SOPs (Brinkmann, 2015).
The identification of all WASP family homologs in all sequenced organisms allows a detailed phylogenetic analysis of the origin of diverse subfamilies evolving differential cellular functions. WASP proteins are multi-domain proteins. They share functions that are encoded by similar domains at the C-termini, whereas different N-terminal domains mainly define their diverse cellular processes. Gene duplication and domain shuffling are two important mechanisms driving novel and increasingly complex developmental programs during evolution. It is thought that this boost in domain shuffling is responsible for the apparent disconnection between greatly increased phenotypic complexity and a relatively small difference in gene number between humans and Drosophila (Brinkmann, 2015).
The whamy gene is an excellent example for how gene duplication and subsequent domain shuffling can create new gene functions after initial gene duplication. It arose through a duplication of wasp at the base of the genus Drosophila. Although the encoded protein has evolved a new function in cell motility, it also functions synergistically with WASP in muscle formation and sensory organ development. In the latter, WHAMY can even partially substitute for WASP, indicating that it has kept functionality following the duplication. This duality is reflected in the sequence of the WHAMY CRIB domains. As there is an overlap in function with WASP, selective pressure has been reduced since the duplication, leading to the observed increase of evolutionary rate. Following the duplication of the CRIB domains within WHAMY, a similar trend can be found. Whereas one domain has kept the function of binding to Cdc42-GTP, the other has lost the ability to interact. This is reflected in domain-specific conserved substitutions. The duplication of the wasp gene and subsequent subneofunctionalization of whamy might have occurred at the same time as the loss of a true WHAMM/JMY ancestor during insect evolution (Veltman, 2010). Like Drosophila WHAMY, the common ancestor of WHAMM/JMY proteins in invertebrates also lacks the characteristic C-terminal tryptophan residue in their VCA domains that is crucial for Arp2/3 binding and activation (Veltman, 2010). This further implies a primary Arp2/3-independent function of the common ancestor of invertebrate WHAMM/JMY proteins (Brinkmann, 2015).
WHAMY shows no Arp2/3-activating nucleation promoting factor (NPF) activity in vitro. However, different from WASP, WHAMY itself is able to promote fast elongation of linear actin filaments from actin-rich clusters. With respect to its activity, WHAMY resembles the WH2-domain containing Ena/VASP polymerases that actively drive processive actin-filament elongation and promote assembly of both lamellipodial and filopodia actin networks. Notably, Ena/VASP proteins are tetramers, and their oligomerization is mandatory to allow for polymerase activity in experiments in solution, as used in this study. Since fast filament elongation was exclusively observed from WHAMY clusters in total internal reflection fluorescence (TIRF) experiments, and consistent with the size exclusion chromatography experiments, it is proposed that WHAMY requires oligomerization to acquire actin polymerase activity. Concerning previously analyzed proteins of the WASP family, the filament elongation activity of WHAMY is therefore rather unique, and when compared to other fast actin polymerases, only the Drosophila formin Diaphanous achieves comparable high elongation activity in vitro. As evidenced from the pyrene data, the activity of WHAMY can further be increased by Rac1 (Brinkmann, 2015).
Rac1 seems to act on both the activity and the localization of WHAMY at lamellipodial tips. Both of the two CRIB domains of WHAMY bind equally to activated Rac1, and only loss of both CRIB domains abolishes Rac1 binding and the localization to the leading edge. Therefore, it currently remains unclear why WHAMY contains two CRIB domains and whether they differentially mediate distinct cellular functions. They might contribute to a local clustering of WHAMY and Rac1 at the leading edge. The most prominent Rac1 effector represents the WAVE regulatory complex (WRC) that drives Arp2/3-mediated branched actin nucleation. Rac1 directly binds and activates the WRC by allosterically releasing the bound Arp2/3-activating WCA domain of WAVE. Overexpression of WHAMY leads to a strong induction of filopodia, presumably due to the filament elongation activity of WHAMY. Additionally, competition between WHAMY and the WRC for Rac1 could disturb the balance between nucleation and elongation activity, and therefore might contribute to the observed overexpression phenotype. Different from WHAMY, WRC function is essential for lamellipodia formation and cell migration in most eukaryotic cells. By contrast, loss of WHAMY function does not impair lamellipodia formation but rather regulates cell spreading and contributes to cell motility (Brinkmann, 2015).
WHAMY does not compete but rather functions together with WASP in Drosophila morphogenesis. Previous studies have revealed that the major established activators of WASP, such as Cdc42 and PIP2, are not required for the function of WASP in sensory organ development or myoblast fusion. This observation already suggests that additional components, such as WHAMY, might act together with WASP in sensory organ development and myoblast fusion. Consistent with this, further reduction of whamy function in wasp mutants was found to phenocopy loss of arp2/3 function, resulting in an excess of neurons and a near absence of bristle sheath, shaft and socket cells. Rescue data further indicate that WHAMY can partially substitute for WASP function. Thus, WHAMY cooperates with WASP rather than acting redundantly in sensory organ development. Based on TIRF microscopy data, it is suggested that WHAMY might potentially generate mother filaments in close vicinity of Arp2/3 complex facilitating Arp2/3-mediated actin assembly (Brinkmann, 2015).
How might WHAMY and WASP act on actin dynamics during sensory organ development? Recent work suggests that the WASP–Arp2/3 pathway is not involved in Notch receptor endocytosis or its processing in the signal-receiving cell (pIIa) but rather plays an important role in the trafficking of Delta-positive vesicles from the basal area to the apical cortex of the signal-sending pIIb cell. This model also implies that recycled Notch ligands such as Delta and Serrate are active at apical junctions with actin-rich structures induced by WASP and the Arp2/3 complex, which in turn activate apical Notch receptor in pIIa. In vivo, WHAMY localizes at dynamic vesicles during sensory organ precursor formation and, together with WASP, becomes strongly enriched at apical junctions shortly after SOP division. Thus, a scenario is proposed in which WASP and WHAMY might act either on the assembly of actin-rich structures or directly promote apical trafficking of Delta through Rab11-recycling endosomes (Brinkmann, 2015).
A dynamic reorganization of the actin cytoskeleton into distinct cellular structures is also necessary to ensure successful myogenesis. Filopodial protrusions are crucial for the attachment of FCMs to the founder cell and growing myotube, and for the initiation of the fusion process. The recognition and adhesion of myoblasts depends on members of the immunoglobulin superfamily (IgSF) that are expressed specifically in myoblasts in a ring-like structure. The interaction of these proteins leads to the formation of a cell communication structure, which has been termed fusion-restricted myogenic adhesive structure (FuRMAS) or podosome-like structure. The cytodomains of the IgSFs trigger the activation of WAVE in founder cells, and of WAVE and WASP in FCMs. In FCMs, WAVE- and WASP-mediated Arp2/3 activation results in the formation of a dense F-actin focus that accumulates at the interface of adhering myoblasts. Electron microscopy studies have revealed that WASP is required for the formation of fusion pores at apposing myoblasts during embryonic and indirect flight muscle development. These fusion pores expand until full cytoplasmic continuity is achieved, and WASP has implicated to be required for fusion pore expansion. It has been discussed that WASP is required for the removal of membrane residuals during membrane vesiculation. WHAMY might contribute to this process, but the detailed mechanistic contribution of WHAMY in fusion pore formation needs to be addressed in future studies by ultrastructural analyses (Brinkmann, 2015).
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