See the embryonic expression pattern of Cdc42 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
The Rho GTPases Rac1 and Cdc42 have been implicated in the regulation of axon outgrowth and guidance. However, the downstream effector pathways through which these GTPases exert their effects on axon development are not well characterized. Axon outgrowth defects within specific subsets of motoneurons expressing constitutively active Drosophila Rac1 largely persist even with the addition of an effector-loop mutation to Rac1 that disrupts its ability to bind to p21-activated kinase (Pak) and other Cdc42/Rac1 interactive-binding (CRIB)-motif effector proteins. While hyperactivation of Pak itself does not lead to axon outgrowth defects as when Rac1 is constitutively activated, live analysis reveals that Pak can alter filopodial activity within specific subsets of neurons similar to constitutive activation of Cdc42. Moreover, the axon guidance defects induced by constitutive activation of Cdc42 persist even in the absence of Pak activity. These results suggest that (1) Rac1 controls axon outgrowth through downstream effector pathways distinct from Pak, (2) Cdc42 controls axon guidance through both Pak and other CRIB effectors, and (3) Pak's primary contribution to in vivo axon development is to regulate filopodial dynamics that influence growth cone guidance (Kim, 2003).
These studies support the idea that Rac1 mediates axon outgrowth through downstream effector pathways that are distinct from those that mediate Cdc42-dependent guidance. This is based on the following: (1) by using mutants of Rac1 and Cdc42 that render them constitutively active, the downstream cellular events that each is responsible for during axon development can be effectively isolated. Rac1 and Cdc42 likely work through different effector pathways, since constitutive activation of each leads to distinct phenotypes. (2) An effector-loop mutation that disrupts Cdc42's ability to bind CRIB-motif proteins can effectively suppress axonal defects resulting from constitutively active Cdc42. In contrast, the parallel mutation in Rac1 only partially suppresses defects resulting from constitutive activation of Rac1. Finally, activation of Pak, a CRIB effector for both Rac1 and Cdc42, leads to guidance defects and increased filopodial activities similar to those seen in activated Cdc42 mutants, but not activated Rac1 mutants (Kim, 2003).
A model is favored in which Rac1 mediates axon outgrowth mainly through effector proteins that do not possess the CRIB-motif, while Cdc42 mediates growth cone guidance primarily through CRIB proteins. Since activation of the CRIB protein Pak does not lead to axon outgrowth, but rather, guidance defects, Pak does not seem to play a direct role in Rac1-dependent outgrowth. This model is consistent with reports demonstrating that Pak is not required for axon outgrowth, at least during its initial phase, but is required during proper guidance and targeting of axons in a later phase. This, however, does not necessarily exclude Pak from playing a significant role in other Rac signaling pathways that mediate axon development (Kim, 2003).
Closer examination of growth cone behavior begins to reveal nonlinear signaling events. The ability of activated Pak to enhance filopodial activity similar to what activated Cdc42 achieves suggests that Pak likely plays a role in Cdc42-mediated growth cone guidance that depends on filopodial activity control. In this context, however, Pak's contribution to filopodial regulation is thought necessary, but not entirely sufficient, to dictate proper guidance. Also, Pak may not be the sole effector responsible for Cdc42-dependent filopodial activity. CRIB proteins affected by the Y40C mutation in constitutively active Cdc42 do not play a role in filopodial regulation, suggesting that other effectors are likely playing redundant roles in mediating filopodial activity. In addition, the persistence of guidance defects in motoneurons expressing Dcdc42V12 in a Pak null genetic background further suggests that Cdc42 is likely working through other CRIB effectors, in addition to Pak, to mediate its effects on growth cone behavior and guidance (Kim, 2003).
By isolating cellular events downstream of Rac1 and Cdc42 through the use of constitutively active mutants, the effector pathways responsible for axon development have become amenable to dissection. Since constitutively active mutants lock the GTPases in an active GTP-bound state, downstream events can be examined irrespective of GTPase activation. Furthermore, by coupling effector-loop mutations with the constitutively active mutation, a role for specific effectors becomes more evident. More studies that isolate effector pathways are necessary to complement existing studies (Kim, 2003).
In conclusion, studies using in situ analysis support the general view that cytoskeletal dynamics, outgrowth, and guidance are not necessarily coupled directly under the activation of Rac1 or Cdc42. Instead, these studies imply that a complex repertoire of growth cone behaviors are being mediated in parallel as a meshwork of events, and that the workings of Pak and other Rac1 and Cdc42 effector proteins, many of which are yet to be examined in situ, collectively contribute to proper axon development (Kim, 2003).
The Pak kinases are effectors for the small GTPases Rac and Cdc42 and are divided into two subfamilies. Group I Paks possess an autoinhibitory domain that can suppress their kinase activity in trans. In Drosophila, two Group I kinases have been identified, dPak and Pak3. Rac and Cdc42 participate in dorsal closure (DC) of the embryo, a process in which a hole in the dorsal epidermis is sealed through migration of the epidermal flanks over a tissue called the amnioserosa. DC is driven in part by an actomyosin contractile apparatus at the leading edge (LE) of the epidermis, and is regulated by a Jun amino terminal kinase (JNK) cascade. Impairment of dPak function using either loss-of-function mutations or expression of a transgene encoding the autoinhibitory domain (AID) of dPak leads to disruption of the LE cytoskeleton and defects in DC does not affect the JNK cascade. Group I Pak kinase activity in the amnioserosa is required for correct morphogenesis of the epidermis, and may be a component of the signaling known to occur between these two tissues. It is concluded that DC requires Group I Pak function in both the amnioserosa and the epidermis (Conder, 2004).
The first accumulations of LE actin at the onset of DC occur at the phosphotyrosine-rich adherens junctions, forming puncta termed actin-nucleating centers (ANCs) that extend apically in the dorsal-most epidermal cells (DMEs). Disruption of the LE contractile apparatus either through loss of maternal and zygotic dPak, or expression of dPak-AID in DME cells is accompanied by loss of phosphotyrosine nodes marking adherens junctions. dPak may be functioning in the assembly and/or regulation of the adherens junctions/ANCs at the LE and could control the LE cytoskeleton in this manner. dPak accumulates at the LE in response to Cdc42 signaling, and the ability of Cdc42 to drive accumulation of phosphotyrosine at the LE is blocked in dpak6 mutant embryos. The Drosophila Group II Pak Mbt is recruited to adherens junctions by Cdc42 in developing photoreceptor cells (Schneeberger, 2003). In mbt mutants, photoreceptor morphogenesis is disrupted and adherens junctions become patchy and disorganized. In both DC and developing photoreceptors, localization of Pak kinases to adherens junctions may be controlled by localized Cdc42 activation (Schneeberger, 2003). Cdc42 has been shown to be activated in live cells by E-cadherin, an adherens junction protein. E-cadherin is a component of the phosphotyrosine-rich adherens junctions at the LE regulated by Group I Pak kinase activity. In addition to adherens junction proteins, βPS integrin and talin, components of integrin-mediated adhesions, accumulate at the LE during DC. Work in cultured cells indicates that one means by which Pak1 can be localized to the cell periphery is through recruitment to integrin-based focal adhesions/complexes by interaction with Pak-interacting exchange factor (PIX). The PIX binding site of Pak1 is conserved in dPak, and a Drosophila PIX homologue, dPix, is required for dPak localization at the NMJ. It is interesting to note that the truncated protein encoded by the dpak6 allele, which fails to localize properly at cell peripheries, including the LE of the DMEs, is missing the PIX binding site (Conder, 2004).
Various lines of evidence from mammalian tissue culture suggest that Cdc42 functions in regulating the JNK signaling cascade (Coso, 1995). In Drosophila, the JNK pathway plays an integral role in dorsal closure, a morphogenetic process involving cell shape changes and local signaling events that occurs late in embryogenesis. One demonstrated function of the JNK pathway is to promote expression of the morphogen Decapentaplegic in cells at the leading edge of the lateral epidermis during dorsal closure. Consistent with this notion, previous studies have shown that dpp expression in the leading-edge epidermal cells is disrupted in embryos carrying mutations in members of the JNK signaling pathway. Reduction but not complete loss of maternally contributed hemipterous results in complete loss of dpp expression while loss of a negative regulator, puckered, results in increased dpp expression (Genova, 2000 and references therein).
Thus dpp expression at the leading edge is sensitive to the level of JNK pathway function. Previous studies using ectopic expression of dominant Cdc42 alleles have suggested that Cdc42 is necessary for dorsal closure and functions upstream of the JNK pathway at the leading edge (Agnes, 1999; Harden, 1999; Riesgo-Escovar, 1996). If this is so, then loss of Cdc42 function should disrupt JNK signaling and therefore dpp expression in these cells. To test this hypothesis, in situ hybridization to dpp mRNA was performed on embryos derived from Cdc424/Cdc426 mothers. Although ~70% of embryos produced by these females displayed epithelial defects and lethality, normal levels of dpp expression were observed in all embryos that developed to the onset of dorsal closure, including those that had arrested development due to insufficient levels of Cdc42 function. Thus, unlike known upstream components of the JNK pathway, reduction in Cdc42 function has no apparent effect on dpp expression by leading-edge cells at the time of dorsal closure (Genova, 2000).
Cdc42 and Rac1 contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Rac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Rac1 dominant-negative mutants is associated with increased cell death. Cdc42 is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Cdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Cdc42 remain short and are abnormally shaped. Cdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types (Eaton, 1995).
Rac1 and CDC42 control actin-dependent processes in the fly's wing imaginal disc epithelium, and plays a role in the formation of the polarized outgrowth of wing hairs. At approximately 35 hours after puparium formation each wing epithelial cell forms a hair by extending a single process from its apical membrane. The emergence of a wing hair is presaged by the accumulation of actin on the distal side of the cell. Outgrowth initiates from this site and is oriented distally. At this stage, the outgrowth is termed a prehair. Subsequently, the prehair elongates and tilts up out of the plain of the epithelium so that its base comes to lie in the center of the apical membrane. Tissue polarity mutants such as frizzled, dishevelled, and prickle interfere with the choice of the site at which actin begins to accumulate before wing hair formation. Because these genes act early in the polarization pathway, they are likely candidates to act as molecules that transmit the polarization signal. Frizzled is unlikely to be involved in structural aspects of hair formation; rather it must polarize some feature of the cell that can later be used to determine either the site of hair outgrowth or cellular orientation. The tissue polarity genes inturned, fuzzy, and multiple wing hair act downstream of frizzled and are required only for hair polarity. These genes may help translate the cellular polarity generated by the Frizzled pathway into polarized hair outgrowth (Eaton, 1996 and references therein).
Rac and the other Ras family GTPases can be mutated to dominant negative forms that interfere with the functioning of wild type GTPases. When a dominant negative Rac1 is expressed in the wing, a polarity phenotype similar to that of inturned and fuzzy is produced. Cells that express a dominant negative Cdc42 often make no hair at all. Expression of dominant negative Cdc42 interferes with actin polymerization in wing hairs. In wild-type cells prior to hair formation, actin is distributed around the periphery in the apical junction region. Actin fibers are observed running across the cells in many directions, often with a radial arrangement. The first sign of hair formation is the accumulatin of actin on the distal side of the cell. At the level of the intercellular junction, actin filaments are often observed extending from the distal vertex into the center of the cell. As the hairs extend, actin disappears from the junctional region. Dominant negative Cdc42 causes dramatic defects in the actin organization that correlate with prehair outgrowth. By the time their wild-type neighbors have extended actin-filled prehairs, cells expressing dominant negative Cdc42 have not polarized the distribution of actin filaments distally, and no sign of outgrowth is evident. These data suggest that Cdc42 is specifically required for actin polymerization in developing wing hairs and that actin polymerization is required for outgrowth (Eaton, 1996).
While Cdc42 is critical for the outgrowth but not the placement of wing hairs, Rac1 is involved in the placement but not outgrowth. Dominant negative Rac1 causes duplication or triplication of wing hairs, but no defects in the appearance of the hairs themselves. Normally there is a continuous band of junctional actin around the perimeter of the apical aspect of wing epithelia. Upon expression of dominant negative Rac1 the normal continuous band of junctional actin is reduced in amount and fraught with gaps. The process by which duplicate hairs are filled with normal amounts of actin is delayed. During formation of hairs the majority of microtubules (see ß1 tubulin for related information) span the cell at the level of apical junctions, while 30 hours later, most microtubules comprise a longitudinal array that runs from the apex to the base of each cell. This suggests that microtubule organization is developmentally regulated during wing formation. Dominant negative Rac1 expression disorganizes the apical microtubule web. It is concluded that Rac1 is needed to maintain the proper organization of the apical microtubule web before prehair formation, at the time planar polarization is thought to occur. It is likely that Rac1 acts at the sites of cell-cell contact where junctions are located, and that dominant negative Rac1 disrupts planar polarization because of the disorganization of junctional actin and microtubules (Eaton, 1996).
The Drosophila homolog of mammalian Jun-N-terminal kinases has been cloned and characterized. JNK is encoded by basket (bsk). Like hemipterous (hep), which encodes the Drosophila JNK kinase, bsk is required in the embryo for dorsal closure, a process involving coordinate cell shape changes of ectodermal cells. Dorsal closure can also be blocked by dominant negative Drosophila cdc42, which has been shown to act upstream of JNKK in vertebrates. Therefore it appears that the JNK pathway is conserved and that it is involved in controlling cell morphogenesis in Drosophila. Although Drosophila JNK efficiently phosphorylates DJun in vitro, bsk function is not required for the specification of cell fate in the developing eye, a process that requires MAP kinase and DJun function (Riesgo-Escovar, 1996).
The Rho subfamily of GTPases has been shown to regulate cellular morphology. A new member of the Rho family, named RhoL, is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary causesnurse cells to collapse and fuse together. Mutant forms of Cdc42 mimic this effect. Expression of constitutively active RhoL leads to nurse cell subcortical actin breakdown and disruption of nurse cell-follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity is specifically required for migration of a subset of follicle cells called border cells. All three activities were necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that Rho protein activities have cell type-specific effects on morphogenesis (Murphy, 1996).
Regulation of cytoskeletal dynamics is essential for cell shape change and morphogenesis. Drosophila embryos offer a well-defined system for observing alterations in the cytoskeleton during the process of cellularization, a specialized form of cytokinesis. During cellularization, the actomyosin cytoskeleton forms a hexagonal array and drives invagination of the plasma membrane between the nuclei located at the cortex of the syncytial blastoderm. Rho, Rac, and Cdc42 proteins are members of the Rho subfamily of Ras-related G proteins that are involved in the formation and maintenance of the actin cytoskeleton. To investigate how Rho subfamily activity affects the cytoskeleton during cellularization stages, embryos were microinjected with C3 exoenzyme from Clostridium botulinum or with wild-type, constitutively active, or dominant negative versions of Rho, Rac, and Cdc42 proteins. C3 exoenzyme ADP-ribosylates and inactivates Rho with high specificity, whereas constitutively active dominant mutations remain in the activated GTP-bound state to activate downstream effectors. Dominant negative mutations likely inhibit endogenous small G protein activity by sequestering exchange factors. Of the 10 agents microinjected, C3 exoenzyme, constitutively active Cdc42, and dominant negative Rho have a specific and indistinguishable effect: the actomyosin cytoskeleton is disrupted, cellularization halts, and embryogenesis arrests. Time-lapse video records of DIC imaged embryos show that nuclei in injected regions move away from the cortex of the embryo, thereby phenocopying injections of cytochalasin or antimyosin. Rhodamine phalloidin staining reveals that the actin-based hexagonal array normally seen during cellularization is disrupted in a dose-dependent fashion. Additionally, DNA stain reveals that nuclei in the microinjected embryos aggregate in regions that correspond to actin disruption. These embryos halt in cellularization and do not proceed to gastrulation. It is concluded that Rho activity and Cdc42 regulation are required for cytoskeletal function in actomyosin-driven furrow canal formation and nuclear positioning (Crawford, 1998).
The Rho subfamily of Ras-related small GTPases participates in a variety of cellular events including organization of the actin cytoskeleton and signaling by c-Jun N-terminal kinase and p38 kinase cascades. These functions of the Rho subfamily are likely to be required in many developmental events. The participation of the RHO subfamily in dorsal closure of the Drosophila embryo, a process involving morphogenesis of the epidermis, has been studied. Drac1, a Rho subfamily protein, is required for the presence of an actomyosin contractile apparatus believed to be driving the cell shape changes essential to dorsal closure. Expression of a dominant negative Drac1 transgene causes a loss of this contractile apparatus from the leading edge of the advancing epidermis, and consequently, dorsal closure fails. Two other Rho subfamily proteins, Dcdc42 and RhoA, as well as Ras1 are also required for dorsal closure. Dcdc42 appears to have conflicting roles during dorsal closure: establishment and/or maintenance of the leading edge cytoskeleton versus its down regulation. Down regulation of the leading edge cytoskeleton may be controlled by the serine/threonine kinase DPAK, a potential Drac1/Dcdc42 effector. RhoA is required for the integrity of the leading edge cytoskeleton specifically in cells flanking the segment borders. The interactions of the various small GTPases in regulating dorsal closure have been characterized and no evidence is found for the hierarchy of Rho subfamily activity described in some mammalian cell types. Rather, the results suggest that while all Rho subfamily p21s tested are required for dorsal closure, they act largely in parallel (Harden, 1999).
A model is given of the control of DC by the Drac1/JNK and Dcdc42/Dpp pathways. Drac1/JNK signaling, initiated by an as yet unknown factor, assembles cytoskeletal components (F-actin, myosin and focal complexes) and other proteins (Dpp, Puckered and Pak) in the leading edge cells and initiates the cellular migration that characterizes DC. Dpp-activated signaling controls the dynamics of epidermal migration, via Dcdc42 and the Dpp pathway, through the serine/threonine kinase Pak, which transiently downregulates the leading edge cytoskeleton at the segmental borders. Transient downregulation of the actin cytoskeleton and focal contacts near the segment border cells is likely to cause local relaxation of the anterior-posterior tension along the LE. Such transient relief of tension may then limit excessive migration of leading edge cells toward each other and prevent the bunching and shearing of epidermal segments that occurs following impairment of Dpp/Dcdc42 signaling. Segment borders cells are potential regions of highest Dpp signaling, because they are adjacent to the highest local concentrations of Dpp protein, and they have high levels of Pak protein and transcripts for the Tkv receptor. Segmental border cells are the only places where transient downregulation of the leading edge cytoskeleton is ever seen in wild-type embryos during DC. As such, it is proposed that the role of Dcdc42/Dpp signaling is the induction of Pak to downregulate the leading edge cytoskeleton at the segment borders, introducing a degree of flexibility to the leading edge during the dorsal closure process (Ricos, 1999).
The Rho sub-family of GTPases (comprising Rho, Rac and Cdc42) regulates many biological processes, including morphogenesis, cell polarity, migration, the cell cycle and gene expression. It is important to develop genetic approaches to allow the dissection, in vivo, of the mechanisms of GTPase regulation and signal transmission, and their biological consequences. In this regard, wing development in Drosophila melanogaster is an excellent model system. To investigate the functions of the Drosophila Cdc42 GTPase (Dcdc42), phenotypes were generated during wing development by expression of the dominant-negative N17 and L89 mutants of Dcdc42. Roles have been identified for Dcdc42 in wing growth, and in cell fate choice during the development of the wing veins and the peripheral nervous system. Reduction of Dcdc42 signaling following over-expression of Dcdc42N17 resulted in a broader but more diffuse domain characterized by wing-margin sensory bristles. This was correlated with a broadened stripe of wingless expression along the dorsal-ventral boundary of third-instar wing imaginal discs. Together with genetic interactions with loss- and gain-of-function Notch alleles, these data support a role for wild-type Dcdc42 as a negative regulator of Notch signaling (Baron, 2000).
Coordination between cell-cycle progression and cytoskeletal dynamics is important for faithful transmission of genetic information. In early Drosophila embryos, increasing maternal cyclin B leads to higher Cdk1-CycB activity, shorter microtubules, and slower nuclear movement during cycles 5-7 and delays in nuclear migration to the cortex at cycle 10. Later during cycle 14 interphase of six cycB mutant embryos, patches of mitotic nuclei, chromosome bridges, abnormal nuclear distribution, and small and large nuclei were observed. These phenotypes indicate disrupted coordination between the cell-cycle machinery and cytoskeletal function. Using these sensitized phenotypes, a dosage-sensitive genetic screen was performed to identify maternal proteins involved in this process. Ten suppressors classified into three groups were identified: (1) gene products regulating Cdk1 activities (cdk1 and cyclin A); (2) gene products interacting with both microtubules and microfilaments (Actin-related protein 87C); and (3) gene products interacting with microfilaments (Chickadee, Diaphanous, Cdc42, Quail, Spaghetti-squash, Zipper, and Scrambled). Interestingly, most of the suppressors that rescue the astral microtubule phenotype also reduce Cdk1-CycB activities and are microfilament-related genes. This suggests that the major mechanism of suppression relies on the interactions among Cdk1-CycB, microtubule, and microfilament networks. These results indicate that the balance among these different components is vital for normal early cell cycles and for embryonic development. These observations also indicate that microtubules and cortical microfilaments antagonize each other during the preblastoderm stage (Ji, 2002).
Although evidence exists that activation of the Rho family GTPase Cdc42 affects axonal development, its specific roles within a growth cone are not well delineated. To evaluate the model that Cdc42 activation regulates growth cone navigation by promoting filopodial activity, a live analysis strategy was adopted that uses transgenic Drosophila lines in which neurons coexpressed constitutively active Cdc42 (Cdc42V12) and membrane-targeted green fluorescent protein. Growth cones that displayed pathfinding defects exhibited little change in their filopodial activity, whereas others without pathfinding defects exhibited an ~50% increase in their filopodial activity. Moreover, effector loop mutations that were added to the constitutively active Cdc42 (Cdc42V12C40 and Cdc42V12A37) exerted little influence over filopodial activity caused by Cdc42 activation but suppressed the pathfinding defects of the growth cones. Together, these data suggest that Cdc42 controls filopodial activity in axonal growth cones independently of its effects on their pathfinding (Kim, 2002).
Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. These data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho (Fritz, 2002).
Thus, when expressed alone, only ctDrac and ctDcdc42 cause midline crossing errors. However, the mutant GTPases interact genetically with mutations in robo, Sos, and chic and with overexpression of ctMLCK. The interactions are surprisingly specific. Midline crossing errors caused by expression of ctDrac or ctDcdc42 are suppressed by heterozygous loss of Profilin and enhanced by expression of ctMLCK. These results indicate that Drac1 and Dcdc42 encourage axons to cross the midline by regulating actin polymerization and/or myosin activity. CtRho and dnRho interact strongly with expression of ctMLCK or heterozygous loss of Robo, which suggests that regulation of myosin activity by Rho is crucial for midline repulsion. This work demonstrates that Rho, Drac1, and Dcdc42 are involved in dictating which axon may cross the midline, presumably by aiding in the transduction of attractive and/or repulsive cues operating at the midline. By using mutations in signaling molecules known to prevent pCC/MP2 axons from crossing the midline, this analysis concentrates on how Rho, Drac1, and Dcdc42 may regulate cytoskeletal dynamics in response to midline repulsive cues (Fritz, 2002).
The Rho family of GTPases was first studied in fibroblasts where activation of Cdc42, Rac, or Rho results in production of filopodia, lamellapodia, and stress fibers, respectively. In wound-healing assays, Rac appears to control actin polymerization to provide the protrusive force needed for movement, while Cdc42 determines cell polarity to localize Rac activity to the leading edge of the cell. Rho seems to play a role in adhesion and spreading during cell migration. These same processes are involved in growth cone motility, which makes the Rho GTPases candidates for regulation of cytoskeletal dynamics during axon guidance. Experiments in neurons, both in vitro and in vivo, indicate that activation of Rac and/or Cdc42 increases axon outgrowth and this is opposed by activation of Rho, which leads to growth cone collapse or retraction. This is consistent with findings that expression of ctDcdc42 or ctDrac allow axons to ignore repulsive signals at the midline and continue extending across the midline (Fritz, 2002).
Rho family GTPases activate a number of effectors that may affect axon outgrowth by regulating adhesion, myosin force generation, and/or actin polymerization. The ctDrac- and ctDcdc42-induced midline crossing errors are suppressed by heterozygous loss of Profilin, an actin-binding protein, which stimulates actin polymerization. Since reducing actin polymerization partially rescues the ctDrac and ctDcdc42 phenotypes as well as errors caused by heterozygous loss of Robo, it is likely that the midline crossing errors are caused by excessive actin polymerization. Increased actin polymerization may produce more filopodia to explore the midline, which leads to midline crossing. There are several pathways through which Drac1 and Dcdc42 might affect actin polymerization. The Cdc42/ Rac effector p21-activated kinase (PAK) activates LIM kinase to phosphorylate cofilin, an actin-depolymerizing factor required for neurite outgrowth. Cdc42 also activates actin polymerization through WASP, which stimulates polymerization by binding to the Arp2/3 complex. The activation of WASP by Cdc42 is enhanced by Profilin, which may explain why the suppression of the ctDcdc42 phenotype is stronger than that of the ctDrac-induced errors. However, actin polymerization may not be the only process regulated by Rho family GTPases to increase outgrowth (Fritz, 2002).
The interactions between the Drac1 and Dcdc42 and ctMLCK indicate that misregulation of myosin activity may contribute to ctDrac- and ctDcdc42-induced axon guidance errors. Coexpression of ctMLCK with ctDrac or ctDcdc42 results in a strong enhancement of midline crossing errors, while expression of dnDrac or dnDcdc42 suppresses the defects caused by increased myosin activity. This suggests that Drac1 and/or Dcdc42 activate myosin activity in the growth cone to increase outgrowth. One mechanism may be through activation of PAK, which leads to phosphorylation of myosin regulatory light chains (MLC) to increase myosin activity. However, it has been shown that PAK also phosphorylates and inactivates MLCK, resulting in less myosin activity. In vitro, PAK phosphorylates MLCK at serine 439, which is present in ctMLCK, and serine 991, which has been removed from ctMLCK, so the impact of this pathway on the truncated ctMLCK protein is uncertain. Alternatively, it is possible that the interaction of Drac1 or Dcdc42 and ctMLCK is a secondary effect to increased actin polymerization. If increased actin polymerization is causing more filopodial exploration of the midline, increasing myosin activity through ctMLCK expression could cause axons to cross the midline before they can retract filopodia encountering repulsive signals. Separating the relative contributions of Drac1 and Dcdc42 to actin polymerization and myosin activity will require more specific experiments involving the effectors of Drac1 and Dcdc42 (Fritz, 2002).
The data suggest that Drac1 and Dcdc42 activation must be prevented or reduced for axons to respond to repulsive signals at the midline. The midline crossing errors seen in Drac1 mutants are strongly enhanced by a partial loss of Robo, which suggests that midline repulsion requires a down-regulation of Drac1 activity. Down-regulation of Rac activity occurs in response to other repulsive signals, such as Ephrin and Semaphorin. A mechanism for this is suggested by experiments showing that Plexin-B, the receptor for Semaphorin, binds specifically to activated Rac, most likely to prevent it from activating effectors. Experiments in cell culture systems have confirmed that Robo-mediated signaling involves down-regulation of Cdc42. Activation of Robo by Slit recruits srGAP1 to the CC3 domain of Roboís cytoplasmic tail, where it interacts with and inactivates Cdc42. Although srGAP1 does not affect the activity of Rac, srGAP2 and srGAP3 also bind to Robo, and one of these may regulate Rac activity. Down-regulation of Cdc42 and Rac by Robo-dependent repulsive signals is consistent with recent experiments showing that activation of DCC by chemoattractive Netrins stimulates neurite outgrowth and results in activation of Cdc42 and Rac1. Together, these data and the literature led to the hypothesis that Robo prevents axons from crossing the midline by decreasing Drac and Dcdc42 activity so that actin polymerization and myosin force generation are reduced (Fritz, 2002).
Down-regulation of Dcdc42 and Drac1 by Robo may also repel axons by preventing coupling of the actin cytoskeleton to the substrate. Rac is required for localization of E-Cadherin to cell-cell contacts and recruiting actin to Cadherin binding sites. Cdc42 and Rac promote Cadherin-mediated adhesion by preventing IQGAP, a CaM-binding Ras GAP, from interfering with the interaction of ß-catenin with alpha-catenin. Integrin-mediated adhesion also involves signaling through Rho family GTPases. By reducing actin and myosin dynamics and decoupling the cytoskeleton from the substrate, downregulation of Drac and Dcdc42 by repulsive guidance receptors would prevent axons from extending across the midline (Fritz, 2002).
Clearly, regulation of Rho family GTPase activity is necessary to prevent axons from crossing the midline inappropriately. Midline repulsive signaling involves regulation of all three GTPases; Drac1 and Dcdc42 are likely downregulated, while Rho seems to be activated downstream of repulsive signals. The Rho family GTPases influence actin polymerization and/or myosin force generation to regulate the processes of growth cone motility that are required for proper response to axon guidance signals (Fritz, 2002).
Rho family small GTPases are thought to be key molecules in the regulation of cytoskeletal organization, especially for actin filaments. In order to examine the functions of Rac1 and Cdc42 in axon guidance at the midline of the central nervous system in Drosophila embryos, Rac1 and Cdc42 were either activated or inactivated in all postmitotic neurons. The phenotypes of Cdc42 activation and Rac1 inactivation were similar to those of roundabout mutants, in that many extra axons crossed the midline. Rac1 inactivation is dominant over Roundabout receptor activation. These observations indicate that Rac1 and Cdc42 have distinct functions in downstream signalling events triggered by Roundabout receptors. In order to further examine the functional difference between Rac1 and Cdc42 in the growth cone morphogenesis, primary embryonic cultures were used to closely observe neurite formation. Activation of Rac1 and Cdc42 is shown to have distinct effects on neurite formation, particularly on growth cone morphology and the actin filaments within. Both Rac1 and Cdc42 activation induced large growth cones and long filopodia, but Cdc42 did so more efficiently than Rac1. Only Rac1 activation, however, induced thick actin bundles in the filopodia. A clear difference was found between Rac1 and Cdc42 in terms of the response to an inhibitor of actin polymerization. These results suggest that Cdc42 is specifically involved in the regulation of actin filaments in growth cones, whereas Rac1 is involved in additional functions (Matsuura, 2004).
Development of the ectodermally derived Drosophila tracheal system is based on branch outgrowth and fusion that interconnect metamerically arranged tracheal subunits into a highly stereotyped three-dimensional tubular structure. Recent studies have revealed that this process involves a specialized cell type of mesodermal origin, termed the bridge-cell. Single bridge-cells are located between adjacent tracheal subunits and serve as guiding posts for the outgrowing dorsal trunk branches. Bridge-cell-approaching tracheal cells form filopodia-like cell extensions, which attach to the bridge-cell surface and are essential for the tracheal subunit interconnection. The results of both dominant-negative and gain-of-function experiments suggest that the formation of cell extensions require Cdc42-mediated Drosophila fibroblast growth factor activity (Wolf, 2002).
Drosophila FGF signalling is used reiteratively during the different developmental steps of tracheal organogenesis. It triggers primary branch outgrowth, controls secondary branch sprouting and mediates terminal branching in response to the signals produced by oxygen-starved cells. Evidence is provided that FGF also acts as a growth factor that stimulates the development of tracheal cell extensions necessary for tracheal branch fusion. This conclusion is based on the observations that tracheal cell extensions are missing in FGF-signalling mutants while the formation of ectopic extensions is induced by ectopic FGF/Bnl. Gain-of-function experiments suggest that the FGF/Bnl-dependent cell extension formation is mediated via the Rho-like GTPase Cdc42 (Wolf, 2002).
What is the function of the tracheal cellular extensions? During early tracheal development, FGF/Bnl is instructive for tracheal branch outgrowth. However, gain-of-function experiments indicate that the dorsal trunk forms independently of FGF/Bnl-guidance, suggesting that FGF/Bnl provides a permissive rather than an instructive signal for the dorsal trunk formation. The mesodermal bridge-cell guides the dorsal trunk branches. The results establish that the FGF/Bnl-induced tracheal cell extensions are necessary for bridge-cell-mediated dorsal trunk formation. Several observations support this conclusion. (1) Bridge-cells are in direct contact with the leading edges of the outgrowing dorsal trunk branches that form cell extensions. (2) While the extensions grow out in an anterior or a posterior direction, they are in direct association with the bridge-cells. (3) The cell extensions interconnect adjacent tracheal metameres ~2.5 h before the dorsal trunk branches fuse. (4) Ectopic expression of dominant-negative Cdc42, which represses the formation of cell extensions, frequently induces the lack of dorsal trunk branch interconnections. (5) Cdc42-activated ectopic extensions partially rescue fusion of dorsal trunk rudiments in embryos that lack FGF/Bnl (Wolf, 2002).
The ability of cell extensions to mediate branch fusion via bridge-cells is restricted to dorsal trunk formation. This specific function is likely to be essential since dorsal trunk branch fusion is a multicellular process while all other tracheal interconnections are single cell fusions. Furthermore, dorsal trunk fusion precedes the other fusion processes although all branches bud out at the same time. In addition, different surrounding cell matrices may require various mechanisms for branch outgrowth. In fact, previous work has shown that dorsal branch cells follow a path along a pattern of grooves left between the muscle precursor cells of adjacent metameres, whereas the dorsal trunk branches remain in association with a contiguous population of mesodermal cells (Wolf, 2002).
Interestingly, FGF signalling has also been implicated in the outgrowth of cytonemes, which are thought to function in the distribution of morphogens during Drosophila imaginal disc development. However, cytonemes are remarkably long and microtubule-free cell extensions with a diameter of 0.2 microm. Thus, they differ from the cell extensions described here in size and cytoskeletal composition, i.e., tracheal cell extensions are more than double in diameter and contain microtubules in addition to filamentous actin (Wolf, 2002).
It is speculated that the bridge-cell recognition, the sliding of the tracheal cell extensions along the bridge-cell surface and finally the fusion process are likely to involve extracellular matrix and/or cell adhesion molecules that are associated with the tubular cell extensions and the bridge-cell surface. The functional characterization of such proteins will provide further insights into the guidance mechanisms of cell extensions along specialized cells (Wolf, 2002).
Transport of macromolecules into and out of the nucleus is a highly regulated process. The RanGTP/RanGDP gradient controls the trafficking of molecules exceeding the diffusion limit of the nuclear pore across the nuclear envelope. Genetic interaction was found between genes establishing the Ran gradient, nuclear transport factor 2 (ntf-2), Ran GTPase activating protein (Sd), and the gene encoding Drosophila Profilin, chickadee (chic). The severe eye phenotype caused by reduction of NTF2 is suppressed by loss of function mutations in chic and gain of function mutations in Sd (RanGAP). In chic mutants, as in Sd-RanGAP, nuclear export is impaired. These data suggest that Profilin and the organization of the actin cytoskeleton play an important role in nuclear trafficking (Minakhena, 2005).
ntf-2 is an X-linked essential gene. Depending on the allele, animals die between the 2nd larval instar and the pupal stage. Some alleles have an adult survival rate of 8%-15% of expected, and all survivors show a small eye phenotype, strongly reduced numbers of ommatidia. The eye phenotype varies from 30% of normal size to a more severe phenotype displaying one or two small patches of 10-40 ommatidia (Minakhena, 2005).
The mutant eye-imaginal discs are smaller than wild-type and are often abnormally shaped. Overall, the structure of the mutant eye discs is perturbed and the organization of the actin cytoskeleton is strongly altered. Only few disorganized, irregularly spaced rabdomere-like structures are apparent in the posterior compartment of the eye disc (Minakhena, 2005).
Advantage was taken of the partial loss of function eye phenotype of ntf-2 alleles to identify genes functioning with ntf-2, and a dominant suppressor screen of the eye phenotype was performed. Males from 2nd and 3rd chromosomal deficiency stocks (deficiency/balancer) uncovering 70% to 80% of the two autosomes, or about 60% of the Drosophila genome, were crossed with ntf-2P7/FM7 females. In the next generation the number of surviving ntf-2 males also carrying a deletion was counted and the survivors monitored for their eye phenotype. For this screen, 136 individual crosses were set up, many of them repeatedly in order to obtain at least 150 adult progeny to screen for the eye phenotype. Deletions and rearrangements were identified in only four regions of the second chromosome that showed suppression. The suppression was confirmed using a second ntf-2 (P49) allele (Minakhena, 2005).
DNA rearrangements affecting regions 22A and 60B-D showed different results with the two ntf-2 alleles tested and were not pursued. Df(2l)cl-h2 (25D-F) appeared to rescue both viability and the eye phenotype, but the gene responsible for the suppression could not be identified. Df(2L)GpdhA (25D-26A) rescued the eye phenotype, but not viability. To identify the gene(s) responsible for the suppression of the eye phenotype, mutations were tested in several genes that are uncovered by Df(2L)GpdhA and are available from the Drosophila stock center (Minakhena, 2005).
Mutants in one gene, chickadee (chic), encoding Drosophila Profilin, uncovered by Df(2L)GpdhA, showed suppression of the ntf-2 eye phenotype. Several loss-of-function alleles of chic were tested, including a complete lethal null allele (chic221) and other partially viable alleles, that are either female, or male and female sterile. All chic alleles were crossed with at least 2 ntf-2 alleles, except chic221 that was tested with 4 different ntf-2 alleles. The suppression of the eye phenotype was observed in all crosses and the majority of surviving trans-heterozygous males showed suppression of the ntf-2 eye phenotype, restoration of wild-type eyes. The percent of males with wild-type eyes varied in different allele combinations. Surprisingly, the eye phenotype was usually either small or wild-type and virtually no eyes of intermediate size were observed (Minakhena, 2005).
To investigate the cause underlying the suppression of the ntf-2 phenotype and possible function of Profilin in nuclear transport, a reporter gene approach was used. Nuclear transport was assayed using UAS-NLS-NES reporter constructs C-terminally tagged with GFP in different mutant backgrounds. One construct contains a wild-type NLS and NES (UAS-NLS-NES-GFP), the other a wild-type NLS but a mutant NES that is not recognized by the nuclear export machinery (UAS-NLS-NESP12-GFP). Expression of the transgenes was driven by a heatshock-GAL4 driver, and the distribution of GFP was analyzed in salivary glands. The activity of the wild-type NES is stronger then that of the NLS. Hence, in wild-type the NLS-NES-GFP is usually localized in the cytoplasm. In contrast, NLS-NESP12-GFP has impaired nuclear export and strongly accumulates in nuclei. In homozygous chic01320 and the hetero-allelic combination chic2/chic221, the distribution of the GFP reporter is altered. In contrast to the cytoplasmic distribution of NLS-NES-GFP in wild-type, in the chic mutant salivary glands the GFP reporter is found predominantly in the nucleus. The localization of NLS-NESP12-GFP is similar in chic and wild-type, indicating that NLS-mediated import is not affected (Minakhena, 2005).
RanGAP functions in nuclear export of cargo and in Sd-RanGAP mutants the NLS-NES-GFP is found in the nucleus and NLS-NESP12-GFP is distributed the same as in wild-type. This failure of exporting NLS-NES-GFP in Sd-RanGAP mutants is reminiscent of what was observed in chic alleles (Minakhena, 2005).
Given the similarity in nuclear export phenotypes in Sd and chic mutants, tests were performed to see if Sd would also suppress the eye phenotype of ntf-2 alleles. The Sd (Sd72) chromosome was crossed with two ntf-2 alleles and it was found that the eye phenotype was suppressed in both of them. To confirm that the SD-RanGAP mutation, and not other genes on the Sd chromosome, is responsible for the suppression, a mutated Sd-RanGAP transgene (UAS-Sd-RanGAP12A-6) was expressed driven by hsp70-GAL4 or arm-GAL4 in ntf-2P7 and ntf-2P49 males and similar levels of suppression was observed as seen with Sd72 (Minakhena, 2005).
The genetic interaction between Sd-RanGAP and ntf-2 is not altogether surprising because both RanGAP and NTF2 are known to function in the formation of the RanGTP-GDP gradient. To investigate if RanGAP is affected in ntf-2 mutants the distribution of RanGAP was studied in eye discs (Minakhena, 2005).
In wild-type cells Ran-Gap is present in low levels in the cytoplasm and forms a clearly visible punctuated circle around the nucleus. The punctuate pattern of RanGAP is due to its association with nuclear pores. This distribution is different in ntf-2 discs. Patches of cells are observed in which RanGAP aggregates in small or large clumps near the nuclei, but in other cells the distribution of the protein looks relatively normal. This observation suggests, that the clumping of RanGAP is an effect of the abnormal organization of the cells within the ntf-2 disc. The cells with clumped RanGAP are usually in close proximity to cells with high levels of F-actin (Minakhena, 2005).
To investigate a connection between Profilin, RanGAP, and actin, it was next asked whether the function of Profilin or actin polymerization might have an effect on RanGAP localization. Clones were generated in eye discs of null alleles of the two genes chic (chic221) and, as a control, act up/capulet (acuE636). Acu participates in actin de-polymerization, the opposite function of Profilin (Minakhena, 2005).
In chic clones RanGAP protein is increased around the nuclear envelope and its distribution is uneven and patchy on the nuclear envelope surface. In wild-type even, punctuated circles are observed. This abnormal distribution was found in 100% of examined clones. In chic clones the level of F-actin was reduced. In the acu control clones high levels of F-actin are detected as expected, but the distribution of RanGAP is not significantly changed (Minakhena, 2005).
To test whether this patchy protein distribution of RanGAP on nuclear pores of chic22 cells is caused by problems in nuclear envelope assembly, the distribution of Lamin and nuclear pore proteins (Nups) was analyzed in chic221 clones. The distribution of both Lamin and Nups is affected in about 30% of clones. This is likely due to the mislocalization of RanGAP. It has been shown previously that RanGTPase functions in nuclear pore and envelope formation (Minakhena, 2005).
The staining experiments show higher levels of RanGAP around nuclei in chic eye disc clones. Whether this is due to overall higher levels of RanGAP in mutant cells was examined. The chic alleles used in the clonal analysis are homozygous lethal; therefore extracts were prepared from wild-type and mutant 1st instar larvae. In Western blots from extracts of chic221 (lethal at first and early second larval instar) and chic01320 (viable and female sterile) larvae, the amount of RanGAP present in mutants is not dramatically changed compared to wild-type. This may be because RanGAP and Profilin are maternally contributed and therefore at these early stages a difference in levels is not detected. Eye-antennal discs were dissected from normal larvae and larvae with chic clones. The dissected tissues also contained some brain material because eye-antennal discs are next to the brain hemispheres and are difficult to separate. In two separate experiments an increase of 30%-50% was seen in the intensity of the RanGAP band in extracts from discs carrying chic221 somatic clones compared to normal eye discs from chic221/+ larvae. The intensity of the RanGAP bands were normalized to that of the control Bic-D band and equals 2.6 for discs with clones and 1.8 for wild-type discs (Minakhena, 2005).
Why lowering the level of Profilin, which functions in actin polymerization, suppresses the ntf-2 phenotype is not immediately apparent, but there are several possible explanations. Lower levels of Profilin may result in reduction of the abnormal actin polymerization in ntf-2 mutant eye discs. But the finding that the ntf-2 eye phenotype is suppressed by the over-expression of RanGAP suggests that the disorganized appearance of F-actin is an indirect result of abnormal nuclear trafficking. Therefore lowering Profilin seems to also affect the abnormal nuclear trafficking inherent to ntf-2 eye discs. This supposition is bolstered by the finding that Profilin is essential for normal nuclear export. The results are consistent with F-actin being regulated by nuclear transport, and in turn, Profilin and Actin controlling aspects of nuclear trafficking (Minakhena, 2005).
Unpolymerized actin is found on NPC-attached nucleoplasmic filaments. It has been shown to function in the nuclear export of proteins and RNA. Unpolymerized actin also associates with Profilin and is exported from the nuclei in a Ran-dependant manner. It is not thought that these processes have a primary role in the mutant phenotypes because staining of ntf-2 eye discs and chic clones with anti-actin antibody display no obvious difference in the distribution of non-polymerized actin. Nevertheless, these processes have to be considered as part of the crosstalk between the actin cytockeleton and Ran-mediated nuclear trafficking (Minakhena, 2005).
That Profilin controls the localization of RanGAP is evident from the abnormal distribution of the protein in chic clones. The uneven distribution of RanGAP at the nuclear envelope is not due simply to higher levels of protein. In Sd transgenic lines that express wild-type or mutant RanGAP, higher levels of protein are found uniformly distributed in the cytoplasm and nucleus. In chic mutant cells, the RanGAP level is about doubled, but the protein distribution is different than that observed in the over-expressing lines (Minakhena, 2005).
Cdc42 recruits Par-6-aPKC to establish cell polarity from worms to mammals. Although Cdc42 is reported to have no function in Drosophila neuroblasts, a model for cell polarity and asymmetric cell division, this study shows that Cdc42 colocalizes with Par-6-aPKC at the apical cortex in a Bazooka-dependent manner, and is required for Par-6-aPKC localization. Loss of Cdc42 disrupts neuroblast polarity: cdc42 mutant neuroblasts have cytoplasmic Par-6-aPKC, and this phenotype is mimicked by neuroblast-specific expression of a dominant-negative Cdc42 protein or a Par-6 protein that lacks Cdc42-binding ability. Conversely, expression of constitutively active Cdc42 leads to ectopic Par-6-aPKC localization and corresponding cell polarity defects. Bazooka remains apically enriched in cdc42 mutants. Robust Cdc42 localization requires Par-6, indicating the presence of feedback in this pathway. In addition to regulating Par-6-aPKC localization, Cdc42 increases aPKC activity by relieving Par-6 inhibition. It is concluded that Cdc42 regulates aPKC localization and activity downstream of Bazooka, thereby directing neuroblast cell polarity and asymmetric cell division (Atwood, 2007).
Little is currently known about how the Par complex is localized or regulated in Drosophila neuroblasts, despite the importance of this complex for neuroblast polarity, asymmetric cell division and progenitor self-renewal. This study shows that Cdc42 plays an essential role in regulating neuroblast cell polarity and asymmetric cell division. Baz localizes Cdc42 to the apical cortex where it recruits Par-6-aPKC, leading to polarization of cortical kinase activity that is essential for directing neuroblast cell polarity, asymmetric cell division, and sibling cell fate (Atwood, 2007).
Asymmetric aPKC kinase activity is essential for the restriction of components such as Mira and Numb to the basal cortex. The aPKC substrates Lgl and Numb are thought to establish basal polarity either by antagonizing activity of myosin II or by direct displacement from the cortex. This study found that Cdc42 recruits Par-6-aPKC to the apical cortex and that Cdc42 relieves Par-6 inhibition of aPKC kinase activity. In the absence of Cdc42, aPKC is delocalized and has reduced activity, resulting in uniform cortical Mira. Expression of Cdc42-DN leads to cortical overlap of inactive Par-6-aPKC and Mira indicating the importance of Cdc42-dependent activation of aPKC kinase activity. Expression of Cdc42-CA leads to cortical aPKC that displaces Mira from the cortex, presumably because Lgl is phosphorylated at the entire cell cortex. This is similar to what is seen when a membrane-targeted aPKC is expressed (Atwood, 2007).
Baz, Par-6 and aPKC have been considered to be part of a single complex (the Par complex). This study found that, when Cdc42 function is perturbed, Par-6 and aPKC localization is disrupted but Baz is unaffected. Why is Baz unable to recruit Par-6-aPKC in the absence of Cdc42? One explanation is that Cdc42 modulates the Par-6-Baz interaction, although Cdc42 has no direct effect on Par-6-Baz affinity. Alternatively, Baz might only be transiently associated with the Par-6-aPKC complex (e.g. as an enzyme-substrate complex); this is consistent with the observation that Baz does not colocalize with Par-6-aPKC in Drosophila embryonic epithelia and its localization is not dependent on either protein. How does Baz recruit Cdc42 to the apical cortex? Like other Rho GTPases, Cdc42 is lipid modified (prenylated), which is sufficient for cortical localization. Baz is known to bind GDP-exchange factors (GEFs), which may induce accumulation of activated Cdc42 at the apical cortex (Atwood, 2007).
The requirement of Par-6 for robust Cdc42 apical enrichment suggests that positive feedback exists in this pathway, a signaling pathway property that is also found in polarized neutrophils. More work is required to test the role of feedback in neuroblast polarity but one attractive model is that Baz establishes an initial polarity landmark at the apical cortex in response to external cues, which leads to localized Par-6-aPKC activity through Cdc42. Phosphorylation of Baz by aPKC might further increase asymmetric Cdc42 activation, perhaps by increased GEF association, thereby reinforcing cell polarity. Such a mechanism could generate the robust polarity observed in neuroblasts and might explain why expression of dominant Cdc42 mutants late in embryogenesis does not lead to significant defects in polarity (Atwood, 2007).
This study argues that Cdc42 functions downstream of Baz. Cdc42 is required for Baz-Par-6-aPKC localization in C. elegans embryos and mammalian neural progenitors. In C. elegans embryos, RNA interference of cdc42 disrupts Par-6 localization, whereas PAR-3 localization is slightly perturbed. In this case, Cdc42 is required for the maintenance but not establishment of PAR-3-Par-6 asymmetry; however, other proteins have been shown to localize Par complex members independently of Cdc42. Conditional deletion of cdc42 in the mouse brain causes significant Par-3 localization defects, although this may be caused by the loss of adherens junctions. More work will be required in these systems to determine if the pathway that has been proposed is conserved (Atwood, 2007).
This study has identified at least two functions of Cdc42 in neuroblasts: first, to recruit Par-6-aPKC to the apical cortex by direct interaction with its CRIB domain and, second, to promote aPKC activity by relieving Par-6 repression. aPKC activity is required to partition Mira and associated differentiation factors into the basal GMC; this ensures maintenance of the apical neuroblast fate as well as the generation of differentiated neurons. Polarized Cdc42 activity may also have a third independent function in promoting physically asymmetric cell division, because uniform cortical localization of active Cdc42 leads to same-size sibling cells. Loss of active Cdc42 at the cortex by overexpression of Cdc42-DN still results in asymmetric cell division, suggesting that other factors also regulate cell-size asymmetry, such as Lgl and Pins. In conclusion, these data show that Cdc42 is essential for the establishment of neuroblast cell polarity and asymmetric cell division, and defines its role in recruiting and regulating Par-6-aPKC function. These findings now allow Drosophila neuroblasts to be used as a model system for investigating the regulation and function of Cdc42 in cell polarity, asymmetric cell division and neural stem cell self-renewal (Atwood, 2007).
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