BIOLOGICAL OVERVIEW

Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. This study shows that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled (ena) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization (Lin, 2009).

The in vivo role of Abi with respect to Abl has remained enigmatic. Abi was first identified as an Abl kinase substrate, functioning in modulating the transformation activity of oncogenic Abl in human cancers. Intriguingly, Abi also functions as an activator of Abl kinase activity. Moreover, the interaction of Abl and Abi can trigger an array of biochemical and functional changes in Abi, including protein phosphorylation, stability and subcellular localization, which might ultimately lead to the control of a particular biological process in vivo. Although both Abi and Abl proteins are highly expressed in the mammalian and Drosophila nervous systems, the role of Abi in modulating the function of Abl in developing nervous systems has remained unclear. In this investigation, genetic and functional studies were conducted to advance the understanding of how abi and Abl interact in vivo. To do this, abi loss-of-function alleles were generated and characterized for genetic and functional studies in Drosophila. Immunohistochemical analysis revealed that Abi is primarily expressed in the developing CNS. Consistent with this finding, phenotypic analysis suggested that mutations in abi resulted in axonal guidance defects in the CNS. In an analysis of Abl mutants, it was found Abl to be crucial for restricting synaptic overgrowth in the larval NMJ. Importantly, further studies of the genetic interaction found a functional link between abi and Abl in axonogenesis and synaptogenesis. Moreover, Abi and Ena were found to cooperate in modulating the function of Abl in NMJ growth. Finally, based on additional cellular biology studies, it is proposed that the functional interactions between Abi, Ena and Abl might be mediated through the modulation of actin cytoskeleton reorganization (Lin, 2009).

Accumulated evidence suggests that the highly conserved actin-regulatory pathways are essential for synaptogenesis and synaptic plasticity. Abi, Ena and Abl proteins are all involved in actin dynamics. Using the Drosophila NMJ as a model system, it is proposed that the abi-ena-Abl interaction in synaptogenesis might be associated with actin cytoskeleton reorganization. In fact, several actin regulatory molecules associated with both Abl and Abi have been implicated in synaptic growth. For example, Wiskott-Aldrich Syndrome protein (Wasp) is a kinase substrate of Abl and also a binding partner for Abi. The mutations in wasp result in phenotypes very similar to those present in Abl mutants, with synaptic overgrowth and hyperbranching at the NMJ. Another example is that of Diaphanous (Dia), which also interacts with both Abl and Abi, and has recently been found to modulate synaptic growth of the Drosophila NMJ. dia mutant heterozygotes have been found to be able to enhance the cellularization phenotype of an Abl maternal-null mutant, suggesting that Dia might be involved in the regulation of Abl signaling for actin reorganization. Consistent with this idea, the interaction of Dia with Abi protein has been found to be important in regulating the formation and stability of cell-cell junctions in mammalian cells. Future studies investigating whether and how Wasp and/or Dia can participate in Abl-Abi signaling for the regulation of Drosophila synaptogenesis could be interesting (Lin, 2009).

Besides Wasp and Dia, other actin regulators might also contribute to Abl-Abi signaling during nervous system development, for, as another study has suggested, Abl may be a key regulator in modulating different types and sites of actin polymerization within the cells. Abi has been shown to play a key role in the activation of the SCAR/WAVE complex, which relays signaling from Rac1 to the Arp2/3 complex for actin cytoskeleton remodeling. Genetic studies have shown that the heterozygosity of scar, but not of kette, suppressed Abl NMJ phenotypes. A current model suggests that eliminating any component from the SCAR/WAVE complex induces the breakdown of other complex components and subsequently results in abnormal lamellipodia formation. Genetic study using the Drosophila NMJ as a model does not appear to fully support this idea. The results suggested that only a subcomplex of SCAR/WAVE might be involved in synaptogenesis. In fact, recent studies have demonstrated that some components of the SCAR/WAVE complex might work outside the complex to regulate various biological processes, including neutrophil chemotaxis, cell motility and adhesion, and the formation of cell-cell junctions. Thus, it is possible that Kette is not in a complex with Abi and Scar to modulate the function of Abl in NMJ growth. To explore this hypothesis, it will be important to examine the genetic interactions between abi and scar or kette in NMJ morphogenesis (Lin, 2009).

This study found strong in vivo evidence for an antagonistic relationship between Abl and Abi in axonogenesis and synaptogenesis. Supporting this model, one very recent study has demonstrated that Abl can inhibit the role of Abi in the engulfment of apoptotic cells in C. elegans (Hurwitz, 2009). Given that Abi is the Abl kinase substrate and that it also functions as an adaptor protein for Abl in regulating other downstream effectors, it is feasible that Abi might act downstream of Abl in modulating NMJ growth. If so, the removal of both copies of abi could conceivably further suppress Abl^-/- NMJ phenotypes. Preliminary morphological and functional data both suggest that the minor NMJ defects of Abl^-/- abi^+/- are further rescued in Abl^-/- abi^-/- mutants. However, Abl^-/- abi^-/- double mutants showed early lethality and defects in axonal innervations, rendering the finding inconclusive. Further epistasis analysis combining abi and abl gain-of-function and loss-of-function mutations are needed to test this hypothesis (Lin, 2009).

Since the data suggest an antagonistic interaction between abi and Abl for the CNS and NMJ phenotypes, it is speculated that Abl heterozygosity would suppress the semilethal phenotype of abi mutants. Surprisingly, preliminary data showed that the lethality of abi hypomorphic mutants (abi^P1/KO and abi^P1/Df) is further increased by Abl^+/-. This result does not seem to support a general bidirectional antagonistic relationship between Abl and abi for the biological processes involved during development. Thus, a complex genetic interaction network between Abl and abi might be present in development processes (Lin, 2009).

Another interesting issue is that the abi mutants did not display obvious defects in synaptic bouton number or synaptic transmission, although they exhibited midline crossing defects in the embryonic CNS. Because other members of SCAR complex, including Scar, Kette, Sra-1 and HSPC300, exhibit both CNS and NMJ phenotypes, it is still possible that abi mutants might show minor morphological or functional abnormalities if different phenotypic characteristics are studied. Detailed morphological assays are required to investigate other phenotypic traits of the NMJ in larval or later developmental stages. Alternatively, one could reason that the roles of Abi in synaptic growth and axonal guidance are not exactly identical. Results similar to this finding have been observed for the loss of spastin, a gene enriched in axons and synaptic connections, as spastin mutants only exhibit NMJ but not CNS defects (Lin, 2009).

This work also suggested that the synaptic overgrowth phenotypes in Abl mutants could be completely rescued by expressing Abl in the presynaptic nerve cells but not in the postsynaptic muscles, suggesting that the presynaptic Abl is more crucial than the postsynaptic population for Drosophila larval NMJ formation. However, studies in mammalian Abl and Arg (also known as Abl2) have shown that both proteins localize to the presynaptic terminals and dendritic spines of synapses in the hippocampal CA1 area. Abl and Arg have also been shown to be essential for the agrin-induced clustering of acetylcholine receptors (AChRs) on the postsynaptic membrane of the mammalian NMJ, suggesting that Abl function is required in the postsynaptic region of the mammalian NMJ. However, these reports do not exclude the possibility that Drosophila Abl might also function in postsynaptic regions of the developing brain. The reason for this speculation is that the mammalian NMJ uses acetylcholine as the neurotransmitter, unlike the Drosophila NMJ, which uses glutamate as a transmitter. Since acetylcholine receptors also function in the developing brain of Drosophila, it would be important to investigate whether Drosophila Abl also plays a role in the postsynaptic region of the neurons, where acetylcholine receptors are expressed (Lin, 2009).

In conclusion, these genetic studies in Drosophila suggest that Abi and Abl play opposing roles in axonogenesis and synaptogenesis. This conclusion is further supported by a series of biochemical, immunocytochemical and morphological studies in cultured cells. These findings offer new insights into the functional interaction between Abl, Abi and Ena in nervous system development (Lin, 2009).

Enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons

The golgi apparatus is optimized separately in different tissues for efficient protein trafficking, little is known of how cell signaling shapes this organelle. This study finds that the Abl tyrosine kinase signaling pathway controls the architecture of the golgi complex in Drosophila photoreceptor (PR) neurons. The Abl effector, Enabled (Ena), selectively labels the cis-golgi in developing PRs. Overexpression or loss-of-function of Ena increases the number of cis and trans-golgi cisternae per cell, and Ena overexpression also redistributes golgi to the most basal portion of the cell soma. Loss of Abl, or of its upstream regulator, the adaptor protein Disabled, lead to the same alterations of golgi as does overexpression of Ena. The increase in golgi number in Abl mutants arises in part from increased frequency of golgi fission events and a decrease in fusions, as revealed by live imaging. Finally, it was demonstrated that the effects of Abl signaling on golgi are mediated via regulation of the actin cytoskeleton. Together, these data reveal a direct link between cell signaling and golgi architecture. Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion (Kannan, 2014).

The Abl tyrosine kinase signaling pathway controls golgi morphology and localization in Drosophila photoreceptors through its regulation of the actin cytoskeleton. Ena, the main effector of Abl in morphogenesis, is associated with the cis-golgi compartment, and it regulates golgi localization and dynamics under the control of Abl and its interacting adaptor protein, Dab. Reducing the levels of Abl or Dab, or overexpressing Ena, led to similar defects in golgi fragmentation state and subcellular distribution. During golgi biogenesis, Abl increases the frequency of fusion of golgi cisternae, and decreases fission events. Abl evidently controls golgi organization through its regulation of actin structure, as the effect of Abl signaling on golgi could be blocked by modulating actin structure genetically or pharmacologically. Collectively, these data reveal an unexpected link between a fundamental tyrosine kinase signaling pathway in neuronal cells and the structure of the golgi compartment (Kannan, 2014).

The data reported here suggest that the Abl signaling pathway controls golgi morphology and localization through its control of actin structure. This is consistent with previous reports that altering the levels of actin modulators perturbs the structure and function of the golgi apparatus. A variety of proteins that modulate actin dynamics have been localized to golgi. Ultra-structural studies established the association of actin filaments with golgi membranes and the association of β and γ actin with the golgi. In cultured cell models, including neurons, actin depolymerization leads to golgi compactness, fragmentation and altered subcellular distribution. It is noted, moreover, that the reported golgi-associated signaling proteins include several that have been linked to Abl signaling, including the Abl target Abi, the Abi binding partner WAVE, and various effectors of Rac GTPase including ADF/cofilin, WASH and Arp2/3. Thus, for example, Abi and WAVE have been implicated in actin dependent golgi stack reorganization and in scission of the golgi at cell division to allow faithful inheritance of golgi complex to daughter cells in Drosophila S2 cell cycles (Kondylis, 2007). These data reinforce the importance of actin-regulating signaling pathways for controlling golgi biogenesis (Kannan, 2014).

Two lines of evidence suggest that the increase observed in golgi number in Abl pathway mutants is due primarily to net fragmentation of pre-existing golgi cisternae and not to de novo synthesis of golgi. First, live imaging of golgi dynamics in neurons of the Drosophila eye disc reveals that the steady-state number of golgi cisternae reflects an ongoing balance of fusion and fission events, much as observed previously in yeast. Quantification of these events in wildtype vs Abl mutant tissue demonstrated directly that loss of Abl significantly increased the frequency of fission events, and reduced the frequency of fusions. Second, the absolute volume of cis-golgi in Abl mutant photoreceptors was not substantially greater than that in wildtype, as judged by direct measurement of the volume of GM130- immunoreactive material in deconvoluted image stacks of photoreceptor clusters. While a small apparent increase was observed in golgi volume in the mutants (~55%, based on pixel counts), it is noted that golgi cisternae are small on the length scale of the point spread function of visible light, such that the fluorescent signal from a single cisterna extends into the surrounding cytoplasm. The increase in apparent golgi volume is therefore within the range expected due simply to fluorescence 'spillover' from the three-fold greater number of separate golgi cisternae in the mutants (Kannan, 2014).

It is striking that both increase and decrease of Ena led to net fragmentation of golgi. Why might this be? It is known that both fission and fusion of membranes requires actin dynamics: at scission, polymerization provides force for separating membranes, while in fusion, actin polymerization is essential for bringing membranes together and for supplying membrane vesicles, among other things. As a result, altering actin dynamics is apt to change the probabilities of multiple aspects of both fission and fusion events, making it impossible to predict a priori how the balance will be altered by a given manipulation, just as either increase or decrease of Ena can inhibit cell or axon motility, depending on the details of the experiment, due to the non-linear nature of actin dynamics. Indeed, this study also observed net golgi fragmentation when actin was stabilized with jasplakinolide, just as was done from depolymerization with cytochalasin or latrunculin. More direct experiments will be necessary to fully understand this dynamic, however. deficits selectively disrupt dendritic morphogenesis but not axogenesis, and perhaps consistent with this, Abl/Ena function is essential for dendrite arborization in these cells but has not been reported to affect their axon patterning. Finally, in some contexts, neuronal development requires local translation of guidance molecules in the growth cone rather than translation in the cell soma. It is likely that the need for actin dynamics to target different subcellular compartments in different cell types will be reflected in different patterns of Abl/Ena protein localization (Kannan, 2014).

This study reports the role of Abl/Ena-dependent regulation of actin structure on overall golgi structure and localization but there may be more subtle effects on golgi function as well. For example, recent evidence supports a role for actin-dependent regulation of the specificity of protein sorting in the golgi complex. Preferential sorting of cargos is achieved by nucleation of distinct actin filaments at the golgi complex. In Hela cells, for example, Arp2/3 mediated nucleation of actin branches at cis-golgi regulates retrograde trafficking of the acid hydroxylase receptor CI-MPR, while Formin family mediated nucleation of linear actin filaments at golgi regulates selective trafficking of the lysosomal enzyme cathepsin D. Similarly, the actin-severing protein ADF/cofilin, the mammalian ortholog of Drosophila twinstar, sculpts an actin-based sorting domain at the trans-golgi network for selective cargo sorting. It will be important to investigate whether the effects of Abl/Ena on golgi morphology have functional consequences on bulk secretion or protein sorting (Kannan, 2014).

Protein trafficking and membrane addition in neurons need to be coordinated with the growth requirements of the axonal and dendritic plasma membranes, but the mechanisms that do so have been obscure. Abl pathway proteins associate with many of the ubiquitous guidance receptors that direct axon growth and guidance throughout phylogeny, including Netrin, Roundabout, the receptor tyrosine phosphatase DLAR, Notch and others. The data therefore suggest a potential link between the regulatory machinery that senses guidance information and the secretory machinery that helps execute those patterning choices. Indeed, preliminary experiments suggest that some of the axonal defects of Abl pathway mutants may arise from alterations in golgi function. Beyond this, Abl signaling is essential in neuronal migration, epithelial polarity and integrity, cell adhesion, hematopoiesis and oncogenesis, among other processes The data reported in this study now compel a reexamination of the many functions of Abl to ascertain whether some of these effects arise, at least in part, from regulation of secretory function (Kannan, 2014).

Ena/VASP proteins cooperate with the WAVE complex to regulate the actin cytoskeleton

Ena/VASP proteins and the WAVE regulatory complex (WRC) regulate cell motility by virtue of their ability to independently promote actin polymerization. This study demonstrates that Ena/VASP and the WRC control actin polymerization in a cooperative manner through the interaction of the Ena/VASP EVH1 domain with an extended proline rich motif in Abi. This interaction increases cell migration and enables VASP to cooperatively enhance WRC stimulation of Arp2/3 complex-mediated actin assembly in vitro in the presence of Rac. Loss of this interaction in Drosophila macrophages results in defects in lamellipodia formation, cell spreading, and redistribution of Ena to the tips of filopodia-like extensions. Rescue experiments of abi mutants also reveals a physiological requirement for the Abi:Ena interaction in photoreceptor axon targeting and oogenesis. These data demonstrate that the activities of Ena/VASP and the WRC are intimately linked to ensure optimal control of actin polymerization during cell migration and development (Chen, 2014).

Ena/VASP proteins regulate cell migration by promoting actin polymerization at the plasma membrane via antagonizing actin filament capping and acting as processive actin polymerases. Each family member consists of an N-terminal EVH1 domain, a central proline-rich region, and a C-terminal EVH2 domain. The EVH2 domain, which contains monomeric and F-actin binding sites, is responsible for promoting actin polymerization. In contrast, the EVH1 domain mediates intracellular targeting of Ena/VASP proteins by interacting with a sequence (D/E)FPPPPX(D/E)(D/E), which is referred to as the 'FPPPP' motif because these residues are essential for binding. Ena/VASP proteins are recruited to focal adhesions by zyxin, which contains four 'FPPPP' motifs. The ability of Ena/VASP proteins to control cell migration, however, depends on their recruitment to the leading edge, by 'FPPPP' motif containing MRL proteins (Mig10, RIAM, and Lamellipodin) (Chen, 2014).

Of all the proteins interacting with the EVH1 domain of Ena/ VASP proteins, Testin (Tes), a focal adhesion protein, stands out as the only one that lacks an 'FPPPP' motif. Tes negatively regulates the localization of Mena at focal adhesions and also inhibits Mena-dependent cell migration. Tes interacts with Mena via its C-terminal LIM3 domain and is unique in being the only protein that binds a single Ena/VASP family member. Given the interaction of Tes with Mena, additional atypical EVH1 binding partners that also lack 'FPPPP' motifs were sought. The EVH1 domain interacts directly with Abi, a component of the WAVE regulatory complex (WRC), which plays an essential role in driving cell migration by activating the Arp2/3 complex in response to Rac signaling. These observations demonstrate that the EVH1:Abi interaction enhances cell migration and the ability of Rac-activated WRC to promote Arp2/3- mediated actin polymerization as well as the function of WRC in vivo in Drosophila (Chen, 2014).

The WRC binds and activates the Arp2/3 complex to drive actin polymerization at the plasma membrane in response to Rac signaling during cell migration (Bisi, 2013). In contrast, Ena/VASP proteins stimulate cell migration by antagonizing actin filament capping and acting as processive actin polymerases. This study has now demonstrated that Ena/VASP proteins can be linked to the function of WRC by virtue of a direct interaction between their EVH1 domains and Abi, an integral component of the WRC (Chen, 2014).

The results have confirmed and extended previous yeast two-hybrid data and pull-downs from cell lysates demonstrating that the EVH1 domains of Mena and VASP can interact with human and mouse Abi1. The structure of several EVH1:FPPPP complexes reveals that the 'FPPPP' motif adopts a type II polyproline helix that is coordinated by three aromatic residues present in all Ena/VASP family members. In contrast, the EVH1 domain interacts with an extended proline-rich binding site in human Abi1. Consistent with their ability to bind, Abi2 has an almost identical sequence whereas Abi3 has two 'LPPPP' motifs in this region. In many respects, the extended nature of the Abi1 interaction resembles that of the N-WASP WH1 binding site in WIP, which also involves three regions of contact. In classical EVH1 interactions, the acidic residues flanking the 'FPPPP' motif play an important role in determining the affinity, orientation and specificity of EVH1 binding. In contrast, the EVH1 binding site in human Abi1 contains two pairs of aspartic acid residues flanking the central phenylalanine in the middle of the motif as well as a downstream acidic patch (DYEDEE). The molecular basis of the EVH1 human Abi1 interaction, including the extended peptide orientation and role of acidic residues, must await structural determination of the complex. Nevertheless, the data clearly demonstrate that the EVH1 domain can bind additional proline rich ligands beyond 'FPPPP' motifs (Chen, 2014).

Interestingly, the meander region of WAVE1 contains an 'LPPPP' motif that is capable of interacting with Mena. The ability of Mena to bind Abi in the WRC presumably explains why it still associates with WAVE lacking its proline rich region. Consistent with the presence of 'LPPPP' motifs pull-downs with recombinant proteins demonstrate that the EVH1 domain of Mena can interact with WAVE 1 and 2, but not WAVE 3. These observations, however, suggest that the interaction with Abi is more important for Mena interactions with the WRC than WAVE. Moreover, in vitro assays clearly demonstrate that the ability of Rac to activate WRC-mediated actin polymerization via the Arp2/3 complex is significantly enhanced by VASP binding to Abi. In contrast to the full-length protein, monomeric VASP or its isolated EVH1 domain is unable to activate the WRC to stimulate Arp2/3-mediated actin polymerization even at high concentrations. This difference may reflect the ability of the VASP tetramer to induce oligomerization of the WRC, an effect that would enhance WRC potency toward the Arp2/3 complex. It is possible that the simultaneous engagement of a VASP tetramer with Abi and the 'LPPPP' motif in WAVE increases the activity of the WRC. However, oligomerization alone cannot account for the data because mutating the actin binding elements of VASP, which should have no effect on tetramerization, abrogates activity. Furthermore, the VASP effect does not appear to be simple allosteric activation of the WRC (i.e., release of the VCA), because this should produce activity equal to that of the VCA alone. While not definitive, the collective data are most consistent with a model in which VASP binds the Rac-activated WRC with high affinity based on tetramerization-mediated avidity and also interacts with actin filaments, thus increasing the association of the WRC with filaments. Because both the released WAVE VCA and actin filaments activate the Arp2/3 complex, assembling these two elements should enhance their cooperative actions and increase actin assembly (Chen, 2014).

In contrast to the situation in humans, the interaction between the EVH1 domain of Ena and Abi in Drosophila is mediated by two 'LPPPP' motifs located in a proline rich region of Abi. The loss of these two 'LPPPP' motifs increases the dynamics of the WRC at the plasma membrane but does not affect lamellipodium formation in S2 cells in culture. In contrast, the consequences of disrupting the interaction of Ena with Abi in vivo are more dramatic, as primary macrophages expressing Abi- DEna have reduced lamellipodial membrane protrusions and defects in cell morphology. Unlike the situation in S2 cells, which have been treated with dsRNA and transiently transfected with GFP-tagged expression constructs, the abi transgenes (Abi and AbiDEna) are expressed from the same genomic locus (Chen, 2014).

These in vivo rescue experiments therefore allow for a more precise analysis of the requirement of the interaction between Ena and Abi rather than in the hypomorphic situation in S2 cells. The ability of AbiDEna to rescue lamellipodium formation in S2 cells might reflect an incomplete abi knockdown or a difference in its expression level compared to endogenous Abi in untreated cells. Consistent with this, in macrophages, this study found that strong expression of Abi in earlier larval stages using the da-Gal4 driver results in a more robust rescue of lamellipodia protrusion and cell morphology defects as compared to macrophage-specific expression (hmlD-gal4) at late larval stages. Given that in vitro actin polymerization assays indicate that VASP (Ena) is not an essential activator but rather acts cooperatively with Rac1 to promote WRC activation, it is likely that in vivo the requirement for this interaction depends on the level of Abi. This explanation may also partially account for the more dramatic phenotypes observed in the multicellular context (Chen, 2014).

Remarkably, this study found that the loss of the ability of Abi to interact with Ena resulted in a similar defect in R-cell targeting as the absence of the complete protein. This suggests that Ena has a nonautonomous role in the larval brain, as has been previously shown for WRC function in targeting of early retinal axons (Stephan, 2011). Mosaic mutant analysis further supports a nonautonomous function for Ena in retinal axon targeting. Thus, it is proposed that the interaction between Ena and the WRC is required to regulate actin dynamics in the target area neurons. However, since the precise projection pattern of early retinal axons depends on complex interactions between different populations of glia cells and neurons in the target field, it remains unclear how Ena and the WRC function together in this developmental context. In contrast, Drosophila oogenesis provides an excellent model to study the cell autonomous function of the interaction between Ena and the WRC (Chen, 2014).

Previous phenotypic analyses of mutant egg chambers suggest Ena and WRC have both distinct and overlapping functions during oogenesis. Both are required for the integrity of the cortical actin in nurse cells and mutant egg chambers become multinucleated as the plasma membrane breaks down due to a loss of cortical actin integrity. In contrast, to wave mutant egg chambers, disruption of ena function does not affect ring canal morphology but rather leads to a reduced and delayed formation of cytoplasmic actin filament bundles. Similar to wave germline clones, the loss of abi in the germline results in a dumpless mutant phenotype and female flies are sterile (Zobel, 2013). This study has found that these defects in egg morphology and female fertility cannot be rescued by reexpression of a full-length Abi deficient in Ena binding. AbiDEna mutant egg chambers have defects in the integrity of the nurse cell cortical actin resulting in detached cytoplasmic actin bundles and ring canals. The rupture of nurse cell membranes is even more obvious at later stages when the fast transport of nurse cell contents starts, as recently observed for ena, wave, and abi mutants (Chen, 2014).

In addition to nurse cell dumping defects, a striking egg chamber elongation defect was also observed. Mutant eggs lacking the interaction between Abi and Ena fail to elongate and remain spherical as similarly found in rac or pak mutants. The round egg phenotype observed in flies expressing AbiDEna suggests that there might be a defect in the basal actin cytoskeleton of the follicle cells that drives egg chamber elongation. Consistently, reexpression of AbiDEna in somatic follicle cells (abi, da > UASt-AbiDEna) also results in a round-egg phenotype. These data suggest a requirement of WRC function in follicle cells during egg elongation. Supporting this notion, this study found that a follicle cell-specific knockdown of Sra-1 function results in a strong round-egg phenotype (Chen, 2014).

The rescue experiments additionally imply a more complex interaction network among Ena, Abi, and SH3 interacting proteins. Whereas a minimal Abi fragment lacking the Ena-binding or proline-rich region and the C-terminal SH3 domain is able to rescue substantially abi mutant traits in Drosophila and Dictyostelium, the disruption of Ena-binding alone completely abolishes Abi activity. Thus, a scenario is proposed in which the influence of Ena on WRC activity depends on additional proteins interacting with the Abi-SH3 domain. The most prominent candidate is the nonreceptor tyrosine kinase Abelson (Abl) that binds Abi and Ena proteins. Based on the antagonistic genetic interaction between ena and abl, it has been hypothesized that a precise balance between Abl and Ena activity is required for fly viability. However, it is still unclear how Abl affects the function of Ena, because mutation of all known Abl phosphorylation sites only has a modest effect on Ena function in vivo. Similarly, Abl and Abi have opposing roles in Drosophila. Thus, a model is proposed in which Ena synergizes with Rac to activate the WRC, but also inhibits Abl function. Abl in turn inhibits WRC function. Thus, the disruption of Ena binding to dAbi would simultaneously decrease WRC stimulation by Ena and increase its inhibition by Abl. Such a scenario would explain why loss of Ena binding to Abi (WRC) phenocopies the abi mutants. This also suggests that the interaction among WRC, Abl, and Ena function is of more general relevance for actin-based processes in multicellular contexts. Furthermore, recent data also suggest that lamellipodin, which cooperates with the WRC to promote cell migration in vivo, is also likely to be part of this complex regulatory network, because it can bind both the EVH1 domain of VASP and the SH3 domain of Abi. In summary, these in vitro data clearly demonstrate that Ena/VASP proteins can directly affect the activity of the WAVE complex, whereas the observations in Drosophila have revealed that, in vivo, the function and activity of Ena/VASP proteins and the WAVE complex are intimately linked (Chen, 2014).

A modifier screen identifies regulators of cytoskeletal architecture as mediators of Shroom-dependent changes in tissue morphology

Regulation of cell architecture is critical in the formation of tissues during animal development. The mechanisms that control cell shape must be both dynamic and stable in order to establish and maintain the correct cellular organization. Previous work has identified Shroom family proteins as essential regulators of cell morphology during vertebrate development. Shroom proteins regulate cell architecture by directing the subcellular distribution and activation of Rho-kinase, which results in the localized activation of non-muscle myosin II. Because the Shroom-Rock-myosin II module is conserved in most animal model systems, Drosophila melanogaster was used to further investigate the pathways and components that are required for Shroom to define cell shape and tissue architecture. Using a phenotype-based heterozygous F1 genetic screen for modifiers of Shroom activity, several cytoskeletal and signaling protein were identified that may cooperate with Shroom. Two of these proteins, Enabled and Short stop, are required for ShroomA-induced changes in tissue morphology and are apically enriched in response to Shroom expression. While the recruitment of Ena is necessary, it is not sufficient to redefine cell morphology. Additionally, this requirement for Ena appears to be context dependent, as a variant of Shroom that is apically localized, binds to Rock, but lacks the Ena binding site, is still capable of inducing changes in tissue architecture. These data point to important cellular pathways that may regulate contractility or facilitate Shroom-mediated changes in cell and tissue morphology (Hildebrand, 2021).

Tissue architecture is typically defined during specific stages of embryonic development and errors in these processes can result in human disease. One example is formation of the vertebrate neural tube. The neural tube is formed via the concerted effort of many cellular pathways that functionally convert a plate of neural ectoderm into a closed tube. Errors in this process can result in birth defects such as spina bifida, exencephaly, or craniorachischisis. One cellular pathway that controls this process is regulated by the Shroom3 cytoskeletal adaptor protein. Shroom3 controls neural tube morphogenesis via the formation of apically positioned contractile networks of actomyosin and these networks facilitate neural tube closure by inducing apical constriction and the anisotropic contraction of actin filaments. This is accomplished via the modular nature of Shroom3. Shroom3 localizes to the apical compartment of epithelial adherens junctions via a direct interaction with F-actin. This interaction is mediated by the Shroom Domain (SD) 1, a unique actin-binding motif present in most Shroom proteins characterized to date. Shroom3 function is also dependent on Rho-kinase (Rock), such that Shroom3 directly binds to Rock and regulates both its localization and catalytic activity. The interaction between Shroom and Rock has been elucidated at the molecular level and is mediated by the conserved SD2 region of Shroom and a conserved coiled-coil region of Rock. The interaction between Shroom and Rock results in the localized activation of non-muscle myosin II (myosin II) contractility, which provides the mechanical force needed to facilitate neural tube morphogenesis. The regulation of myosin II activity by Rock and other cellular pathways has been well described. Rock modulates myosin II activity in two ways. First, Rock can directly phosphorylate the associated regulatory light chain (RLC), which modulates the actin-associated ATPase activity and the conformation of myosin II. Secondly, Rock negatively regulates the phosphatase that dephosphorylates the RLC, thus preventing the inactivation of myosin II (Hildebrand, 2021 and references therein).

Shroom proteins are required for numerous biological processes and are associated with several human diseases. In mammals, there are three definitive Shroom proteins, Shroom2, Shroom3, and Shroom4, each of which contains an N-terminal PDZ domain, the centrally located SD1, and the C-terminally located SD2. All three proteins can directly interact with F-actin and regulate cell morphology via Rock. In humans, SHROOM2 has been linked to neural tube morphogenesis, colorectal cancer, and medulloblastoma, while in vitro studies indicate it is important for cell migration, vasculogenesis, metastasis, and melanosome biogenesis. SHROOM3 mutations have been implicated in chronic kidney disease, heart morphogenesis, and neural tube closure in humans. Using model organisms or cell culture, Shroom3 has been shown to control neural tube closure, axon growth, intestine architecture, eye morphogenesis, thyroid budding, and kidney development. Finally, SHROOM4 mutations have been associated with X-linked mental defects (Hildebrand, 2021).

The Shroom gene is conserved in Drosophila and encodes multiple protein isoforms that have different subcellular distributions and activities in vivo. The most highly conserved region of Drosophila Shroom is the SD2, the region that binds to Drosophila Rho-kinase (Rok). Drosophila Shroom also contains a divergent SD1 motif and this appears to mediate localization to adherens junctions in polarized epithelia. Consistent with the known activities of mammalian Shroom3, expression of Drosophila Shroom in epithelial cells induces apical constriction in a Rok and myosin II dependent manner. While Shroom3 is essential for mouse and human development, Shroom is not absolutely essential for Drosophila viability, as Shroom null flies can be recovered, albeit with significantly reduced frequency. In Drosophila embryos, Shroom is planarly distributed and works in a complicated network with RhoA, Rok, and myosin II to control convergent extension movements. These elegant studies showing the role of Shroom in regulating directional contractility are supported by observations that Shroom proteins can be polarly distributed in mammalian tissues and cells (Hildebrand, 2021 and references therein).

To better understand the mechanisms that control Shroom-regulated changes in cell and tissue morphology, this study has established tools to perform genetic screens for modifiers of Shroom activity in Drosophila. Shroom gain-of-function phenotypes in the eye and wing can be suppressed or enhanced by known components of the Shroom pathway. Using a candidate approach, several cytoskeletal regulators were identified, including Short stop and Enabled, as participants in Shroom-mediated changes in cell morphology. Shroom regulates the distribution of Ena and this is likely mediated by conserved proline-rich sequences in Shroom and the EVH1 domain of Ena. This study further shows that while Ena is required for the Shroom gain-of-function phenotypes, apical recruitment of Ena is not sufficient to cause changes in cell morphology. Additionally, by using an isoform of Shroom that does not bind Ena, but still engages Rok, this study showed that apical constriction can be modulated by different cellular pathways depending on the context (Hildebrand, 2021).

This study describes a genetic approach to identify cellular pathways that participate in tissue morphogenesis. This method takes advantage of the observation that ectopic Shroom protein can utilize the endogenous contractile machinery within epithelial cells to induce apical constriction and disrupt normal tissue morphology. While this work focuses on candidate genes that encode known regulators of epithelial and tissue architecture, it is predicted these tools can be used to perform unbiased, genome-wide screens to identify novel participants in Shroom-mediated cellular processes. Two different tissues, eye and wing imaginal discs, were used for these studies, and these screens can identify factors that are used in a wide range of tissues and cells to control cell dynamics. This is based on the observations that ShroomA, the isoform most similar to mammalian Shroom3, induces similar cellular phenotypes in both types of imaginal discs, and the phenotypes can be modified in both tissues. A powerful aspect of this screen is that these processes are functionally conserved in vertebrate cells and tissues. Additionally, the simplified nature of the Drosophila genome makes these screens possible. Due to genetic and functional redundancy, it is predicted that the analysis performed in this study would be more complicated using vertebrate or cell culture model systems. Drosophila have single genes for Shroom, Rok, myosin II, and Ena while mammals possess gene families for these factors. In support of this, previous work has shown that both Rock1 and Rock2 must be inhibited to prevent Shroom3-mediated apical constriction in cell culture. This screening approach should allow for the identification of novel genetic interactions in Drosophila that can be further verified in mammalian model systems to define their potential role in human disease (Hildebrand, 2021).

Most of the modifiers identified in this study participate in defining actin or microtubule architecture. Of these, several regulate actin dynamics at the level of polymerization or stability, including Ena, Diaphanous, Chickadee, and Slingshot. Interestingly, three of these proteins can be linked, directly or indirectly, to neural tube formation in mice. It should be noted that several classes of actin regulators did not appear to modify the Shroom phenotypes, including nucleators, binding proteins, or adaptors, suggesting that specific types of actin organization are required for Shroom-induced perturbation of cell architecture. This is further supported by the observation that Tropomyosin was also identified in the screen. Tropomyosin regulates the structure of actin filaments and the binding of other proteins, including myosin II and cofilin, that in turn modulate cell architecture or behavior. It is particularly intriguing to note that Tropomyosin mutations can suppress phenotypes caused by the loss of Flapwing, presumably caused by increased myosin II activity. In addition to the actin cytoskeleton, these studies also support a role for microtubules in Shroom-induced phenotypes. This is consistent with the role of microtubules in apical constriction in Drosophila. Recent evidence indicates that apical-medial microtubules play an important role in ventral furrow invagination and this is mediated by Patronin, a protein known to interact with Shot. These studies show that microtubules stabilize the connection of contractile networks to cell junctions to facilitate tissue morphogenesis. These studies are consistent with the current results in relation to Shroom function and Shot distribution in the wing epithelium. It will be interesting to determine if the identified proteins act upstream or downstream of Shroom. While the data suggest Ena acts downstream of Shroom, proteins such as Tropomyosin could function upstream by regulating the amount of Shroom that can bind to F-actin or downstream by modulating the amount of myosin II that can be recruited or activated by the Shroom-Rok complex. It was surprising that determinants of cell adhesion or polarity, such as cadherins or Par complex proteins, were not identified in this screen. It is possible that these proteins are present in sufficient quantity and reducing the dosage is unable to modify the Shroom overexpression phenotype and thus other genetic approaches will be needed to assess the role of these pathways (Hildebrand, 2021).

The data show that endogenous Shroom protein is expressed in epithelial cells during wing and eye development, suggesting it functions in these tissues under normal circumstances. Shroom null flies that survive to adults do not exhibit significant defects in the eyes or wings, although null embryos do exhibit defects in convergent extension and perhaps this could contribute to the observed reduction in viability. In embryos, Shroom is important for the polarized distribution of contractile myosin II needed for convergent extension. It is possible that Shroom activity in disc epithelial cells is redundant to other pathways that regulate Rok and myosin II and Shroom normally functions to make these pathways more robust or function with higher fidelity. Uncovering these subtle interactions will require additional genetic approaches. The localization of Shroom in the eye and wing disc appears to be highly regulated and is reminiscent of that exhibited by myosin II and phosphorylated Sqh, particularly in the eye imaginal disc. A dramatic increase was observed in Shroom protein in cells that are exiting the morphogenetic furrow and forming the pre-clusters that will give rise to the ommatidia. As the ommatidia form, Shroom expression becomes restricted to the R3/4 cells and eventually is lost from these cells. This distribution is essentially the inverse to that of E-cadherin, which is highest in the radial junctions and lower in the circumferential junctions. This could reflect differences in adhesive interactions between the ommatidia pre-clusters and the inter-ommatidia cells, which facilitates rotation of the ommatidia. This hypothesis is supported by previous studies demonstrating that differential adhesion generates specific cellular organization and compartmentalization in the developing eye. Interestingly, the PCP protein Flamingo is also expressed in R3 and R4 and previous studies have identified interactions between the Shroom3 and PCP pathways in the neural tube. As eye development continues, this study observed Shroom expression in the pigment cells of the pupal retina. In both the imaginal disc and the retina, Shroom distribution is restricted to specific cell junctions, suggesting there are differential adhesive or contractile forces associated with these membranes (Hildebrand, 2021).

In the wing imaginal disc, expression of Shroom protein was observed in rows of cells that border the anterior half of the wing margin. Consistent with the genetic interactions, a similar expression pattern was observed for both Ena and Shot in these cells. It is currently unclear if the co-expression of Shroom, Ena, and Shot is controlled pre- or post-transcriptionally. It is possible that the expression of Shroom, Ena, and Shot is coordinately regulated in a gene network. Alternatively, the stability or apical localization of these proteins may be interdependent or closely orchestrated. This expression pattern in the anterior wing margin is similar to members of the Irre cell Recognition Module (IRM), including cell surface receptors Roughest, Hibris, and Kirre, which help position the sensory organs. This is particularly interesting in light of the fact that the vertebrate orthologs of these genes, Neph and Nephrin-1, and Shroom3 are all involved in formation of podocytes in the glomerulus of the mammalian kidney. It will be exciting to apply genetic analysis to investigate if these pathways cooperate to regulate tissue morphology (Hildebrand, 2021).

Ena and Shroom show extensive co-expression and colocalization in both the wing and eye imaginal disc, although Ena is more widely expressed than Shroom. In both the wing and eye imaginal disc, Ena is expressed in most cells and is localized primarily in the tricellular junctions with lower expression in the adherens junctions. However, as seen in the wing margin and the morphogenetic furrow, cells that express Shroom protein also exhibit high levels of Ena in the cell junctions. Importantly, reducing the amount of Shroom protein perturbs the localization of Ena in the anterior wing margin. The relationship between Ena, Shroom, Rok, and myosin II in defining cell shape is likely to be complicated. This stems from the observations that these factors could be placed both upstream and downstream of Shroom. For example, it has been previously shown that Shroom distribution to the apical adherens junctions is mediated, at least in part, by direct binding to F-actin. However, it has also been established that RhoA and Rok regulate F-actin architecture to influence Shroom distribution, which then facilitates the polarized distribution of Rok and myosin II. Ena has been shown to have multiple roles in Drosophila development, including axon guidance, collective cell migration, and epithelial morphogenesis. The role Ena plays in Shroom-mediated apical constriction is unclear. The current data suggest that Ena functions downstream of Shroom and is recruited to adherens junctions via an LPPPP-EVH1 interaction. Ena is primarily defined as a modulator of F-actin dynamics that facilitates the formation of long filaments by competing with barbed-end capping and promoting the addition of actin monomers to the barbed end. This activity may be important for providing the substrate for activated myosin II to drive cell contraction. This is consistent with studies in vertebrate cells showing that Diaphanous 1, is also required for contractility in adherens junctions and that this study has also identified Dia as a potential modifier of Shroom activity (Hildebrand, 2021).

Elegant studies from several groups have identified many other signaling pathways that control the distribution of contractile myosin II networks during Drosophila development, including the Fog, PCP, HH, Dpp, EGF, Toll, and integrin signaling pathway. How all these signaling pathways are orchestrated and converge on myosin II at the cellular and tissue level is a fascinating question. It has been shown that the above processes use a variety of methods to regulate the small GTPase RhoA, which activates Rok, including several GTP exchange factors or GTPase Activating Proteins. It should be noted that other GTPases such as Rap1 or CDC42 also regulate apical constriction. This work has shown that Shroom3 may activate Rock independent of RhoA, suggesting that there as mechanisms to bypass small GTPases in the activation of myosin II. It will be informative to utilize this screening approach to further test how these pathways might work with ShroomA to control cell morphology (Hildebrand, 2021).

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, the localization was analyzed of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). The composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. The data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. An explanation is provided for the observed heterogeneity and it is suggested that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways (Dobramysl, 2021).

The regulation of actin polymerization is crucial for numerous cell functions, including cell migration, adhesion, and epithelial closure and is often disrupted in disease, such as cancer metastasis and intracellular infection by pathogens. Micron-scale actin superstructures and their associated regulators form transient membrane-bound complexes that orchestrate large-scale cytoskeletal remodeling and provide the mechanical infrastructure for the cell. One of the best examples is filopodia, with their characteristic membrane-associated 'tip complex' where new actin monomers are incorporated, leading to rapid extension of the filopodia from the cell surface. The tip complex contains many components, including formins such as diaphanous-related formin 3 (Diaph3), barbed-end polymerases Enabled (Ena), vasodilator-stimulated phosphoprotein (VASP), actin bundling proteins including Fascin, and the molecular motor myosin X. There are currently three main models for filopodia formation, each identifying specific tip complex proteins as the key players: (1) formins mediating de novo actin nucleation; (2) a preexisting actin network generated by the Arp2/3 complex becoming bundled by Fascin; and (3) membrane-bound adaptor proteins recruiting Ena/VASP, which could coexist with either formin or Arp2/3 complex-based mechanisms. One way to reconcile these models is to postulate the existence of subtypes of filopodia on the basis of their mechanism of formation. What is not yet clear is whether the subtypes reflect differences between cell types or coexist in the same cell and whether they impart particular properties to the growing filopodia. This question was recently examined by measuring whether the amount of Ena and VASP at the tip complex correlated with the protrusion velocity of filopodia, using cultured Xenopus retinal ganglion cells. A correlation was observed in only a subset of filopodia, suggesting that the accumulation of Ena/VASP proteins is not essential and there are diverse molecular mechanisms that lead to filopodial elongation (Dobramysl, 2021).

This study comprehensively analyzed the role of heterogeneity in the filopodial tip complex. By measuring endogenously tagged actin regulators in Drosophila, similar heterogeneity to exogenous expression in Xenopus retinal ganglion cells was confirmed. This study found that a cell-free system of filopodia-like structures (FLSs) is characterized by similar heterogeneities, and it allowed making of large-scale combinatorial measurements of the correlations of actin regulators with each other and with the morphology of the actin bundle. The emergence of FLSs and their resulting lengths are remarkably insensitive to the presence or absence of any individual tip complex protein. By measuring the momentary rates of growth and shrinkage of the actin bundle and incorporating theoretical modeling, a simple theory was identified that suggests a mechanistic role for tip complex heterogeneity, and its predictions were tested in vitro and in vivo. This work explains how diverse combinations of tip complex proteins give rise to filopodia (Dobramysl, 2021).

This study found that actin regulatory proteins form heterogeneous semidynamic assemblies on membranes composed of at least three or four different subcomplexes where actin bundles nucleate and grow. The resulting actin bundles grow and shrink with velocities that fall on a Laplace distribution, which results in exponentially distributed filopodial lengths. Using the mathematics governing probability distributions, it was possible to link the observed velocity distributions to pairs of fluctuating actin regulators. The subcomplexes are reminiscent of proteins and interactions that were previously thought to be important in filopodium formation. Cdc42-GTP was most highly correlated with VASP and N-WASP; Ena and VASP correlated with each other, and Diaph3, previously implicated in de novo filopodia nucleation, correlated with membrane-adaptor protein TOCA-1, although not with Cdc42-GTP. However, with the complex composition of the extracts and multiple interaction partners for all the proteins involved, it is not yet concluded that no correlation means no relevant interaction (Dobramysl, 2021).

Previous theoretical work considered actin filament length distributions resulting from monomer addition-removal processes together with fragmentation driven by gelsolin and how length control can emerge from other properties of cytoskeletal regulation (such as limited monomer availability, active transport of monomers, capping protein, and formin inhibitors). In contrast, long-tailed exponential length distributions were observed both in vitro and in vivo, suggesting that stochastic processes are governing filopodial dynamics. The primary result shows that FLS and filopodial growth velocities follow a Laplace distribution. These observations are not compatible with simple monomer addition/removal processes, yet still point to a simple emergent dynamic arising from molecular complexity. The fluctuations of components on which the theory depends may originate from many different biochemical possibilities. For example, ubiquitination cycles of VASP have been observed to alter its dynamics within the tip complex, together with filopodial properties, downstream of netrin-1 signaling. Other possible molecular candidates include phosphorylation cycles, GTP/GDP exchanges, or specific protein-protein interactions (Dobramysl, 2021).

The heterogeneity reported resembles observations made for clathrin-mediated endocytosis in mammalian cells and components of the adhesome present in filopodia, suggesting that a similar mechanism based on a heterogeneity of multiple players is a more general feature of cell regulation. The redundancy in molecular composition allows a robustness and may also allow a variety of upstream and downstream components to intersect with the control of filopodia and co-opt them in diverse biological contexts. A multicomponent system could also ensure that signals regulating filopodia must be multiple and coincident, as only rarely will a single input be sufficient to cause an effect, and it takes an overexpression scenario to subvert the normal homeostatic mechanisms, such as Fascin in cancer. In FLSs, the membrane interactions together with SH3 domain and proline-rich regions in Ena, N-WASP, VASP, and Diaph3 are similar to observations with N-WASP and Nck in purified systems that have phase separation properties. It may be that a Laplace-distributed output and the harnessing of fluctuations is the reason for such organization. This study shows that in spite of a dynamic and heterogeneous tip complex, a constraint emerges in the resulting activity, which may be what allows actin machinery to be co-opted in a stereotypical manner, accommodating different tissue regulatory programs without any alteration to its underlying functional properties (Dobramysl, 2021).

The branching code: A model of actin-driven dendrite arborization

The cytoskeleton is crucial for defining neuronal-type-specific dendrite morphologies. To explore how the complex interplay of actin-modulatory proteins (AMPs) can define neuronal types in vivo, this study focused on the class III dendritic arborization (c3da) neuron of Drosophila larvae. Using computational modeling, the main branches (MBs) of c3da neurons were demonstrated to follow general models based on optimal wiring principles, while the actin-enriched short terminal branches (STBs) require an additional growth program. To clarify the cellular mechanisms that define this second step, this study concentrated on STBs for an in-depth quantitative description of dendrite morphology and dynamics. Applying these methods systematically to mutants of six known and novel AMPs (Arp2/3, Capu, Ena, Singed, and Twinstar), the complementary roles were revealed of these individual AMPs in defining STB properties. These data suggest that diverse dendrite arbors result from a combination of optimal-wiring-related growth and individualized growth programs that are neuron-type specific (Sturner, 2022).

Neurons develop their dendrites in tight relation to their connection and computation requirements. Thus, dendrite morphologies display sophisticated type-specific patterns. From the cell biological and developmental perspective, this raises the question of at which level different neuronal types might use shared mechanisms to assemble their dendrites. And, conversely, how are specialized structures achieved in different neuronal types? To start addressing these question computational and comparative cell biological approaches were combined. It was found that two distinct growth programs are required to achieve models that faithfully reproduce the dendrite organization of c3da neurons. The models single out the STBs that are also molecularly identifiable as unique structures, displaying specific localization of actin and Singed. By combining time-lapse in vivo imaging and genetic analyses, this study sheds light on the machinery that controls the dynamic formation of those branchlets (Sturner, 2022).

The complex interplay of AMPs generates highly adaptive actin networks. In fact, in contrast to earlier unifying models, it is now clear that even the same cell can make more than one type of filopodium-like structure. This study characterized the effect of the loss of six AMPs on the morphology and dynamics of one specific type of dendritic branchlet, the STB of c3da neurons. With this information, a molecular model for branchlet dynamics in vivo is delineated in the developing animal. Similar approaches to model the molecular regulation of actin in dendrite filopodia have been taken recently for cultured neurons. The advantage of the present approach is that it relies directly on the effect of the loss of individual AMPs in vivo, preserving the morphology, dynamics, and adhesive properties of the branchlets, and non-cell-autonomous signals remain present (Sturner, 2022).

The combination of FRAP experiments and the localization of Singed/Fascin on the extending STBs indicated that actin is organized in a tight bundle of mostly uniparallel fibers in the STBs. This organization is thus very different from that of dendritic filopodia of hippocampal neurons in culture. The actin filaments in the bundle appear to be particularly stable in the c3da-neuron STBs, as the actin turnover that this study revealed by FRAP analysis was 4 times slower than that reported in dendrite spines of hippocampal neurons in vitro and 20-fold slower than in a lamellipodium of melanoma cells in vitro. It is nonetheless in line with previous data on stable c3da-neuron STBs and with bundled actin filaments of stress fibers of human osteosarcoma cells. Treadmilling was observed, similar to that of filopodia at the leading edge, with a retrograde flow rate 30 times slower than in filopodia of hippocampal cells and comparable to rates observed for developing neurons in culture lacking the mammalian homologues of Twinstar and actin-depolymerization factor (ADF)/Cofilin. Slower actin kinetics could be related to the fact that neurons differentiating in the complex 3D context of a developing animal are being imaged. Recent quantification of actin treadmilling in a growth cone of hippocampal neurons in 3D culture, however, did not produce differences with 2D-culture models(Sturner, 2022).

The alterations of MB and STB morphology and dynamics caused by the loss of individual AMP functions reported in this study can now be combined with preceding molecular knowledge about these conserved factors to produce a hypothetical model of the actin regulation underlying STB dynamics. Dendrite structure and time-lapse imaging point to an essential role of Twinstar/Cofilin for the initiation of a branchlet, in agreement with previous literature. Drosophila Twinstar/Cofilin is a member of the ADF/Cofilin protein family, with the capacity of severing actin filaments but with poor actin-filament-depolymerizing activity. It is thus proposed that Twinstar/Cofilin localized at the base of c3da STBs can induce a local fragmentation of actin filaments that can then be used as substrate by the Arp2/3 complex. In fact, in c4da neurons, Arp2/3 localizes transiently at the site where the branchlets will be formed, and its presence strongly correlates with the initiation of branchlet formation. Previous and present time-lapse data point to the role of Arp2/3 in the early phases of branchlet formation. Thus, it is suggested that localized activity of Arp2/3 generates a first localized membrane protrusion (Sturner, 2022).

Given the transitory localization of Arp2/3, this study interrogated the role of additional actin nucleators in this context. From an RNAi-supported investigation, Capu was identified as potential modifier of c3da STBs. Capu displays complex interactions with the actin-nucleator Spire during oogenesis, involving cooperative and independent functions of these two molecules. An increase in Spire levels correlates with a smaller dendritic tree and inappropriate, F-actin-rich, and shorter dendrites in c4da neurons. In this study, though, the loss of Spire function did not yield a detectable phenotype in c4da neurons. In c3da neurons, it was found that Capu and Spire support the formation of new branchlets and display a strong genetic interaction in the control of the number and length of MBs and STBs and surface area. Thus it is suggested that they cooperatively take over the nucleation of linear actin filaments possibly producing the bundle of uniparallel actin filaments. Mutants for capu showed changes in the positioning of dendritic branches, not observed in spire mutants, which could mean that Capu localization defines the sites of Capu/Spire activity. However, Spire seems to promote branch dynamics, suggesting additional independent functions of Spire possibly not related to nucleation, given that Spire itself is a weak actin nucleator. While there is no clear indication in vivo for the molecular mechanisms supporting this function, an actin-severing activity of Spire was reported in vitro. The role of Spire on STB dynamics appears to be consistent with favoring actin destabilization or actin dynamics (Sturner, 2022).

Singed/Fascin bundles actin filaments specifically in the c3da neuron STBs and gives these branches their straight conformation. The localization of Singed/Fascin in the c3da STBs correlates with their elongation. While the complete loss of singed function suppressed dynamics, the mild reduction in protein levels analyzed in this study led to more frequent STB elongations and retractions. Further, the branchlets extended at the wrong angles and displayed a tortuous path. Singed/Fascin controls the interaction of actin-filament bundles with Twinstar/Cofilin and can enhance Ena binding to barbed ends. Thus, in addition to generating mechanically rigid bundles, it can modulate actin dynamics by regulating the interaction of multiple AMPs with actin. It is speculated that the retraction and disappearance of the STB could be due to Singed/Fascin dissociating from the actin filaments, possibly in combination with Spire and Twinstar/Cofilin additionally severing actin filaments. In fact, the presence of detectable Twinstar/Cofilin along the c3da STBs was recently reported (Sturner, 2022).

Ena is important for restricting STB length, and it inhibits the new formation and extension of STBs. This appears to be a surprising function for Ena that is in contrast to its role in promoting actin-filament elongation or to its capacity of supporting the activation of the WAVE regulatory complex. Similar to what was previously reported for ena-mutant c4da neurons, a balance between elongation and branching was also observed in c3da neurons. In Drosophila macrophages, Ena was shown to associate with Singed/Fascin within lamellipodia. In line with these recent data, it is suggested that Ena might closely cooperate with Singed to form tight actin bundles that slow down STB elongation (Sturner, 2022).

Taken together, a comprehensive molecular model of dendrite-branch dynamics for the STBs of c3da neurons was put forward. In this analysis, the role of extracellular signals on the regulation of the dynamics of STBs was excluded, for simplicity. Nonetheless, such signals are likely to have a profound effect, particularly on the regulation of elongation and stabilization of STBs in relation to their target substrate. In addition, similar to what has been suggested for c1da neurons, the distribution of MBs in the target area might follow guidance cues that were not included in the analysis, such as permissive signals that specifically guide c4da neurons to tile the body wall or promote appropriate space filling (Sturner, 2022).

The investigation of morphological parameters in combination with genetic analysis has proven extremely powerful to reveal initial molecular mechanisms of dendrite differentiation. Early studies, though, have been limited in the description power of their analysis concentrating on just one or two parameters (e.g., number of termini and total dendrite length). This limitation has been recognized and addressed in more recent studies (Sturner, 2022).

A major outcome of the present and previous work is the establishment of powerful tools for a thorough and comparative quantitative morphological analysis of different mutant groups. A detailed tracing of neuronal dendrites of the entire dendritic tree or a certain area of the tree in a time series with a subsequent automatic analysis allows a precise description of mutant phenotypes. This study additionally generated tools for extracting quantitative parameters of the dynamic behavior of dendrite branches from time-lapse movies based on a novel branch registration software. This time-lapse tool yields an automated quantification after registration detecting branch types and their dynamics. Moreover, the tool operates in the same framework as the tracing and morphological analysis. These tools available within the TREES toolbox, and their use to support comparative analysis among datasets is encouraged (Sturner, 2022).

What are the fundamental principles that define dendrite elaboration and which constraints need to be respected by neurons in establishing their complex arbors? Models based on local or global rules have been applied to reproduce the overall organization of dendritic trees, including da neurons. The c3da model is based on the fundamental organizing principle that dendrites are built through minimizing cable length and signal conduction times. This general rule for optimal wiring predicts tight scaling relationships between fundamental branching statistics, such as the number of branches, the total length, and the dendrite's spanning field (Sturner, 2022).

This study found that c3da neurons respect the general developmental SFGT or MST models when stripped of all their STBs. However, the characteristic STBs of c3da dendrites did not follow this scaling behavior. Instead, a second growth program had to be applied to add the STBs to this basic structure, respecting their number, total length, and distribution. The two-step model developed in this work suggests that while main dendritic trees have common growth rules, the dendritic specializations of different neuronal cell types do not necessarily have the same constraints. This view is compatible with findings in a companion paper showing, in c1da neurons, a specialized branch-retraction step following an initial growth step. In the two-step c3da dendrite model, the resulting synthetic morphologies resemble the real dendritic trees including those of five out of the six AMP mutant dendritic trees without any changes to the model parameters. The two-step model uses, for example, the reduced total length and reduced surface area of mutants for singed and twinstar and grows synthetic trees that have the same distribution of branch lengths and amounts as expected for those mutants. The synthetic trees corresponding to the twinstar mutant have less STBs than any other AMP mutant synthetic tree, consistent with the real mutant phenotypes (Sturner, 2022).

This work indicates that a combination of thorough statistical analysis (such as using the presented morphometrics) and models, like the one developed in this study, can help capture the fundamental principles that govern dendrite differentiation. Together with genetics analysis and systematic cell biology approaches, this type of study can deliver quantitative predictions for molecular models of dendrite elaboration (Sturner, 2022).

In conclusion, this study has put forward the hypothesis that neuronal dendrites are built based on common, shared growth programs. An additional refinement step is then added to this scaffold, allowing each neuron type to specialize based on its distinctive needs in terms of number and distribution of inputs. In the exemplary case of c3da neurons, this study investigated molecular properties of these more-specialized growth programs and proposed a first comprehensive model of actin regulation that explains the morphology and dynamics of branchlets (Sturner, 2022).

Most of the AMPs studied are essential, and all perform multiple functions during the course of development. Clearly, in these experiments, the acute function of each AMP in the process of STB formation and during STB dynamics has not been isolated. Rather, the progressive reduction of functional protein in MARCM clones or during the development of homozygous animals might represent a confounding factor. Future studies will be aimed at using and developing tools for acute protein-function inactivation in vivo to add to the toolbox (Sturner, 2022).

Earlier Summaries

Enabled is cytoskeletal regulator that facilitates continued actin polymerization at the barbed ends of actin filaments, induces cellular projections when overexpressed, and functions together with several different receptors (including Robo and UNC-40/DCC) implicated in axon guidance. This overview of Enabled starts with ABL, one of a number of mammalian cancer causing proteins (oncoproteins). ABL is a highly conserved nonreceptor tyrosine kinase containing SH3 and SH2 protein interaction domains. In less technical terms, ABL functions to modify other proteins by phosphorylation, and it attaches to other proteins by means of SH3 and SH2, two different protein interaction domains. One target of Drosophila ABL is Enabled, a protein that binds to ABL and other oncoproteins; Ena possess the Src homology 3 (SH3) protein interaction domain. SH2 and SH3 domains are found in many "adaptor" proteins that bind to other proteins in the progress of carrying out their function.

Recently a breakthrough in understanding Enabled and Abl has come from the cloning of a Mouse homolog of Enabled (Mena). Both Enabled and Mena are relatives of VASP (Vasodilator-Stimulated Phosphoprotein). A conserved domain of Mena targets it to proteins containing a specific proline-rich motif. The association of Mena with the surface of the intracellular pathogen Listeria monocytogenes and the G-actin binding protein Profilin suggests that Mena may participate in bacterial movement by facilitating actin polymerization. That is, Enabled and Mena, and indirectly ABL, serve to modify the actin based cytoplasm, an important agent in maintaining cell shape, regulating cell migration, and many other cell functions. In Drosophila, ABL and ENA act in the developmental processes of axon pathfinding and eye morphogenesis.

First the relationship of ABL to cancer will be examined because of the insight this provides to an understanding of ABL and Enabled function. Derivatives of ABL protein tyrosine kinase are involved in Philadelphia chromosome positive chronic myelogenous leukemia and acute lymphocytic leukemia in humans, and the pre-B-cell leukemia caused by Abelson murine leukemia virus in mice (from which ABL derives its name). The Philadelphia chromosome is generate by fusion between two normal human chromosomes. These translocations are not found at random, but in the middle of two genes, Abl and Bcr (named after the breakpoint cluster region). The product of this gene fusion is BCR-ABL protein, a hybrid protein able to confer a leukemia phenotype on cells in which it is expressed (Gertler, 1995 and references).

BCR is a multifunctional protein with a pseudo Kinase domain (apparently non-functional), a CDC24 domain and a GAP domain. CDC24 is a yeast gene that when mutated results in defective bud formation. It is believed that CDC24 plays a critical role in the establishment of cell polarity, development of normal cell shape, localization of secretion, and cell-surface deposition. The GAP (GTPase-activating protein) function targets p21 RAC, an important signaling protein in determining and modifying cell shape. Not only does BCR form a hybrid protein with ABL in the Philadelphia translocation, but BCR itself serves to target ABL, resulting in ABL activation (Arlinghaus, 1992 and references). When occuring on a composite protein, the interaction of BCR with ABL causes cell transformation. The chimeric protein products of these oncogenes exhibit elevated tyrosine kinase activity, oligomerization and association with the actin cytoskeleton, tyrosine phosphorylation of BCR sequences, association with signal transducing proteins GRB2, SHC, CBL, SYP and members of the 14-3-3 protein family (Gertler, 1995 and references).

The discovery that the bacterium Listeria recruits the cellular protein VASP, generated great intrigue in the scientific community. Using VASP, Listeria co-opts the cell's cytoskeleton to transport itself within the host cell it infects. VASP is a ligand for profilin, an actin-monomer binding protein that can stimulate the formation of filamentous actin, the non-muscle form of actin that serves as the basis for the actin based cytoskeleton of the cell. Listeria motility results from the rapid polymerization of F-actin at one pole of the bacterium, a process enhanced by host profilin. The properties of VASP make it a candidate host factor that can mediate the recruitment of profilin to the surface of Listeria, and thus promote F-actin assembly. Mena and Evl are two murine proteins highly related to Enabled as well as to VASP. A conserved domain in the N terminal portion of Mena and ENA targets these proteins to proline rich regions of other proteins. VASP interacts with zyxin, a LIM-domain protein localized in focal adhesions that shares a proline-rich motif with vinculin and ActA, two other cytoskeletal proteins. The proline rich region in VASP, which is shared with ENA and Mena, is most likely the profilin-binding site shared by these proteins. Mena itself has been shown to bind to the actin-binding protein Profilin. Localization of Mena to focal adhesions is mediated by its conserved N-terminus, and Mena, like VASP is recruited to the surface of Listera. Mena produces multiple isotypes. One is widely expressed, and a second is enriched in, or specific to, neural cells types. Thus Mena lends some understanding to the possible role of Drosophila ENA in axon pathfinding. (Gertler, 1996).

The well studied migration of neuronal growth cones serves as a paradigm for the actin-driven formation of membrane protrusions (Forscher, 1992). Establishment of proper connections in the central nervous system depends on the ability of neuronal growth cones to guide neurites to their final targets. The ABL-ENA-Profilin pathway is implicated in the process of axonal outgrowth and fasciculation. The mechanism by which the growth cone advances is based on dynamic rearrangement of the actin based cytoskeleton, and it is in this process that Enabled and ABL affect axonogenesis.

Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identify alleles of only one gene, enabled. The ENA protein contains proline-rich motifs and binds to ABL and Src SH3 domains. ENA is also a substrate for the Abl kinase. Tyrosine phosphorylation of ENA is increased when it is coexpressed in cells with human or Drosophila Abl, and endogenous ENA tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos have defects in axonal architecture. Therefore, it has been concluded that a critical function of Drosophila ABL is to phosphorylate and negatively regulate ENA protein during neural development (Gertler, 1995).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 684

Structural Domains

The most notable feature of the predicted ENA protein is a proline-rich core, with 58 proline residues located between amino acids 340-480 and seven matches to the proline-rich consensus site for binding the ABl SH3 domain (Gertler, 1995).

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. E/DFPPPPXD/E, a motif repeated four times within the primary sequence of ActA, is identified as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling (Niebuhr, 1997).

The Enabled/VASP homology 1 (EVH1; also called WH1) domain is an interaction module found in several proteins implicated in actin-based cell motility. EVH1 domains bind the consensus proline-rich motif FPPPP and are required for targeting the actin assembly machinery to sites of cytoskeletal remodeling. The crystal structure of the mammalian Enabled (Mena) EVH1 domain complexed with a peptide ligand reveals a mechanism of recognition distinct from that used by other proline-binding modules. The EVH1 domain fold is unexpectedly similar to that of the pleckstrin homology domain, a membrane localization module. This finding demonstrates the functional plasticity of the pleckstrin homology fold as a binding scaffold and suggests that membrane association may play an auxiliary role in EVH1 targeting (Prehoda, 1999).

date revised: 25 August 2023 

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