Rac1
puckered encodes a VH1-like phosphatase that acts directly as a Jun kinase
phosphatase to down-regulate Jun kinase (JNK) activity during dorsal closure
of the Drosophila embryo. puc plays a role in
follicle cell morphogenesis during oogenesis. The follicle cells (FCs) form an epithelial sheet around a cyst of germ cells (the oocyte and 15 nurse cells)
that then develops as an egg chamber unit. Late in oogenesis,
specific groups of follicle cells become distinct. During stage
9, most follicle cells migrate posteriorly to cover the oocyte
surface in a columnar epithelium. Approximately
50 cells -- the nurse cell FCs (NCFCs) -- flatten to form
a squamous domain over the nurse cells. Subsequently,
subgroups of the columnar FCs change shape and migrate. Beginning in stage 10B, the centripetally migrating FCs (CMFCs) move inward between the oocyte and nurse
cells. These cells will create anterior eggshell
structures such as the operculum. The main body FCs
(MBFCs) stretch during stage 11, as the oocyte rapidly enlarges with the transfer of the nurse cell contents, a process called nurse cell dumping. During stages 12
through 14, two dorsal anterior groups of FCs migrate anteriorly
to create the dorsal appendages. The posterior pole FCs (PPFCs) include cells that produce the aeropyle of the eggshell. The FCs that will create a specific eggshell
structure are fated earlier in oogenesis, as revealed by specific
patterns of gene expression (Dobens, 2001 and references therein).
The Rho family of small GTPases are important modulators of
cell shape and epithelial cell reorganization. Specific Rho
proteins can manifest specialized cellular functions. The cell-type
specific effects extend to their abilities to stimulate the
JNK pathway. The differing requirements for Puc in different regions of the
follicular epithelium suggest that these regions show
distinct sensitivities to the actions of Rho family GTPases.
Because of evidence that Rac1 and Cdc42 regulate
expression of A251-lacZ during embryogenesis, the ability of these small GTPases to regulate
A251-lacZ expression in the FC was tested. Two opposing
effects were found: activated Rac1 and dominant negative Cdc42
activate A251-lacZ expression in posterior cells; dominant
negative Rac1 and activated Cdc42 repress A251-lacZ
expression in anterior cells. Clones for activated Rac1 were
not recovered in the anterior nurse cell FCs, suggesting that
these cells are highly sensitive to increased Rac activity. These
data suggest that a balance between these two small GTPases
may define the level of A251-lacZ expression in the nurse cell
FCs and the posterior pole FCs. This may reflect the shared
origins of these distinct cell groups as terminal follicle cells (Dobens, 2001).
Neither Rac1 nor Cdc42 appeared to regulate A251-lacZ
expression in the main body FCs or the centripetally migrating
FCs. This latter observation is surprising, given the strong
requirement for Puc activity in these cells. Perhaps a distinct
Rho family GTPase is active in these cells, such as Rho1.
Consistent with this, mutants in PAK kinase, which acts in
parallel to JNK signaling during dorsal closure, have a variable
defect in centripetal migration (Dobens, 2001).
Activated GTPase expression in the follicle cells results in
additional phenotypes. Activated Rac1 has non-autonomous
phenotypes in the posterior pole FCs. Similarly,
activated Rac1 has non-autonomous effects on gene
expression in the dorsal ectoderm of the embryo. This suggests that in both tissues, a short-range
signal dependent on Rac1 signaling can upregulate
expression of these intron II enhancer traps in adjacent cells.
Dpp is a local signal to the dorsal ectoderm during dorsal
closure; however, the data suggest that Dpp is not the key
signal to induce A251-lacZ in the posterior FC. It is noted that loss of puc in marked clones never results in upregulation of A251-lacZ
expression in adjacent cells, indicating that whatever its
identity, the short range signal is not regulated by puc (Dobens, 2001).
Expression of activated Rac1 in the posterior pole FCs
causes domain-specific disorganization of the epithelium.
Dominant negative Cdc42 also causes this phenotype, similar
to genetic loss of Cdc42 activity. Similar phenotype are seen for activated RhoL. Thus the balance of Rho GTPases in the posterior follicular epithelium may be
important to maintain epithelial structure, similar to roles
ascribed to Rac in metastasis (Dobens, 2001).
Based on data presented here, it is proposed that puc functions
as a rheostat to modulate gene expression responses to Rho
family GTPases. This modulation is critical to
coordinate morphogenesis in distinct FC domains. Because Puc can act as a Jun kinase
phosphatase in the embryo, it has been speculated that Puc modulates
the activity of similar kinases that act downstream of the small
GTPases at the FC termini. It is likely that puc modulates Jun
kinase signal in the anterior FCs, since the Jun kinase kinase
encoded by hemipterous is required for anterior A251-lacZ expression. However, puc
is also required at the posterior, whereas late A251-lacZ
expression is not dependent on hep. Components of the
posterior pathway remain to be identified (Dobens, 2001).
The insensitivity of main body FCs to stimulation by Rac or
Cdc42 might suggest that these cells are incompetent to
respond to small GTPase activation during the late stages of
oogenesis. However, responses are suppressed by an independent mechanism. The dynamic
pattern of Jun and Fos and puc itself, suggests
that FCs may vary greatly, both in time and in space, in their
competence to respond to the JNK pathway. To complicate this
picture, the data indicates that puc regulates levels of Fos and
Jun, even in the main body FCs. Loss of puc leads to elevation
of Fos/Jun protein levels. Conversely overexpression of either
Puc or DN-Rac1 lowers Fos/Jun levels. Currently the direct mechanism for puc regulation of Fos/Jun levels is unknown, but these results recall observations that stability of Fos and Jun depends on phosphorylation. In cell culture, site-specific
mutations in Fos or Jun that block phosphorylation confer
instability; conversely, phosphate-mimetic mutations confer
greater stability. It is concluded that proper levels of Puc
phosphatase modulates Rho family GTPase signal output to
coordinate follicle cell morphogenesis. These results are
similar to the effect of both loss- and gain-of-function Puc in
blocking dorsal closure and a role for puc in disc epithelial morphogenesis. The precise regulation of Puc activity levels is critical to coordinate epithelial cell sheet spreading in at least three tissues (Dobens, 2001 and references therein).
Border cell migration in the Drosophila ovary is a relatively simple and genetically tractable model for studying the conversion of epithelial cells to migratory cells. Like many cell migrations, border cell migration is inhibited by a dominant-negative form of the GTPase Rac. To identify new genes that function in Rac-dependent cell motility, a screen was performed for genes that when overexpressed suppressed the migration defect caused by dominant-negative Rac. Overexpression of the Drosophila inhibitor of apoptosis 1 (DIAP1), which is encoded by the thread (th) gene, suppresses the migration defect. Moreover, loss-of-function mutations in th cause migration defects but, surprisingly, did not cause apoptosis. Mutations affecting the Dark protein, an activator of the upstream caspase Dronc, also rescues RacN17 migration defects. These results indicate an apoptosis-independent role for DIAP1-mediated Dronc inhibition in Rac-mediated cell motility (Geisbrecht, 2004).
The work reported here demonstrates a new function for DIAP1 in promoting cell migration. The strongest evidence for this is that border cells lacking DIAP1 fail to migrate. This finding is surprising since there has been no previous indication that IAP proteins contribute to cell motility. However, in most cells, it would be difficult or impossible to uncover a requirement for DIAP1 in cell migration because loss of the protein typically results in cell death (Geisbrecht, 2004).
The effect of DIAP1 on cell migration appears to be through the small GTPase Rac and its effects on the actin cytoskeleton based on genetic, biochemical, and cell culture experiments. First, overexpression of DIAP1 suppresses RacN17 migration defects specifically and does not rescue border cell migration defects that are due to other causes. Moreover, overexpression of either actin5C or profilin, both of which would be expected to increase the amount of polymerization competent G-actin in the cell, also rescue RacN17 border cell migration defects. The association of DIAP1 protein with Rac and profilin in S2 cells together with the finding that overexpression of DIAP1 can enhance activated Rac's effects on the actin cytoskeleton in cultured cells further support the conclusion that DIAP1 affects cell migration via Rac and the actin cytoskeleton. Additional support is provided by the finding that overexpression of Rac in border cells results in increased accumulation of DIAP1 protein and F-actin in vivo (Geisbrecht, 2004).
The effect of DIAP1 on border cell migration is clearly independent of its role in preventing apoptosis. The lack of apoptosis in th mutant follicle cell clones is striking since at other stages of Drosophila development, cells fail to survive in the absence of DIAP1. However, IAP proteins are thought to play a less critical role in survival of certain mammalian cells as well, where the current view is that the balance between proapoptotic and antiapoptotic BCL-2 family proteins is the deciding factor between life and death. The results presented here suggest that DIAP1 is not required for survival of every cell and tissue of the fly either. It may be that in both flies and mammals, different cell types have distinct requirements for particular classes of survival molecules (Geisbrecht, 2004).
Although the ability of DIAP1 to rescue RacN17 border cell migration defects is independent of its function in preventing cell death, and independent of its inhibition of effector caspases, the effects result from inhibition of the initiator caspase Dronc. The finding that inhibition of Dronc can rescue RacN17 border cell migration defects indicates that Dronc activity has a negative effect on migration. Since Dronc is a protease, the most parsimonious hypothesis would be that Dronc cleaves one or more proteins required for Rac-mediated cell motility. Previous studies have shown that Rac can be cleaved and inactivated by caspase 3 in lymphocytes. Therefore one possibility is that Rac itself is a Dronc substrate in border cells. A number of cytoskeleton-associated proteins are cleaved by caspases, including actin. Since reduced profilin levels was observed in th mutant follicle cells, it is also possible that profilin is a Dronc substrate, though no increased accumulation of actin or profilin was detected in cells overexpressing DIAP1. Further study will be required to pinpoint the physiologically relevant Dronc substrate in border cells (Geisbrecht, 2004).
The observation that two different dark mutant alleles cause mild border cell migration defects suggests that Dronc, which is thought to be constitutively active at a low level in most cells, contributes to normal migration. In fact, caspases have been shown to function in cell proliferation and differentiation in a variety of cell types, in addition to their better known role in promoting apoptosis. In some cases, caspase activity is required for terminal differentiation events that resemble incomplete apoptosis. For example, terminal differentiation of Drosophila sperm requires removal of much of the cytoplasm and requires caspase activity. Similarly, differentiation of mammalian lens cells and erythrocytes requires caspase activity. Other differentiation events, such as those of macrophages and skeletal muscle, do not overtly resemble apoptosis and yet require caspase activity. There must be some mechanism in such cells, and in border cells, to restrict the caspase activity to selected substrates so that apoptosis does not occur (Geisbrecht, 2004).
DIAP1 is a member of an evolutionarily conserved family of proteins that contain BIR domains. BIR domain-containing proteins are found in organisms from yeast to man and seem to have arisen in evolution prior to the apoptotic machinery. For example, in yeast, BIR1p is a protein required for proper chromosome segregation and cytokinesis. Yet the yeast genome does not encode an obvious caspase and yeast are not known to undergo apoptosis. In C. elegans, there is a BIR domain protein that does not suppress apoptosis when overexpressed. Reduction in the expression of this protein by RNA interference leads to defective cytokinesis and a phenotype that is very similar to loss of the worm formin protein. This is interesting since formin homology proteins can bind Rac, stimulate actin polymerization in concert with profilin, and promote cell migration. However it is not known if the C. elegans BIR domain protein interacts with formin or profilin, or whether it functions downstream of Rac. Taken together, these observations suggest that a primitive function of BIR domain proteins may have been regulation of cell division and the cytoskeleton (Geisbrecht, 2004).
It is well known that growth factors promote both survival and proliferation, as well as migration of specific cells. For example, Steel factor acting through the c-kit receptor tyrosine kinase regulates survival and proliferation of primordial germ cells and melanocytes in the mouse embryo. In addition, Steel factor and c-kit may contribute to guiding the embryonic migrations of these two cell populations. Conversely, overexpression of a factor that functions in repulsive guidance of Drosophila primordial germ cells causes excessive germ cell death. This intimate relationship between guidance and survival may exist to ensure that only those cells that migrate to the appropriate location survive and proliferate (Geisbrecht, 2004).
Rho family GTPases have also been demonstrated to affect both migration and survival. By activating gene expression through the JNK pathway, Rac1 protects COS 7 cells from apoptosis induced by ultraviolet light. Rac is also required for survival of cerebellar granule neurons. Another pathway required for both cell survival and Rac-mediated cell migration is the phosphatidylinositol 3-kinase pathway (PI3K). Activation of PI3K, and its downstream effector Akt, is capable of promoting neuronal survival in the absence of growth factors. Akt is also essential for Rac-mediated motility in mammalian fibroblasts. Akt is activated by Rac, and phosphorylated Akt colocalizes with Rac at the leading edge of fibroblasts. Therefore, there are several biochemical pathways that control, and possibly coordinate, cell survival and cell motility.
The mammalian formin homology protein FRL functions as a survival signal, in addition to its role in Rac-mediated regulation of the cytoskeleton. Overexpression of a truncated form of FRL, containing only the N-terminal Rac binding site, results in inhibition of cell growth and apoptosis in the macrophage cell line P388D1. These lines of evidence support the view that regulation of the cytoskeleton and cell survival are intertwined. The present study demonstrates that the inhibitor of apoptosis proteins, well known for their role in cell survival, can also promote cell migration, thus demonstrating a new and unexpected molecular link between survival and migration (Geisbrecht, 2004).
Paxillin is a prominent focal adhesion docking protein that regulates cell adhesion and migration. Although numerous paxillin-binding proteins have been identified and paxillin is required for normal embryogenesis, the precise mechanism by which paxillin functions in vivo has not yet been determined. An ortholog of mammalian paxillin in Drosophila (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, 2004).
Paxillin is a scaffolding protein found in focal adhesions. Targeted disruption of paxillin in mice results in an early embryonic lethal phenotype with defects in multiple mesodermally derived structures. The recent completion of the Drosophila genome revealed the evolutionary conservation of many of the key molecules found in focal adhesions, including integrins, paxillin, vinculin, FAK, p130CAS, and ILK. The Drosophila paxillin is predominantly expressed in embryos, pupae, and male adults. In situ analysis of staged embryos reveals a restricted expression pattern of Dpax. In particular, Dpax is highly expressed in tissues undergoing cell shape changes or cell migration. Overexpression of Dpax in late larval stages results in a pupal lethal phenotype with few escapers bearing malformed phenotypes, suggesting that Dpax also plays an important role during later stages of development (Chen, 2004).
A loss-of-function mutant of Drosophila paxillin has not yet been reported. Therefore, the UAS/GAL4 system was employed to investigate the function of Dpax in the later stages of development. As has been reported for Drosophila FAK, overexpressing Dpax results in a blistered-wing phenotype. In mammals, paxillin is a substrate of FAK in transducing signals from integrins. FAK regulates focal adhesion disassembly and has been shown to be involved in Drosophila Wnt4-mediated cell movement during ovarian morphogenesis and is also required for border cell migration during oogenesis. The function of Dpax in oogenesis is not clear; however, Dpax is also highly expressed in the border cells (Chen, 2004).
The blistered-wing phenotype is also found in integrin mutant flies. In the prepupal stage, the wing is a single epithelial sheet, and integrins have been suggested to play a regulatory role. As development progresses this sheet folds into a dorsal and ventral side, and the integrins play an adhesive role at these later stages. Using drivers that are expressed at different stages of development, the studies suggest that paxillin could be important for both the regulatory and adhesive functions of the integrins. Such functions would be consistent with studies of mammalian systems in which paxillin functions downstream of multiple integrins and can regulate both inside out and outside in signaling. In addition, both paxillin and FAK are important for focal adhesion turnover. Thus, too much paxillin or FAK may increase the turnover of focal complexes and perturb the stable adhesion between two epithelia, thereby resulting in the blistering phenotype (Chen, 2004).
Using a gain-of-function screen for modifiers that can rescue the Dpax-induced wing blistering, Cdi/TESK was identified. Like LIMK, Cdi/TESK phosphorylates the actin-depolymerizing factor cofilin and stabilizes F-actin. Cdi/TESK is highly homologous to LIMK in the kinase domain; however, a recent study has demonstrated that Cdi/TESK functions downstream of Rac1 during spermatogenesis (Raymond, 2004). Drosophila LIMK functions downstream of Rho1 in regulating disk morphogenesis (Chen, 2004). Dlimk and components in the Rho-LIMK pathway, including ssh, tsr, and bs/DSRF, also rescue the blistering phenotype. In addition, another regulator of SRF and actin, diaphanous, also shows genetic interactions with Dpax. Diaphanous is a direct effector of Rho which cooperates with LIMK to regulate SRF activation. All of these components play important roles in regulating F-actin synthesis. Taken together, these data indicate that it is possible that an increase in actin levels can prevent the increase in focal adhesion turnover caused by the excess level of paxillin, therefore suppressing the blistering phenotype. It is possible that simply overexpressing actin might be sufficient to rescue the blistering phenotype, although the results suggest that paxillin itself does not affect F-actin synthesis or actin organization. The ability of paxillin, however, to coimmunoprecipitate with LIMK and the increased cofilin phosphorylation in Pxl–/– MEFs suggests that paxillin can modulate LIMK function. These data, combined with the genetic and biochemical evidence that paxillin can regulate Rho, suggest that paxillin could act at multiple points to regulate the Rho pathway (Chen, 2004).
Interestingly, while modulation of some components downstream of Rho is able to suppress the blistering phenotype, overexpression of other components such as ROK does not alter this phenotype. While this could reflect insufficient expression levels or more complex regulation of ROK, the data suggest that paxillin's regulation of the Rho pathway may involve either modulation of only certain downstream components or a lack of function for these components in the paxillin-induced phenotypes (Chen, 2004).
Rho GTPases play an important role in regulating actin cytoskeleton organization. Genetic and biochemical analysis reveal that paxillin activates Rac signaling but inactivates Rho signaling. Previous binding and localization studies suggest that mammalinan paxillin may regulate Rac through its indirect association with at least two Rac exchange factors. Pix/Cool is linked to paxillin via PKL/Git2, the ARF-GAP, and overexpression studies with mutants of paxillin and other members of this complex have led to the suggestion that paxillin may be important for recruiting this complex to focal contacts. A second binding partner, Crk, can also link paxillin to Rac activation via a nontraditional exchange factor, Dock180. Mislocalization of one or both complexes in Pxl–/– mouse embryo fibroblasts (MEF)s could therefore lead to defects in Rac activation and subsequent defects in lamellipodium dynamics and migration. Both Pix/Cool and Crk localization were examined in rescued and Pxl–/– MEFs and only a minor decrease was detected in Cool and Crk positive peripheral adhesions in Pxl–/– cells. In MEFs, therefore, paxillin is not required for localization of these proteins to peripheral adhesions. This may be due to functional redundancy, as the paxillin family member Hic-5 can also bind the PKL-Pix complex and Crk can bind to other focal adhesion proteins, including p130Cas. In any case, mislocalization of these complexes is unlikely to account for the differences in Rac activation. In contrast, genetic studies of Drosophila have shown that deletion of a region encompassing the Drosophila homolog of Cool was able to suppress the Dpax-induced blistering. Thus, one potential mechanism by which paxillin may control Rac activation in Drosophila is through regulation of Pix/Cool. Since Rac and Rho have been shown to antagonize each other, it remains possible that in higher eukaryotes, paxillin could indirectly regulate Rac via regulation of Rho (Chen, 2004).
It is not clear how paxillin down-regulates Rho activity. Paxillin might be important for spatial regulation of Rho activity and/or controlling the activity or localization of a Rho GAP or GEF. Two Rho GAPs have been linked to mammalian paxillin. Graf is a Rho GAP that was originally identified as a Fak-binding partner, and a homolog of this protein has been identified in Drosophila studies. Since paxillin can interact with Fak, it is possible that loss of paxillin may somehow affect Graf localization or activation. While Fak localization to focal adhesions is less efficient in Pxl–/– MEFs, the effects are minimal and thus this is unlikely to account for the enhanced Rho activity. It is worth noting that it has recently been reported that mammalian paxillin binds to the p120 RasGAP and competes with p120 RasGAP for binding to p190 RhoGAP. It has been suggested that paxillin inhibits Rho by promoting the formation of free p190 RhoGAP. The Drosophila ortholog of p190 RhoGAP does not bind to the Drosophila p120 RasGAP. In addition, only minor changes in p190 localization to the leading edge were detected in Pxl–/– MEFs. Thus, paxillin may antagonize Rho function through multiple distinct regulatory mechanisms (Chen, 2004).
Taken together, these data suggest that while paxillin has the ability to interact with multiple proteins involved in diverse signaling pathways, a major function of this scaffolding protein in vivo is to regulate Rho family GTPases. Thus, misregulation of these GTPases is likely to account for the adhesion defects observed during development in mouse and Drosophila studies (Chen, 2004).
The Drosophila homolog of the proto-oncogenic RAC protein kinase
(DRAC-PK) gives rise to two transcripts with the same coding potential,
generated by the use of two different polyadenylation signals. Because of the presence of a weaker initiator ACG codon upstream from the major
AUG, each transcript encodes two
polypeptides, such that the larger protein contains an N-terminal extension. Like the human isoforms,
DRAC-PKs possess a novel signaling region, the pleckstrin homology domain. DRAC-PK proteins
have a similar expression pattern, being regulated both maternally and zygotically, and are expressed
throughout Drosophila development. Antisera specific for recombinant DRAC-PK and for its C
terminus detect two polypeptides of 66 and 85 kDa in Drosophila extracts. The antirecombinant
antisera also recognize a polypeptide of 120 kDa from Drosophila, which apparently shared an epitope
related to DRAC-PK sequences. The role of p120 appears to be restricted compared with that of
DRAC-PK, since it is not detected in larvae or adult flies. There is no spatial restriction of
DRAC-PK expression during embryogenesis, suggesting that localized activation might be a regulatory
mechanism for its function. DRAC-PK possesses an intrinsic kinase activity that is approximately
8-fold higher in adult flies than in 0-3-h embryos undergoing rapid mitotic cycles (Andjelkovic, 1995).
A Drosophila homolog of the
serine/threonine kinase PAK (DPAK, now termed PAK-kinase) is a target of the Rho subfamily proteins Rac and Cdc42.
Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases.
DPAK binds to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities
in the distributions of DPAK, integrin, and phosphotyrosine suggest an association of DPAK with
focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK is
elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of
the embryo. The accumulation of cytoskeletal elements initiating cell
shape changes in these cells can be inhibited by expression of a dominant-negative DRacA
transgene. Leading-edge epidermal cells, which segment borders, express
particularly large amounts of DPAK. These cells undergo transient losses of cytoskeletal structures during dorsal
closure. DPAK may be regulating the cytoskeleton through its association with focal
adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase
signaling pathway recently demonstrated to be required for dorsal closure (Harden, 1996).
The rotund (rn) locus of Drosophila at cytogenetic position 84D3,4 has been isolated. Rotund protein 1.7 is similar to the product of the human n-chimaerin gene, which is
expressed in brain and testes. Recently, the GAP activity of n-chimaerin has been demonstrated and shown
to be specific for the Rac subfamily of the Ras oncoproteins. The Rac proteins have been implicated in
the regulation of secretory processes. In addition to being expressed in the imaginal discs, the m1.7
racGAP transcript is detected in developmentally specific germ line cells of the testes, the primary spermatocytes (Agnel, 1992).
The rotund gene in Drosophila melanogaster is associated with a gene coding for a RacGTPase-activating protein,
RnRacGAP. Cellular studies have shown that RacGAP proteins function as negative regulators of
substrate Rac proteins which, in turn, control the localization and polymerization state of actin within
the cell. Previous sequence analysis of rn RacGTPase genomic DNA and incomplete cDNA clones suggests that
at least two differentially spliced forms of the transcript exist: rnRacGAP(1) and rnRacGAP(2). Using
nested reverse transcription-polymerase chain reaction (RT-PCR) methods, the missing
exon and intron sequences have been cloned, and differences have been detected between rnRacGAP(1) and rnRacGAP(2)
involving 24 nucleotides (nt) of coding sequences and 119 nt of 3'UTR. This translates to a difference
of seven amino acids at the C-termini of the polypeptide products. In RT-PCR analysis, utilization of
form-specific primers provides a simple assay for the tissue specificity of expression of the two forms.
rnRacGAP(1) is the predominant species in the testes and is expressed at a low level in the ovary and
somatic tissues. rnRacGAP(2) is only very weakly expressed and is detectable solely in the testes (Hoemann, 1996).
RacGAP proteins have been shown to down-regulate members of the Rho/Rac subfamily, small
GTPases controlling actin network organization. Only one RacGAP protein, RnRacGAP, has been
identified in Drosophila. To examine RnRacGAP function, transgenic strains were generated expressing
RnRacGAP under the control of the heat-shock promoter hsp70. In cellularising embryos, ectopic
RnRacGAP induces lethality, associated with radical cell-shape changes, apical F-actin delocalization,
and inhibition of basal actin polymerization. Overexpression of RnRacGAP in pupae induces a number
of phenotypes with distinct critical periods of induction. These include wing shape and margin changes,
wing vein defects, disorientation of wing hairs and thoracic bristles, and abdominal segment fusion.
Thus, changes in cell shape/adhesion and reorganization of the actin network are sensitive to
overexpression of RnRacGAP throughout development in Drosophila (Guichard, 1997).
A novel Drosophila gene, DRacGAP/RacGAP50C
has been identified that behaves as a negative
regulator of Rho-family GTPases Rac1 and Cdc42. Reduced function of DRacGAP
or increased expression of Rac1 in the wing imaginal disc cause similar effects
on vein and sensory organ development and cell proliferation. These effects
result from enhanced activity of the EGFR/Ras signalling pathway.
In the wing disc, Rac1 enhances EGFR/Ras-dependent activation of MAP Kinase in
the prospective veins. Interestingly, DRacGAP expression is negatively regulated
by the EGFR/Ras pathway in these regions. During vein formation, local DRacGAP
repression would ensure maximal activity of Rac and, in turn, of Ras pathways in
vein territories. Additionally, maximal expression of DRacGAP at the
vein/intervein boundaries would help to refine the width of the veins. Hence,
control of DRacGAP expression by the EGFR/Ras pathway is a previously
undescribed feedback mechanism modulating the intensity and/or duration of its
signalling during Drosophila development (Sotillos, 2000).
Evidence is presented for the
cooperation of Rac and Ras signaling pathways in the context
of a whole organism. A Drosophila gene,
DRacGAP, is described that encodes a putative GAP for Rac and Cdc42
GTPases. Both DRacGAP and DRac1 are
involved in the control of cell proliferation. Moreover, reduced
activity of DRacGAP or overexpression of DRac1 in the wing
imaginal disc cause similar defects: widening of veins,
development of extra sensory organs (SOs), apoptosis and the
appearance of enlarged cells that differentiate multiple hairs
with abnormal polarity. These phenotypes result
from DRac1 enhancement of epidermal growth factor receptor
(Egfr)/Ras signaling. The Egfr/Ras pathway pathway, which
operates through activation of the Ras/Raf/MEK/MAP kinase
cascade controls multiple developmental processes and is accurately regulated.
Indeed, this pathway controls the expression of its own
negative and positive regulators. Interestingly, expression of DRacGAP is repressed by
Egfr/Ras signaling in the prospective veins and accumulates
at the vein/intervein boundaries. These results suggest that
control of DRacGAP expression by the Egfr/Ras pathway
provides a new mechanism to modulate the intensity of this
pathway during Drosophila development (Sotillos, 2000).
DRacGAP gives rise to a unique transcript of 2.3 kb present
throughout development. The expression pattern of
DRacGAP is highly dynamic. It is ubiquitous during the initial
stages of embryogenesis and becomes restricted, after germ band
retraction, to the central and peripheral nervous system. In the early second instar wing imaginal disc, DRacGAP
expression occurs mostly in the presumptive wing region. Afterwards, DRacGAP mRNA accumulation is widespread
in all imaginal discs. At late third instar
DRacGAP is expressed in the eye-antenna disc in two stripes of
cells located at, and ahead of, the morphogenetic furrow and in
several rings of cells in the presumptive antenna. In
the wing disc, DRacGAP mRNA accumulates in the presumptive
interveins, where it persists in pupae, being stronger at
the vein/intervein boundaries (Sotillos, 2000).
Overexpression of DRacGAP in the wing disc interfers with
vein development. Expression of dominant negative (DN) DRacGap in the wing
disc results in loss of wing tissue (wing
notching), veins of increased width, fusion of adjacent veins
and appearance of vein material connecting neighbouring veins. In addition, wings have extra SOs along the vein L3
and in the proximal regions of veins L4 and L5. DRacGAP DN wings showed enlarged cells (indicated
by low trichome density) and alteration in the number
and polarity of the trichomes. The enlarged cells are
also visible in the imaginal disc (phalloidin staining, which
labels cortical actin) indicating the early onset of this defect (Sotillos, 2000).
By analogy with its closest homologs,
DRacGAP should regulate RacGTPase. Indeed, overexpression of wild-type Rac1 causes vein thickening,
development of extra SOs and wing enlargement. Coexpression of Rac1 and DN DRacGAP
has a synergistic effect,
suggesting that DRacGAP reduces Rac1 activity.
Consistently, the lethality of larvae overexpressing Rac1 is
reversed by coexpression of RacGAP and the
wing size reduction associated with a decreased activity of
Rac1 in DN Rac1 flies
is enhanced by coexpression of overexpression of DRacGAP (Sotillos, 2000).
Overexpression of DN DRacGAP or Rac1
causes vein enlargement and the appearance of extra
SOs, two structures requiring Egfr/Ras/Raf/MAPK activity. Since Rac and
Ras pathways cooperate in mammalian cells in the control of
cell proliferation, an investigation was carried out to see whether the
phenotypes associated with increased Rac signaling could be due
to overactivity of the Ras pathway. This appears to be the case,
since a reduction in Egfr signaling (by expression of DN Raf) reduces vein, wing
notching and large cell phenotypes. Similarly, when levels of the Egfr
activators rhomboid and vein (vn) are decreased, the wing notching associated with DN DRacGAP
expresson is substantially corrected. In contrast,
activation of Egfr signaling enhances the mutant phenotype
of DN DRacGAP flies. Thus, although flies heterozygous for
mutant argos, a repressor ligand of Egfr, are phenotypically wild type, its
combination with DN DRacGAP significatively increases the
number of campaniform sensilla of DN DRacGAP flies. These
results further suggest that the efficacy of the Egfr pathway
is enhanced by increased Rac activity. Rac signaling ultimately
activates MAPK since expression of DN DRacGAP widens
the domains of accumulation of dp-ERK in the presumptive
veins and spreads them into the interveins. This effect
is enhanced by coexpression of Rac1 (Sotillos, 2000).
Expression of DRacGAP appears to be decreased in the
domains of Egfr activation. This is most apparent in the
notum region of second instar wing disc, where the
Egfr activator vein is expressed, and in the
presumptive veins of third instar wing disc, territories
of maximal Egfr signaling. This observation suggests that the Egfr pathway may repress the expression of DRacGAP. In agreement with this notion,
expression of DRacGAP is either decreased or enhanced in
cells ectopically expressing Ras V12 or argos in which
the EFGR/Ras pathway is activated or repressed, respectively (Sotillos, 2000).
The enlarged cell size of wing cells overexpressing Rac
and the small size of wings with reduced Rac function indicate a role for
Rac in cell proliferation control. In mammalian cells, Ras and Rac pathways
cooperate in stimulating cell proliferation. Similarly, Drosophila Rac1 and Ras also appear to cooperate in this process since the reduced size of the
wings of DN Raf-expressing flies is largely normalized by reduction of
DRacGAP function or by overactivity of
Rac1. The induction of cell death by DN DRacGAP, where Rac activity is
upregulated, is in apparent contradiction with these results. However, note that in mammalian cells, quantitative variations in the level of Ras signaling can cause very different effects. Thus, instead of cell proliferation, high levels of
Ras signaling may induce cell cycle arrest
at G1 and apoptosis as part of a cell self
protective mechanism. In that situation, G1 arrest is caused by
Raf-dependent induction of p21 WAF1/CIP1
expression, which inhibits Cdk activity and
indirectly represses CycE transcription. The situation appears to be very similar in
Drosophila. Cell death of DN DRacGAP flies
could be attributed to their arrest at the G1 stage, since the phenotype is
rescued by expression of CycE. Moreover, the accumulation of
p21 causes apoptosis, enlargement of cell size and polarity
defects, phenotypes that are corrected by expression of CycE. Accordingly, it is
hypothesised that induction of cell cycle arrest and apoptosis by
overactivity of the Rac pathway could be a consequence of
increased Ras signaling. Rac would potentiate the reduced Raf
signaling occurring in DN Raf flies, allowing wing disc cells to
proliferate, but in the presence of wild-type Raf, overactivity
of Rac would enhance Raf signal to such a high level as to
induce p21 expression and ultimately, apoptosis. This interpretation is supported
by the partial rescue of the wing notching phenotype of
DN DRacGAP flies in a ve;vn heterozygous background and in
DN Raf flies where Egfr/Ras signaling is reduced (Sotillos, 2000).
Interestingly, in the Drosophila wing disc, DRacGAP
transcription is repressed by the Egfr/Ras pathway.
Activity of this pathway is finely tuned by its control of the
expression of its own inhibitors and activators. The results indicate that Ras signaling can self-stimulate through activation of the Rac pathway by
repression of DRacGAP. During vein formation, the
Egfr/Ras pathway, once it has attained a certain threshold,
should repress expression of DRacGAP in the prospective vein
regions, thus ensuring maximal activity of Rac and, in turn, of
Ras pathways in these territories of the imaginal wing disc,
which should trigger vein differentiation. In contrast, maximal
expression of DRacGAP at the vein/intervein boundaries
should locally decrease Rac and Ras signaling, and in
collaboration with Notch and Dpp pathways help to refine the final width of the veins. Hence, this regulatory loop is another feedback mechanism
modulating the activity of the Egfr/Ras pathway during
Drosophila development (Sotillos, 2000).
A Drosophila gene has been identified that has substantial sequence homology to a distinct class of
proto-oncogenes that includes DBL, VAV, Tiam-1, ost and ect-2. It has predicted Rho or Rac guanine
exchange factor (Rho/RacGEF) and pleckstin homology (PH) domains with the PH immediately
downstream of the Rho/RacGEF. Rho/RacGEFs catalyze the dissociation of GDP from the Rho/Rac
subfamily of Ras-like GTPases, thus activating the target Rho/Rac. Members of the Rho/Rac subfamily regulate organization of the actin
cytoskeleton, which controls the morphology, adhesion and motility of cells.
Message from this gene is found throughout oogenesis and embryogenesis. Of particular interest,
message is most abundant in furrows and folds of the embryo where cell shapes are changing and the
cytoskeleton is likely to be undergoing reorganization (Werner, 1997).
A motif-based search method was used to identify putative effector
proteins for Rac and Cdc42. A search of the GenBankTM data base for similarity with the minimum
Cdc42/Rac interactive binding (CRIB) region of a potential effector protein p65PAK has identified
over 25 proteins containing a similar motif from a range of different species. These candidate
Cdc42/Rac-binding proteins include family members of the mixed lineage kinases (MLK), a novel
tyrosine kinase from Drosophila melanogaster (DPR2), a human protein MSE55, and several novel
yeast and Caenorhabditis elegans proteins. Two murine p65PAK isoforms and a candidate protein
from C. elegans, F09F7.5, interact strongly in
a filter binding assay with the GTP form of both Cdc42 and Rac, but not with Rho. Three additional candidate proteins, DPR2, MSE55, and MLK3 showed binding
to the GTP form of Cdc42 and weaker binding with Rac, and again no interaction with Rho. These
results indicate that proteins containing the CRIB motif bind to Cdc42 and/or Rac in a GTP-dependent
manner, and they may, therefore, participate in downstream signaling (Burbelo, 1995).
Dorsal closure depends on the activities of a Jun amino (N)-terminal
kinase kinase (JNKK) encoded by the hemipterous gene, and of a JNK
encoded by basket. Hep is required for cell determination in the leading edge of
migrating epithelia, by controlling specific expression of the puckered gene in
these cells. puc encodes a protein related to vertebrate dual-specificity MAPK phosphatases of the CL100 family (E. Martin-Blanco, A. Gampel, and A. Martinez Arias, pers. comm. to Glise, 1997). During dorsal closure, decapentaplegic is expressed in the row of cells
making up the leading edge of the epithelia. The small GTPases
Dcdc42, Drac1, and the Hep JNKK control decapentaplegic expression in this migratory process. Activated Drac1 and Dcdc42 induce distinct, although partly overlapping, responses. Dcdc42 appears to be a good inducer of ectopic puc and dpp expression in ectodermal cells located more ventrally, whereas Drac1 seems more active in cells nearest to the leading edge.
Appropriate dpp and puc expression in the leading edge also depends on the inhibitory
function of the puc gene. puc acts as a repressor of dpp expression in the ectoderm, likely acting to inhibit Basket, the Jun N-terminal kinase. In puc mutants ectopic expression of puc and dpp is induced in the ectoderm, that is, outside the normal domain of puc expression in the leading edge. In addition, puc (but not dpp) is expressed ectopically in amnioserosa cells. These observations indicate a cell non-autonomous effect of puc mutations. The data suggest that the leading edge is the source
of a JNK autocrine signal, and exclude a role of Dpp as such a ligand. Dorsal closure
couples JNK and Dpp signaling pathways, a situation that may be conserved in
vertebrate development (Glise, 1997).
Rac activation is thought to be important for stimulating dorsal
closure because expression of dominant negative forms of Rac (DN Rac)
or Cdc42 inhibits dorsal closure in the Drosophila embryo. Since
activated Jra/Djun rescues the defect in dorsal closure induced by
expression of DN Rac, Rac probably functions upstream of
JNK activation to stimulate dorsal closure. To begin
to address the mechanism whereby Rac and Misshapen cooperate to
activate JNK, cultured cells were transfected with either Msn or NIK,
together with DN Rac and an epitope-tagged JNK, and kinase activity
assays were performed on JNK precipitates. Although overexpression of either
NIK or msn leads to a four- to five-fold increase in JNK
activation, coexpression of DN Rac markedly decreases JNK activation. Because PAK
family Ste20 kinases are activated by GTP-bound Cdc42 and Rac, it had been assumed that this family
of Ste20 kinases rather than an SPS1 Ste20 kinase family member would cooperate with Rac to
activate JNK. Thus, discovery of the role of Misshapen in conveying Rac signals to JNK has stimulated consideration of new paradigms for how
Rac functions to activate JNK. It is not thought that Rac activates Msn directly. Unlike PAK family
members, Msn does not contain a consensus Rac-binding motif and no binding of Msn to
activated Rac in vitro can be detected. Rather, it is hypothesized that Rac cooperates with Msn
to activate a downstream MKKK. MKKKs have been shown to bind GTP-bound Cdc42 or Rac. Thus, Rac may cooperate with Msn to regulate a downstream MKKK in a manner similar
to the way Ras cooperates with a yet to be defined kinase to activate RAF. In this model, binding of an
MKKK to activated Rac would facilitate interaction of this MKKK with Msn, thereby enabling its
activation by Msn. However, the possibility cannot be excluded that Rac and Msn activate
parallel pathways converging on JNK activation (Su, 1998).
Among the putative Rho/Rac effector targets in mammals
are the protein kinase
N/protein kinase C-related
kinase (PKN/PRK) family of
serine/threonine kinases. PKN (also referred to as PRK1) and the closely related protein PRK2 together account for the vast majority of Rho-binding autokinase activity detected in most mammalian tissues. The carboxy-terminal catalytic domains of
these kinases are highly homologous to the PKC family kinases, but they
possess unique amino-terminal regulatory sequences, including three
leucine zipper-like repeats shown to be important for the interaction with the Rho GTPase. These proteins also interact detectably with the Rac GTPase, suggesting that they may be shared effector targets of the
Rho and Rac GTPases.
Despite the identification of closely related PKN
homologs in several organisms, the precise biological function of these putative Rho targets
remains unknown. The ability of the Rho GTPases to regulate cell morphology and motility
suggests that these proteins and their associated signaling pathway
components are likely to perform functions essential to the normal
morphogenesis of developing multicellular organisms. Dorsal closure (DC) does
not require new cell divisions, and appears to depend solely on
dramatic cell shape changes within a subset of epidermal cells. These
shape changes are initially restricted to two symmetric rows of
epidermal cells, known as the leading edge (LE) cells, and are followed
by the stretching of the more lateral epidermal cells, ultimately
resulting in the meeting of the two rows of LE cells at the dorsal midline.
Three classes of genes have been implicated in DC; namely, the Rho
family of GTPases; the c-Jun amino (N)-terminal kinase (JNK) cascade
components, including the decapentaplegic (dpp) signaling
pathway genes, and several membrane-associated proteins. The recently described Drosophila loss-of-function Rho1 mutant is defective for DC, and
homozygous mutant embryos exhibit an obvious hole in the
dorsal-anterior portion of the larval epidermis.
Although Rac1 loss-of-function mutants have yet to be
reported, overexpression of Rac1N17, a dominant-negative form
of Rac1, in developing Drosophila embryos, results in a DC defect. Similar results have also been reported
for Cdc42N17 (Lu, 1999 and references).
Four Drosophila
homologs of the mammalian JNK pathway genes [hemipterous (hep; JNKK), basket (JNK), Djun, and kayak (Dfos)] are all required for DC. Mutations in
any of those genes result in a dorsal-open phenotype very similar to
that seen in either the Rho1 mutants or embryos overexpressing
dominant-negative Rac1 or Cdc42. Furthermore, disruption of the JNK
pathway in hep mutants abolishes the expression of two
independent downstream target genes of Djun, dpp and
puckered (a MAP kinase phosphatase), which are also required for DC. The
hep mutation also blocks an increase in dpp
expression in the LE cells induced by expression of activated Rac1V12 in those cells. These results have led to a model for
DC in which Rac1 (and possibly Cdc42) signals through the JNK pathway
to activate the expression in LE cells of Dpp, a secreted ligand of the
TGF-beta receptor. In turn, Dpp relays an instructive signal to
initiate stretching of the more lateral epidermal cells. The signaling role of Rho1 or downstream effector targets
of Rho1 in this process is unknown. A Drosophila homolog of the mammalian PKN family kinases,
Drosophila Pkn is shown to bind specifically to both Rho1 and Rac1 GTPases in a GTP-dependent manner, and its kinase activity is promoted
by both interactions, suggesting that Rho1 and Rac1 GTPases can
utilize Pkn as a downstream effector target. Both Rho1 and
Rac1 bind Pkn through the amino-terminal region of the protein. Significantly, it appears that Rho1 and Rac1 interact with Pkn
through distinct binding sites. A loss-of-function mutation in the Drosophila Pkn gene leads specifically to a DC defect during embryogenesis. However, this Pkn-mediated DC pathway is
independent of the Rac-JNK-Dpp pathway; rather, the Pkn-mediated DC pathway appears to act
coordinately with the Rac-JNK-Dpp pathway to regulate epidermal cell shape changes
during morphogenesis of the Drosophila embryo (Lu, 1999 and references).
The predicted Drosophila Pkn protein sequence is closely
related to both human PKN and PRK2 (60% overall identity to both) and has all of the conserved features found in other PKN family members, including the amino-terminal negative
regulatory pseudosubstrate motif, the three
leucine zipper repeats (HR1a, HR1b, and HR1c) that mediate GTPase
binding, a central conserved
region of unknown function found in the PKC-eta kinases, and the carboxyl-terminal PKC-like kinase domain (Lu, 1999).
The expression pattern of the Drosophila Pkn gene is highly
dynamic during embryogenesis. An in situ hybridization analysis of wild-type embryos reveals that Pkn mRNA is
abundant at the blastodermal stage, suggesting that it is maternally
loaded. At stage 13, when DC is normally initiated, the most prominent expression is seen in the dorsal LE cells and in two pairs of discontinuous stripes on the epidermis of each segment. However, the
expression becomes more restricted in later stages and can only be
detected in the anterior and posterior spiracles, the pharynx, and the
mouth tip at stage 16 (Lu, 1999).
Although homozygous Pkn mutant embryos do not exhibit
obvious developmental defects prior to stage 13, ~10% of them die
as embryos with an obvious hole in the anterior region of the dorsal
epidermis. This dorsal-open phenotype closely resembles the
DC defects observed previously with loss of function of the
Rho1 gene and several components of the Rac-mediated
JNK cascade. To examine the requirement for maternally loaded Pkn mRNA,
germ-line clone (GLC) mutants of Pkn were generated:
~50% of the mutant embryos derived from these clones are found to
display the same DC defect. Phenotypic analysis of these
embryos with various histological markers reveals that they are
correctly patterned and do not exhibit any detectable defects of the
central or peripheral nervous system or the somatic musculature, suggesting that Pkn is not required prior to DC (Lu, 1999).
According to the current working model of DC, the Rac1 and Cdc42
GTPases activate the downstream JNK cascade kinases to induce the
expression of several genes in the LE cells, including dpp. Thus, DPP mRNA expression in the LE cells is a convenient assay of activation of the JNK cascade in
vivo. Because Pkn functions biochemically as a Rac1 effector target,
and mutations in Pkn cause a DC defect similar to that seen
with JNK pathway mutants, it was of interest to determine whether Pkn is
required for the previously established Rac-JNK-Dpp pathway. Therefore, the expression of DPP mRNA was examined in
Pkn mutant embryos. Mutations in the JNK cascade gene hep
result in embryos that fail to express detectable levels of
DPP mRNA in the LE cells. In contrast to the loss of dpp
expression seen in hep mutant embryos, expression of
dpp in the LE cells of Pkn mutants is not detectably
affected, indicating that Pkn is not
required for the previously reported Rac-JNK-Dpp pathway. This
conclusion was also verified by examination of the expression of beta-galactosidase from a puckered-lacZ enhancer trap, which
has been shown previously to be eliminated in a hep mutant
background. Pkn mutant embryos
that exhibit an obvious dorsal-open phenotype retain normal levels of
puckered-lacZ expression, confirming that the
Rac-JNK pathway leading to transcriptional activation is not
detectably affected by the absence of Pkn (Lu, 1999).
The shapes of epidermal cells of Pkn mutant
embryos were examined. All epidermal cells adopt an unstretched polygonal shape following an initial apparently normal LE
cell stretching, similar to that seen in the JNK pathway
mutants, such as hep. Thus, it appears that while
both Rac-JNK- and Pkn-mediated signals are required for the stretching
of epidermal cells required for DC, they are associated with distinct
pathways. Because the expression of Pkn mRNA is enriched specifically
in the LE cells prior to DC, the possibility that Pkn
expression is regulated by the JNK pathway was examined. However, Pkn
expression is unaffected in hep mutant embryos (Lu, 1999).
Accumulating evidence suggests that the Rac1 GTPase, as well as some
of the JNK pathway components, performs a function in the LE cells that
may be distinct from the regulation of gene expression, but is
necessary for the stretching of the LE cells. Because Pkn is a biochemical effector of the
Rac1 GTPase that mediates DC, but is not required for dpp or
puckered gene expression, the possibility was tested that the
Pkn mutant interacts genetically with the JNK pathway
component, basket. Removal of one copy of the
basket gene from a Pkn mutant GLC background
significantly increases the frequency of dorsal-open embryos, suggesting that both Pkn and JNK activities converge at some point to affect a related aspect of the DC process (Lu, 1999).
The Rho1 GTPase has also been implicated in DC.
Null alleles of Rho1 exhibit a DC defect that closely resembles that seen in Pkn mutants although the relevant Rho1-mediated pathway in this process has not been established. Because
the cell shape changes in the LE cells appear to initiate the DC
process, and Pkn expression is enriched in the LE cells just prior to
DC, the possibility was explored that a Rho1-Pkn signal
mediates shape changes in those cells. To start, the
requirement for Rho1 activity was examined specifically in the LE cells. A dominant-negative form of Rho1
(Rho1N19) is expressed in the LE cells of wild-type embryos. More than 60% of these
embryos display a dorsal-open phenotype very similar to that seen in
the Rho1, Pkn, and JNK pathway mutants. Similar to Rho1 and
Pkn mutants, but not to a hep mutant, embryos
expressing Rho1N19 in the LE cells exhibit normal levels of
DPP mRNA expression in the LE cells. Moreover, all
of the epidermal cells in these embryos ultimately adopt an unstretched
polygonal shape following a normal initial LE cell stretching at early
stage 13. This result demonstrates clearly that expression of the dominant-negative Rho1 in the LE cells does not cause a dorsal-open phenotype by nonspecifically blocking the Rac-JNK-Dpp pathway and
suggests that a Rho1-mediated second instructive signal is generated in
the LE cells, which together with Dpp, is required for the stretching
of the more lateral-ventral cells (Lu, 1999).
In light of the fact that Drosophila Pkn interacts equally
well with the activated Rac GTPase, it is possible that a Rac-Pkn interaction contributes to DC. However, the lack of a
Rac1 loss-of-function mutant in Drosophila makes it
difficult to examine the specific role of that interaction. Because the
JNK pathway mutants are also associated with a defect in stretching of
the LE cells, it has been suggested that components of the JNK pathway may mediate a Rac-dependent cell stretching signal that is unrelated to
transcriptional regulation. It is difficult to imagine how Pkn
could transduce a signal from Rac to this JNK-mediated cell shape
change pathway and yet not be required for the Rac-JNK transcriptional
pathway. However, it is possible that Pkn can transmit a Rac signal
independent of the JNK-Dpp pathway. Indeed, recent evidence suggests
that the Drosophila gene Myoblast city, which is
required for DC, encodes a Rac-specific activator that does not appear to regulate dpp expression
(Nolan, 1998). This observation suggests that Rac may perform
multiple functions in dorsal closure (Lu, 1999).
Significantly, there does seem to be some cross-talk between the
Pkn-mediated signaling pathway and the JNK pathway. Removal of one copy of
basket from a Pkn mutant GLC background significantly
increases the frequency of dorsal-open embryos. This result suggests
that some component of JNK cascade signaling is sensitive to the
activity of Pkn. Taken together with the fact that Rho1 generates a
JNK-Dpp independent signal in the LE cells that is required for DC, it is clear from these studies that distinct but coordinated signaling pathways mediated by the Rho and Rac GTPases within the LE cells are
essential for normal DC, and that Pkn is a strong candidate for an
effector that mediates signals downstream of both GTPases (Lu, 1999).
spire is a maternal effect locus that affects both the dorsal-ventral
and anterior-posterior axes of the Drosophila egg
and embryo. It is required for localization of determinants
within the developing oocyte to the posterior pole and to
the dorsal anterior corner. During mid-oogenesis, spire
mutants display premature microtubule-dependent
cytoplasmic streaming, a phenotype that can be mimicked
by pharmacological disruption of the actin cytoskeleton
with cytochalasin D. spire has been cloned by transposon
tagging and is related to posterior end mark-5, a gene from
sea squirts that encodes a posteriorly localized mRNA.
Spire mRNA is not, however, localized to the posterior
pole. Spire also contains two domains with similarity to
the actin monomer-binding WH2 domain, and Spire binds to actin in the interaction
trap system and in vitro. In addition, Spire interacts with
the rho family GTPases RhoA, Rac1 and Cdc42 in the
interaction trap system. This evidence supports the
model that Spire links rho family signaling to the actin
cytoskeleton (Wellington, 1999).
Previous work has shown that in spir mutants, the
microtubules bundle at the cortex prematurely, during stage
8, and this bundling of the microtubules is accompanied by
rapid, microtubule-dependent swirling of the cytoplasm. Both the bundling of
microtubules and cytoplasmic streaming are normally seen
later in stage 10 wild-type oocyte. A bi-directional signaling process occurs between the oocyte
and the posterior follicle cells to establish the posterior pole of
the egg.
Phenotypes indicative of a defect in this signaling process
include transformation of the posterior follicle cells into an
anterior follicle cell fate; misorganization of the microtubules
at stage 6; localization of Oskar mRNA to the center of the
oocyte; localization of Bicoid mRNA to the posterior pole, and
premature cytoplasmic streaming. The posterior follicle cell fates are established correctly in
spir. In contrast to a previous report, no central spot of Oskar mRNA
staining is observed in spir mutants. Finally, Bicoid mRNA localization
appears relatively normal in spir. These results
suggest that signaling between the posterior follicle cells and
the oocyte is not abnormal in spir mutants. In spir mutant oocytes, microtubules sometimes show abnormal
distributions during stage 6, but it is believed that
this probably reflects an earlier manifestation of the known spir
microtubule defect (Wellington, 1999 and references therein).
WH2 domains, like those found in Spir, have been found
in the Wiskott-Aldrich syndrome protein (WASP), verprolin,
Scar-1 (see Drosophila SCAR), and a number of other proteins of unknown function. The WH2 domains of N-WASP and of Scar1
have been shown to bind directly to G-actin in vitro. In
addition, Spir binds to unpolymerized
actin in vitro.
Although Spir is capable of binding to actin monomers
through its WH2 domain, no defects in the
actin cytoskeleton have been observed in spir mutants, suggesting a number of
possibilities. The defects may be in actin structures that are
difficult to observe, such as the cell cortex. In fact, spir phenotypes can be mimicked by treatment with
cytochalasin D, a drug that affects the polymerization state of
actin; defects in the actin
cytoskeleton in cytochalasin D-treated oocytes have not been observed. Alternatively,
spir may act downstream of the actin cytoskeleton and, thus,
not change it. Finally, in vitro experiments have shown that, in
the absence of the neighboring cofilin homology and acidic
domains, the two WH2 domains of N-WASP have either no
effect on or slowly depolymerize filamentous actin. Since Spir is lacking the cofilin homology
and acidic domains, Spir may have only minor or no
effect on filamentous actin (Wellington, 1999 and references therein).
It is becoming more apparent that a relationship exists
between the actin cytoskeleton and premature microtubule-dependent
cytoplasmic streaming. The premature cytoplasmic streaming phenotype of spir can be
mimicked by the addition of cytochalasin D,
a drug which depolymerizes actin filaments. Additional
evidence that the actin cytoskeleton is involved in repressing
microtubule bundling and streaming comes from analysis of
mutant phenotypes of genes linked to the actin cytoskeleton.
In addition to spir,
mutants in chickadee, which encodes profilin and capu, which
is thought to bind to profilin, also exhibit these microtubule
behaviors. While phenotypes for cdc42 and rac1 have been
described during oogenesis, their effects on patterning during
oogenesis are unknown. The
finding that Spir interacts with rho family GTPases suggests
that at least one of the rho family GTPases is functioning in
patterning. Further genetic and biochemical studies will be
required to determine the nature of Spir's interaction with rho
family GTPases in vivo.
The analysis of spir suggests that rho family GTPAses and
actin function with Spir in patterning the Drosophila oocyte.
Further studies on spir should elucidate the role of rho family
GTPases and the actin cytoskeleton in patterning during
oogenesis (Wellington, 1999).
Axon terminals change morphology with differentiation to become mature synapses. A molecule that might regulate this process has been identified by a screen of Drosophila mutants for abnormal motor activities. The still life (sif) gene encodes a protein homologous to guanine nucleotide
exchange factors, which convert Rho-like guanosine triphosphatases (GTPases) from a guanosine diphosphate-bound inactive state to a guanosine triphosphate-bound active state. The Sif proteins are found adjacent to the plasma membrane of synaptic terminals. Expression of a truncated Sif protein results in defects in neuronal morphology and induced membrane ruffling with altered actin localization in human KB cells. Thus, Sif proteins may regulate synaptic differentiation through the organization of the actin cytoskeleton by activating Rho-like GTPases (Sone, 1997).
The structure and localization of Sif suggest that Sif activates
Rho-family G proteins in the periactive zones. However, the
family comprises several members including Rac, Cdc42
and Rho, each playing a distinct role in a variety of cellular
events. It
is therefore important to estimate which member of the family
is activated by Sif in the periactive zone. The
GEF activity of Sif was assayed using Sif's catalytic ability to
dissociate [3H]GDP from mammalian RAC1, CDC42 and
RHOA, employing bacterially expressed a glutathione-S-transferase
(GST)-fusion protein carrying the DH domain and adjacent
Pleckstrin-homology (PH) domain of Sif. The
GST-SIF stimulates the dissociation of GDP from RAC1 both
in a time-dependent and dose-dependent manner, but does not show the activity for CDC42 and RHOA. Thus, these data demonstrate that Sif specifically
activates Rac1 in vitro, suggesting that Sif activates Rac in
the periactive zones (Sone, 2000).
A cell-adhesion molecule Fasciclin 2, which is required for synaptic growth, and Still life (Sif), an activator of Rac, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. betaPS integrin and Discs large, both involved in synaptic development, also decorate the zone. However, Shibire (Shi), the Drosophila dynamin that regulates endocytosis, is found in the distinct region. Mutant analyses show that sif genetically interacts with Fas2 in synaptic growth and that the proper localization of Sif requires Fas2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. It is proposed that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively (Sone, 2000).
To characterize the Sif localization pattern, particularly in reference to synaptic functional domains, the subcellular distribution of Sif in the boutons of larval neuromuscular junctions was examined by concomitant staining with anti-Pak antibody using laser-scanning confocal microscopy. Anti-Sif antibody labels the synaptic boutons in a network-like pattern, which is strikingly complementary with Pak staining in the boutons. The cross-section profile of the fluorescent intensity also shows that the staining patterns of Sif and Pak are mostly complementary to each other. These staining patterns demonstrate that the areas stained for Sif surround the active zones. Close examinations further reveal that anti-Sif and anti-Pak antibodies produce a number of concentric figures that are occasionally separated from each other. These data suggest that the active zone and the outer ring together form a structural unit that constitutes a synapse. The Sif-positive regions around the active zones are referred to as periactive zones (Sone, 2000).
To characterize the periactive zone, especially in identifying its functional significance, the distribution patterns of other molecules were examined with the aid of Pak staining. Monoclonal antibody, MAb1D4, against Fas2 labels the boutons in a complementary pattern with Pak staining. Fas2 staining surrounds the Pak-positive regions and forms concentric patterns as observed for Sif staining. The cross-section profile also shows similar patterns as Sif and Pak double staining. Sif and Fas2 are indeed co-localized in overlapping network-like patterns. Fas2 is involved in synaptic growth, stabilization and structural plasticity, possibly through its homophilic adhesion. These data suggest that Fas2 controls these synaptic events locally in the periactive zones. Thus, the periactive zone is characterized by the specific localization of two distinct types of molecules: a cell adhesion molecule (Fas2) that controls synaptic development and an intracellular molecule (Sif) that is a GEF to Rac (Sone, 2000).
In an attempt to understand the function of the periactive zone further, other molecular markers that stain the zone were sought. MAb6G11, the monoclonal antibody against betaPs integrin (Myospheroid) that is structurally similar to the vertebrate integrin beta1 subunit also shows staining that is complementary to Pak staining. Unlike Sif and Fas2, however, MAb6G11 staining is observed much more diffusely on the muscle surfaces surrounding the outside of the bouton, suggesting the staining in the postsynaptic specialization: the subsynaptic reticulum. In mutants of the mys gene the extent of the cell contact between nerve terminals and muscles is altered by either a primary or secondary effect of the mutation, and the growth of larval neuromuscular synapses is affected. These synaptic defects observed in the betaPs integrin mutants may represent its function in the periactive zones (Sone, 2000).
The polyclonal antibody against Dlg protein stains synaptic boutons in a way similar to MAb6G11. The Dlg staining also appears to be moderately diffused on the muscle surfaces surrounding the bouton. This pattern is complementary with the anti-Pak staining when the bouton is scanned at the surface level. In dlg mutants, the structural properties of synapses, including the formation of subsynaptic reticulum at the postsynapses and the number of active zones at the presynapses, are altered. Furthermore Dlg regulates the synaptic localization of Fas2 by binding directly to the cytoplasmic tail of Fas2. Therefore, one of the roles for Dlg in synaptic development is probably the localization of Fas2 to the periactive zone. These observations indicate that two additional molecules, betaPs integrin and Dlg, are present in synaptic areas including the periactive zones. They are both involved in the structural development of the neuromuscular synapses, and therefore appear to participate in the control of synaptic development in the periactive zones (Sone, 2000).
It has been suggested that a dynamin-rich domain in the presynaptic terminal functions as a site for the vesicular endocytosis. A recent study has also indicated that this domain is distinct from the active zone for exocytosis and instead surrounds the active zone. It is of interest to find out whether the periactive zone is involved in endocytosis and therefore the spatial relationships between the dynamin-rich domain and the periactive zone were examined. Synaptic boutons were co-stained with anti-Fas2 antibody and a polyclonal antibody against the Shi protein, the Drosophila homolog of dynamin. Shi is an essential factor for endocytosis, since endocytosis is completely blocked in the shi mutant. Anti-Shi antibody stains synaptic boutons in donut-like patterns but these patterns are found almost within the holes of the Fas2 rings. This Shi staining may partly overlap with Fas2 staining, but these staining patterns are distinct from each other. Thus, it is concluded that the periactive zone does not coincide with the dynamin-rich zone and therefore does not likely represent a functional domain for endocytosis (Sone, 2000).
Molecules involved in the synaptic development are present in the periactive zone. This implies that the periactive zone is the membrane domain that is specialized for the control of synaptic development. To test this possibility, the NMJ phenotype of the sif mutants was examined. Since the originally identified sif mutation is a hypomorphic allele, stronger sif mutant alleles induced with a chemical mutagen, ethyl methanesulfonic acid, were isolated. Among the alleles newly isolated in this study, one allele, sif ES11, is possibly functionally null, because the wild-type protein of more than 200 kDa has disappeared in the mutant flies, as revealed by a Western blot. Sequence analyses of the cDNAs indicate that the allele produces a truncated protein as a result of a frameshift mutation. The truncated protein lacks the Dbl-homology (DH) domain that catalyzes the GEF activity (Sone, 2000).
The mutant flies exhibit reduced locomotor activity, as observed for the original sif allele. In the mutant larvae, the synaptic bouton number of NMJ is slightly but significantly reduced when compared with the control larvae. Moreover, the reduced bouton number found in the mutant is rescued by expression of a sif minigene in neurons under the control of the elav promoter. These results suggest that sif functions in the regulation of synaptic growth. However, properties of basic synaptic transmission at the NMJs, including the frequency and amplitude of spontaneous MJPs, the amplitude of EJPs and the quantal content, are not significantly altered in the sif ES11 mutants. In addition, repetitive stimulation at 0.35 mM Ca2+ causes a similar decrease of EJPs in the mutants, as has been observed in the wild-type NMJs, and vesicle endocytosis is apparently unchanged, as estimated by the uptake of FM1-43. These data indicate that the lack of GEF activity of Sif results in no apparent effects on basic electrophysiological functions, which is consistent with the separate location of the active and periactive zones in the synaptic terminals (Sone, 2000).
Since Sif and Fas2 are co-localized in the periactive zone, a test was performed to see whether there is a genetic interaction between sif and Fas2 loci. The hypomorphic allele of Fas2, Fas2e76, shows a reduced number of boutons: would changing the dose of sif+ affect this bouton number phenotype? Strikingly, the double mutant of Fas2e76 and sif ES11 recovered the bouton number phenotype to the wild-type level, which suggests the presence of a suppressive genetic interaction between the two loci. To assess this genetic interaction further, the effect of Sif overexpression in the Fas2e76 background was examined. Sif overexpression does not clearly affect the synaptic bouton number in the wild-type background; rather, it causes a significant reduction in Fas2e76. Moreover, NMJs with extremely few synaptic boutons (less than 20) are observed in the Fas2e76 larvae overexpressing Sif, when compared with the NMJs in Fas2e76. Taken together, these data suggest that Sif and Fas2 are the components of related signaling pathways that control synaptic development, and Sif may, in an inhibitory manner, modulate the effect of Fas2 that regulates synaptic growth (Sone, 2000).
The possibility that the molecules in the periactive zone may affect each other in establishing their zonal localization was examined. Because Sif and Fas2 co-localize typically in the periactive zones and interact genetically, focus was placed on these proteins and an investigation was carried out to see if any perturbation occurs in the distribution of several molecular markers in the mutant background of Fas2 or sif. In the sif mutants, the localization of Fas2 is indistinguishable from the wild type. Conversely the Sif localization is normal in a hypomorphic allele of Fas2. However, in the boutons of a more severe hypomorphic allele of Fas2, Fas2e76, the Sif localization to the periactive zones is perturbed. The Fas2e76 mutation reduces Fas2 expression to 10% of the wild-type level. In most Fas2e76 boutons, the network pattern of Sif staining is still observed, but frequently in an irregular or diffused fashion. In more extreme cases, Sif is distributed almost evenly throughout the boutons or is largely concentrated on one side of the boutons so that Sif staining considerably overlaps Pak staining (Sone, 2000).
The mutant larvae could be distinguished from the wild type by the blind test for the Sif and Pak co-staining patterns. Similar results were obtained in the heterozygotes with Fas2e76 and a Fas2 null allele, suggesting that the alteration of Sif localization is not due to a second-site mutation on the Fas2e76 chromosome. To investigate the altered distribution of Sif further, the mutant boutons were examined under the electron microscope. A large number of Sif signals are occasionally present in the medial portions of the electron-dense regions in the Fas2e76 boutons and these signals are still associated with the plasma membrane, as are the signals observed in the wild type. It is therefore concluded that the reduction of Fas2 in the periactive zones results in the improper localization of Sif along the plasma membrane. Previous study has shown that the synaptic localization of Fas2 requires Dlg. Therefore, the localization of Sif was examined in the dlg mutant background, but no apparent alteration was found in the network pattern. A considerable amount of Fas2 is still present in the periactive zones of dlg mutant boutons, while faint or no staining is detected in the Fas2e76 boutons. This residual Fas2 seems to be sufficient to sustain the proper localization of Sif in the dlg mutants (Sone, 2000).
In mice, TIAM1 and STEF have been identified as GEFs
that specifically activate RAC1, and both are highly related to Sif in domain
organization and amino acid sequence in several domains,
indicating that these two proteins are likely to be the mouse
orthologs of Sif. Interestingly, both Tiam1 and Stef are
expressed in the brain, and Tiam1 expression is observed in the adult
hippocampus. Therefore, it would be important
to examine whether TIAM1 and STEF are localized
in the synaptic terminals (Sone, 2000 and references therein).
RAC is well known as a regulator of the actin-based
cytoskeleton and cell adhesion in various cells. RAC is also implicated in the
structural changes of nerve terminals, including growth cones and
dendrites. Therefore, in the periactive zones, activated
RAC may locally regulate the processes of the structural change
in the synaptic terminals, which include reorganization of the
actin-based cytoskeleton and cell adhesion (Sone, 2000 and references therein).
Previous studies have shown that RAC acts in the neurite
outgrowth of neuroblastoma cells that depend on the signal
from integrin on the cell surface. The
mammalian SIF homolog TIAM1, which functions as a RAC
GEF, recruits integrin to specific adhesive contacts at the cell
periphery. Moreover, expression of
TIAM1 increases cadherin-mediated cell adhesion in epithelial
MDCK cells. Therefore, there appear to
be signaling links between the RAC and cell-adhesion
molecules. Sif activates Rac; sif
genetically interacts with Fas2 in synaptic growth and the Sif
localization is perturbed in the Fas2 mutants. Taken together,
these data suggest that the Sif-Rac pathway is linked to the
cell-adhesion molecule Fas2 in close vicinity in the periactive
zone (Sone, 2000).
The periactive zone has been indicated as a region for the
control of synaptic development. The periactive zone
surrounds the active zone, which is the site for vesicle
exocytosis or neurotransmission. This concentric organization
suggests that the two zones specialize for the different cellular
functions and constitute an elemental unit for the presynaptic
structure. Investigation of how these zones are incorporated
into the synaptic bouton during development will be of interest.
The segregated distribution of the two zones suggests that
the mechanisms controlling synaptic development and
neurotransmission may be separable. This view is supported
by the mutant analyses for Fas2 and Sif; both mutations affect
structural properties of synapses without changing basic
electrophysiological functions. In the NMJs of Fas2 mutants,
the bouton number is decreased or increased depending on the
alleles but the total synaptic strength is maintained at the
normal level. Functional strength of the synapse is regulated only through
the activity of a transcription factor, cAMP-response-element-binding
protein (CREB), which functions independently of
Fas2. Also in sif mutants, the basic
electrophysiological properties of NMJs are normal. These
observations clearly contrast with the mutant phenotypes for
the proteins controlling vesicle exocytosis: Synaptotagmin,
Cysteine string protein, n-Synaptobrevin and Syntaxin 1A. Mutants in genes coding for all these proteins show impaired EJPs. Taken together, these results indicate that synaptic development and neurotransmission are genetically
separable phenomena and are regulated by independent
pathways. It is proposed that these genetically separable
phenomena are spatially segregated into the two zones on the
presynaptic plasma membrane, although the possibility that the two zones interact with each other cannot be excluded (Sone, 2000).
Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance. Using yeast two-hybrid screening and in vitro interaction assays, it has been shown that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induces similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. The findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration (Driessens, 2001).
Many previously identified Rac targets contain a distinctive Rac binding site, the CRIB motif, but sequence analysis does not reveal any obvious CRIB-like sequence in plexin-B1. To identify the region of plexin-B1 that contains the Rac interaction site, a series of truncations were expressed as GST fusion proteins in E. coli. These were used in a dot blot assay. Rac interacts with a region encompassing 180 residues (amino acids 1724-1903) of the receptor (Driessens, 2001).
Plexin-B1 is a member of a large family of transmembrane proteins, and based on sequence alignments, four classes of plexins (A, B, C, and D) have been described. To test whether Rac could interact directly with other members of the family, cDNAs were obtained for human plexin-A2 (kiaa0463), plexin-B2 (kiaa0315), and plexin-D1 (kiaa0620). A region corresponding to amino acids 1724-1903 of plexin-B1 was cloned into the pGEX vector; GST fusion proteins were analyzed in the dot blot assay, but under these conditions only plexin-B1 was found to interact (Driessens, 2001).
In Drosophila, two plexins have been identified: Drosophila plexin-A and Drosophila plexin-B. Recombinant Drosophila plexin-B protein (C-terminal 435 amino acids, similar to plexin-B1 two-hybrid clone) interacts strongly with in vitro translated Drosophila L61Rac1 and weakly with wild-type Drosophila Rac1 in a pull-down experiment. Drosophila plexin-A does not interact with Drosophila Rac1 under the same conditions. A Drosophila plexin-B fragment corresponding to amino acids 1724-1903 of human plexin-B1 interacts similarly with Drosophila Rac1, as does a shorter, 149 amino acid region. Partial binding was observed with a 54 amino acid domain (Driessens, 2001).
Two blocks of sequence similarity, of approximately 320 and 150 amino acids each, have been identified in plexin cytoplasmic domains. These two blocks of sequence similarity are separated by a variable linker. This linker region is most divergent between the plexin subfamilies. The minimal Rac binding region in Drosophila plexin-B consists of the last 149 amino acids of the first conserved block but does not contain the linker region. Alignment of this 149 amino acid region of Drosophila plexin-B with other human plexins reveals a sequence highly conserved among all plexin subfamilies (Driessens, 2001).
A mechanism is proposed for plexin-B signaling to the actin cytoskeleton. In this mechanism, clustering of B plexins induces a Rac-dependent activation of Rho. These results provide a framework for the further exploration of the complex mechanisms by which plexins affect the actin cytoskeleton in different cell types, including neurons (Driessens, 2001).
Plexins are neuronal receptors for the repulsive axon guidance molecule Semaphorins. Plexin B (PlexB) binds directly to the active, GTP-bound form of the Rac GTPase. A seven amino acid sequence in PlexB is required for RacGTP binding. The interaction of PlexB with RacGTP is necessary for Plexin-mediated axon guidance in vivo. A different region of PlexB binds to RhoA. Dosage-sensitive genetic interactions suggest that PlexB suppresses Rac activity and enhances RhoA activity. Biochemical evidence indicates that PlexB sequesters RacGTP from its downstream effector PAK. These results suggest a model whereby PlexB mediates repulsion by coordinately regulating two small GTPases in opposite directions: PlexB binds to RacGTP and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA (Hu, 2001).
Plexin B binds to the active form of Rac (RacGTP); the binding maps to a 147 amino acid region, PlexBDelta3 (amino acids 1617 through 1765). To identify the critical binding sequence in PlexBDelta3, small deletions and point mutations were introduced and a seven amino acid sequence NTLAHYG (1722 through 1728) toward the C terminus of PlexBDelta3 has been identified that, when deleted, abolishes Rac binding (PlexBDelta3d7). Deletions in neighboring regions 1743 through 1759 (PlexBDelta3d17) and 1707 through 1714 do not affect Rac binding (Hu, 2001).
The NTLAHYG sequence is highly conserved among Plexin family members. In particular, the tyrosine residue within the sequence is invariable. In human Plexin B1, a putative Cdc42/Rac interactive binding (CRIB)-like motif right after this conserved sequence has been described. Although the CRIB-like motif is not found in Drosophila PlexB, this may reflect a conservation of the binding mechanism at a higher structural level. The Psi blast program predicts two blocks of sequences in the Plexin cytoplasmic domain that share similarity with R-ras family GAP proteins. The sequence needed for RacGTP binding is located between these two GAP-like regions (Hu, 2001).
In the Drosophila genome, there are six Rho family small GTPases: Rac1 (referred to here as Rac), Rac2, Cdc42, RhoA, Mtl, and RhoL. To gain some insight into the specificity of the interaction, the binding of PlexBDelta3 with all six Drosophila Rho-like GTPases was examined. Only Rac and Rac2, which share the highest degree of sequence similarity (93% identity), show strong interactions with the BDelta3 region of PlexB (Hu, 2001).
Several lines of evidence suggest that RhoA is also involved in PlexiB signaling. Clustering of the vertebrate PlexB in Swiss 3T3 cells leads to stress fiber formation, indicative of Rho activation. The response can be blocked by inhibitors of Rho or of its downstream effector Rho kinase. Genetic data also indicate that RhoA mediates part of Plexin B signaling in embryonic axon guidance. It was of interest, then, to enquire whether RhoA may also directly associate with PlexB (Hu, 2001).
PlexBDelta, a larger piece of the PlexB cytoplasmic domain (1617 through 1827) binds to RhoA. In contrast to a preferential binding to GTPgammaS-bound Rac, PlexBDelta binds to the GTPgammaS and GDP-bound forms of RhoA equally well. The binding requires the last 40 amino acids of PlexBDelta. The seven amino acid internal deletion that eliminates PlexBDelta binding to Rac does not affect its binding to RhoA. Thus, two independent regions in PlexB cytoplasmic domain have been defined that are important for PlexB association with Rac and RhoA, respectively. Cdc42, another Rho family GTPase, does not bind to PlexBDelta (Hu, 2001).
Dosage-sensitive genetic interactions suggest Rac antagonizes Plexin B signaling. Since there is no mutant available for PlexB with which to examine genetic interactions, whether the gain of function phenotype of PlexB is sensitive to the level of expression of Rac or RhoA was examined. PlexB is endogenously expressed by CNS neurons. Overexpression of PlexB in all embryonic CNS neurons can be achieved with the UAS-GAL4 binary expression system. Flies containing the UAS-PlexB transgene reporter are crossed to flies carrying a neuron-specific transcriptional control driver, elav-GAL4. With two independent UAS-PlexB transgenic lines, a consistent, GAL4-dependent phenotype was observed in specific motor nerve branches. In particular, a striking defect was observed in the ability of the ISNb (intersegmental nerve b) motor axons to innervate the ventral longitudinal muscles 7, 6, 13, and 12. In wild-type embryos, the ISNb projects into the ventral longitudinal muscles. Particular motor axons innervate specific muscles; for example, the RP3 motor axon innervates muscles 7 and 6, while other ISNb axons innervate muscles 13 and 12. When PlexB is overexpressed in these neurons, two types of phenotypes are observed that are consistent with PlexB being a repulsive guidance receptor for muscle-expressed Semaphorins: (1) the RP3 axon frequently fails to defasciculate from the ISNb motor nerve branch, and as a result muscles 7 and 6 are uninnervated; (2) ISNb axons often fail to reach their distal-most target muscle 12 (scored as 'stall'). The copy number of UAS-PlexB transgene and elav-GAL4 driver was varied to generate embryos with a range of levels of expression of PlexB; 'RP3 missing' and 'stall' phenotypes are dose dependent. This dosage sensitivity suggests that the PlexB gain-of-function phenotype may provide a sensitive background for revealing genetic interactions with genes encoding downstream components involved in Plexin B signaling (Hu, 2001).
Does the binding of PlexB to Rac increase or decrease the output of Rac? It was reasoned that if increasing PlexB expression produces its effect by activating Rac, then genetically limiting Rac gene dose might suppress the PlexB overexpression phenotype. Alternatively, if PlexB signals by turning down Rac activity, then an enhancement of the plexB overexpression phenotypes might result when Rac is reduced. Indeed, the results support the second alternative: PlexB inactivates Rac. Rac protein level was reduced by 50% using a small deficiency line, Df(3L)Ar14-8 (61C04-62A08), in a moderate PlexB overexpression background (one copy transgene, one copy driver). This resulted in a distinct increase in the penetrance of PlexB gain of function phenotypes. A complementary effect results when Rac dosage is increased in the same neurons where PlexB is overexpressed with a UAS-Rac transgene. Under such conditions, a suppression on the PlexB gain of function phenotypes is observed (Hu, 2001).
Consistent with the idea that PlexB signals by downregulating Rac activity, the Plexin gain-of-function stall phenotype is reminiscent of the loss-of-function phenotype of a positive regulator of Rac, the Trio GEF. Trio has been shown to play a role in axon guidance in Drosophila and nematode and has provided additional evidence that, in this capacity, Trio interacts with Rac and regulates PAK activity. Similar to reducing Rac, reducing Trio enhances PlexB gain-of-function phenotype. However, the enhancement caused by reducing Trio is not as great as that caused by reducing Rac. This probably reflects that Trio is not directly coupled to PlexB and is not the only positive regulator (GEF) for Rac in motor axons. Rather, Trio is likely to be one of many positive regulators of Rac in these axons (Hu, 2001).
The role of RhoA in PlexB signaling was examined by reducing RhoA gene dosage with two different RhoA mutant alleles, Rhorev220 and RhoAl(2)k07236. Instead of enhancing the PlexB gain-of-function phenotypes as the Rac deficiency does, partially removing RhoA suppresses the PlexB gain-of-function phenotypes. This result suggests that RhoA acts antagonistically to Rac and, moreover, that RhoA partially mediates Plexin B signaling (Hu, 2001).
To further test the model that PlexB downregulates Rac output, the effect of increasing Plexin was examined in Rac dominant-negative embryos. No mutant for Drosophila Rac has yet been published, but a loss-of-function analysis for Rac, achieved by overexpressing a dominant-negative form of Drac (N17Rac) in neurons, has revealed dramatic defects in motor axon guidance. The same ISNb nerve branch that is affected by PlexB overexpression is also sensitive to overexpression of dominant-negative Rac (N17Rac). The predominant ISNb defect in N17Rac embryos occurs at an earlier target entry point, where the whole ISNb branch normally branches off from ISN nerve. In N17Rac embryos, the ISNb fails to enter the ventral muscles and instead follows the ISN distally toward dorsal muscles (scored as 'bypass'). The difference in the quality of the ISNb phenotype of N17Rac and Plexin B gain of function embryos may likely reflect the fact that Rac is downstream of multiple guidance receptors (Hu, 2001).
The penetrance of the N17Rac bypass phenotype is very sensitive to gene dosage. When the N17Rac transgene is expressed using drivers of different strengths, different frequencies of defects result. This suggests that N17Rac only partially knocks out the wild-type gene function and that expressing N17Rac with a driver of medium strength may provide a sensitized background for testing genes that regulate the remaining Rac activity. It was reasoned that if the Plexin and Rac interaction regulates Rac activity, then it might be possible to alter the penetrance of the N17Rac bypass phenotype by simultaneously increasing PlexB gene dose in the same neurons. Indeed, coexpressing PlexB and RacN17 results in a distinct enhancement of the ISNb bypass phenotypes. N17Rac embryos also show bypass defects in the SNa motor axons that project to lateral muscle targets. This SNa bypass phenotype has never been observed in any other mutant background, and it also turns out to be enhanced by simultaneously overexpressing PlexB in these neurons (Hu, 2001).
In a reciprocal experiment PlexB was reduced in Rac dominant-negative embryos to see if this had an opposite effect. This was done by injecting double-strand RNA of PlexB into N17Rac embryos. N17Rac embryos injected with Plexin B dsRNA show distinct reduction in bypass defects compared with N17Rac embryos injected with buffer. Thus, reducing PlexB and increasing PlexB in Rac dominant embryos produces opposite modulations, consistent with the model that PlexB downregulates Rac activity (Hu, 2001).
To test whether the PlexB gain-of-function and the genetic interactions depend on the direct association between PlexB and Rac, a mutant PlexB transgene, UAS-Plex Bd7, was constructed containing the seven amino acid NTLAHYG deletion in the Rac binding region of an otherwise wild-type PlexB. The same enhancement test on N17Rac embryos was performed with this mutant transgene, and no enhancement was observed. The PlexB gain-of-function phenotypes also seem to be dependent on this Rac binding region. In contrast to wild-type PlexB, when the d7 mutant transgene is overexpressed under the control of the same neuronal GAL4 driver elav, the frequency of ISNb phenotypes is significantly lower. This low-penetrance phenotype is not enhanced by removing one copy of Rac (Hu, 2001).
In vivo expression and targeting of PlexBd7 transgene could not be examined due to the lack of PlexB antibody. Nevertheless, the seven amino acid deletion does not affect protein expression and stability of PlexBDelta3 in in vitro experiments. Three independent lines of PlexBd7 transgenes show consistent behavior when tested for their phenotypes and interactions with Rac, arguing that the negative result is not caused by the insertion site (Hu, 2001).
In light of the genetic interactions between PlexB and Rac, the biochemical nature of this negative regulation was investigated. PlexB was found to compete with the Rac downstream effector PAK (p21-activated kinase) for binding to RacGTP. PAK is a serine/threonine kinase that mediates a major part of Rac signaling output to actin polymerization. Upon binding to RacGTP, PAK undergoes a conformational change that releases an autoinhibition on the kinase domain and becomes active. Since Rac binding is critical for PAK activation and also because PlexB and PAK bind to Rac in the same GTP-dependent manner, it was asked whether PlexB and PAK may bind to the same region of Rac and whether their binding to RacGTP is mutually exclusive (Hu, 2001).
An in vitro pull-down competition assay was used in which in vitro translated L61Rac was incubated with bead-bound GST-PAK1-141 in the presence or absence of soluble PlexB protein fragment: PlexBDelta2(1619-1753). (PlexBDelta2 is a 135 amino acid fragment of PlexB. It binds to RacGTP equally well as PlexBDelta3, but it can be expressed at a higher level.) When Plexin BDelta2 is present in the binding solution, the amount of RacL61 pulled down by PAK1-141 is greatly reduced. The extent of reduction is dependent on the amount of PlexBDelta2 used. At the 48:1 molar ratio of PlexBDelta2 to PAK1-141, the reduction is close to complete. PlexBDelta2d7, a deletion PlexB fragment that is incapable of binding to Rac, does not compete with PAK for the Rac binding. Conversely, the presence of PAK protein fragment PAK78-151 also reduces RacL61 binding to PlexBDelta3. This shows that the binding of the two proteins to RacGTP is indeed mutually exclusive (Hu, 2001).
Does this competition exist in vivo? If it does, then it may be expected that overexpressing PAK together with PlexB in embryos will cancel out PlexB gain-of-function effect. Indeed, overexpressing PAK in a PlexB gain-of-function background suppresses the phenotypes of the latter, demonstrating that PlexB signaling can be antagonized by the Rac effector PAK in vivo (Hu, 2001).
It is concluded that PlexB mediates repulsion in vivo in part by binding to active Rac (RacGTP) and downregulating its effector output and in part by binding to and activating RhoA. Biochemical analysis shows that PlexB binds to RacGTP. A seven amino acid sequence in the cytoplasmic domain of PlexB is required for this binding. Genetic analysis shows that PlexB downregulates the output of RacGTP. Removal of one copy of Rac enhances a PlexB gain-of-function phenotype, while overexpression of PlexB enhances a Rac dominant-negative phenotype in motor axon guidance. Overexpression of a mutant form of PlexB that lacks the seven amino acid sequence required for Rac binding does not generate its own gain-of-function phenotype, and it does not enhance a Rac dominant-negative phenotype. It is also shown that PlexB binds to RhoA through a different region of its cytoplasmic domain. Although the biochemical mechanism is not known, genetic analysis suggests that PlexB increases the output of RhoA (Hu, 2001).
The results presented here allow a confirmation and extension of a current model concerning the role of GTPases in axon guidance. This model suggests that attractive guidance cues locally activate Rac or Cdc42 in the growth cone while repulsive guidance cues locally activate RhoA. It is argued that what is important is the relative balance in the output of Rac versus RhoA. An example is provided in which the PlexiB receptor mediates repulsive axon guidance by downregulating RacGTP output and simultaneously upregulating RhoA output. A coordinate regulation of these two small GTPases may allow the receptor to have a finer control over actin regulatory machinery. Semaphorin signaling can be converted from repulsion to attraction by changes in cGMP level. It would be interesting to test whether and how the cGMP signaling can affect this Rac/Rho balance (Hu, 2001).
Drosophila has two Plexins: A and B. Both Plexin A and B are highly expressed in the central nervous system. The two proteins share high sequence similarity in their cytoplasmic domain, indicating a similar mode of signaling shared by the two. A direct physical association of RacGTP with PlexB but not with PlexA has been demonstrated. However, genetic interactions have been found between Rac and both Plexins. For example, increasing PlexA also enhances the Rac dominant-negative phenotype as does PlexB. In COS cell and DRG neurons, Rac shows coclustering with PlexA upon Sema3A ligand treatment. It is likely that ligand binding to PlexA causes Rac binding (and subsequent inactivation of Rac) just as with PlexB, but it may be that PlexA requires an unknown third protein to help mediate or facilitate this physical interaction. From a genetic perspective, they both appear to function in the same way, mediating repulsion at least in part by inactivating Rac (Hu, 2001).
The transmembrane protein OTK associates with Plexin A and contributes to the Sema 1a/Plexin A signaling pathway. Mammalian Plexin B1 also coimmunoprecipitates with OTK. In the future, it will be interesting to test whether PlexB also interacts with OTK in vivo and to what degree the Rac/Rho GTPases and OTK signaling pathways function together or in parallel downstream of Plexins (Hu, 2001).
Mammalian Vav signal transducer proteins couple receptor tyrosine kinase signals to the activation of the Rho/Rac GTPases, leading to cell differentiation and/or proliferation. The unique and complex structure of mammalian Vav proteins is preserved in the Drosophila homologue, Vav. Drosophila Vav functions as a guanine-nucleotide exchange factor (GEF) for DRac. Drosophila cells overexpressing wild-type (wt) Vav exhibit a normal morphology. However, overexpression of a truncated Vav mutant (that functions as an oncogene when expressed in NIH3T3 cells) results in striking changes in the actin cytoskeleton, resembling those usually visible following Rac activation. Dominant-negative Rac abrogates these morphological changes, suggesting that the effect of the truncated Vav mutant is mediated by activation of Rac. In Drosophila cells, stimulation of the Drosophila EGF receptor (DER) increases tyrosine phosphorylation of Vav, which in turn associates with tyrosine-phosphorylated DER. In addition, the following results imply that Vav participates in downstream DER signalling, such as ERK phosphorylation: (1) overexpression of Vav induces ERK phosphorylation; and (2) 'knockout' of Vav by RNA interference blocks ERK phosphorylation induced by DER stimulation. Unlike mammalian Vav proteins, Drosophila Vav was not found to induce Jnk phosphorylation under the experimental circumstances tested in fly cells. These results establish the role of Vav as a signal transducer that participates in receptor tyrosine kinase pathways and functions as a GEF for the small RhoGTPase, Rac (Hornstein, 2003).
The receptor tyrosine kinases (RTKs) play an important role in the control of most fundamental cellular processes including the cell cycle, cell migration, cell metabolism and survival, as well as cell proliferation and differentiation. RTK stimulation leads to the deployment of signalling proteins that relay the appropriate specific signals, resulting in the desired cell fate. Many of the signal transducing proteins, including RTKs, are conserved throughout evolution. In the past few years, genetic and biochemical studies in Drosophila have revealed the identity and function of many signalling cascade molecules that are also shared by mammals. However, there are still many signalling proteins whose roles are still unknown. One such signal transducer protein, the Drosophila melanogaster homologue of mammalian Vav proteins, has been isolated and partially characterized (Hornstein, 2003 and references therein).
Vav proteins represent a novel family of signal transducers that couple tyrosine kinase signals with the activation of the Rho/Rac GTPases and are likely to play an integral role in the regulation of cell differentiation in many tissues. The first member of the mammalian Vav family of cytoplasmic signal transducer proteins to be identified, Vav1, was isolated as an oncogene. Removal of its amino terminus activates Vav1 as a transforming protein. Likewise, the corresponding molecular lesions in Vav2 and Vav3, two other members of the mammalian Vav family, render these proteins transforming. Unlike Vav1, which is exclusively expressed in hematopoietic cells, Vav2 and Vav3 are expressed in both hematopoietic cells and in many cells of nonhematopoietic origin. Numerous biochemical and overexpression experiments revealed that tyrosine phosphorylation of Vav1 in response to activation by one of several cytokines, growth factors or antigen receptors regulates its activity as a GEF for the Rho/Rac family of GTPases, RhoA, Rac1 and RhoG. Activation of these GTPases leads to cytoskeletal reorganization and activation of stress-activated protein kinases (SAPK/JNKs) in T cells. Vav2 and Vav3 function in a similar but not identical fashion. While both Vav2 and Vav3 also act as GEFs, there are conflicting reports regarding which GTPases are activated by them, and whether these GTPases are distinct from those activated by Vav1. Knockout experiments revealed that in T cells, Vav1 integrates signals from lymphocyte antigen receptors and costimulatory receptors to control differentiation, proliferation and the response to activation. Thus, mice deficient in Vav1 exhibit defects in numerous responses to T-cell stimulation, including capping of the T-cell receptor (TCR) postactivation, recruitment of the actin cytoskeleton to the CD3 chain of the TCR, interleukin-2 (IL-2) production and proliferation, cell cycle progression, activity of NF-AT, phosphorylation of SLP-76 and increase in Ca2+ influx. Mice deficient in Vav2 display no obvious defects in T-cell development yet exhibited some defects in B-cell function. Mice lacking both Vav1 and Vav2 displayed major defects in B-cell function that are as dramatic as the defects in T-cell development and activation observed in Vav1-/- mice. Since there are no reports regarding Vav3-/- mice at the present time, the picture of the intricate signalling network induced by the Vav proteins is incomplete. However, it is obvious that the redundancy and complexity of the mammalian Vav proteins even in hematopoietic cells together with the possibility that they differ both in their proteinñprotein interactions and in their activation of various GTPases, makes it difficult to clearly interpret the results of knockout and other experiments in mammals (Hornstein, 2003 and references therein).
In Drosophila, only one Vav homologue is present (Dekel, 2000). The highly conserved and unique structure of Vav suggests that the vav genes probably evolved from one ancestral gene and that they are important regulatory molecules in flies as well as in mammals. Drosophila Vav encodes a protein whose similarity with hVav1 is 47% and with hVav2 and hVav3 is 45%. Like mammalian Vav proteins, Droosophila Vav encodes a 'calponin-homology' (CH) region, a dbl homology (DH) domain, a pleckstrin homology (PH) domain and both an Src Homology 2 (SH2) and an Src Homology 3 (SH3) domains. However, unlike mammalian Vav proteins, Drosophila Vav lacks an amino-SH3 region. Vav is the only known Drosophila Rho GEF that encodes in addition to a DH region, both SH2 and SH3 domains, attesting that it may be a uniquely versatile signal transducer. The fact that only one homologue of Vav is present in flies, combined with the unique and highly conserved structure of the protein, promises that any study of Vav in flies should be highly beneficial and instructive (Hornstein, 2003).
A hallmark of Vav signal transducer proteins is that their tyrosine residues become phosphorylated on tyrosine residues in response to EGF stimulation. Furthermore, Vav proteins are known to bind to the stimulated EGFR through their SH2 region. In mammalian cells, Drosophila Vav is tyrosine phosphorylated in response to EGFR induction; in vitro, the Drosophila Vav SH2 region is associated with tyrosine-phosphorylated EGFR (Dekel, 2000). These results combined with the encoded domain structure of Vav strongly suggest that Drosophila Vav may function as a signal transducer protein in the Drosophila EGF receptor (DER) signalling cascades. This study investigated the role of Vav in downstream signalling from the DER, its interaction with the Drosophila Rac pathway, and its ability to effect cytoskeletal changes in Drosophila cells (Hornstein, 2003).
This study demonstrates that Vav can activate Rac in vivo. Rac is involved in Drosophila in various cellular processes including cell shape, cell adhesion, gene transcription, protein trafficking and cell cycle progression, as well as numerous developmental processes. One of the best known characteristics of Rac is that it is involved in actin cytoskeletal organization. Indeed, the expression of a constitutively activated form of DRac (V12DRac) in cultured S2 cells causes marked changes in the morphology of the cells, leading to lamellipodia and microspikes. These results indicate that overexpression of oncVav (Drosophila Vav that lacks 214 residues of its amino-terminus), but not wild-type Vav, can induce a morphology similar to that obtained with V12DRac (constitutively active mutant Rac). The fact that N17DRac inhibits the changes in the morphology obtained with oncVav further substantiates the conclusion that the activation of Rac by Vav is responsible for the observed cytoskeletal reorganization. Correspondingly, an inactive hVav1 variant defective in its ability to activate Rac inhibited the ability to induce actin cytoskeletal organization, thus further supporting the tight association between Vav, Rac and actin organization. Notably, coexpression of oncVav and V12DRac leads to more profound changes in cytoskeleton organization compared to those observed in cells overexpressing each protein alone. This result could be explained by the fact that the sum of activation reached by both the endogenous Rac activated by oncVav as well as the constitutively activated Rac yields a more striking morphology. Consistent with these results with wild-type Vav, wt hVav1 does not cause any change in morphology of NIH3T3 cells and COS cells. Conversely, a remarkable change in actin organization has been observed following overexpression of hVav1 in T cells. These conflicting results obtained with mammalian wtVav1 could stem from the use of different experimental systems, including the strong possibility that different levels of endogenous Vav2 and Vav3 exist in these systems. The cytoskeletal changes in S2 cells transfected with oncVav are compatible with a previous study demonstrating that an amino-terminus-truncated hVav1 caused depolarization of fibroblasts and triggered the bundling of actin stress fibers in NIH3T3 cells. Taken together, these results support a pathway in which Drosophila Vav serves as a GEF for Rac, thereby triggering the reorganization of the actin cytoskeleton (Hornstein, 2003).
Drosophila Vav is tyrosine phosphorylated in response to stimulation of DER and it also associates with the stimulated receptor. This result is compatible with the known characteristics of mammalian Vav proteins. For example, when ectopically expressed in nonhematopoietic cells, Vav1 associates with the EGFR through its SH2 region and becomes tyrosine phosphorylated upon induction with EGF. A similar result was reported for the ubiquitously expressed Vav2 and Vav3 proteins. Although it is well established that the Vav proteins are tyrosine phosphorylated upon EGFR stimulation, the exact contribution of the various mammalian Vav proteins to the EGFR signalling pathway is not understood. These studies with Drosophila Vav shed some light on these events. Thus, this study demonstrates that overexpression of Vav in D2F cells leads to increased ERK phosphorylation. Moreover, its elimination by the use of dsRNAi blocks phosphorylation of ERK even following stimulation of DER. There are opposing results regarding the link between Vav and ERK activation in mammals. Overexpression of Vav1 in Jurkat T cells induced 3-4-fold activation of ERK activation. Vav1 activates ERK when ectopically expressed in NIH3T3 or CHO cells. Furthermore, Vav1 coimmunoprecipitates with ERK in a human myeloma cell line stimulated with interleukin-6. Finally, T cells deficient in Vav1 exhibit defects in ERK phosphorylation. Conversely, the activation of Ras and ERK following stimulation of the TCR in Vav1 null Jurkat T cells appears normal. Without the complication of multiple homologues, these studies strongly suggest a role for Drosophila Vav in the ERK pathway in S2 cells (Hornstein, 2003).
Despite the existence of several studies that point to a link between hVav1 and Ras, it is still unclear how Vav proteins can affect ERK. Drosophila Vav may affect the Ras/ERK pathway through its function as a GEF towards DRac. Cross talk between the Rac and Ras pathways has been shown to exist in mammals. Rho family small GTPases were found to play an important role in mediating the activation of Raf by Ras. Thus, a dominant-negative mutant of Rac can block Raf activation by Ras. Additionally, the effect of Rac can be substituted by the PAK kinase, which is a direct downstream target of Rac. Moreover, PAK directly associates with Raf-1 under both physiological and overexpressed conditions. The extent of interaction between PAK and Raf-1 is correlated with the ability of PAK to phosphorylate Raf and induce mitogen-activated protein kinase activation. These studies strongly suggest that cross talk between Rac and Ras exist and it is mediated through activation of downstream effectors of Rac, such as PAK. Furthermore, it was demonstrated that MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42. A novel Drosophila gene, DRacGAP, has been identified which behaves as a negative regulator of the GTPases, DRac1 and DCdc42. Reduced function of DRacGAP or increased expression of DRac1 in the wing imaginal disk causes effects on vein and sensory organ development and cell proliferation as a result of enhanced activity of the EGFR/Ras signalling pathway. Thus, DRac and DRas are involved in cross-talk mechanisms that modulate Drosophila development (Hornstein, 2003 and references therein).
Drosophila Vav might also activate ERK in a GEF-independent manner. For instance, Vav might stimulate the Ras/ERK pathway via PLC activation, just as Vav1 was shown to activate PLC. PLC contributes to the activation of Ras, probably by stimulating the activity of the diacylglycerol-dependent exchange factor, Ras GRP. It is highly conceivable that the activity of hVav1 towards PLC is mediated in a GEF-independent mode. Whether such a mechanism is also elicited in flies remains to be determined. Drosophila Vav contains several protein-binding domains (SH2, SH3) that might participate in various pathways that result in activation of the Ras/ERK pathway. For instance, Vav may bind to the adapter molecule DShc, that was shown to be associated with the Grb2/Drk proteins leading to DRas activation. Indeed, mammalian Shc binds mammalian Vav proteins. Collectively, the current results clearly illustrate that Vav influences both the DRac and ERK pathways. However, it is not clear yet whether it exerts its influence on ERK by an exclusive inducement of the DRac pathway and/or through a GEF-independent activity (Hornstein, 2003).
The involvement of mammalian Vav proteins, by functioning as GEFs towards Rac in the JNK signalling cascade, is well establishe. Moreover, Vav proteins mediate this response through their function as GEFs towards Rac. This pathway is highly conserved between mammals and flies. In Drosophila, it can transduce signals of a diversified nature, leading to changes in cell polarity and mediating immunity in the adult. It is also required for dorsal closure during embryonic development. Genetic studies focusing on these processes placed the Rho family small GTPases in the JNK signalling cascade. However, although this study demonstrated that Vav functions as a GEF towards DRac in Drosophila, Vav does not seem to be involved in the sorbitol-induced JNK activation. In accordance, no effect has been detected of Rho family small GTPases on sorbitol-induced JNK activation in S2 cells. It is therefore conceivable that in S2 cells, the sorbitol-induced activation of JNK is not mediated through activation of DRac, and therefore does not require Vav. The possibility that Vav is involved in JNK activation under other physiological pathways in Drosophila, such as dorsal closure, still exists; however, this question merits further investigation (Hornstein, 2003).
In summary, these results show that, in fly cells, Vav functions in various signalling cascades in which it can play a role as a GEF or participate as an adapter protein. A P-element insertion has recently been reported to inactivate Vav, leading to lethality of flies (Bourbon, 2000). Further genetic experiments will be required to better understand the physiological function of Vav in developmental systems (Hornstein, 2003).
The capacity of stem cells to self renew and the ability of stem cell daughters to differentiate into highly specialized cells depend on external cues provided by their cellular microenvironments. However, how microenvironments are shaped is poorly understood. In testes of Drosophila, germ cells are enclosed by somatic support cells. This physical interrelationship depends on signaling from germ cells to the Epidermal growth factor receptor (Egfr) on somatic support cells. Germ cells signal via the Egf class ligand Spitz (Spi), and evidence is provided that the Egfr associates with and acts through the guanine nucleotide exchange factor Vav to regulate activities of Rac1. Reducing activity of the Egfr, Vav, or Rac1 from somatic support cells enhanced the germ cell enclosure defects of a conditional spi allele. Conversely, reducing activity of Rho1 from somatic support cells suppresses the germ cell enclosure defects of the conditional spi allele. It is proposed that a differential in Rac and Rho activities across somatic support cells guides their growth around the germ cells. These novel findings reveal how signals from one cell type regulate cell-shape changes in another to establish a critical partnership required for proper differentiation of a stem cell lineage (Sarkar, 2007).
In the male gonad of Drosophila, germ cells are surrounded by somatic cells that define their cellular microenvironmen. Germline stem cells (GSCs) are attached to a cluster of nondividing cells at the apical tip, called hub cells, and associated with cyst progenitor cells (CPCs) that act as stem cells for the somatic support cell lineage. Two CPCs extend their cytoplasm around one GSC, toward the hub, and toward each other such that each GSC appears to be completely enclosed in its cellular microenvironment. GSCc and CPCs generate differentiating daughters, called gonialblasts and cyst cells, respectively. The gonialblasts undergo transit amplification divisions to produce 16 spermatogonia, which become spermatocytes, grow in size, undergo the meiotic divisions, and differentiate into sperm. Two cyst cells grow cytoplasmic extensions around one gonialblast to form the germ cell cellular microenvironment that controls various aspects of germ cell differentiation (Sarkar, 2007).
Germ cells associated with somatic cells mutant for the Map-Kinase Raf fail to differentiate and accumulated as early-stage germ cells instead. A similar accumulation of early-stage germ cells was observed in Egfrts mutant testes shifted to nonpermissive temperature, and in testes from animals mutant for Stem cell tumor (Stet; Rhomboid2), a protease that cleaves Egfr ligands. However, stet mutant germ cells in addition fail to associate with somatic support cells, suggesting that the Egfr pathway is required for setting up the critical cellular microenvironment (Sarkar, 2007).
Loss of spi results in a failure of germ cells to differentiate, similar to the effects of loss of stet or the Egfr. Wild-type testes are long (∼2 mm) tubular structures that contain germ cells in a spatio-temporal order along the apical-to-basal axis. Early germ cells (GSCs, gonialblasts, and spermatogonia) are small and have small, densely packed nuclei in DAPI-stained preparations. Spermatocytes are located basal to the spermatogonia, and differentiating spermatids fill the distal part of the testis (Sarkar, 2007).
Animals carrying a temperature-sensitive allele of spi, spi77-20, die when raised at 29°C. However, spi77-20 animals raised at a slightly permissive temperature (27°C) survive and have tiny testes. Most of these testes (40 of 50) contain only small cells, as seen at the tip of wild-type testes, and do not have spermatocytes or differentiating spermatids. Staining with molecular markers revealed that the testes contains increased numbers of GSCs, gonialblasts, and spermatogonia compared to wild-type. The remaining testes (10 of 50) have high numbers of early germ cells and a few spermatocytes, but no differentiating spermatids (Sarkar, 2007).
Testes from spi77-20 animals raised at an intermediate permissive temperature (25°C) are longer than testes from animals raised at 27°C, but significantly shorter (500 μm-1.5 mm) than wild-type testes. A substantial part of the testes is occupied by tumor-like aggregates of early-stage germ cells. However, spermatocytes and differentiating spermatids are also present (Sarkar, 2007).
spi activity is both sufficient and required within the germ cells. Expression of a cleaved version of Spi (sSpi) in germ cells but not in somatic support cells of spi77-20 testes restores the wild-type phenotype, and germ cell clones mutant for spi accumulate at early stages based on phase-contrast microscopy and DAPI-stained preparations) (Sarkar, 2007).
spi was also required for somatic support cells to associate with and enclose the germ cells. Germ cell clones mutant for the conditional spi77-20 allele from animals raised at 27°C either do not associate with somatic support cells or associated with only one somatic support cell (4 of 20 clones), based on staining with soma-specific antibodies, such as the transcription factor Traffic Jam (Tj). Tj labels the nuclei of somatic support cells that are normally associated with early-stage germ cells (Sarkar, 2007).
Germ cell enclosure can be investigated by labeling testes with molecular markers such as antibodies against the membrane-bound β-catenin Armadillo (Arm) that labels the cell membranes of somatic support cells as they surround the germ cells. In wild-type testes, each GSC, gonialblast, and cluster of developing germ cells is associated with and surrounded by two somatic support cells. In testes from spi77-20 animals raised at 27°C, Tj-positive cells did not form cytoplasmic extensions around the germ cells. Similar results were obtained with other markers, including