Rho1
The steroid hormone 20-hydroxyecdysone (ecdysone) is the key regulator of postembryonic developmental transitions in insects and controls metamorphosis by triggering the morphogenesis of adult tissues from larvae. The Rho GTPase, which mediates cell shape change and migration, is also an essential regulator of tissue morphogenesis during development. Rho activity can modulate gene expression, in part, by activating LIM kinase (LIMK) and consequently affecting actin-induced SRF transcriptional activity. A link has been established between Rho-LIMK-SRF signaling and the ecdysone-induced transcriptional response during Drosophila development. Specifically, Rho GTPase, via LIMK, regulates the expression of several ecdysone-responsive genes, including those encoding the ecdysone receptor itself, a downstream transcription factor (Br-C), and Stubble, a transmembrane protease required for proper leg formation. Stubble and Br-C mutants exhibit strong genetic interactions with several Rho pathway components in the formation of adult structures, but not with Rac or Cdc42. In cultured SL2 cells, inhibition of Rho, F-actin assembly, or SRF blocks the transcriptional response to ecdysone. Together, these findings indicate a link between Rho-LIMK signaling and steroid hormone-induced gene expression in the context of metamorphosis and thereby establish a novel role for the Rho GTPase in development (Chen, 2004).
Metamorphosis in Drosophila is stringently controlled by pulses of the steroid hormone ecdysone at discrete developmental stages. During larval-pupal transition, ecdysone triggers coordinated changes in tissue morphology that involve histolysis of larval tissues and the initiation of adult structures. Rho GTPase-mediated signaling pathways have been implicated in several aspects of morphogenesis during Drosophila embryo formation. However, a role for Rho signaling in metamorphosis has not yet been reported. Among the downstream mediators of Rho signaling are the LIM kinases, and a closely related Drosophila ortholog of mammalian LIM kinases (designated Dlimk) is specifically expressed at relatively high levels in late larval and pupal stages, suggesting a potential role in Rho-LIMK signaling during this transition. In adult flies, Dlimk is expressed at substantially higher levels in males than in females, consistent with a potential evolutionarily conserved role in spermatogenesis, a process in which mammalian LIMK2 has been implicated. Dlimk mRNA is uniformly expressed throughout eye, wing, and leg imaginal discs (Chen, 2004).
The GAL4/UAS transgene system was used to examine Dlimk function in vivo. Overexpression of Dlimk in the imaginal wing disc via several different wing-specific GAL4 drivers causes notched wings, missing wing veins, vein fusion, and blistered wings. The notched wing phenotype appears to reflect an increase in apoptosis and is rescued by the p35 viral caspase inhibitor. In addition, wings exhibit enlarged cells (indicated by low wing hair density) and alterations in the number and polarity of wing hairs. Notably, similar defects in wing hair number and polarity are also seen in rho1 and Drok (the Rho effector kinase that activates LIMK) mutants, suggesting that Dlimk functions in the same pathway. Mammalian LIMKs promote actin assembly in cultured cells, and prominent F-actin accumulation and aberrant actin organization are observed in the wing discs of transgenic flies specifically overexpressing Dlimk. Thus, Dlimk can regulate actin assembly in developing tissues (Chen, 2004).
To verify that Dlimk normally regulates morphogenesis during the larval-pupal transition, a kinase-deficient form of Dlimk (DlimkD522A) was used as a dominant-negative protein. An analogous mutation in mammalian LIMK1 gives rise to a protein that specifically interferes with LIMK function in cultured cells. Use of a T80-GAL4/UAS-DlimkD522A transgenic line to express DlimkD522A during development results in viable and fertile animals, with approximately 85% of adults exhibiting malformed wings and legs, consistent with a normal requirement for Dlimk in proper disc morphogenesis. In wild-type adult legs, the femur and tibia are elongated and slender structures; however, in DlimkD522A mutant flies, the femur is bent and twisted, and the tibia is often shorter and twisted. In addition, wings are malformed and are approximately 40% smaller than those of wild-type flies. Coexpression of DlimkD522A and wild-type Dlimk results in flies whose wings appear normal, indicating that the effects of dominant-negative Dlimk result from specific inhibition of the endogenous wild-type Dlimk as opposed to nonspecific interference with an unrelated signaling pathway (Chen, 2004).
The malformed legs in DlimkD522A flies closely resemble leg defects in flies in which Rho signaling is perturbed through genetic disruption of Rho1, DrhoGEF2 (a guanine nucleotide exchange factor for Rho1), sqh (myosin light chain), and zipper (nonmuscle myosin heavy chain). Sqh and zipper are downstream targets of Drok and regulate actomyosin contractility. Loss-of-function mutants of Rho1 or DrhoGEF2 strongly suppress the severity of wing defects associated with Dlimk expression. Reducing Rho activity by overexpressing the potent Rho inhibitor, p190 RhoGAP, also efficiently suppresses Dlimk-induced wing defects. Moreover, reducing levels of Diaphanous or Drok, two Rho targets that promote actin assembly, also substantially reduces the severity of Dlimk-induced wing defects. A loss-of-function allele of blistered, the Drosophila SRF ortholog, also suppresses the Dlimk-induced wing defects, suggesting that regulation of SRF-dependent transcription by Rho-LIMK signaling plays a role in wing morphogenesis. Significantly, in mammalian cells, LIMK and Diaphanous cooperate to regulate SRF activity (Geneste, 2002). Reducing levels of the Rho-related GTPases, Rac1, Rac2, and Cdc42, or the Rac activator, Myoblast city (Mbc), or the Rac/Cdc42 effector target, PAK, has very little effect on the Dlimk-induced wing phenotype. Thus, it appears that in the developing leg and wing, Dlimk specifically mediates a Rho-actin signaling pathway required for imaginal-disc morphogenesis (Chen, 2004).
Defects in leg morphogenesis resembling those in DlimkD522A flies are seen in mutants of several ecdysone-inducible genes, including those encoding the transcription factor, Broad-complex (BR-C or br), and Stubble (Sb), a transmembrane serine protease (Appel, 1993). Both genes are required for disc morphogenesis during larval-pupal transition. Significantly, br mutants interact genetically with mutants of Sb, zipper, and blistered during imaginal-disc morphogenesis (Beaton, 1988: Gotwals, 1991), suggesting that the observed role for a Rho-Dlimk pathway in leg morphogenesis could reflect a requirement for this pathway in the response to ecdysone (Chen, 2004).
To determine if the Rho-Dlimk pathway interacts genetically with br or Sb, a heat-shock-inducible DlimkD522A transgene that exhibits a low-penetrance malformed leg phenotype was crossed with br and Sb mutants. DlimkD522A and mutants of several Rho signaling components strongly interact with Sb63b and Sb70, two dominant-negative alleles of Stubble, to produce malformed legs at a high frequency. However, mutants of the Rho-related GTPases, Rac1, Rac2, and Cdc42, do not enhance the frequency of leg defects. Similarly, several components of the Rho-Dlimk pathway, but not Rac and Cdc42, strongly interact with br1 in an analogous genetic interaction test (Chen, 2004).
The observed interactions among Rho1, Dlimk, br, and Sb support a role for Rho signaling in ecdysone-regulated metamorphosis. However, neither Rho1 expression nor activation is ecdysone inducible. In light of studies linking Rho-LIMK signaling to effects on gene expression (Sotiropoulos, 1999), BR-C and Sb expression were examined in flies overexpressing Rho1, Dlimk, or p190 RhoGAP during early puparium stages, when disc morphogenesis is underway. Expression of BR-C and Sb mRNA normally peaks approximately 2-4 hr after puparium formation. However, in flies overexpressing Rho1 or Dlimk, expression of these genes persists well beyond the normal peak of expression seen in 'driver-only' control flies (approximately 8-10 hr after puparium formation. Moreover, expression of these genes is greatly reduced at all stages of pupation in flies expressing p190 RhoGAP. Significantly, although most of the transgenic flies that overexpress p190 RhoGAP die at a late pupal stage, the few 'escapers' that eclose exhibit malformed wings and twisted and bent leg phenotypes that are very similar to those seen in flies expressing DlimkD522A . In addition, the pupal lethality that is frequently observed with overexpression of p190 RhoGAP is efficiently rescued by coexpressing Dlimk, indicating that the late developmental defects that arise as a consequence of Rho inactivation largely reflect defects in Rho-LIMK signaling (Chen, 2004).
Expression of the ecdysone receptor (EcR) itself is similarly regulated by Rho1 and Dlimk. However, loss-of-function alleles of the EcR or Sb fail to rescue the effects of overexpressing Dlimk, suggesting that Rho-LIMK signaling controls additional aspects of metamorphosis independently of its effects on the ecdysone response. Thus, Rho-LIMK signaling may play a role in coordinating Rho-directed cell shape changes and movements with ecdysone-induced gene expression during tissue morphogenesis (Chen, 2004).
Many of the identified transcriptional targets of the ligand-activated ecdysone receptor are, themselves, transcription factors, which are not the actual effectors of tissue morphogenesis. However, the Stubble gene, which is highly sensitive to Rho-LIMK signaling, encodes a protein that participates directly in morphogenesis through its ability to promote remodeling of the extracellular matrix. The ability of Rho to direct both actin-mediated cell shape changes and the expression of a cell surface protease provides a potential mechanism for coordinately regulating these two major components of tissue morphogenesis during development (Chen, 2004).
To examine more directly a requirement for a Rho-actin-SRF pathway in the transcriptional response to ecdysone, Drosophila SL2 cells were used. In SL2 cells, as in developing discs, ecdysone induces the expression of EcR mRNA. Transfection of cells with the Rho-inhibitory C3 toxin or pretreatment with the actin polymerization inhibitor, latrunculin B, substantially reduces the ecdysone-induced increase in EcR mRNA but does not affect transcription of the ecdysone-insensitive gene rp49 or the Rho1 gene. As expected, latrunculin B completely inhibits morphogenesis of leg appendages, indicating a requirement for F-actin assembly. To examine the role of SRF in ecdysone-induced EcR expression, SL2 cells were treated with RNAi corresponding to the blistered gene. RNAi-treated cells exhibit reduced SRF expression and an absence of ecdysone-induced EcR mRNA expression. Together, these results suggest that the ability of Rho and Dlimk to promote F-actin assembly and SRF activation is responsible for their effects on ecdysone-responsive gene expression and tissue morphogenesis. In addition, the findings in SL2 cells indicate that the observed effects of Rho-SRF signaling on the ecdysone response are cell-autonomous effects. Interestingly, genetic interactions have been observed between zipper and sb and between zipper and br, suggesting that Rho-regulated actomyosin contractility, in addition to F-actin assembly, may also influence the ecdysone response. In this regard, it is interesting to note that mechanical stretching of cells reportedly promotes SRF activity. Alternatively, actomyosin contractility may play a parallel role in disc morphogenesis that is independent of any direct regulation of the ecdysone response (Chen, 2004).
No motif has been identified within the 5' and 3' regulatory sequences (2 kb each) of the EcR gene has been identified that matches the reported SRF binding consensus site. Hence, it remains possible that an SRF-regulated coactivator of ecdysone receptor gene expression is a primary target of Rho-Dlimk signaling. It is interesting to note that the Drosophila transcription factor, Crooked legs, regulates expression of ecdysone receptor mRNA and is encoded by an ecdysone-inducible gene that is also required for wing and leg morphogenesis. Such findings highlight the complexity of the gene expression hierarchy involved in the morphogenetic response to ecdysone and indicate a likely role for transcriptional feedback mechanisms (Chen, 2004).
It is concluded that Rho GTPase-mediated signal transduction to the actin cytoskeleton and ecdysone-induced gene expression are both critical regulatory components of tissue morphogenesis during Drosophila development. A direct relationship has been described between these two pathways in the context of metamorphosis. Specifically, these findings indicate that Rho, through its ability to activate LIMK and promote actin polymerization, regulates the expression of several ecdysone-responsive genes, including the ecdysone receptor itself. By modulating the expression of ecdysone-responsive genes, including a cell surface protease, the Rho-LIMK signaling pathway appears to play a critical role in regulating the proper morphogenesis of adult structures from the imaginal discs of larvae. This connection represents a previously unrecognized link between Rho GTPase signaling and nuclear hormone signaling that potentially plays a broader role in additional developmental contexts (Chen, 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 (Dpax) 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, 2005).
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, 2005).
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, 2005).
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, 2005).
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, 2005).
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, 2005).
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, 2005).
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, 2005).
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, 2005).
The correct localization of myosin II to the equatorial cortex is crucial for
proper cell division. A collection of genes was examined that causes defects
in cytokinesis and revealed (with live cell imaging) two distinct phases of myosin
II localization. Three genes in the rho1 signaling pathway, pebble (a Rho
guanidine nucleotide exchange factor), rho1, and rho kinase, are
required for the initial recruitment of myosin II to the equatorial cortex. This
initial localization mechanism does not require F-actin or the two components of
the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and
racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin
(diaphanous), and profilin (chickadee) are required to stably
maintain myosin II at the furrow. In the absence of these latter genes, myosin
II delocalizes from the equatorial cortex and undergoes highly dynamic
appearances and disappearances around the entire cell cortex, sometimes
associated with abnormal contractions or blebbing. These findings support a model
in which a rho kinase-dependent event, possibly myosin II regulatory light chain
phosphorylation, is required for the initial recruitment to the furrow, whereas
the assembly of parallel, unbranched actin filaments, generated by
formin-mediated actin nucleation, is required for maintaining myosin II
exclusively at the equatorial cortex (Dean, 2005).
This study has discovered three steps in the myosin II
localization/activation process that involve distinct groups of genes:
(1) an initial recruitment of myosin II to the equatorial cortex that is
independent of F-actin and centralspindlin but requires rho1 signaling;
(2) a secondary stabilization of myosin II at the midzone that requires
F-actin and a second set of genes that are likely involved in building a
specific type of actin network, and (3) the activation of furrowing
once myosin II is localized that depends on centralspindlin (Dean, 2005).
Rho1, its
activating guanidine nucleotide exchange factor pebble, and rho kinase are each
required for the initial recruitment of myosin II to the equatorial cortex. Rho1
has been implicated in two pathways that are important for cytokinesis.
In the first pathway, rho1 signals to F-actin
through the formin diaphanous. However, proteins on this F-actin
pathway, including F-actin itself, are not essential for the initial myosin II
recruitment to the equatorial cortex. However, rho kinase, another
downstream target of rho1, is essential. Because rho kinase phosphorylates the
myosin II RLC, it is possible that
phosphorylation of the RLC is essential for myosin II recruitment to the furrow.
This hypothesis could not be directly tested, because the myosin II heavy
chain forms large aggregates when the RLC is depleted by RNAi (Dean, 2005).
Phosphorylation of the RLC both activates the motor domain and, in some myosins,
increases bipolar thick filament formation. Because
F-actin is not required for myosin II recruitment, activation of the motor is
unlikely to be the mechanism by which phosphorylation of the RLC would cause
recruitment of myosin II to the equatorial cortex. It is quite possible,
however, that the rho kinase-mediated myosin II phosphorylation leads to thick
filament assembly and that this assembly is important for localization of myosin
to the equatorial cortex. Indeed, in Dictyostelium, it is clear that
bipolar thick filament formation is sufficient for myosin II localization to the
midzone of a mitotic cell. The nonactin-based mechanism of recruitment of myosin II filaments remains
unknown (Dean, 2005).
In contrast to the lack
of F-actin involvement in the early recruitment of myosin II to the equatorial
cortex at anaphase, F-actin disruption by Latrunculin A results in a failure to maintain
myosin II in the equatorial region. Interestingly, the downstream rho1 effectors
diaphanous/formin and chickadee/profilin are also necessary for myosin II
maintenance at the equatorial midzone. Although the loss of these genes could
deplete F-actin, phalloidin staining has shown that F-actin is still present in all
of the RNAi-treated cells. In addition, these RNAi-treated
cells still contract, unlike when F-actin is completely disrupted with LatA.
Thus, myosin II appears to be interacting with F-actin in the cortex as it
disperses in dynamic patches throughout the cortex of these diaphanous- or
chickadee-depleted cells (Dean, 2005).
It is suggested that the role of diaphanous/formin and chickadee/profilin in
maintaining the myosin II contractile ring is through the creation of specific
F-actin structures. In particular, formin- and profilin-mediated nucleation
results in unbranched actin filaments because profilin
promotes the barbed-end growth of formin-capped actin filaments. Indeed, electron microscopy has
shown that F-actin in the cleavage furrow mainly consists of unbranched, bundled
filaments. These parallel
filaments contrast with Arp2/3-mediated nucleation, which creates a highly
branched actin filament network. Indeed, Arp2/3, although essential for
lamellipodia formation, is not required for cytokinesis in Drosophila
cells. The hypothesis here
is that once myosin II is recruited to the equatorial cortex of
the cell by a rho kinase-dependent mechanism, possibly localized activation of
RLC phosphorylation, it is retained there because of its higher affinity for
parallel, unbranched actin filaments than to branched actin networks. Consistent
with this hypothesis, myosin II is depleted from the lamellipodia in migrating
cells where Arp2/3 is localized and branched F-actin networks are formed but is enriched in the lamella where F-actin filaments are
more likely to be aligned in parallel bundles. Thus,
it is proposed that high rho1 signaling to Diaphanous at the cleavage furrow
maintains a higher concentration of parallel actin filaments in this region
compared with the rest of the cortex, and these parallel filaments serve to
selectively retain myosin II at the equator to form a stable contractile ring.
In the absence of these parallel actin filaments, myosin II can bind branched
F-actin throughout the cortex, perhaps occasionally organizing them into
parallel bundles that cause increased myosin recruitment corresponding to the
flashes of cortical myosin accumulation, but these interactions are unstable (Dean, 2005).
Live-cell imaging shows that when pavarotti or racGAP50c
are depleted, the cells do not display significant contractions despite
recruiting myosin II to the equatorial cortex. Although there is some modest
membrane contractile activity in these cells, it is clear that significant
contraction or furrowing requires both components of the centralspindlin
complex. It is surprising that only these proteins were found to be necessary
for cortical contraction at sites of myosin II localization. Data from fixed
cells, as well as earlier studies, indicated that Drosophila cells do not
undergo equatorial contractions during mitosis when Diaphanous or Chickadee is
depleted. However, live-cell imaging shows that
when either of these two genes is depleted in S2 cells, not only is myosin II
transiently localized to the equatorial cortex before dispersing, but cells do
indeed display transient equatorial contraction. It is difficult to recognize
these events in fixed cells because of their transient nature and the somewhat
irregular shapes of cells depleted of these proteins. This work highlights the
importance of live-cell imaging in the study of dynamic processes such as
cytokinesis (Dean, 2005).
In addition to the suppression of furrowing, depletion of centralspindlin also
leads to an inability to retain F-actin exclusively at the equatorial cortex
during cytokinesis. This similar phenotype of the centralspindlin complex and
the F-actin affecting proteins suggests that centralspindlin may be an upstream
regulator of F-actin filament formation. Indeed kinase-dead mutants of Pavarotti
have been shown to accumulate at the spindle poles and are associated with an
abnormal accumulation of F-actin near the centrosomes.
Centralspindlin may be acting indirectly by helping to localize an important
actin-affecting protein at the central spindle, or it may act more directly on
the cortex. Because RacGAP50c has been shown to bind Pebble in vitro, it has been
hypothesized that centralspindlin affects the F-actin cortex through rho1
signaling by the localization and/or activation of Pebble. However, RacGAP50c depletion does
not lead to a lack of myosin II recruitment as does Pebble or Rho1 depletion,
and, thus, centralspindlin must act in a rho1-independent manner. For instance, the racGAP activity of
centralspindlin may itself be important for signaling to the F-actin cortex.
Finally, centralspindlin cannot be the major actomyosin ring positioning signal
because myosin II is properly recruited in its absence (Dean, 2005).
Morphogenesis of the Drosophila embryo is associated with a dynamic reorganization of the actin cytoskeleton that is mediated by small GTPases of the Rho family. Often, Rho1 controls different aspects of cytoskeletal function in parallel, requiring a complex level of regulation. The guanine triphosphate (GTP) exchange factor DRhoGEF2 is apically localized in epithelial cells throughout embryogenesis. DRhoGEF2, which has previously been shown to regulate cell shape changes during gastrulation, recruits Rho1 to actin rings and regulates actin distribution and actomyosin contractility during nuclear divisions, pole cell formation, and cellularization of syncytial blastoderm embryos. It is proposed that DRhoGEF2 activity coordinates contractile actomyosin forces throughout morphogenesis in Drosophila by regulating the association of myosin with actin to form contractile cables. These results support the hypothesis that specific aspects of Rho1 function are regulated by specific GTP exchange factors (Padash Barmchi, 2005; full text of article).
Guanine nucleotide exchange factors regulate the activity of the small GTPase Rho1, which is thought to act as a molecular switch in a broad spectrum of morphogenetic processes that require a complex reorganization of the actin cytoskeleton. However, the manner in which different aspects of Rho1 function are regulated by RhoGEFs is not well understood. This study found that DRhoGEF2 protein is broadly distributed in epithelia during oogenesis and embryonic development and concentrated at the apical surface of cells, suggesting that it may regulate Rho1 throughout morphogenesis. The defects of DRhoGEF2 mutants are less severe than those of Rho1 mutants, suggesting that DRhoGEF2 regulates specific aspects of Rho1 function (Padash Barmchi, 2005).
DRhoGEF2 has been shown to regulate cell shape changes during gastrulation, and DRhoGEF2 is implicated in epithelial folding during imaginal disc development, a process that depends on cell shape changes that are similar to those driving invagination of the germ layers. This paper shows that DRhoGEF2 regulates cytoskeletal reorganization and function during pole cell formation and blastoderm cellularization. All of these processes require the contraction of actomyosin rings. It is proposed that DRhoGEF2 regulates Rho1 activity during cell shape changes requiring actomyosin contractility. The results support the hypothesis that individual RhoGEFs may regulate specific aspects of Rho1 function during development (Padash Barmchi, 2005).
Interestingly, DRhoGEF2 has been found to be nonessential during cytokinesis, which also involves the function of contractile actin rings. The function of Rho1 during cytokinesis is regulated by the RhoGEF pebble that initiates actin ring assembly. In pebble mutants, cytokinesis is blocked at mitotic cycle 14 and subsequent mitoses occur without cytokinesis, creating polyploid, multinucleated cells. Although large multinucleated cells are also observed in DRhoGEF2 mutants at the extended germ band stage it is not clear whether these cells are caused by a block in cytokinesis or are caused by earlier defects during cellularization. In contrast to pebble, DRhoGEF2 may not be required for the assembly of actin rings, but may play a nonessential role in the separation of daughter cells. This is reminiscent of observations during cellularization. Although the function of actin rings appears compromised throughout cellularization, the data suggest that some contractile activity remains that leads to the basal closure of blastoderm cells and is responsible for the cellularized appearance of DRhoGEF2 mutants at the onset of gastrulation (Padash Barmchi, 2005).
At the retracted germ band stage, DRhoGEF2 is enriched at the apical cortex of cells in the leading edge of the lateral epidermis, which is consistent with the view that it may regulate Rho1 during dorsal closure. Rho1 function is essential for dorsal closure, and the cuticles of zygotic Rho1 mutants show dorsal holes. In DRhoGEF2 mutants, the lateral epithelial sheets closed the embryo dorsally. This does not exclude the possibility that constriction of actin cables may contribute to dorsal closure and that DRhoGEF2 may play a role in this process. Overall, the data suggest that DRhoGEF2 function may not be essential for the generation of contractile force, but rather regulate the temporal and spatial coordination of actomyosin contractility (Padash Barmchi, 2005).
During syncytial nuclear divisions and cellularization, DRhoGEF2 is localized specifically at the invaginating furrows. In DRhoGEF2 mutants, actin is irregularly distributed and metaphase furrow formation is less uniform than in the wild type. The defects in furrow formation lead to mitotic defects and the subsequent elimination of abnormal nuclei from the cortex so that, at the onset of cellularization, ~20% of the nuclei have been lost. These phenotypes are reminiscent of the defects seen in mutants of the nonreceptor tyrosine kinase Abelson (Abl). The abnormalities in actin distribution observed in abl mutants are likely caused by the mislocalization of Dia, which leads to ectopic actin polymerization at the apical end of cells. Changes in Dia distribution were not observed in DRhoGEF2 mutants, suggesting that DRhoGEF2 may regulate actin distribution by a different mechanism . Perturbations in actin distribution are observed throughout early development in DRhoGEF2 mutants. During cellularization, significant amounts of actin fail to redistribute to the base of the furrow canal. These observations show that one of the roles of DRhoGEF2 is to regulate furrow assembly. The defects in actin distribution also affect the pole cells, which fail to reorganize their cortical actin cytoskeleton and remain embedded in the somatic nuclear layer rather than sitting on top of it. Consequently, they are obliterated during invagination of the cellularization front (Padash Barmchi, 2005).
It is speculated that DRhoGEF2 may have a function in the assembly of actin cables by regulating the association of actin with other proteins such as myosin II. The mislocalization of actin observed in DRhoGEF2 mutants may be caused by failure of actin to associate with myosin. Interestingly, although myosin II is present at the metaphase furrows, it plays no essential role in their formation, and this suggests that the function of DRhoGEF2 in furrow assembly may be independent of actomyosin contractility (Padash Barmchi, 2005).
Phenotypic analysis suggests that DRhoGEF2 regulates actomyosin contractility during cellularization. Previously, the actin-binding protein Bnk has been implicated in the regulation of contractile forces. In bnk mutants, actin hexagons detach from each other and constrict prematurely. Based on this phenotype, it is suggested that, during the slow phase, cortical actin hexagons are linked to each other through Bottleneck (Bnk), and that actomyosin constriction causes the network to contract as a whole, thereby pulling the membrane front inwards. Once the cellularization front has reached the base of the nuclei and Bnk is degraded, actin hexagons detach from each other and contract as individual rings, thereby closing the blastoderm cells basally. It is proposed that DRhoGEF2-mediated activation of Rho1 may regulate the force that keeps actin hexagons under tension. Bnk counteracts contraction during the slow phase by linking individual actin rings to each other. Degradation of Bnk during the fast phase releases individual actin rings, and the DRhoGEF2-mediated contractile force now contributes to basal closure. Therefore, DRhoGEF2 and bnk act in concert to coordinate actin ring contraction during cellularization. In DRhoGEF2-bnk double mutants, the actin network disintegrates progressively, suggesting that DRhoGEF2 and bnk may play an additional role in the assembly or stabilization of actomyosin filaments (Padash Barmchi, 2005).
It has been proposed that actin network contraction contributes to the inward movement of the furrow canal. Although the data suggest that network tension is severely reduced in DRhoGEF2 mutants, the rate of membrane invagination is unaffected. This is consistent with reports on the role of myosin II during cellularization, suggesting that network tension may not contribute to membrane invagination. In the wild type, actin rings squeeze the nuclei slightly and push them basal-wards as the actin network moves over them. This may contribute to the parallel alignment of astral microtubules surrounding the nuclei and to nuclear elongation. In DRhoGEF2 mutants, nuclei are wider than in the wild type and irregularly aligned. It is proposed that network tension may create an ordered hexagonal array of actin rings that contributes to a parallel alignment of nuclei during cellularization. The force moving the actin network inward may be created by plus end-directed tracking of actin on astral microtubules and by membrane insertion as previously suggested. These observations suggest that actomyosin contractility plays a role in the spatial coordination of cytoskeletal function during cellularization (Padash Barmchi, 2005).
Two effector pathways have been implicated in the transduction of Rho1 activation to the actin cytoskeleton. During cytokinesis, which is mechanistically related to cellularization, a linear pathway including profilin and Dia have been proposed to link Rho1 to the contractile actomyosin ring. The maternally supplied Dia plays a role in a spectrum of cytoskeletal functions during early embryogenesis that also require DRhoGEF2 function, such as metaphase furrow formation, pole cell formation, and cellularization. Dia is localized at the cellularization front and is necessary for the recruitment of cytoskeletal components such as the actin-binding protein anillin and the septin homologue Pnut. The phenotypes of dia mutants suggest that dia is necessary for the assembly of contractile actin rings at sites of membrane invagination (Padash Barmchi, 2005).
The similarities between dia and DRhoGEF2 mutants might suggest dia as a downstream effector of DRhoGEF2. However, the defects of dia mutants are morphologically different from those of DRhoGEF2 mutants. In dia mutants, metaphase furrows do not form and contractile rings at the base of polar cytoplasmic buds fail to assemble. During cellularization, actin fails to condense into individual rings, and the network disintegrates during the second phase of cellularization. In DRhoGEF2 mutants actin rings form and remain largely intact but fail to constrict. In addition, the temporal and spatial localization of Dia and Pnut to the cellularization front was unaffected in DRhoGEF2 mutants and dia was not required for the localization of DRhoGEF2. These findings do not exclude that DRhoGEF2 activity may in part be mediated by dia, however, they suggest that some dia-dependent aspects of Rho1 function are still active in DRhoGEF2 mutants and that another pathway may be involved in transduction of the DRhoGEF2 signal (Padash Barmchi, 2005).
A well-characterized pathway regulating actomyosin contractility in mammalian cells and in C. elegans links Rho1 to actin via Rho kinase, the regulatory subunit of myosin light chain phosphatase (MBS) and myosin II. Rho kinase-mediated phosphorylation inhibits the activity of MBS and induces a conformational change in myosin II allowing it to form filaments that promote sliding of antiparallel actin filaments. The data are consistent with a model in which DRhoGEF2 regulates the association of actin with myosin II, thereby stabilizing actomyosin cables. It is proposed that failure to activate the Rho kinase pathway may compromise the recruitment of actin into contractile cables. This may destabilize actin cables and lead to the mislocalization of actin and to the defects in actomyosin contractility observed in DRhoGEF2 mutants. The Drosophila homologue of Rho kinase, Drok, and myosin II have recently been identified as downstream effectors of DRhoGEF2 during the regulation of actomyosin contractility in Schneider (S2) cells. In addition, myosin II is required for basal closure of blastoderm cells and the myosin II heavy chain encoded by zipper (zip) interacts genetically with DRhoGEF2. These data support the model that DRhoGEF2 may regulate actomyosin contractility through the Rho kinase pathway. Mutants in Drok and Drosophila myosin light chain phosphatase have been identified, however, their role during early embryogenesis has not been reported. Interestingly, inhibition of Drok activity by injection of the specific Rho kinase inhibitor Y-27632 into embryos before cellularization disrupts the localization of myosin II. Similar observations have been made in Drok mutant cell clones in imaginal discs. By contrast, DRhoGEF2 mutants reveal no significant changes in the localization of myosin II during cellularization. It is possible that the differences in myosin II localization between DRhoGEF2 and Drok mutants are due to different mechanisms of action at the molecular level. In mammalian cells myosin II phosphorylation is required for the generation of contractile force but not for its localization. Further investigations will be necessary to resolve how the DRhoGEF2 signal is transduced to the cytoskeleton (Padash Barmchi, 2005).
Little is known about the events that regulate the specific subcellular localization and activation of DRhoGEF2. It has recently been shown that DRhoGEF2 particles are transported from the cytoplasm to the cell periphery by tracking microtubule plus ends in Drosophila S2 cells. DRhoGEF2 particles have been observed during syncytial development that may be involved in a similar process in the embryo. It is speculated that DRhoGEF2 may be delivered to specific membrane subdomains at the cellularization front by microtubules. The G-protein α-subunit encoding gene concertina (cta) has been shown to regulate the dissociation of DRhoGEF2 from microtubules. cta has been implicated in the activation of DRhoGEF2 during gastrulation, but is not required during cellularization. It has been suggested that the force moving the actin network inward may be generated by plus end-directed crawling of actin on astral microtubules. It is speculated that DRhoGEF2 may regulate actin ring constriction during cellularization while associated with the tip of astral microtubules by recruiting Rho1 to the site of actin rings (Padash Barmchi, 2005).
DRhoGEF2 is concentrated in actin-rich regions throughout development and the human orthologue of DRhoGEF2, PDZ-RhoGEF, has been shown to bind to actin directly. Although the domain structure of DRhoGEF2 and PDZ-RhoGEF is very similar, the actin-binding region of PDZ-RhoGEF is not conserved in DRhoGEF2. Nevertheless, the localization of DRhoGEF2 is consistent with the view that it may associate with actin, however, further experiments are needed to corroborate this theory (Padash Barmchi, 2005).
Actin cytoskeletal networks push and pull the plasma membrane (PM) to control cell structure and behavior. Endocytosis also regulates the PM and can be promoted or inhibited by cytoskeletal networks. However, endocytic regulation of the general membrane cytoskeleton is undocumented. This study provides evidence for endocytic inhibition of actomyosin networks. Specifically, it was found that Steppke, a cytohesin Arf-guanine nucleotide exchange factor (GEF), controls initial PM furrow ingression during the syncytial nuclear divisions and cellularization of the Drosophila embryo. Acting at the tips of ingressing furrows, Steppke promotes local endocytic events through its Arf-GEF activity and in cooperation with the AP-2 clathrin adaptor complex. These Steppke activities appear to reduce local Rho1 protein levels and ultimately restrain actomyosin networks. Without Steppke, Rho1 pathways linked to actin polymerization and myosin activation abnormally expand the membrane cytoskeleton into taut sheets emanating perpendicularly from the furrow tips. These expansions lead to premature cellularization and abnormal expulsions of nuclei from the forming blastoderm. Finally, consistent with earlier reports, it was also found that actomyosin activity can act reciprocally to inhibit the endocytosis at furrow tips. It is proposed that Steppke-dependent endocytosis keeps the cytoskeleton in check as early PM furrows form. Specifically, a cytohesin Arf-GEF-Arf G protein-AP-2 endocytic axis appears to antagonize Rho1 cytoskeletal pathways to restrain the membrane cytoskeleton. However, as furrows lengthen during cellularization, the cytoskeleton gains strength, blocks the endocytic inhibition, and finally closes off the base of each cell to form the blastoderm (Lee, 2013).
Coupling actomyosin networks to the plasma membrane (PM)
is essential for cells to migrate, interact, change shape,
and divide. As examples, actin networks form and function
at the leading edge of migratory cells, at cell-substrate
adhesion complexes, and at cell-cell adhesion complexes in
multicellular tissues. To assemble these complexes, receptors
can physically engage the actin cytoskeleton and also induce
cytoskeletal assembly via Rho- family guanosine triphosphate
(GTP)ases and phosphoinositide signaling. Inversely,
endocytosis can remove receptors from the PM promoting
the turnover of adhesion and signaling complexes.
More generally, the close links of both actin networks and
endocytic machinery with the PM suggest possible crosstalk
between these subsystems. Indeed, endocytic signaling
nucleates local actin networks to help drive membrane invagination
and scission (Mooren, 2012; Anitei, 2012). In contrast, more widespread
membrane cytoskeleton activity can create tension that
inhibits membrane invagination. Conceivably, endocytosis
could also inhibit the membrane cytoskeleton, but such
activity is undocumented (Lee, 2013).
The syncytial Drosophila embryo is a well-established
model for studying actomyosin networks and membrane trafficking
during PM furrow ingression. In the early syncytial
embryo, nuclei divide synchronously just beneath the PM.
At each division cycle, the activities of Rho-family GTPases, the Arp2/3 complex, and the formin Diaphanous
(Dia) organize actomyosin-based PM ingressions
(pseudocleavage furrows) that surround each nucleus to prevent
nuclear collision and loss. Once ~6,000 nuclei form,
similar mechanisms induce a final round of PM ingressions.
These furrows persist and elongate through membrane trafficking
to apical and lateral sites, and with support of
actomyosin networks at their basal tips (the furrow canals). This massive PM growth cellularizes the first embryonic
epithelium, a process completed with constriction of
actomyosin rings formed at the base of each cell.
Recently, endocytic events were detected at the tips of
pseudocleavage furrows and early cellularization furrow canals
by the presence of Amphiphysin (Amph)-positive tubules
and the internalization of labeled PM (Sokac, 2008). These events have
provided a model for studying how the actin cytoskeleton
can both promote and inhibit endocytosis (Sokac, 2008; Yan, 2013). However,
the role of this endocytosis is unclear, and paradoxically, it
would appear to counteract membrane growth. This study examined
how Arf G protein (Arf) activation might be involved. In other
contexts, Arfs promote endocytosis by recruiting coat proteins,
activating lipid signaling, and triggering actin polymerization. Like other G proteins, Arfs are activated
by guanine nucleotide exchange factors (GEFs). Cytohesins
are a major class of PM Arf-GEFs (Donaldson, 2011), and roles for cytohesin
Arf-GEFs have been documented at migratory leading edges,
focal adhesions, and adherens junctions in mammalian cell
culture (Santy, 2005; Torii, 2010; Ikenouchi, 2010). Drosophila contains one cytohesin, called
Steppke (Step). Step is known to function in postembryonic
insulin and EGF signaling, which mammalian
cytohesins do as well, but its contributions to
the Drosophila embryo and to other cellular processes are
unknown. This study shows that Step promotes endocytosis
at pseudocleavage furrows and furrow canals to restrain actomyosin
networks at these sites (Lee, 2013).
These data provide the first description of cytohesin function in
a developing embryo. Drosophila Step promotes
a subset of endocytic events at the tips of ingressing PM
furrows during embryo cellularization. Endocytosis has been
documented previously at these sites (Sokac, 2008), but its role has
been unclear. By manipulating a conserved upstream activator
of endocytosis, this study has identified an important role of endocytosis
in controlling the membrane cytoskeleton. The data argue
that Step acts at furrow tips to induce local Arf-dependent
endocytosis, which in turn antagonizes Rho1-dependent
actomyosin network assembly at these sites.
It was also found that the cytoskeleton can inhibit endocytosis at
the furrow tips, as has been previously shown in this system
(Sokac, 2008; Yan, 2013) and in other contexts. An overall
model is proposed in which this reciprocal relationship is one-sided at specific
developmental stages. At newly forming PM furrows, Step
dominates, promoting endocytosis that keeps cytoskeleton
activity in check for proper pseudocleavage and cellularization
furrow architecture and growth. During later cellularization,
the cytoskeleton dominates. Zygotic expression of actin regulators
such as Nullo normally increases actomyosin activity
as cellularization proceeds and appears to work in
conjunction with Dia to block endocytic events at the furrow
tips (Sokac, 2008; Yan, 2013)]. By counteracting the inhibitory endocytosis,
cytoskeletal activity would elevate further but at these later
stages is locally restrained by a distinct mechanism requiring
Bottleneck (Schejter, 1993). To form the blastoderm, this second restraint
mechanism is removed, and contractile rings close off the
base of each cell. In the absence of the initial step-mediated
restraint mechanism, it is proposed that the cytoskeleton abnormally
dominates the relationship at all early PM furrows.
Without Step-based endocytic inhibition, it is speculated that
actomyosin networks abnormally expand and inhibit other
endocytic events leading to coexpansion of cytoskeletal polymers
and PM from the furrow tips (Lee, 2013).
An important element of the model is the local induction
of endocytic events. The data localize Step to the tips of
ingressing PM furrows, and both the loss and overexpression
of Step alter membrane organization specifically at these
sites. This localized Step-regulated activity occurs in a dynamic
global membrane trafficking system within each forming
cell. During the peripheral nuclear divisions, each nucleus
acquires its own endoplasmic reticulum and Golgi apparatus
that function with recycling endosomes to direct exocytosis
to growing PM furrows at cellularization. Simultaneously,
endocytic events occur over the apical PM and at
the furrow tips, with endocytosed material recycled
to the growing furrows. Thus, the overall membrane system
is in continual flux, and coordination by local regulation
would be expected. The data identify a polarized endocytic
activator required for the process. Step Arf-GEF activity is
critical for restraining the membrane cytoskeleton at furrow
tips, and a subset of AP-2 activities is involved as well (Lee, 2013).
How could endocytosis and actomyosin networks impact
each other at the tips of PM furrows or elsewhere? This question
can be considered from several levels of organization.
First, a simple and direct connection could be endocytic
removal of one or more PM actomyosin regulators. This work
identifies Rho1 or an upstream regulator as a candidate.
Intriguingly, membrane trafficking has been previously
linked to the Rho1 pathway in this context. Specifically, recycling
endosomes have been implicated in the trafficking of
RhoGEF2 to the PM (Cao, 2008). It was hypothesized that RhoGEF2
might also be a target of Step for its removal from the PM
but no difference was found in RhoGEF2 levels at furrow canals
in step loss-of-function embryos. Thus, Rho1 may be a more
specific target of Step, although a direct connection to the
Rho1 pathway remains to be determined. Of note, a number
of septins have also recently been observed on the Amph-positive tubules (Lee, 2013).
Second, interplay between different pools of actin is
possible. For example, actin contributes to the invagination
and scission of endocytic vesicles, and thus, endocytic
actin and other PM actin networks could compete for regulators
or components. Additionally, there could be signaling
crosstalk between regulators of the different networks. For
example, Arf signaling often elicits local Rac or Cdc42 activity, and this might trigger crosstalk affecting Rho activity. Interestingly, overexpression of Cdc42-interacting protein
4 (Cip4) appears to antagonize Dia at furrow canals,
although Cip4 mutants have no cellularization phenotype on
their own. Significantly, however, this study found that Step
acts with AP-2 to control the membrane cytoskeleton. This
Step-AP-2 cooperation suggests that clathrin-coated pits are
involved in the antagonism, although it does not exclude the
possibility of separate cytoskeletal crosstalk (Lee, 2013).
Third, larger scale interactions should be considered. Endocytosis
could remove membrane in bulk that would otherwise
support the membrane cytoskeleton, although observations
of residual furrow canal endocytic activity with step
loss of function suggest a more specific mechanism. Inversely,
the membrane cytoskeleton could block endocytosis by
elevating PM tensio or possibly by sterically blocking
endocytic machinery from accessing the PM (Lee, 2013).
Endocytic-cytoskeletal crosstalk is relevant to many cellular
processes. For example, receptor endocytosis occurs in proximity
to actomyosin networks in various contexts, including
migratory leading edges, focal adhesions, and adherens junctions. However, these endocytic events and actomyosin
networks have mainly been studied independently,
and thus their functional integration is not understood. This study highlights the possibility that endocytic activity at such assemblies could simultaneously remove receptors and
antagonize local cytoskeletal networks, with both effects promoting
complex turnover and cellular dynamics (Lee, 2013).
During development, small RhoGTPases control the precise cell shape changes and movements that underlie morphogenesis. Their activity must be tightly regulated in time and space, but little is known about how Rho regulators (RhoGEFs and RhoGAPs) perform this function in the embryo. Taking advantage of a new probe, which recognizes the active, GTP-bound form of Rho1 and that can be expressed in vivo using the UAS/GAL4 system, thus facilitating the visualisation of RhoGTPase, evidence is presented that Rho1 is apically activated and essential for epithelial cell invagination, a common morphogenetic movement during embryogenesis. In the posterior spiracles of the fly embryo, this asymmetric activation is achieved by at least two mechanisms: the apical enrichment of Rho1; and the opposing distribution of Rho activators and inhibitors to distinct compartments of the cell membrane. At least two Rho1 activators, RhoGEF2 and RhoGEF64C are localised apically, whereas the Rho inhibitor RhoGAP Cv-c localises at the basolateral membrane. Furthermore, the mRNA of RhoGEF64C is also apically enriched, depending on signals present within its open reading frame, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to spatially restrict Rho1 activity during epithelial cell invagination (Simoes, 2006).
Using a probe that allows the visualisation of Rho1 activity in the course
of normal development, evidence is presented that this GTPase is active at the
apical side during the process of cell invagination. In the spiracles Rho1
activity is essential to control this movement, similarly to that previously
shown during Drosophila gastrulation, when mesodermal cells fail to
invaginate after inhibition of Rho1 function. An apical enrichment is observed of Myosin II, a possible target of
activated Rho1, analogous to that reported in other tissues of the fly embryo
where this type of movement occurs, such as the mesoderm and the salivary
glands. Inhibition of Rho1 activity results in a disorganised
pattern of apical Myosin II and F-Actin in the spiracle cells. It is suggested that
concentration of active Rho1 at the apical side organises the Actin
cytoskeleton and promotes high Myosin II accumulation/activity in this region,
leading to a contractile Actin-Myosin based force to produce a wedge-shaped
cell (Simoes, 2006).
These data show that spatial restriction of Rho1 activity is achieved by
distinct mechanisms. (1) Albeit ubiquitous, Rho1 protein is strongly
enriched on the apical side of the invaginating spiracle cells. (2) To
ensure that this GTPase is active exclusively on that side of the cell,
opposing Rho regulators are differentially distributed in two distinct
membrane domains: two Rho activators, RhoGEF64C and RhoGEF2, are apically
localised, whereas a Rho inhibitor, the RhoGAP Cv-c, occupies the
complementary, basolateral domain. Cell shape changes and
inward cell movements driving invagination are impaired if Rho1 becomes
activated in a spatially unrestricted manner. These observations stress the
importance of finely tuning Rho1 localisation and activation during normal
tissue morphogenesis (Simoes, 2006).
Several mechanisms might be at work to achieve the specific localisation of
the Rho regulators that direct cell invagination. In the case of RhoGEF64C, its mRNA and protein are apically localised, suggesting that apical
transport of RhoGEF mRNA followed by local translation is a mechanism to
activate Rho1 in a spatially restricted manner. Recent studies show that the
mRNA of RhoA can also be transported and locally translated in the axons and
growth cones of embryonic rat neurons, where RhoA controls growth cone
collapse in response to Semaphorin 3A. This shows that intracellular mRNA transport of Rho GTPases and of their regulators may be an important mechanism to control
spatial GTPase activation (Simoes, 2006).
Loss of function of the RhoGEFs involved in spiracle invagination leads to
variable apical defects, which are compatible with a partial loss of Rho1
function: knocking out RhoGEF64C resulted in a mild disruption of cortical
Actin without blocking invagination, while the absence of RhoGEF2 could result
in a complete failure of the invagination process. These results suggest that
several RhoGEFs are required to properly activate Rho1 during spiracle cell
movement and organ shaping (Simoes, 2006).
One interesting observation from these studies is the fact that mutants for
the RhoGAP Cv-c did not show ectopic activated Rho1 on the basolateral
membrane where this RhoGAP was localised. Thus, several mechanisms must be at
work to ensure that Rho1 activity is excluded from the basolateral domain
during cell invagination: the presence of at least one RhoGAP on the basal
membrane, the apical restriction of RhoGEFs and the existence of low levels of
Rho1 protein on the basolateral side of the cells. In addition, it was also
observed that spiracles from severe cv-c mutants showed lower levels
of apical Rho1-GTP than their wild-type counterparts, correlating with the
disruption of their apical Actin. Defects in apical Actin/Myosin II have also been
reported during invagination of the tracheal pits in cv-c mutants.
Taken together, these observations suggest that GTP hydrolysis is a necessary
step in the regulation of Rho1 function during cell invagination and the
RhoGAP Cv-c may help to maintain a steady state level of apical Rho1-GTP (Simoes, 2006).
Based on the differential distribution of Rho1 GEFs and GAPs, a
model is proposed in which Rho1 must shuttle back and forth between two membrane
compartments, being GTP-bound on the apical cell membrane and GDP-bound on the
basolateral side.
Thus, during tissue morphogenesis, epithelial cells can couple their
apical-basal polarity to the spatial control of small RhoGTPase function (Simoes, 2006).
RhoGTPases act as dynamic switches in many developmental and cellular
contexts. In order to understand how they orchestrate these dynamic processes,
their activity states needs to be visualised over time. It is anticipated that
this work and the tools described will provide a basis for studying Rho1
activity in vivo. It will be interesting to extend this analysis to other
contexts in which Rho GTPases are known to act -- such a dorsal closure,
neurulation, wound healing -- and to identify the Rho regulators involved in
each case, relating their spatial/temporal distribution with the patterns of
Rho GTPase activity (Simoes, 2006).
The construction and maintenance of normal epithelia relies on local signals that guide cells into their proper niches and remove unwanted cells. Failure to execute this process properly may result in aberrant development or diseases, including cancer and associated metastasis. This study shows that local environment influences the behavior of dCsk-deficient cells. Broad loss of dCsk leads to enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion. Loss of dCsk in discrete patches leads to a different outcome: epithelial exclusion, invasive migration, and apoptotic death. These latter phenotypes required sharp differences in dCsk activity between neighbors; dE-cadherin, P120-catenin, Rho1, JNK, and MMP2 mediate this signal. Together, these data demonstrate how the cellular microenvironment plays a central role in determining the outcome of altered dCsk activity, and reveal a role for P120-catenin in a mechanism that protects epithelial integrity by removing abnormal cells (Vidal, 2006).
The mechanisms that regulate organ size and shape are not well understood, but recent studies have pointed to the importance of local interactions between neighboring cells. For example, in the process known as 'cell competition', cells with relatively higher proliferative rates actively eliminate their neighbors by programmed cell death. Conversely, apoptotic cells send proliferative signals to their neighbors to compensate for their loss. In this way, normal tissue size is achieved. The misregulation of such mechanisms may contribute to the development of cancer, since most solid tumors arise from intact epithelia and are resistant to size-control signals. Tumors are particularly dangerous when linked to metastasis, a process in which cells leave the primary tumor and invade distant tissues. These processes are best understood within the context of an intact epithelium, in which the full range of cell interactions is retained. Work in Drosophila has provided an important in situ view of the action of oncogenes within epithelia (Vidal, 2006).
Src family kinases (SFKs) are active in a broad range of cancer types, including tumors of the breast, colon, and hematopoietic systems. SFK activity typically increases as tumorigenesis progresses and is associated with metastatic behavior. The major inhibitor of SFK activity is C-terminal Src kinase (Csk) and its paralog Chk; these may act as tumor suppressors in, e.g., breast cancer, presumably through their ability to inhibit Src activity and perhaps other pathways. Drosophila Csk acts primarily or exclusively through Src pathway regulation, and the reduction of dCsk activity by itself led to increased organ size, organismal lethality, and increased cell proliferation due to a failure to exit the cell cycle. However, neither Csk loss nor Src activation has been clearly linked to early events in tumorigenesis, bringing into question the role of Csk/Src in proliferation in vivo. Instead, Src is currently thought to be a major player in the metastatic events that occur later in oncogenesis. How Csk or Src promotes the metastatic behavior of cells in situ remains largely unknown (Vidal, 2006).
This study analyzed the phenotypes of dCsk in the context of developing epithelia. The outcome of a cell's loss of dCsk is linked to its cellular microenvironment. When dCsk activity is reduced broadly in the developing eye or wing, the result is overproliferation, inhibition of apoptosis, and decreased cell adhesion. Tissue integrity is retained, but dCsk cells become inappropriately mobile and fail to maintain their appropriate contacts. The outcome of these effects is an overgrown and mispatterned adult tissue. By contrast, loss of dCsk in discrete patches results in epithelial exclusion, invasive migration through the basal extracellular matrix, and eventual apoptotic death; these events occur exclusively at the boundary between dCsk and wild-type cells. Further emphasizing the unique nature of cells at this boundary, a specific requirement was found for a signal that includes Drosophila orthologs of E-cadherin, P120-catenin, RhoA, JNK, and the metalloprotease MMP2. Hence, this study explores the mechanisms by which the cellular microenvironment can direct different behaviors of cells, both in the regulation of apoptosis and epithelial integrity. It also uncovers a mechanism for the removal of abnormal cells from a normal epithelium (Vidal, 2006).
This study shows that reducing dCskactivity results in a blockade of apoptosis and downregulation of cellular adhesion. The work is consistent with the view that Csk is a tumor suppressor that acts at multiple steps. Mutations in the locus encoding the Csk paralog Chk have been described in breast tumors, and, in this study, it has been observed that human Chk can functionally replace dCsk. Therefore the experimental advantages of developing Drosophila imaginal epithelia were used to explore specific aspects of dCsk function that are relevant to the behavior of tumor cells (Vidal, 2006).
Visualization studies suggest that a reduction in dCsk activity leads to a failure of cells to stably retain associations with their neighbors, resulting in prolonged cell movement as cells slide across each other in a manner not observed in wild-type tissue. This may reflect a failure to establish stable junctions, excess cell motility, or both. Recent work has demonstrated a critical and dynamic role for the cadherin-based apical junctions in patterning the Drosophila retina. Misexpressing dE-cadherin prevents patterning defects in GMR>dCsk-IR retinas, suggesting that dCsk cells have reduced dE-cadherin function. Links between Csk, Src, cadherins, and junctional integrity have been reported in mammalian cell culture, and an association has been observed between Drosophila Src42A and dE-cadherin during embryonic development (Takahashi, 2005). The data are consistent with this view: misexpression of a kinase-dead form of Src42A leads to a disruption in the localization of the dE-cadherin-associated protein Armadillo; also, reduced Armadillo levels observed in dCsk retinas is rescued by dE-cadherin misexpression. Together, these data suggest that altering dCsk/Src activity affects cell movements by decreasing dE-cadherin adhesion (Vidal, 2006).
The mechanism by which dCsk alters dE-cadherin function is not clear, but it is relevant to note that Src activation can shift cadherin-based cell adhesion from a 'strong' to a 'weak' adhesive state in mammalian cultured cells. Phosphorylation of cadherins and catenins may mediate 'inside-out' signaling that can alter the adhesive strength of the homophilic bond between cells (reviewed in Gumbiner, 2005). Evidence for such a mechanism has been provided for integrin-mediated focal adhesions (reviewed in Hynes, 2002), and Src activity can alter focal adhesions (Yeatman, 2004). However, normal basal membrane architecture was observed in dCsk cells, as assessed both by anti-integrin staining and by transmission electron microscopy, indicating that at least the gross structure is not affected (Vidal, 2006).
The ability of dCsk to influence cell proliferation, apoptosis, and cell adhesion is consistent with its ability to direct tissue overgrowth: reducing dCsk activity throughout a tissue (or the entire organism) leads to significantly enlarged tissues. This ability demonstrates that dCsk can participate in the mechanisms that set tissue size. A small number of other proteins have been implicated in this process, including Salvador, Hippo, and Lats/Warts, which show phenotypes that are strikingly similar to dCsk. Furthermore, dCsk can directly phosphorylate Lats/Warts (Stewart, 2003) in vitro (Vidal, 2006).
However, reduction of dCsk activity shows some important differences. Mutations in salvador, hippo, or lats/warts lead to an increase in Diap1 levels, which, in turn, blocks apoptotic cell death. By contrast, reductions in dCsk does not significantly alter Diap1 protein levels. Furthermore, although both Hippo and dCsk are required to exit the cell cycle, the cell cycle profile from hippo mutant cells is normal, while dCsk cells contain a significant shift toward G2/M (Read, 2004; Stewart, 2003). Perhaps the most striking difference is the effects of these factors on discrete mutant patches. While broad loss of dCsk activity leads to expanded tissues, surprisingly discrete patches of dCsk tissue are eliminated by neighboring cells. Unlike salvador, hippo, or lats/warts, clonal patches of dCsk cells fail to survive to adulthood. The effects of dCsk reduction are more similar to those reported for the tumor suppressor gene scribble. The scribble locus encodes a component of the septate junction that regulates cell polarity and proliferation; mutant cells display neoplastic overgrowth in a homotypic environment, but are removed by JNK-dependent apoptosis in discrete clonal patches abutting wild-type tissue (Vidal, 2006).
This work provides evidence that neighboring wild-type tissue provides a locally nonautonomous signal that leads to the removal of dCsk mutant cells. For example, FRT-derived clones of dCsk cells were out-competed by neighbors with normal levels of dCsk: this was most easily seen by the clonally related 'twin spot' of wild-type tissue that was consistently larger than the few surviving dCsk clones. In contrast, FRT-mediated dCsk clones that encompassed the entire eye survived and overproliferated. In the developing wing, cells at the periphery of sd>dCsk-IR or ptc>dCsk-IR expression domains were preferentially removed by apoptosis. This death is dependent not on absolute dCsk activity, but on the juxtaposition of cells that are starkly different in their levels of dCsk. Small differences, for example across the ptc>dCsk-IR or omb>dCsk-IR graded expression domains, did not trigger cell death (Vidal, 2006).
This translocation and death of dCsk-IR cells at the patched/wild-type boundary requires at least two steps. At boundaries with wild-type tissue, dCsk cells initially lose their apical profile, shift downward, and eventually become basally excluded from the epithelium. Such excluded cells then migrate away from the boundaries in both directions and eventually die by apoptosis. These events are strikingly reminiscent of those described for tumor cells undergoing metastasis. Altered activity of both Csk and Src has been implicated in a broad variety of tumors. Typically, however, increased Src activity is associated with later events in tumorigenesis, particularly metastasis. Although the connections between high Src activity and metastases are not understood, they likely include Src's ability to break cell-cell junctions and increase cell motility. Another hallmark of metastatic behavior is the ability to degrade basal extracellular matrix: this study also demonstrate a functional requirement for MMP2 activity during the translocation of mutant cells out of the wing epithelium (Vidal, 2006).
While evidence supports the view that the activity of Csk -- and presumably Src and perhaps other effectors -- can regulate metastatic behavior, it alone is not sufficient. First, reducing dCsk activity by itself is not sufficient to allow migrating cells to survive; the data suggest that most or all eventually die. This is consistent with previous work highlighting the importance of a 'two-hit' model to allow for stable tumor overgrowth and metastasis. A second mutation that prevents apoptotic cell death would be minimally required. Second, all cells within a discrete dCsk patch are not equivalent: cells at the boundary of the clone that border cells of strongly differing dCsk levels are exclusively prone to release from the epithelium. This work predicts that cells at the borders of some human tumors are especially prone toward metastatic behavior. Metastasis is often the most serious aspect of a tumor, and approaches that address the metastatic behavior of cells may need to take into account the properties of cells at the periphery. Understanding whether and how these cells are unique may help to more effectively target therapeutic intervention (Vidal, 2006).
In addition to enabling a detailed examination of dCsk cells and their behavior within an epithelium, this model system permitted identification of signaling components that are necessary to execute the aberrant cell mobility and cell death. The results indicate important roles for dE-cadherin, dP120ctn, Rho1, dJnk, and MMP2 (Vidal, 2006).
JNK-dependent apoptosis is required for a broad palette of related mechanisms such as cell competition in developing tissues and the removal of scribble mutant cells. JNK signaling is also associated with the movement of cells within epithelia, including dorsal closure in Drosophila and in mammals. Interestingly, JNK activity is required for the synthesis of MMP2 by v-Src-transformed mammalian cells (Vidal, 2006).
JNK activity can be triggered by several upstream signaling factors, including the small GTPases of the Rho family, and genetic data provide a link between dCsk, dJnk, and Rho1. Rho family proteins are key regulators of cell shape and motility. They also promote the cytoskeletal rearrangements required for epithelial-to-mesenchymal transitions (EMTs), and it is noted that dCsk boundary cells show a number of features that are reminiscent of EMTs. In Drosophila, Rho1 was found to induce an 'invasive' phenotype in wing disc cells, but, in this study, it was demonstrated that, similar to dCsk boundary cells, ptc>Rho1 misexpressing cells also undergo apoptotic death. Most importantly, halving the genetic dose of Rho1 strongly suppresses discrete loss of dCsk, but does not appreciably affect broad loss. Thus, Rho1 activity is linked to dCsk, and activation of Rho1 is sufficient to phenocopy both the apoptotic and migratory phenotypes of dCsk cells located near wild-type tissue (Vidal, 2006).
Previous work in mammalian cell culture has provided direct links between Src and P120-catenin, between cadherins and P120-catenin, and between RhoA and P120-catenin; the latter two interactions have been reported in Drosophila tissue culture systems as well. This study further supports links between these factors in dCsk boundary cells. Interestingly, although normal levels of both dP120ctn and Rho1 were required for the efficient removal of dCsk boundary cells, they were not required for the phenotypes resulting from broad loss of dCsk. The requirement for p120ctn specifically in boundary cells may explain why, although it is the only ortholog present in Drosophila, dP120ctn (Drosophila p120-catenin) is not required for organism viability (Vidal, 2006).
Both Src and P120-catenins are known to directly interact with cadherins, and, in fact, a role was demonstrated for dE-cadherin/Shotgun in the removal of dCsk cells. A model is postulated in which the loss of dCsk results in the remodeling of the zonula adherens, presumably by the phosphorylation of catenins and dE-cadherin itself by Src. Src activation is known to switch cadherin from a strong adhesive state to a weak one, providing one potential explanation for why dCsk retinal cells displayed reduced cell adhesion in situ. One critical question regarding cadherins is whether they have signaling roles that are independent of their adhesive properties. Perhaps relevant to this point, it was surprising to find that reducing dE-cadherin function leads to a suppression of the effects of dCsk-IR at the boundary. A simple dCsk-IR-mediated reduction in dE-cadherin adhesion would be enhanced by further reducing dE-cadherin activity, suggesting that dE-cadherin may provide an active signal that promotes boundary cells' release from the epithelium. If such a signal does exist, neighboring wild-type cells must trigger it, either through their own endogenous dE-cadherin or through a separate, local signal. Why are multiple (3-4) rows affected? The results are consistent with the creation of a successive new boundary as the previous row of cells descends, although other longer-range signals cannot be ruled out (Vidal, 2006).
It is noted that reducing dCsk activity by itself is not sufficient to direct stable tumor overgrowth, supporting the importance of a 'two-hit' model in Drosophila. Loss of the junction protein Scribble showed similar phenotypes to dCsk, including apoptosis, but was found to confer survival and metastatic-like behavior to cells in the presence of an activated Ras isoform. Interestingly, coexpression of dE-cadherin prevents this metastatic behavior (Vidal, 2006).
Finally, how can dP120ctn and Rho1 promote release of dCsk near wild-type boundaries but not act similarly with other dCsk cells? One source of information is the cadherins themselves: the boundary creates an interface of cadherins that have been exposed to different levels of Csk and, presumably, Src activity. This unusual interface may generate the needed dE-cadherin signal. Importantly, recent work has noted a change in the subcellular localization of P120-catenin and E-cadherin specifically at the border of human tumor tissues. At the time that ptc>dCsk-IR boundary cells lose their apical profiles, this study found that dP120ctn is relocalized to the cytoplasm. These results again emphasize the possibility that cells at tumor boundaries pose a special risk of undergoing epithelial-to-mesenchymal-like transitions and metastatic behavior. Metastasis is often the most serious complication of progressing tumors. Targeting therapies to this aspect of cancer may benefit from considering boundary cells and their potentially distinctive properties (Vidal, 2006).
Rho GTPase and its upstream activator, guanine nucleotide exchange factor 2 (RhoGEF2), have emerged as key regulators of actin rearrangements during epithelial folding and invagination (Nikolaidou, 2004). A Rho-GTPase-signaling pathway is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation. This study shows that Drosophila 18 wheeler (18W), a Toll-like receptor protein, is a novel component of the Rho-signaling pathway involved in epithelial morphogenesis. 18w mutant embryos have salivary gland invagination defects similar to embryos that lack components of the Rho pathway, and ubiquitous expression of 18W results in an upregulation of Rho signaling. Transheterozygous genetic interactions and double mutant analysis suggest that 18W affects the Rho-GTPase-signaling pathway not through Fog and RhoGEF2, but rather by inhibiting Rho GTPase activating proteins (RhoGAPs). RhoGAP5A and RhoGAP88C/Crossveinless-c (CV-C) are required for proper salivary gland morphogenesis, implicating them as potential targets of 18W (Kolesnikov, 2007).
Based on microarray experiments, 18w is downregulated in Scr mutants, implicating it in salivary gland development. To follow 18w expression in more detail, RNA in situ hybridizations were performed. The results show that in wild-type embryos 18w RNA is not maternally contributed but is expressed in salivary gland cells prior to and throughout their invagination. Within the salivary placode, 18w expression first becomes evident at stage 11 as a small spot in the dorsal posterior region. During early stage 12, following the beginning of invagination, 18w expression spreads throughout the placode. During the remainder of stage 12, 18w transcripts can be detected in both gland cells that have yet to invaginate as well as those that have already internalized, albeit at a reduced intensity. At stage 13, 18w transcripts cease to be expressed in salivary gland cells but are evident in salivary duct cells. In addition to salivary glands and ducts, 18w RNA is also detected in other tissues undergoing morphogenesis, including the tracheal placodes and the hindgut. As anticipated from microarray experiments, 18w transcripts are absent exclusively from parasegment two in Scr mutants while expression in the rest of the embryo remains unaltered. Overall, performing microarray experiments with Scr mutant embryos has proven to be an effective method for identifying salivary gland genes (Kolesnikov, 2007).
While 18w is expressed in several tissues undergoing morphogenesis, embryonic defects have yet to be identified in 18w mutant embryos. The striking 18w expression within the invaginating salivary gland prompted a more carefully investigation of the role of 18w in embryonic salivary gland development. In examining a null allele of 18w, 18wΔ21, it was discovered that initiation of invagination in the dorsal posterior region of the placode appears normal. However, during the next phase of invagination, in which the remaining cells of the salivary placode normally internalize in a strict sequential order, defects become apparent. In 18w mutants invagination is less synchronized; too many placode cells invaginate simultaneously rather than sequentially, thereby causing a wider lumen in 66% of 18w mutants when compared to wild-type embryos. Thus, it is the timely progression of invagination, not the invagination process itself, that is affected in 18w mutants. Due to the lack of proper coordination in 18w, the internalization of the gland cells appears to be delayed causing the cells that internalize last to remain near the ventral surface instead of reaching their final, more dorsal, position within the embryo. Eventually all of the salivary gland cells do internalize in 18w mutants, but the proximal part of the gland, which remains abnormally close to the ventral surface, becomes caught in the anterior movement of the ectoderm during head involution. As a result, the glands end up much closer to the anterior end of the embryo than in wild-type embryos. Similar defects are seen in mutants homozygous for the loss of function 18w allele, 18wΔ7–35, and in 18wΔ21, 18wΔ7–35 transheterozygous mutant embryos. Based on these results, 18w is an important component in coordinating salivary gland invagination (Kolesnikov, 2007).
Activation of the Rho-signaling pathway results in the phosphorylation of the myosin II regulatory light chain, encoded by the spaghetti squash (sqh) gene. The phosphorylated form of Sqh can interact with actin and cause actomyosin-based contractility at the apices of cells. To verify that 18W is part of the Rho pathway, whether overexpression of 18W results in an upregulation of Rho signaling, evident as an increase in phosphorylation of Sqh, was checked. Immunoblot analysis on embryonic extracts using anti-phospho-Sqh antibody reveals that overexpressing 18W throughout the embryo results in a two-fold increase of P-Sqh when compared to wild type. Similar results are seen when a constitutively active form of Rho is overexpressed ubiquitously in the embryo. Moreover, introducing one copy of a phosphomimetic sqh transgene, sqhE20E21, into an 18w mutant rescues the 18w invagination defects, indicating that 18W acts upstream of Sqh phosphorylation. Thus, both genetic and biochemical evidence indicate that 18W is a novel component of the Rho signaling pathway (Kolesnikov, 2007).
To determine whether 18W negatively regulates the RhoGAP branch of the Rho pathway, it was necessary to identify the RhoGAPs involved in salivary gland development. Of the 20 distinct RhoGAPs encoded by the Drosophila genome, 17 UAS-RhoGAP dsRNA lines were examined. Each of these lines was crossed to flies containing a salivary gland-expressing driver, scabrous-GAL4, and the progeny were screened for salivary gland defects. Only RhoGAP5A dsRNA expression resulted in salivary gland defects. Invagination defects are evident from more anteriorly placed glands, while migration defects resulted in wavy glands. Similar defects are seen upon expressing high levels of constitutively active Rho in the salivary gland with the scabrous-GAL4 driver. Overexpressing low levels of Rho in the gland results in mostly migratory defects, indicating that gland migration is more sensitive to the levels of Rho signaling than is the process of invagination. Invagination defects are seldom accompanied by migration defects, presumably because glands that have not properly internalized do not reach the visceral mesoderm upon which they normally migrate (Kolesnikov, 2007).
Since the GAL4/UAS dsRNA system may not result in a complete loss-of-function phenotype, strong alleles of two GAPs, RhoGAP68F and RhoGAP88C/Cv-c, which will be referred to simply as Cv-c. These have previously been shown to regulate Rho activity but lack salivary defects using the dsRNA interference technique. While neither rhoGAP68F nor cv-c mutations cause invagination defects, the cv-c mutants do display migration defects similar to those seen in embryos expressing RhoGAP5A dsRNA. To determine whether RhoGAP5A and Cv-c act redundantly within the salivary gland to regulate Rho activity, RhoGAP5A dsRNA within the gland was examined in a cv-c mutant. These embryos should have reduced activity of both RhoGAPs. They have gland invagination and migration defects that are more severe and penetrant than embryos that lack just one of them, indicating that these RhoGAPs are, in fact, partially redundant during salivary gland development (Kolesnikov, 2007).
In situ hybridization in wild-type embryos shows that cv-c RNA is not maternally contributed but is expressed in several tissues undergoing morphogenesis. In addition to its expression within the developing trachea and mesoderm, cv-c, similar to 18w, is expressed in the salivary glands prior to and during their invagination. Unlike 18w, however, cv-c expression does not appear to originate at the initial invagination site but rather initiates expression throughout most of the placode. During the onset of invagination at stage 12, cv-c expression intensifies and continues to be expressed within cells that have internalized until the conclusion of invagination at stage 13 (Kolesnikov, 2007).
To examine whether 18W regulates Rho activity by inhibiting Cv-c, several overexpression and genetic interaction experiments were performed. As might be expected if 18W negatively regulates Cv-c, overexpressing 18w within the salivary gland results in migratory defects similar to those seen in cv-c mutant embryos. In addition to migratory defects, however, some embryos overexpressing 18w also exhibit invagination defects, suggesting that Cv-c may not be the only RhoGAP negatively regulated by 18W. Moreover, lowering the dose of cv-c enhances the defects caused by 18w overexpression and suppresses the 18w mutant invagination defects, further supporting the role of 18W as a negative regulator of RhoGAP signaling. Therefore, genetic interaction experiments indicate that 18W regulates Rho signaling in the salivary gland by inhibiting at least one known RhoGAP (Kolesnikov, 2007).
Previous studies have shown that the Fog ligand activates RhoGEF2 through an as yet unidentified receptor, leading to the apical constriction of cells that form the ventral furrow and posterior midgut. Similar to salivary gland cells in 18w mutants, cells of the ventral furrow and posterior midgut in fog mutants do eventually invaginate but in an uncoordinated and delayed fashion. Since 18w and fog mutants have similar invagination defects, and 18W is a receptor protein that activates Rho signaling, whether 18W might be the FOG receptor was examined. This seems unlikely, however, because FOG overexpression within the salivary gland rescues the 18w mutant salivary gland defects. Since fog mutations do not completely eliminate apical constriction during ventral furrow and posterior midgut formation but RhoGEF2 mutations do, it has been argued that additional pathways must regulate apical constrictions via RhoGEF2. However, since 18w RhoGEF2 double mutants have more severe defects than either of the single mutants, 18W is not one of the additional upstream activators of RhoGEF2. Although neither present downstream of FOG nor upstream of RhoGEF2, 18W does appear to be positioned upstream of Sqh phosphorylation since the 18w salivary gland mutant phenotype can be rescued by introducing one copy of a phosphomimetic allele of sqh (Kolesnikov, 2007).
One possible way that 18W might activate Rho signaling is by negatively regulating RhoGAPs. Two RhoGAPs, RhoGAP5A and Cv-c, were identified that function partially redundantly during salivary gland morphogenesis. Embryos defective for both RhoGAPs exhibit invagination and migration defects similar to those observed when 18W is overexpressed within the salivary glands. Comparable defects are also seen upon expression of activated Rho, supporting the role of both RhoGAPs and 18W in Rho signaling (Kolesnikov, 2007).
Although overexpression and genetic interaction data demonstrate that 18W does indeed work in opposition to Cv-c activity, whether 18W actually negatively regulates RhoGAPs or if it controls Rho signaling through an alternate and unknown pathway has yet to to be deciphered. Another RhoGAP, RhoGAPp190, is regulated by the Src family of tyrosine kinases in both mammals and Drosophila. Depending on the site of phosphorylation, mammalian RhoGAPp190 can be either activated or inhibited by Src, while the Drosophila RhoGAPp190 appears to be only negatively regulated by the Drosophila Src homolog, Src64B. Genetic interactions and double mutant analysis with 18w and either Src64B or the other Drosophila Src gene, Src42A, however, suggest 18W does not regulate RhoGAPs via Src kinases in Drosophila (Kolesnikov, 2007).
Considering that 18W is a member of the Toll family of receptors, it might signal through the pathway used by Toll itself. Upon activation by its ligand, Spätzle, Toll signals via the cytoplasmic proteins MyD88, Tube, and Pelle to promote the degradation of the Cactus protein. This degradation releases the sequestered transcription factor Dorsal, allowing it to enter the nucleus and activate transcription. Although both 18w and Toll are expressed in the salivary gland, no evidence was found to suggest that 18W signals through the Toll-pathway or that it functions redundantly with Toll. Zygotic tube, pelle, MyD88, or Toll mutant embryos do not have salivary gland defects and MyD88 does not physically interact with any of the Toll-like receptors except for Toll itself. Similarly, there are no obvious genetic interactions between mutant alleles of 18w and Toll based both on lethality and salivary gland abnormalities (Kolesnikov, 2007).
Similar to the Toll family of receptors, many RhoGAPs and RhoGEFs are found in both mammals and flies. The Drosophila genome encodes 21 RhoGAPs and 20 RhoGEFs but only seven Rho-family GTPases. Since a specific RhoGTPase can be regulated by multiple RhoGAPs, there may be some redundancy in the function of the RhoGAPs. This appears to be the case during salivary gland development. Of the 17 RhoGAPs analyzed by RNAi, by available alleles, or by both, two resulted in distinct defects in the salivary glands. Mutant embryos that lack both of these RhoGAPs have more severe and penetrant gland defects than embryos that only lack one, indicating that the two have redundant roles during gland development (Kolesnikov, 2007).
Since 18W is expressed in several tissues undergoing morphogenesis, it will be interesting to establish whether it is important for the development of additional tissues other than the salivary gland. It will also be interesting to determine whether 18W functions in opposition to the particular RhoGAPs that are active within these other tissues. Overall, since very little is known about pathways controlling RhoGAP activity during apical constriction, identifying additional genes that interact with 18W may prove to be important not only in elucidating RhoGAP regulation but also in understanding the process of epithelial invagination (Kolesnikov, 2007).
Despite significant progress in identifying the guidance pathways that control cell migration, how a cell starts to move within an intact organism, acquires motility, and loses contact with its neighbors is poorly understood. This study shows that activation of the G protein-coupled receptor (GPCR) Trapped in endoderm 1 (Tre1) directs the redistribution of the G protein Gβ as well as adherens junction proteins and Rho guanosine triphosphatase from the cell periphery to the lagging tail of germ cells at the onset of Drosophila germ cell migration. Subsequently, Tre1 activity triggers germ cell dispersal and orients them toward the midgut for directed transepithelial migration. A transition toward invasive migration is also a prerequisite for metastasis formation, which often correlates with down-regulation of adhesion proteins. Uniform down-regulation of E-cadherin causes germ cell dispersal but is not sufficient for transepithelial migration in the absence of Tre1. These findings therefore suggest a new mechanism for GPCR function that links cell polarity, modulation of cell adhesion, and invasion (Kunwar, 2008).
Cell migration plays a important role during a variety of processes such as development, immune defense, and metastasis. The coordinated migration of different kinds of cells in space and time gives rise to the three germ layers and the three-dimensional architecture of different organs and organisms. Cells of the immune system migrate through blood vessels and tissues to reach infected sites; and cancer cells migrate away from their tissues of origin to ectopic places during metastasis. Thus far, the basic mechanisms of cell migration have been elucidated mostly from in vitro studies in solitary cells. Cell migration in living, multicellular organisms, however, is likely much more complex. At the onset of directed migration, cells not only have to acquire motility but also have to be able to perceive specific, directional migration cues. During their journey, migrating cells may be required to detect and interpret multiple, possibly conflicting guidance cues, and must coordinate their adhesion to surrounding cells to reorient, pause, and move in a directed fashion while targets change. Finally, at the end, cells have to know when they have reached their target and cease their motility (Kunwar, 2008).
Significant progress has been made in identifying guidance molecules, receptors, and intracellular mediators that act during directed migration. G protein-coupled receptors (GPCRs) have been widely studied for their role in directional migration. Cells use GPCRs to detect and migrate toward higher concentrations of chemoattractants. Immune cells and germ cells, for example, express the chemokine receptor CXCR4 and follow the distribution of the chemokine SDF1 (stromal cell-derived factor 1) (Kunwar, 2008).
Lymphocytes use sphingosine-1-phosphate receptors to egress from lymphoid tissues, where S1P levels are higher. Despite significant progress in identifying the guidance molecules, receptors, and intracellular mediators that act during directed migration, the cellular and molecular mechanisms that initiate cell migration are only poorly understood. At the start of migration, cells need to acquire motility, may lose cell adhesion with neighboring cells, and are required to gain the ability to respond directionally to external cues. The detailed cellular transformations, the temporal sequence of these events, and the relative influence caused by intrinsic and extrinsic cell information are the focus of this study (Kunwar, 2008).
Drosophila germ cells provide a genetically tractable system to visualize and follow individual germ cells as they start directed migration. The onset of directed germ cell migration coincides with the transepithelial migration of germ cells through the primordium of the future midgut. Evidence for a germ cell autonomous function for transepithelial migration came from the identification of a novel GPCR trapped in endoderm 1. Maternal tre1 RNA is present in germ cells, and tre1 function is required there. General cell motility and the movements of germ cells toward the gonad do not depend on Tre1, which suggests that Tre1 specifically regulates the onset of migration (Kunwar, 2008).
To understand the cellular mechanisms underlying the onset of directed migration, two-photon imaging was used to visualize the cellular transformations that occur in vivo as germ cells migrate through the midgut epithelium. Comparison of wild-type and tre1 mutant germ cells suggests that regulated activation of the Tre1 GPCR controls three phases of early migration: polarization of germ cells, dispersal into individual cells, and transepithelial migration. Germ cell polarization leads to a redistribution of cell-cell adherens proteins, such that Drosophila E-cadherin (DE-cadherin) levels are reduced from the leading edge of the migrating cells and accumulate in the tail region. Tre1 likely signals via the G proteins Gγ1 and Gβ13f as well as Rho-1, since Gβ and Rho-1 protein localization is detected in the tail region, and deletion of their function specifically in germ cells causes the same phenotype as mutation in tre1. These results suggest a novel function for GPCR signaling in initiating cell migration by polarizing the migrating cell. This polarization leads to the redistribution of signaling components and adherens proteins and may trigger cell dispersal and directed migration (Kunwar, 2008).
To visualize germ cell migration in developing embryos, a germ cell-specific expression system, which translates the actin-binding domain of Moesin fused to EGFP under the control of nanos regulatory sequences, was used (Sano, 2005). Germ cells appear motile soon after their formation at the blastoderm stage (stage 5, 2 h and 10 min to 2 h and 50 min after egg laying [AEL]), as they produce small protrusions away from their neighbors. Despite this apparent motility, germ cells only rarely (1-2 germ cells per embryo) separate from their neighbors and migrated directly through the underlying blastoderm cells. Subsequently, during gastrulation (stage 7-8, 3 h to 3 h and 40 min AEL), as germ cells are internalized together with the invaginating posterior midgut primordium, they round up and show less protrusive activity. At stage 9 (3 h and 40 min to 4 h and 20 min AEL), germ cells are found inside the midgut primordium in a tight cluster; they are in close contact with each other and show little contact with the surrounding somatic midgut cells. During this stage, germ cells started to reorganize, changed their shape, and take on a highly polarized morphology. Using electron microscopy, a radial organization of germ cells within the midgut is clearly visible, with the large germ cell nuclei pointed toward the midgut while fine membranous material, apparently corresponding to the tail region, fills the inside of the cluster. This organization orients the leading edge of each germ cell toward the surrounding midgut primordium. Next, the germ cells lose adhesion to each other, and extensions reach from the germ cells toward the midgut epithelium (Kunwar, 2008).
Subsequently, germ cells disperse as they migrated through the midgut primordium to reach the basal side of the midgut cells by stage 10 (4 h and 20 min to 5 h and 20 min AEL). Long cytoplasmic extensions connected germ cells with each other immediately after the onset of transepithelial migration. As germ cells transmigrate through the midgut epithelium, they appear completely individualized, display amoeboid behavior, and are polarized with a broad lagging edge and actin localized at the leading edge. On average, individual germ cells transmigrate the midgut within 40 min from the onset of polarization. Tracking of individual germ cells showed that they disperse radially and transmigrate in all directions through the pocket of the midgut epithelium After transmigration, germ cells reorient on the midgut toward the dorsal side of the embryo, sort into two bilateral groups, and migrate toward the gonadal mesoderm, which forms on either side of the embryo (Kunwar, 2008).
Tre1 encodes an orphan GPCR that is required maternally in germ cells for their migration through the midgut epithelium. In embryos from tre1 mutant females, (hereafter referred to as 'mutant embryos'), germ cells fail to cross the midgut epithelium. This phenotype could result from a defect in the acquisition of motility by germ cells or in their ability to polarize, disperse, or transmigrate. To distinguish between these possibilities, tre1 mutant germ cells were observed live by in vivo imaging. At stage 5, tre1 germ cells show small protrusions and sporadically cross the blastoderm with a similar frequency to the wild type. In striking difference to the wild type, however, the tre1 germ cell cluster does not reorganize at stage 9 and fails to transmigrate to the midgut. Mutant germ cells do not polarize, and remain in a tight, disorganized group in which germ cells fail to interact with the surrounding midgut cells (Kunwar, 2008).
To begin to understand how Tre1, an orphan GPCR, initiates germ cell migration, it was asked whether Tre1 function is mediated by trimeric G protein activation in germ cells. It was found that only a single Gγ (Gγ1) and a single Gβ (Gβ13f) subunit are provided maternally. Loss of maternal Gβ13f or Gγ1 function causes defects in gastrulation, which precluded an immediate analysis of germ cell migration (Kunwar, 2008).
However, it was possible to rescue the gastrulation defect through early zygotic, soma-specific expression of the respective G protein. This genetic manipulation allowed testing for a germ cell-specific role of these G proteins, since early Drosophila germ cells are transcriptionally silent, and germ cells thus depend completely on the maternally provided G proteins. In embryos rescued for the gastrulation defect, Gβ13f mutant germ cells are unable to disperse and migrate through the midgut epithelium, and thus resembled the tre1 phenotype. Gγ1 mutants showed a similar although slightly weaker phenotype likely caused by residual function of the Gγ1N159 allele used, which lacks the C-terminal isoprenylation site required for membrane anchoring. These results suggest that germ cell transepithelial migration requires Tre1-mediated canonical G protein signaling (Kunwar, 2008).
For Gα proteins, focus was placed in particular on the role of the single D. melanogaster Gα12/13A homologue, encoded by concertina (cta), because this subfamily of G proteins has been shown to regulate cell migration and metastatic invasion and to directly interact with E-cadherin and Rho1. Cta protein is present in the germ cells and maternal loss of cta causes a gastrulation defect similar to Gβ13f and Gγ1. Again, it was possible to rescue the gastrulation phenotype by early, somatic Cta expression, as described for Gγ1 and Gβ13f. In contrast to findings with Gβ and Gγ mutants, however, cta mutant germ cells migrated normally to the gonad. To confirm this result, mutant cta germ cells derived from cta mutant mothers were transplanted into wild-type embryos. It was found that cta germ cells migrated to the gonad with similar efficiency as transplanted wild-type control germ cells. Thus, Gα12/13 does not act as the sole mediator of Tre1 GPCR activation. Analysis of the available mutants in other Gα proteins did not reveal a single Gα protein that mediates the Tre1 signal, which perhaps indicates that redundant or overlapping functions of Gα proteins act downstream of Tre1 (Kunwar, 2008).
The observation that both Gβ13f and Gγ1 are required for germ cell dispersal and transepithelial migration suggests that Tre1 function in germ cells is mediated by a G protein-dependent pathway, akin to the requirement for GPCR signaling seen during the directed migration of Dictyostelium discoideum amoeba and in neutrophils toward a chemokine gradient. To determine how Tre1 signaling may affect downstream components, the localization of Gβ13f protein was analyzed, as well as the localization of Rho1, which has been shown to affect germ cell transepithelial migration in wild-type and tre1 mutant germ cells. It was found that Gβ13f and Rho1 proteins were localized uniformly along the cell membrane of wild-type germ cells at the blastoderm stage (Kunwar, 2008).
At stage 9, as wild-type germ cells polarize, Gβ13f and Rho1 proteins are down-regulated along the germ cell membranes facing the midgut, and become highly enriched in the tail region. In early germ cells, Gβ13f and Rho1 proteins are uniformly distributed in tre1 mutants similar to the wild type; in contrast to the wild type, however, this uniform distribution persists during stage 9. These results suggest that Tre1 receptor activation leads to germ cell polarization in part by causing the redistribution of downstream signaling molecules away from the leading edge and accumulation in the tail (Kunwar, 2008).
tre1 mutant germ cells fail to disperse at the onset of the migration, which suggests that tre1 regulates adhesion molecules in germ cells. DE-cadherin is a good candidate, since it is deposited maternally in the early embryo. The role of DE-cadherin was first tested in the adhesion of wild-type germ cells. For this analysis, a newly identified partial loss-of-function allele of Drosophila E-cadherin encoded by the shotgun (shg) gene, which allows normal oogenesis, was used. In embryos derived from shgA9-49 mutant ovaries, germ cells did not organize into a radial cluster. Instead, germ cells separated from one another prematurely, at early stage 8 (3 h and 10 min to 3 h and 40 min AEL) compared with stage 10 in the wild type (4 h and 20 min to 5 h and 20 min AEL). This dispersal phenotype was observed in embryos from homozygous germ line clones, in which embryonic patterning defects were rescued by a wild-type shg+ copy from the father (M-Z+). This suggests that DE-cadherin is required autonomously in germ cells, since they are transcriptionally quiescent and thus likely depend exclusively on maternally contributed DE-cadherin. These results indicate that DE-cadherin is required for germ cell-germ cell adhesion in the wild-type embryo (Kunwar, 2008).
To understand how DE-cadherin is regulated in the dispersal step, the distribution of DE-cadherin was analyzed in wild-type germ cells. It was found that DE-cadherin as well as α and β catenins were initially uniformly present along the germ cell membrane but become enriched in the tail region during germ cell polarization (Kunwar, 2008).
In stark contrast, DE-cadherin remained uniformly distributed along the cell surface in tre1 mutant embryos. To quantitate the levels, the fluorescent intensity of DE-cadherin staining on the cell membrane of wild-type and tre1 mutant germ cells was compared. It was found that DE-cadherin is distributed uniformly and that levels are similar in wild-type and mutant germ cells at stage 5, before migration, whereas the levels are reduced along the leading edge membrane of wild-type germ cells compared with tre1 mutant germ cells at stage 9. These results suggest that tre1 activation leads to a reduction of DE-cadherin along the leading edge and restricts it in the tail region (Kunwar, 2008).
In shg mutants, early dispersal of germ cells does not lead to premature migration through the midgut, as would be expected if release of germ cell-germ cell adhesion via E-cadherin was the only trigger for transepithelial migration. Instead, shg mutant germ cells moved through the midgut slightly later during stage 10 than wild-type germ cells. This delay phenotype is less penetrant compared with the precocious dispersal phenotype and could be caused by an impaired ability of the shgA9-49 mutant germ cell to migrate at this and subsequent stages (Kunwar, 2008).
To test directly if Tre1 acts via DE-cadherin in transepithelial migration, embryos were generated that lacked tre1 and maternal shgA9-49 function. The germ cells in these embryos dispersed early, thus displaying a phenotype similar to shgA9-49 mutants; 80% of tre1, shgA9-49 double mutant embryos showed precocious dispersal as opposed to 0% in the tre1 mutant embryos (Kunwar, 2008).
However, even these dispersed germ cells were not able to transmigrate through the midgut in tre1, shgA9-49 double mutant embryos, thereby resembling tre1 mutant germ cells. This suggests that loss of germ cell-germ cell contact may not be sufficient to trigger transepithelial migration. To test this idea further, germ cell-germ cell contact was disrupted independent of E-cadherin function by reducing the germ cell number. Alleles of the maternal effect gene tudor (tud) were used to reduce the number of germ cells in the embryo to a single germ cell. Such single, tud mutant germ cells migrated through the midgut and invariably reached the gonad. These germ cells had normal morphology and appeared polarized. Next, mutant embryos lacking both tre1 and maternal tud were analyzed. In the absence of tre1, single germ cells were left inside the midgut and did not migrate to the gonad. Thus, whereas germ cell individualization requires Tre1-mediated down-regulation of DE-cadherin, Tre1 activity has additional roles in transepithelial migration (Kunwar, 2008).
This study has used live imaging to explore the mechanisms by which germ cells acquire motility and traverse the midgut epithelium. It was found that before transepithelial migration, germ cells polarize toward the midgut and down-regulate E-cadherin from the leading edge and accumulate E-cadherin in the tail region. This polarization requires Tre1 GPCR activity. It is proposed that GPCR-mediated polarization triggers germ cell dispersal and orients germ cells toward the midgut for directed transepithelial migration (Kunwar, 2008).
A requirement for GPCR signaling during the directed migration toward a chemokine gradient has been described in detail in D. discoideum amoeba and in mammalian neutrophils. The events underlying signal transduction leading to the polarization of migrating cells have been worked out extensively in these cells. The first localized response to receptor activation is the enrichment of the activated G protein βγ subunits, which results in the activation of phosphoinositide 3 (PI3) kinase. As a consequence of chemokine sensing, the PI3 kinase product phosphatidylinositide 3,4,5-tris phosphate (PIP3) becomes localized to the leading edge, and the phosphatase PTEN (phosphatase and tensin homolog) moves to the lagging edge in a Rho dependent manner (for review see Affolter, 2005). These signaling events organize the cytoskeleton leading to cellular polarization and directional movement. The current studies suggest a new mechanism by which GPCR signaling initiates directed cell migration. Activation of Tre1 causes a redistribution of G protein β, the GTPase Rho1, DE-cadherin, and other adherens junction components to a small region in the tail of the germ cells. The decrease in DE-cadherin from the leading edge of germ cells causes a loss of adhesion across the broad leading edge of the germ cells and causes germ cell polarization toward the midgut. This localization event may thereby convert an adherent group of cells into directionally migrating individuals. Tre1 belongs to a family of GPCRs that includes Moody in D. melanogaster and GPR84 in mouse and human (Bainton, 2005; Bouchard, 2007). Based on the results with Tre1, this family may act to regulate cellular polarity and adhesion, a view in line with the proposed function of Moody in epithelial morphology at the blood-brain barrier, and with GPR84, which was recently described to be up-regulated in microglia upon infection (Kunwar, 2008).
How could Tre1 activation cause DE-cadherin redistribution? Regulation of E-cadherin is widely attributed to play an important role in metastasis and in the epithelial-to-mesenchymal transition that occurs during gastrulation and neural crest migration. In these systems, it has been proposed that E-cadherin is regulated by transcriptional repression or by Gα12/13-mediated uptake and turnover. The data suggest the presence of a different mode of regulation, since neither transcriptional regulation nor Gα12/13 activity seem to be required for the regulation of DE-cadherin in germ cells. An attractive mechanism for DE-cadherin down-regulation could be the control of its endocytosis by Tre1. During zebrafish gastrulation, Rab GTPases have been shown to control E-cadherin turnover and the adhesion of mesendodermal cells (Ulrich, 2005). A role for Rab proteins in germ cell migration has yet to be demonstrated. This study found the same localization pattern for Gβ13f, Rho1, and DE-cadherin in the wild type, and this pattern is disrupted in tre1 mutant germ cells. This suggests a role for G protein and Rho1 activation in the polarization of DE-cadherin in germ cells (Kunwar, 2008).
Tre1 also affects transepithelial migration independently of global DE-cadherin regulation. Uniform down-regulation of DE-cadherin or loss of germ cell-germ cell contact in single cells are neither sufficient to trigger precocious transepithelial migration in the wild type nor able to suppress the tre1 transepithelial migration phenotype. One possibility is that the localized activation of Tre1 and polarized down-regulation of DE-cadherin at the leading edge would orient germ cells radially toward the midgut. This radial orientation would allow germ cells to respond to additional guidance cues required for directed transepithelial migration. Although these additional guidance cues may not depend on DE-cadherin, they require G protein signaling and Tre1 (Kunwar, 2008).
A function for E-cadherin in controlling adhesion and migration has been studied extensively in the progression of tumor metastasis and the development of epithelial-mesenchymal transitions (EMTs). Cells undergoing metastasis and EMTs express lower levels of E-cadherin, and the loss of E-cadherin promotes invasion of tumor cells. The loss of E-cadherin in these cases promotes the disruption of E-cadherin-mediated cell adhesion between epithelial cells, allowing these cells to spread and migrate, and is often triggered through induction of the transcriptional repressors Twist and Snail in response to inductive signals. However, in the case of germ cell dispersal, the effect of Tre1 on DE-cadherin is not transcriptional because DE-cadherin is provided maternally in the germ cells. These data suggest that Tre1 GPCR signaling might regulate the turnover or cellular distribution of DE-cadherin-mediated adhesion complexes in a polarized fashion. It is possible that in addition to transcriptional mechanisms, such a polarized regulation also functions during EMT and metastasis (Kunwar, 2008).
Morphogenesis is largely driven by changes in the shape of individual cells. However, how cell shape is regulated in developing animals is not well understood. This study shows that the onset of TGFbeta/Dpp signaling activity correlates with the transition from cuboidal to columnar cell shape in developing Drosophila melanogaster wing disc epithelia. Dpp signaling is necessary for maintaining this elongated columnar cell shape and overactivation of the Dpp signaling pathway results in precocious cell elongation. Moreover, evidence is provided that Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, it was demonstrated that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. These results identify a cell-autonomous role for Dpp signaling in promoting and maintaining the elongated columnar shape of wing disc cells and suggest that Dpp signaling acts by regulating Rho1 and MRLC (Widmann, 2009).
Cell extrusion was observed when Dpp signaling was locally reduced in tkva12 bsk- clones, but not when it was reduced throughout the dorsal compartment by expression of Dad. This indicates that cell extrusion is a consequence of the sharp boundary of Dpp signaling at the clone border. One of the first morphological consequences of the loss of Dpp signaling in tkva12 bsk- clones was the apical constriction of mutant cells and surrounding control cells. Apical constriction correlated with increased staining intensities of F-actin and P-MRLC, a marker for active non-muscle myosin II, at the apicolateral side of tkva12 bsk- and neighboring wild-type cells. The formation of a similar actin-myosin ring has been previously demonstrated during the extrusion of apoptotic cells, and it has been proposed that contraction of this ring squeezes cells out of the epithelium. It is currently unclear whether these increased staining intensities reflect an increase in the total amount of F-actin and P-MRLC in tkva12 bsk- mutant clones, or whether they are instead merely a consequence of the apical constriction of cells. Nevertheless, these findings are consistent with the view that contraction of an actin-myosin ring might contribute to the extrusion of tkva12 bsk- cells. Apical cell constriction was paralleled with cell shortening along the apical-basal axis. Based on the observation that reduction in Dpp signaling throughout the wing disc pouch resulted in apical-basal cell shortening, but not in apical cell constriction, it is speculated that cell shortening, and thus the formation of an inappropriate cell shape, might be an initial event leading to the extrusion of tkva12 bsk- cells. If so, cell extrusion might not represent a specific response to eliminate slow-growing or apoptotic cells, but rather represents a general response to inappropriate cell function or morphology. In the wild type, cell extrusion might be instrumental in maximizing tissue fitness by removing cells with inappropriate function or morphology (Widmann, 2009).
The basal membrane of tkva12 bsk- cells and neighboring control cells, identified by PSβ-integrin labeling, became apposed. Since this led to a reduction in the lateral contact between mutant and neighboring control cells, this apposition might help to dislodge tkva12 bsk- cells from the remaining epithelium, and thereby, might aid the extrusion process. It is also noted that extruded tkva12 bsk- cells displayed features reminiscent of epithelial-to-mesenchymal transition (EMT). In particular, a strong decrease in E-cadherin, a hallmark of EMT and actin-rich processes were observed in extruded tkva12 bsk- cells. Interestingly, a role for Dpp/BMPs in preventing EMT has also been identified in vertebrates. Mouse BMP7, which is related to Dpp, for example, is required for counteracting EMT associated with renal fibrosis. Decreased E-cadherin levels have also recently been reported following the extrusion of cells deficient for C-terminal Src kinase from Drosophila epithelia, indicating that this might be a more common consequence of cell extrusion (Widmann, 2009).
Reduced apical-basal cell length was observed when Dpp signaling was severely reduced, either in clones or throughout the wing disc pouch; however, apical cell constriction, fold formation and cell extrusion were only detected by clonal reduction of Dpp signaling. Instead, cells were apically widened and did not extrude when Dpp signaling was reduced throughout the dorsal compartment. These experiments therefore allowed the effects of sharp boundaries of Dpp signaling at clone borders to be separated from cell-autonomous functions of Dpp signaling. They demonstrate that the cell-autonomous function of Dpp signaling is not to prevent apical cell constriction, folding and cell extrusion, but rather to maintain proper columnar cell shape. Moreover, three further observations suggest that Dpp signaling has an instructive role that drives cell elongation. (1) In the wild type, an increase in Dpp signal transduction activity correlated with apical-basal cell elongation in second instar larval discs. (2) In wing discs of late third instar larvae, Dpp signal transduction activity correlated with apical-basal cell length along the anteroposterior axis. (3) Activation of Dpp signaling, by expressing the constitutively active Dpp receptor TkvQ-D, resulted in precocious cell elongation and apical cell narrowing during early larval development. These findings indicate that Dpp signaling is an important trigger for the cuboidal-to-columnar transition in cell shape that occurs during mid-larval development (Widmann, 2009).
How does Dpp signaling promote the apical-basal elongation of wing disc cells? Compartmentalization of Rho1 activity has been recognized as being important for shaping cells and tissues. In the wild-type wing disc, Rho1 protein is enriched and the activity of the Rho1 sensor is increased at the apicolateral side, and more moderately at the basal side, of elongated cells. By contrast, Rho1 activity is more uniform in cuboidal cells, and overexpression of RhoGEF2, which leads to uniform distribution of this protein and presumably also uniform Rho1 activity, resulted in a cuboidal cell shape. Rho1, when present at the apicolateral side of cells, might therefore have a function in stabilizing or promoting cell elongation. Since the apicolateral increase in Rho1 sensor activity correlated with an increase of P-MRLC at a similar location, this function of Rho1 might be mediated by myosin II. The observation that a decrease in the bulk of Rho1 activity, either through expression of Rho1N19 or rho1dsRNA, resulted in cell elongation rather than in cell shortening, further suggests that the compartmentalization of Rho1 activity is important for shaping wing disc cells. Future studies will need to examine the morphogenetic consequences of locally modulating the activity of Rho1 (Widmann, 2009).
The results provide strong evidence for a functional link between Dpp signaling and Rho1-myosin II. Shortening of cells with compromised Dpp signaling could be rescued by a decrease in Rho1 or MRLC activity. In particular, the expression of MbsN300, an activated form of a subunit of myosin light chain phosphatase, which in wild-type wing discs did not significantly alter cell length, did rescue the shortening of Dpp-compromised cells. This indicates a specific interaction between Dpp signaling and Mbs-myosin II. The data further suggest that Dpp signaling controls apical-basal cell length by compartmentalizing Rho1 protein abundance and/or activity. (1) In late third instar wing discs, apicolateral enrichment of Rho1 protein and Rho1 sensor activity directly correlated with the local level of Dpp signal transduction activity. (2) Rho1 protein abundance and Rho1 sensor activity were decreased at the apicolateral side of cells when Dpp signal transduction was compromised by expression of Dad. (3) Rho1 protein and Rho1 sensor activity were increased at the apicolateral side and also at the basal side of cells when Dpp signal transduction was activated during early development by expression of TkvQ-D (Widmann, 2009).
Local activation of Rho1 and myosin II can lead to contraction of actin-myosin filaments, which can increase the cortical tension that is important for the shaping of cells during various developmental processes. By compartmentalizing Rho1 activity, Dpp signaling might promote both apical-basal cell elongation and apical cell narrowing. An increase in tension at the apicolateral cell cortex might promote apical cell narrowing. At the same time, a relative decrease in cortical tension laterally, compared with that on the apicolateral side, might allow cells to elongate through intrinsic cytoskeletal forces and/or extrinsic forces imposed by the growth of the epithelium. In this model, Dpp signaling directs the cuboidal-to-columnar shape transition of wing disc cells by increasing the Rho1 and myosin II activities at the apicolateral side of cells. The local increase of Rho1 and myosin II activities might then shift the balance of tension between the apicolateral cell cortex and the lateral cell cortex towards an increased tension at the apicolateral cell cortex (Widmann, 2009).
The results identify a Dpp-Brk-Rho1-myosin II pathway controlling cell shape in the wing disc epithelium. The elimination of Brk function in mad- mutant cells allowed these cells to maintain a normal columnar cell shape, indicating that Dpp controls epithelial morphogenesis through repression of Brk. Since Brk acts as a transcriptional repressor, the link between Brk and Rho1 is most probably established through an unknown Brk-repressible gene. The identification of genes transcriptionally repressed by Brk will thus be important for determination of how Dpp signaling controls Rho1 and thereby, epithelial cell shape. The finding that Dpp signaling has a cell-autonomous morphogenetic function indicates that Dpp signaling provides a connection between cell-fate specification, cell growth and the control of morphogenesis. It, thereby, might help to facilitate the coordination of these processes during wing disc development (Widmann, 2009).
Given the evolutionary conserved functions of Rho and myosin II, it is anticipated that the mechanisms regulating columnar cell shape, which are describe in this study for the wing disc, will also operate in a wide range of other epithelia. Moreover, the role of TGFβ/Dpp signaling in patterned morphogenesis appears to be conserved in vertebrates, raising the possibility that Rho and myosin II are common mediators of TGFβ/Dpp signaling (Widmann, 2009).
Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of any given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. This study isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis it was observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. This work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. Pkn was shown to negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. It is proposed that Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase (Ferreira, 2014).
Drosophila pole (germ) plasm contains germline and abdominal determinants. Its assembly begins with the localization and translation of oskar (osk) RNA at the oocyte posterior, to which the pole plasm must be restricted for proper embryonic development. Osk stimulates endocytosis, which in turn promotes actin remodeling to form long F-actin projections at the oocyte posterior pole. Although the endocytosis-coupled actin remodeling appears to be crucial for the pole plasm anchoring, the mechanism linking Osk-induced endocytic activity and actin remodeling is unknown. This study reports that a Golgi-endosomal protein, Mon2, acts downstream of Osk to remodel cortical actin and to anchor the pole plasm. Mon2 interacts with two actin nucleators known to be involved in osk RNA localization in the oocyte, Cappuccino (Capu) and Spire (Spir), and promotes the accumulation of the small GTPase Rho1 at the oocyte posterior. This study also found that these actin regulators are required for Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. It is proposed that, in response to the Osk-mediated endocytic activation, vesicle-localized Mon2 acts as a scaffold that instructs the actin-remodeling complex to form long F-actin projections. This Mon2-mediated coupling event is crucial to restrict the pole plasm to the oocyte posterior cortex (Tanaka, 2011).
In many cell types, asymmetric localization of specific RNAs and proteins is essential for exhibiting proper structure and function. These macromolecules are transported to their final destinations and anchored there. This latter step is particularly important for the long-term maintenance of cell asymmetry. A genetically tractable model for studying intracellular RNA and protein localization is the assembly of the pole (germ) plasm in Drosophila oocytes and embryos. The pole plasm is a specialized cytoplasm that contains maternal RNAs and proteins essential for germline and abdominal development. It is assembled at the posterior pole of the oocyte during oogenesis. Drosophila oogenesis is subdivided into 14 stages, with pole plasm assembly starting at stage 8. The functional pole plasm is assembled by stage 13, stably anchored at the posterior cortex of the oocyte and later inherited by the germline progenitors (pole cells) during embryogenesis (Tanaka, 2011).
Pole plasm assembly begins with the transport of oskar (osk) RNA along microtubules to the posterior pole of the oocyte. There, the osk RNA is translated, producing two isoforms, long and short Osk, by the alternate use of two in-frame translation start sites. Although short Osk shares its entire sequence with long Osk, the isoforms have distinct functions in pole plasm assembly. Downstream, short Osk recruits other pole plasm components, such as Vasa (Vas), to the oocyte posterior, presumably through direct interactions. By contrast, long Osk prevents pole plasm components from diffusing back into the cytoplasm. Intriguingly, embryonic patterning defects are caused by either the ectopic assembly of pole plasm [elicited by Osk translation at the oocyte anterior directed by the osk-bicoid (bcd) 3'UTR] or the leakage of pole plasm activity into the bulk cytoplasm (induced by overexpressing osk). Thus, the pole plasm must be anchored at the posterior cortex for proper embryonic development (Tanaka, 2011).
Short and long Osk also differ in their subcellular distributions. Short Osk is located on polar granules, specialized ribonucleoprotein aggregates in the pole plasm, and long Osk is associated with endosome surfaces. Intriguingly, the oocyte posterior, where endocytosis is increased, is highly enriched with markers of early, late and recycling endosomes (Rab5, Rab7 and Rab11, respectively). osk oocytes, however, do not maintain either the accumulation of endosomal proteins or the increased endocytic activity at the posterior. Furthermore, the ectopic expression of long Osk at the anterior pole of the oocyte results in the anterior accumulation of endosomal proteins along with increased endocytosis. Thus, long Osk regulates endocytic activity spatially within the oocyte (Tanaka, 2011).
The endocytic pathway has two separate roles in pole plasm assembly (see Tanaka, 2008). First, it is required for the sustained transport of osk RNA by maintaining microtubule alignment. For example, in oocytes lacking Rabenosyn-5 (Rbsn-5), a Rab5 effector protein essential for endocytosis, the polarity of the microtubule array is not maintained, disrupting osk RNA localization. A similar defect occurs in hypomorphic rab11 oocytes. Second, the endocytic pathway acts downstream of Osk to anchor the pole plasm components. In rbsn-5 oocytes aberrantly expressing osk at the anterior, Osk and other pole plasm components diffuse from the anterior cortex into the ooplasm, indicating that endocytic activity is essential for stably anchoring them to the cortex (Tanaka, 2011).
The endocytic pathway is thought to anchor pole plasm components by remodeling the cortical actin cytoskeleton in response to Osk. Pole plasm anchoring is sensitive to cytochalasin D, which disrupts actin dynamics, and requires several actin-binding proteins, such as Moesin, Bifocal and Homer. Osk induces long F-actin projections emanating from cortical F-actin bundles at the posterior pole of the oocyte. Ectopic F-actin projections are also induced at the anterior pole when long Osk is misexpressed at the oocyte anterior (Tanaka, 2008). However, when the endocytic pathway is disrupted, F-actin forms aggregates and diffuses into the ooplasm, along with pole plasm components (Tanaka, 2008). These observations led to the hypothesis that Osk stimulates endocytosis, which promotes actin remodeling, which in turn anchors the pole plasm components at the posterior oocyte cortex. However, the molecular mechanism linking Osk, the endocytic pathway and actin remodeling is still unknown (Tanaka, 2011).
This study has identified Mon2, a conserved Golgi/endosomal protein, as an essential factor in anchoring pole plasm components at the oocyte posterior cortex. Oocytes lacking Mon2 did not form F-actin projections in response to Osk, but neither did they exhibit obvious defects in microtubule alignment or endocytosis. It was also shown that two actin nucleators that function in osk RNA localization in the oocyte, Cappuccino (Capu) and Spire (Spir), play an essential role in a second aspect of pole plasm assembly: the Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. Finally, it was found that Mon2 interacts with Capu and Spir, and promotes the accumulation of the small GTPase Rho1 at the oocyte posterior. These data support a model in which Mon2 acts as a scaffold, linking Osk-induced vesicles with these actin regulators to anchor the pole plasm to the oocyte cortex (Tanaka, 2011).
To learn more about how the pole plasm is assembled and anchored during Drosophila oogenesis, a germline clone (GLC) screen was conducted for ethyl methanesulfonate-induced mutations showing the abnormal localization of GFP-Vas, a fluorescent pole plasm marker (Tanaka, 2008). In a screen targeting chromosomal arm 2L, six mutants were identified that mapped into a single lethal complementation group, which was named no anchor (noan). In wild-type oocytes, GFP-Vas was first detectable at the posterior pole at stage 9, where it remained tightly anchored, with a progressive accumulation of protein until the end of oogenesis. In the noan GLC oocyte, GFP-Vas initially localized to the oocyte posterior during stages 9-10a, but its level gradually decreased, becoming undetectable in the mature oocyte. Similarly, the localization of Staufen (Stau) and Osk at the posterior pole, which occurs prior to that of Vas, was not maintained in the noan oocytes. Although the noan oocytes developed into normal-looking mature oocytes, the eggs were fragile and did not develop. Therefore, it was not possible to analyze the effects of the loss of maternal noan activity on the formation of abdomen or germ cells in embryos. Nevertheless, these results indicated that noan mutations cause defective anchoring of pole plasm components to the posterior pole of the oocyte (Tanaka, 2011).
The genetic mapping and subsequent DNA sequencing of the noan locus revealed that all the noan alleles had a nonsense mutation in CG8683, which encodes a homolog of a budding yeast protein, Mon2p, also termed Ysl2p. noan is referred to as mon2. All the mon2 alleles showed identical defects in the posterior localization of GFP-Vas with full penetrance. As the mutation in the mon2K388 allele was the most proximal to the translational initiation site among the six alleles identified, mon2K388 was primarily used to characterize the mon2 phenotype (Tanaka, 2011).
em>Drosophila Mon2 consists of 1684 amino acids and represents a highly conserved protein among eukaryotes. It has two Armadillo (ARM) repeat domains, which are likely to mediate protein-protein interactions, and a DUF1981 domain, which is functionally uncharacterized. In budding yeasts, mon2 (ysl2) was identified as a gene whose mutation increases sensitivity to the Na+/H+ ionophore monensin, and is synthetically lethal with a mutation in ypt51, which encodes a Rab5 homolo. Yeast Mon2p (Ysl2p) forms a large protein complex on the surface of the trans-Golgi network and early endosomes, and it is proposed to act as a scaffold to regulate antero- and retrograde trafficking between the Golgi, endosomes and vacuoles (Tanaka, 2011 and references therein).
This study found that Capu and Spir act together to form long F-actin projections and to anchor pole plasm components at the oocyte cortex, and that Mon2 is essential to these processes. Capu and Spir also regulate the timing for initiating ooplasmic streaming and microtubule array polarization in the oocyte (Qualmann, 2009). However, the polarity of microtubule arrays was not affected in mon2 oocytes. Therefore, Mon2 is not always required for Capu and Spir to function. Rather, it appears to regulate specifically these actin nucleators through the Osk-induced endocytic pathway (Tanaka, 2011).
Mon2 is required for the formation of Osk-induced long F-actin projections at the oocyte posterior. Interestingly, ectopic overexpression of Osk at the anterior pole in the mon2 oocyte induced granular, albeit faint, F-actin structures, indicating that Osk-induced actin remodeling does not totally cease in the mon2 oocyte. Ectopic Osk at the anterior of capu spir double-mutant oocytes also induced faint F-actin granules in the cytoplasm. Thus, additional, as yet uncharacterized, actin regulators appear to function in response to Osk. Notably, two actin-binding proteins, Bifocal and Homer, play redundant roles in anchoring Osk to the cortex. Although the precise roles of Bifocal and Homer in this process remain elusive, they might function independently of Mon2 (Tanaka, 2011).
Oocytes lacking Rab5 showed disrupted posterior cortical F-actin bundles, which was suppressed by the simultaneous loss of Osk. These results reconfirm that the endocytic pathway needs intact Osk function for actin remodeling (Tanaka, 2008). This study also found that the F-actin disorganization in rab5 oocytes is Mon2-dependent. Therefore, Mon2 can facilitate actin remodeling even when Rab5 is absent, but endosomal trafficking, in which Rab5 is involved, is crucial for regulating Mon2. Mammalian Rab5 is also involved in actin remodeling. For example, Rac1 GTPase, a regulator of F-actin dynamics, is activated by Rab5-dependent endocytosis, and the local activation of Rac1 on early endosomes and its subsequent recycling to the plasma membrane spatially regulate actin remodeling. Thus, local endocytic cycling provides a specific platform for actin remodeling in a wide range of cell types (Tanaka, 2011).
There is growing evidence that endosomes act as multifunctional platforms for many types of molecular machinery. Intriguingly, Mon2 is located on the Golgi and endosomes, without entirely accumulating at the oocyte posterior. It is therefore proposed that the Osk-induced stimulation of endocytic cycling at the oocyte posterior leads to the formation of specialized vesicles, which instruct a fraction of Mon2 to regulate the activity of Capu, Spir and Rho1 to form long F-actin projections from the cortex. Although the functional property of Osk-induced endocytic vesicles has yet to be ascertained, long Osk is known to associate with the surface of endosomes. Therefore, long Osk might modify endosome specificity to recruit and/or stabilize the machineries responsible for actin remodeling (Tanaka, 2011).
Oocytes lacking Mon2 can mature without morphological abnormalities, but their eggs are nonviable. Furthermore, Drosophila mon2 mutations show recessive lethality, indicating that Mon2 has additional functions in somatic cell development. It might function in regulating vesicle trafficking or protein targeting, as reported in yeasts. As vesicle trafficking is often linked with establishing and maintaining cell polarity, it is an attractive idea that Mon2 might regulate the polarity protein localization and/or mediate the signal transduction for cell polarization in somatic cells, as well as in germ cells. Supporting this idea, a Mon2 homolog in C. elegans has been implicated in the asymmetric division of epithelial stem cells (Kanamori, 2008; Tanaka, 2011 and references therein).
It has been proposed that long Osk localizes to the endosomal membrane and generates a positive-feedback loop for cortical anchoring of pole plasm components. Osk is also thought to generate another positive-feedback loop to maintain the polarity of microtubule arrays, and the process appears to be endosomal protein-dependent. Although Rbsn-5 is required for both feedback loops, Mon2 acts specifically in the loop regulating actin remodeling for pole plasm anchoring, indicating that the two feedback loops are regulated by distinct mechanisms. The endocytic pathway consists of multiple vesicle trafficking steps, including endocytosis, endosomal recycling, late-endosomal sorting and endosome-to-Golgi trafficking. Therefore, determining which steps in the endocytic pathway are used by the two Osk-dependent positive-feedback loops is an important aim for future exploration (Tanaka, 2011).
The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. A unique embryonic tracheal tubulogenesis phenotype has been described caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. This study demonstrates that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, it was shown that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. It was also shown that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways (Jeon, 2012).
The Drosophila tracheal system is an intricate branched network of air-filled tubes that delivers oxygen to tissues. Tube formation in the tracheal system involves complex morphogenetic events that differ between tube types. Multicellular tubes have lumens that are surrounded by the apical surfaces of several cells. Unicellular tubes are formed by rolling up of single cells to form junctions with themselves. Seamless tubes are intracellular structures within terminal cells. Many genes have been identified that affect the formation and morphology of tracheal tubes (Jeon, 2012).
The absence of the two Type III RPTPs, Ptp4E and Ptp10D, changes the geometries of the tubes in unicellular and terminal branches, so that they form spherical cysts in place of continuous tubular lumen. The phenotype involves a loss of negative regulation of the Egfr RTK, and Ptp10D physically associates with Egfr (Jeon, 2009; Jeon, 2012).
One of the mammalian Ptp4E/Ptp10D orthologs, PTPRJ (DEP-1), is a direct regulator of multiple growth factor receptor TKs (Arora, 2011; Berset, 2005; Chabot, 2009; Lampugnani, 2003; Kappert, 2007; Palka, 2003; Tarcic, 2009). This led to a test of the hypothesis that Btl and Pvr, the other two Drosophila growth factor receptor TK orthologs that are expressed in embryonic tracheae, are also required for the Ptp4E Ptp10D phenotype (Jeon, 2012).
A quantitative analysis of cyst size at the TC/LT junction showed that tracheally expressed DN mutants of Egfr, Btl, and Pvr all suppress the Ptp4E Ptp10D phenotype almost to wild-type, suggesting that dysregulation of all three RTKs is required for the replacement of linear tubes by spherical cysts. Also, CA mutants of each RTK enhance the phenotype, producing enlarged cysts. These effects cannot be produced by all RTKs, since expression of a CA mutant of InR did not enhance the phenotype (Jeon, 2012).
If the Ptp4E Ptp10D cyst phenotype is the consequence of simultaneous deregulation of all three RTKs, it might be possible to generate the phenotype in a wild-type background by expressing multiple RTK CA mutants. This did not work, even when all three CA mutants were expressed at once. Possible explanations include: 1) because PTP activity normally dominates over RTK activity, the effect on PTyr levels of removing negative regulation by the RPTPs is much greater than that produced by expressing CA RTK mutants in the presence of the RPTPs; 2) other TKs are important for the phenotype, and their activities must also be increased; 3) the RPTPs have PTP-independent activities as adhesion molecules, and generation of the phenotype requires both elevation of RTK activity and the absence of the PTP-independent functions of the RPTPs (Jeon, 2012).
Whether Ptp4E and Ptp10D both regulate all three RTKs was investigated. If they have specificity for particular RTKs, one might be able to generate the cyst phenotype by removing only one RPTP in the presence of a CA RTK mutant. Such combinations were made for Egfr (Ptp4E, Btl>Egfr-CA and Ptp10D, Btl>Egfr-CA), but neither of them had cysts. Since the PTP domains of Ptp4E and Ptp10D are 89% identical, it is likely that they have the same enzymatic targets. The idea that the RPTPs have redundant functions is also consistent with the observation that Ptp4E and Ptp10D single mutants have no detectable phenotypes, while the double mutant is lethal and has both tracheal (Jeon, 2009) and nervous system defects (Jeon, 2008: Jeon, 2012).
When each of the DN RTK mutants was expressed together with a CA mutant of one of the other RTKs in the Ptp4E Ptp10D background, the DN mutant was now unable to suppress the phenotype back to wild-type. Instead the phenotype returned to the strength of unmodified Ptp4E Ptp10D mutants. This shows that if the activity of one RTK is sufficiently elevated, it can replace the requirement for another RTK. Thus, none of the three RTKs is uniquely required to generate the phenotype. Rather, the formation of tubes vs. cysts is controlled by the total amount of activity of certain RTKs in tracheal cells. This implies that the downstream pathways whose increased activity causes cyst formation use SH2-domain effectors that can bind to PTyr sites on any of the three RTKs (Jeon, 2012).
It is interesting that the three RTKs can substitute for each other in regulating the tube vs. cyst decision when they are deregulated by loss of the RPTPs, since the RTKs do not seem to have interchangeable activities in wild-type tracheal cells (or in other tissues). Loss or gain of Btl function produces defects in primary, secondary, and terminal tracheal branching. Loss of Egfr function produces a much more subtle tracheal phenotype affecting tissue integrity. Maintenance of tissue integrity requires signaling through the MAP kinase pathway downstream of Egfr, but is unaffected by reduction of MAP kinase signaling downstream of Btl (Jeon, 2012).
These findings can be explained by the fact that growth factor receptor TKs are usually in an inactive state, due to insufficient levels of ligands and to negative regulation by PTPs. They become active only when they come into contact with elevated levels of their ligands at specific times and places. The activities of Type III RPTPs that dephosphorylate the RTKs might also be transiently reduced at some of these times and places, possibly through interactions of their XC domains with as yet unidentified ligands. As a consequence of the tight control of RTK activity, only those downstream signaling pathways that are most responsive to a particular RTK are likely to be activated by that RTK at any time in wild-type embryos, and the outcomes of signaling through these pathways may also be controlled by the subcellular distributions of ligands, RTKs, RPTPs, and downstream effectors. By contrast, in the absence of Ptp4E and Ptp10D, basal levels of RTK ligands may be able to drive all of the growth factor RTKs to a high level of activity, resulting in strong signaling through all of the downstream pathways they can control. Loss of negative regulation might also cause delocalization of signaling, so that effectors whose activity is normally restricted to particular parts of the cell become activated in a cell-wide manner. Under these conditions, a reduction in the activity of any one of the RTKs by a DN mutant will decrease signaling through multiple downstream pathways. Adding a CA mutant of another RTK can then turn signaling through all of these pathways back up, compensating for the effects of the DN mutant (Jeon, 2012).
The ability of RTKs to substitute for each other in control of cyst formation is conceptually similar to cell transformation through elevation of RTK signaling. RTK activity in cultured cells is tightly controlled, and only a few endogenous RTKs are normally involved in cellular responses to the mitogenic growth factors in their culture medium. Many RTKs can signal through the Ras/MAP kinase pathway, however, and elevated Ras/MAP kinase transduction is sufficient to cause transformation of established cell lines. Thus, oncogenic (CA) mutants of a variety of RTKs are able to transform fibroblastic cell lines when expressed at high levels, regardless of whether the endogenous versions of the RTKs are normally used to regulate proliferation in those cell lines (or are even expressed there) (Jeon, 2012).
The observations on the interchangeability of the RTKs suggests that the decision to form cysts rather than tubes in Ptp4E Ptp10D mutants is very sensitive to the levels of PTyr on the effector binding sites on autophosphorylated RTKs, and that cysts appear when total PTyr rises above a critical threshold. This conclusion is based on the complete suppression of the Ptp4E Ptp10D cyst phenotype that is produced by expression of any of the three DN mutants, even though each DN would eliminate only about 1/3 of total PTyr on the RTKs, if they have roughly equal activities. In wild-type embryos, negative regulation by the RPTPs keeps RTK signaling well below this threshold, so the system is insulated against random fluctuations in phosphorylation or downstream signaling (Jeon, 2012).
The Ptp4E Ptp10D GB cyst number phenotype is completely suppressed by Btl-DN, but not suppressed at all by Egfr-DN and only slightly by Pvr-DN. This might be taken as evidence that elevation of Btl activity is uniquely required to replace GB tubes with cysts. However, when Btl-DN is competed against Pvr-CA, it is only able to suppress the Ptp4E Ptp10D, Btl>Pvr-CA phenotype back to that of unmodified Ptp4E Ptp10D, indicating that Btl can be replaced by Pvr if its activity is sufficiently elevated. These findings can be explained if Btl activity is much higher than Egfr or Pvr activity in GB cells, so that a DN mutant that knocks down endogenous Btl activity by a DN mutant has a greater effect on phosphorylation of effector binding sites than mutants that reduce Egfr or Pvr activities. The activity of CA RTK mutants is independent of the endogenous levels of RTKs, so the Pvr CA mutants could still reverse the effect of Btl-DN even if Pvr-DN has no effect on its own (Jeon, 2012).
To evaluate whether the MAP kinase signaling pathway is involved in the determination of tube geometry, a CA mutant of Phl, the Drosophila Raf kinase, was expressed in the Ptp4E Ptp10D background. Phl-CA enhances the TC/LT phenotype almost as strongly as Egfr-CA. When Phl-CA is combined with Egfr or Btl DN mutants, the phenotype is suppressed back to that of unmodified Ptp4E Ptp10D. Since these RTKs are upstream of Raf, the fact that suppression occurs suggests that pathways other than the MAP kinase pathway are required to generate the phenotype. However, these other pathways may be stimulated by elevation of MAP kinase signaling, since one would expect suppression back to a near-wild-type phenotype if they were completely independent of the MAP kinase pathway (Jeon, 2012).
The involvement of Rho GTPases was tested by expressing DN mutants of Rho1, Rac1, and Cdc42 in the Ptp4E Ptp10D background. Rho1-DN and Rac1-DN completely suppress the TC/LT phenotype, and Cdc42-DN and a tracheally expressed Rho1 RNAi construct produces partial suppression. The DN mutant data do not necessarily show that Rho and Rac are both required for generation of the phenotype. DN mutants may occlude binding of wild-type Rho family GTPases to their GEFs, some of which can act on both Rho and Rac. Therefore high-level expression of Rac-DN might inhibit Rho activation, and vice versa. However, the suppression of the TC/LT phenotype by Rho1 RNAi, which is a specific inhibitor, implicates Rho1 itself in the phenotype. Rho1-CA enhances the Ptp4E Ptp10D phenotype, producing cysts on most DBs (Jeon, 2012).
The differences in the abilities of the three RTK DN mutants to suppress the effects of Phl-CA and Rho-CA may provide clues to the pathways that act downstream of these kinases. The MAP kinase and Rho pathways are not necessarily independent, since Ras/MAP kinase pathway activation can increase Rho-GTP in some cell lines. Also, each of the three RTKs is likely to activate both pathways to some extent, as well as many other downstream pathways. Defining the specific outputs of the MAP kinase and Rho pathways that control tracheal tube geometry and identifying other RTK pathways that regulate tube formation is likely to require genome-wide screens for suppressors and enhancers of the Ptp4E Ptp10D phenotype (Jeon, 2012).
The chromosomal passenger complex (CPC), containing Aurora B kinase, Inner Centromere Protein, Survivin, and Borealin, regulates chromosome condensation and interaction between kinetochores and microtubules at metaphase, then relocalizes to midzone microtubules at anaphase and regulates central spindle organization and cytokinesis. However, the precise role(s) played by the CPC in anaphase have been obscured by its prior functions in metaphase. This study identified a missense allele of Drosophila Survivin (FlyBase name: Deterin) that allows CPC localization and function during metaphase but not cytokinesis. Analysis of mutant cells showed that Survivin is essential to target the CPC and the mitotic kinesin-like protein 1 orthologue Pavarotti (Pav) to the central spindle and equatorial cell cortex during anaphase in both larval neuroblasts and spermatocytes. Survivin also enabled localization of Polo kinase and Rho at the equatorial cortex in spermatocytes, critical for contractile ring assembly. In neuroblasts, in contrast, Survivin function was not required for localization of Rho, Polo, or Myosin II to a broad equatorial cortical band but was required for Myosin II to transition to a compact, fully constricted ring. Analysis of this 'separation-of-function' allele demonstrates the direct role of Survivin and the CPC in cytokinesis and highlights striking differences in regulation of cytokinesis in different cell systems (Szafer-Glusman, 2011).
The chromosomal passenger complex (CPC), composed of the Ser-Thr kinase Aurora B and three partner proteins, plays several key roles in mitosis and meiosis, including regulation of attachment of kinetochores to microtubules, the spindle checkpoint that delays anaphase onset until all chromosomes are under tension on the spindle, regulation of sister chromatid cohesion, and cytokinesis. To accomplish these different tasks, the Aurora B kinase must be exquisitely localized in space and regulated in time. During mitosis, Aurora B associates with the microtubule-binding protein Inner Centromere Protein (INCENP), Borealin/DASRA/CSC-1, and the small, multifunctional BIR-motif protein Survivin to form the CPC (Szafer-Glusman, 2011).
Dissecting the role of individual CPC components has been hampered by the extraordinary interdependence of the four subunits; depletion of any single CPC protein by RNA interference knockdown in human cells affected the structural unit, localization, and function of the CPC. The structural basis of this interdependence is evident in the crystal structure of the Survivin-Borealin-INCENP core of the CPC complex, in which Borealin and INCENP associate with the C-terminal helical domain of Survivin to form a tight three-helix bundle (Szafer-Glusman, 2011 and references therein).
Strict localization of Aurora B by the CPC ensures that this kinase, which has multiple substrates, phosphorylates the correct targets at the proper points in cell cycle progression. Concentrated on chromosomes from G2, then at inner centromeres from prometaphase until the metaphase-to-anaphase transition, the CPC is required to regulate chromosome condensation, spindle formation and dynamics, kinetochore maturation, kinetochore-microtubule interaction, correct chromosome alignment, and control of the spindle checkpoint. At anaphase onset the CPC then translocates to the central spindle (CS) midzone and equatorial cortex and is involved in CS formation. At the CS Aurora B phosphorylates the centralspindlin components, Pav/mitotic kinesin-like protein 1 (MKLP1)/Zen-4 and the RacGAP50/MgcRacGAP/Cyc-4. However, the mechanisms that target the CPC to the spindle midzone and equatorial cortex after onset of anaphase and the mechanisms by which the CPC regulates central spindle formation and cytokinesis are not understood. In addition, the requirements for CPC function for critical events in metaphase and at the metaphase-anaphase transition have complicated analysis of how the CPC is localized and functions at later stages for cytokinesis (Szafer-Glusman, 2011).
This study characterized the role of Drosophila Survivin (dSurvivin), previously termed deterin and analyzed in its antiapoptotic activity, as a regulator of cell division, identifying a missense mutation (scapolo) in the Drosophila Survivin BIR domain that allows recruitment and function of the CPC in metaphase but disrupts CPC localization and function in anaphase and telophase. The findings reveal that Survivin plays a role in targeting the CPC and centralspindlin to the central spindle and the equatorial cell cortex during anaphase. In spermatocytes, Survivin function is also required to localize Polo and localize the small GTPase RhoA to set up the contractile ring machinery at the onset of cytokinesis. In larval neuroblasts undergoing mitotic division, however, the scapolo mutant did not block initial accumulation of Rho to a band at the equatorial cortex, although it did cause failure of cytokinesis. The different requirements for Survivin function for equatorial accumulation of Rho in spermatocytes versus neuroblasts may reflect a fundamental difference in the series of steps that lead to formation of the contractile ring in these two cell types (Szafer-Glusman, 2011).
A missense mutation leading to substitution of serine for the wild-type Pro-86 of Drosophila Survivin uncouples the function of Survivin in metaphase from function during anaphase and telophase, indicating a direct requirement for Survivin and the chromosomal passenger complex in orchestrating the profound reorganization of the cortical cytoskeleton at the cell equator at the onset of cytokinesis. This 'separation-of-function' allele allowed analysis of Survivin and CPC function during cytokinesis, which is normally obscured by the better-known roles of the CPC at centromeres during metaphase, when it facilitates alignment of chromosomes to the spindle equator and mediates the spindle checkpoint. The finding that a point mutation in the BIR domain disrupts activity of Survivin during cytokinesis challenges the model that the C-terminal domain of Survivin is sufficient for cytokinesis function (Lens, 2006) and indicates that residues in the BIR domain are important for localization and activity of Survivin at the central spindle (Szafer-Glusman, 2011).
Survivin associates with kinetochores and the central spindle with different dynamics, being highly mobile in prometaphase and metaphase and strongly immobile at the anaphase central spindle. This change in dynamics may underlie the largely normal localization and function of scapolo (scpo) (a missense allele of the Drosophila Survivin) at metaphase but the fully penetrant effect on assembly of the F-actin contractile ring and cytokinesis observed in scpo mutants (Giansanti, 2004; Szafer-Glusman, 2011).
Cytokinesis depends on the assembly of an equatorial actomyosin ring regulated by local activation of the small GTPase RhoA at the cortex, in turn catalyzed by the RhoGEF Ect2/Pebble. It has been proposed that association of RhoGEF/Pebble with centralspindlin promotes local RhoA activation at the cortex. In addition, the kinase polo (PLK1) has been implicated in RhoGEF localization and Rho activation, at least in part by phosphorylation of the centralspindlin component MgcRacGAP. The current observations that the Drosophila RhoA homologue, Rho1, failed to accumulate at the equatorial cortex in scpo mutant spermatocytes implicate Survivin and the CPC in the mechanism(s) that localize and activate RhoA at the equatorial cortex in these cells. This requirement may in part act through effects on Polo kinase. Failure to localize Polo to the central spindle in scpo mutant spermatocytes could prevent localization of RhoGEF by the centralspindlin complex and the consequent activation of Rho at the cortex. In this model, failure to localize Polo may contribute to the failure to form an equatorial ring of localized Rho1 and, in consequence, the inability to form a localized ring of myosin regulatory light chain and F-actin in scpo mutant male germ cells undergoing meiotic division. This mechanism may also explain the failure to maintain pole-to-equatorial microtubules observed in scpo mutant spermatocytes. It is likely that Rho-mediated activation of the Formin Dia helps stabilize microtubule arrays at the equatorial cortex of dividing cells, as active Rho and Formin (mDia) have been shown to regulate stabilization of microtubule arrays at the cortex of migrating fibroblasts. Consistent with this model, this study found that microtubules reached the plasma membrane at the equator of scpo dividing spermatocytes, but the bundles are transient and fail to form stable arrays at the cortex (Szafer-Glusman, 2011).
A striking finding of this work is the difference in requirement for Survivin function for localization of the Polo kinase and RhoA in anaphase neuroblasts versus spermatocytes. This difference raises two possibilities: either Survivin is not part of a universal signaling mechanism that directs cytokinesis, or different semiredundant mechanisms can drive cytokinesis, similar to redundancy between astral pulling and sliding of central spindle microtubules for anaphase B, and different cell types rely more strongly on one mechanism or the other. Indeed, consistent with the latter possibility, spermatocytes and neuroblasts display different cytoskeletal architectures during cytokinesis (Giansanti, 2006). In neuroblasts, actomyosin initially accumulates in a broad cortical band, presumably because this is the region of the cell cortex that escapes repression of Rho associated with the plus ends of astral microtubule. This initial wide band gradually narrows into a tight equatorial ring as the cell progresses into telophase. Thus assembly of the contractile apparatus in neuroblasts proceeds, as proposed for Caenorhabditis elegans embryos, in 'two genetically separable steps' in which localization of contractile machinery is initially independent of the central spindle. In support of this model, this study found that Rho1 accumulates in a broad cortical band in scpo mutant neuroblasts, suggesting that the first stage can occur independent of Survivin and CPC localization to the central spindle (Szafer-Glusman, 2011).
Spermatocytes, in contrast, do not form an initial wide equatorial band of contractile ring components. Instead, from their first appearance in early anaphase, the actomyosin rings in spermatocytes are tightly focused at the cell equator). It is speculated that this restricted initial localization of contractile ring components and the apparent lack of a preceding wide equatorial band may be a consequence of a more stringent global block to Rho1 activation at the cortex in spermatocytes than in neuroblasts. It is proposed that this global block is eventually overridden by positive regulation of Rho1 by local concentration of RhoGEF, in turn facilitated by CPC-dependent events associated with and/or localized by central spindle microtubules. Rho1 activation would then occur within a narrow peak exactly at the site where pole-to-equator microtubules interact to maximize RhoGEF deposition/concentration at the cortex. Indeed, F-actin ring assembly occurs locally and cytokinesis initiates immediately after the pole-to-equator microtubules contact the cortex in Drosophila spermatocytes. It is proposed that, according to this model, the defects in Survivin lead to lack of CPC activity and abnormal centralspindlin, resulting in absence of Rho1 and Polo kinase from the equator of scpo mutant spermatocytes (Szafer-Glusman, 2011).
In neuroblasts, where a more permissive cortex allows a broad belt of Rho1 activation at the cell equator, Survivin and CPC appear to promote gradual convergence of the initial broad band into a narrow ring centered at the maximum of RhoGEF activity at the cortex. In scpo mutants, which display irregular anaphase central spindles devoid of Pav, the broad Rho1 cortical band fails to narrow, the cells fail to form a focused, narrow ring of myosin, and cell division proceeds with inefficient and incomplete constriction (Szafer-Glusman, 2011).
A key difference between neuroblasts and spermatocytes that may account, at least in part, for the differences in behavior of Rho1 and myosin complex proteins is in the relationship between Polo kinase and the CPC observed in scpo mutant mitotic versus male meiotic cells. In spermatocytes, Polo and the CPC are interdependent and Polo colocalizes with the CPC along its full journey from metaphase through anaphase and telophase. In neuroblasts, in contrast, Polo localization during cytokinesis appears to be independent of the CPC and centralspindlin, at least at early stages of cell division, but Polo appears to colocalize with Feo, the Drosophila homolog of PRC1, that required for central-spindle formation and cytokinesis. A second difference between neuroblasts and spermatocytes may be the recently described, spindle-independent backup system that can localize myosin to a broad band at the cell cortex near the future cleavage plane under control of the neuroblast cell polarity system. The broad localization of myosin to the cell cortex observed in ana/telophase neuroblasts in scpo mutants may be in part due to these redundant mechanisms (Szafer-Glusman, 2011).
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).
RhoA, a small GTPase, regulates epithelial integrity and morphogenesis by controlling filamentous actin assembly and actomyosin contractility. Another important cytoskeletal regulator, Moesin (Moe), an ezrin, radixin, and moesin (ERM) protein, has the ability to bind to and organize cortical F-actin, as well as the ability to regulate RhoA activity. ERM proteins have previously been shown to interact with both RhoGEF (guanine nucleotide exchange factors) and RhoGAP (GTPase activating proteins), proteins that control the activation state of RhoA, but the functions of these interactions remain unclear. This study demonstrates that Moe interacts with an unusual RhoGAP, Conundrum (Conu; CG34104), and recruits it to the cell cortex to negatively regulate RhoA activity. In addition, this study shows that cortically localized Conu can promote cell proliferation and that this function requires RhoGAP activity. Surprisingly, Conu's ability to promote growth also appears dependent on increased Rac activity. These results reveal a molecular mechanism by which ERM proteins control RhoA activity and suggest a novel linkage between the small GTPases RhoA and Rac in growth control (Neisch 2013).
Although previous studies have implicated ERM proteins in the regulation of RhoA activity, the molecular mechanisms underlying this function have remained unclear. This work shows that a previously uncharacterized RhoGAP, Conu, physically and functionally interacts with Moe. Consistent with its sequence similarity to other RhoGAP proteins, Conu was found to have GAP activity for Rho1 in vitro and negatively regulates Rho1 in vivo. The data further suggest that Moe promotes Conu's RhoGAP activity, and therefore negatively regulates Rho1 by recruiting Conu to the plasma membrane (Neisch 2013).
Surprisingly, the data suggest that Conu also functions to positively regulate Rac1 activity. Although Conu's ability to promote proliferation is dependent on its RhoGAP activity, this alone is not sufficient to cause overproliferation. Two lines of evidence indicate that this additional function involves positively regulating Rac1. First, coexpression of Conu enhances a rough-eye phenotype associated with Rac1 expression in the eye. This effect is not dependent on Conu GAP activity, indicating that it is not the result of cross-talk between different Rho family small GTPases. Second, Conu acts synergistically with the small GTPase Arf6 in causing overproliferation. Previous studies have shown that Arf6 promotes activation of Rac1 at the plasma membrane. Consistent with the idea that Arf6 promotes Rac1 activity, coexpression of Arf6 strongly enhances the Rac1 eye phenotype. These results suggest that Conu functions to negatively regulate Rho1 activity and positively regulate Rac1 activity, and that the combination of these two effects promotes overproliferation when Conu is activated (Neisch 2013).
Conu's closest orthologue in the mammalian genome is ARHGAP18, also known as MacGAP, with which it shares 40% sequence similarity. ARHGAP18 has been shown to have GAP activity for the Rho1 homologue RhoA. ARHGAP18 was also recently found to promote tumor formation and cell proliferation when ectopically expressed in mammary epithelia, consistent with the observation that Conu promotes proliferation in Drosophila epithelial tissues. Little is currently known about the details of ARHGAP18 function in mammalian cells, but it is interesting to note that ARHGAP18 is also required for cell spreading, a function that is associated with Rac1 activation in many cells. It is also notable that Conu and ARHGAP18 share a region of homology near the N-terminus (aa 45-90) that is separate from the GAP domain and has similarity to the SAM (sterile alpha motif) kazrin repeat 2 domain. SAM domains serve as oligomerization or protein-protein interaction motifs, so it is possible that this domain mediates interactions with one or more Rac1 regulatory proteins. However, preliminary results indicate that this domain alone does not enhance Rac1 in the eye. It will be interesting to determine whether ARHGAP18 also regulates Rac1 activity, and whether both functions are also involved in growth control in mammals (Neisch 2013).
Despite the overproliferation phenotypes observed from activated Conu, loss-of-function conu mutations are viable and lack obvious imaginal disk phenotypes. Functional redundancy with another RhoGAP seems a likely explanation for this result, because the Drosophila genome encodes 21 RhoGAP proteins in addition to Conu. Thus the loss of a single RhoGAP, such as Conu, may have only a very minor effect on overall Rho1 activity in the imaginal epithelium. In addition, the phenotypes exhibited by Moe mutants, which are much more severe, are probably due to Moe's ability to negatively regulate Rho1 activity via Conu, together with its ability to stabilize the apical cell cortex through interactions with F-actin. Indeed, it is speculated that the severe epithelial defects observed in Moe mutants are the combined result of increased apical actomyosin contractility caused by increased RhoA activity and decreased cortical stability caused by the loss of Moe's membrane-cytoskeletal cross-linking function. In this model, loss of either function alone would have relatively minor effects, but the combined defect would result in the severe cortical disorganization that has been described for Moe mutants. A critical future line of investigation is to identify other Rho1 regulatory proteins that function with Moe to regulate the activity of Rho1 in the apical domain (Neisch 2013).
A remaining question is how does Conu contribute to growth control in the imaginal disks? The answer to this important question is unclear, but the data suggest that decreased Rho1 activity functions synergistically with Rac1 in this process. Recent studies in Drosophila suggest that Jun N-terminal kinase (JNK) activation downstream of Rac1 activity can promote increased proliferation and metastatic activity in the presence of activated Ras (Brumby, 2011). Although attempts to suppress Conu-mediated overgrowth by inhibiting the JNK pathway gave ambiguous results, increased expression of the puc-lacZ reporter for JNK activity was observed in cells expressing activated Conu, consistent with a role of JNK pathway activation in Conu-mediated proliferation. In contrast, other signaling pathways involved in growth control (Notch, TGFβ, Wnt, Hippo, and Hedgehog) were unaffected. Ras activation is thought to protect cells from the proapoptotic effects of JNK pathway activation, and it is possible that decreased Rho1 activity caused by Conu activation has similar effects, especially given that it has been shown that Rho1 is proapoptotic in these tissues. While further studies will be required to more firmly establish the mechanistic basis of Conu function in tissue growth, these findings of a role for Moe and Conu in this process highlight the importance of precise regulation of the apical cell cortex and Rho family small GTPases in growth control (Neisch 2013).
Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained as AJ are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through DE-cadherin endocytosis by limiting the Cdc42/Par6/aPKC complex activity. This study demonstrates that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin containing Rab11-positive recycling endosomes in Drosophila post-mitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 may regulate the formation of recycling endosomes thorough localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin containing vesicular trafficking during AJ remodeling in live epithelia (Yashiro, 2014).
Semaphorin family proteins are well-known axon guidance ligands. Recent studies indicate that certain transmembrane Semaphorins can also function as guidance receptors to mediate axon-axon attraction or repulsion. The mechanisms by which Semaphorin reverse signaling modulates axon-surface affinity, however, remain unknown. This study reveals a novel mechanism underlying upregulation of axon-axon attraction by Semaphorin-1a (Sema1a) reverse signaling in the developing Drosophila visual system. Sema1a promotes the phosphorylation and activation of Moesin (Moe), a member of the ezrin/radixin/moesin family of proteins, and downregulates the level of active Rho1 in photoreceptor axons. It is proposed that Sema1a reverse signaling activates Moe, which in turn upregulates Fas2-mediated axon-axon attraction by inhibiting Rho1 (Hsieh, 2014).
The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. This study shows that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppressed Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promoted tissue growth when apoptosis was prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. These results identify a novel molecular link between the Hippo and JNK pathways and implicate the essential role of the JNK pathway in Hippo signaling-related tumorigenesis (Ma, 2005).
Recent studies have revealed a complex interaction network between Hippo and other key signaling pathways, including TGF- β /SMAD and Wnt/β-catenin pathways, whereas its communication with JNK signaling remains elusive. This study provides genetic evidences that impaired Hippo signaling promotes overgrowth through Rho1-JNK signaling in Drosophila. First, loss of Hippo signaling induces JNK activation and its target gene expression. Second, Yki-induced overgrowth is suppressed by blocking Rho1-JNK signaling. Third, ectopic Rho1 expression phenocopies Yki-triggered overgrowth and proliferation when cell death is compromised (Ma, 2005).
Yki/YAP's ability in promoting tissue growth depends on transcription factors, including Sd/TEADs and SMADs. Consistent with this notion, this study found Sd, but not Mad, is essential for Yki-induced JNK activation, whereas ectopic Sd expression is sufficient to activate JNK signaling by itself. The Rho1 GTPase was further implicated as the critical factor that bridges the interaction between Hippo and JNK signaling. Rho1 not only mediates Yki-induced JNK activation and overgrowth, but also serves as a direct transcriptional target of Yki/Sd complex. Intriguingly, Rho1 activation was also found to promote nuclear translocation of Yki in wing discs, and reducing Yki activity significantly impeded Rho1 induced growth, implying the existence of a potential positive feedback loop to amplify Yki-induced overgrowth and to help maintain signaling in a steady state. Consistent with thi observation, recent studies
reported that GPCRs could activate YAP/TAZ through RhoA in mammals, whereas elevated JNK signaling in Drosophila could stimulate Yki nuclear translocation during regeneration and tissue growth. Thus, these results provide the other side of the story about a novel cross-talk between Hippo and JNK signaling (Ma, 2005).
Intriguingly, it was found that ectopic Yki expression driven by ptc-Gal4 induced MMP1 activation, puc-LacZ expression, rho1 transcription, and Yki target gene transcription predominantly in the proximal region of wing disk, but not that of the dorsal/ventral boundary. This is consistent with a recently published paper showing that tension in the center region of Drosophila wing disk is lower than that in the periphery, which correlates with lower Yki activity. It is also worth noting that despite the requirement of JNK signaling in Yki-induced wing overgrowth, JNK was not activated strictly in an autonomous manner upon Yki overexpression. This could be caused by supercompetitive activity of Yki expression clones, or, alternatively, through a propagation of JNK signal into neighboring cells, which would be very interesting to study further (Ma, 2005).
Apart from its role in growth control, the Hippo pathway also regulates tumor invasion and metastasis. Similarly, JNK signaling plays a major role in modulating metastasis in both flies and mammals. Rho1 was also reported to cooperate with oncogenic Ras to induce large invasive tumors. Hence, it is likely that Rho1 also acts as the molecular link between Yki and JNK signaling in modulating metastasis as well (Ma, 2005).
Rho1:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
| References
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