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).
Regulation of synaptic morphology depends on endocytosis of activated growth signal receptors, but the mechanisms regulating this membrane-trafficking event are unclear. Actin polymerization mediated by Wiskott-Aldrich syndrome protein (WASp) and the actin-related protein 2/3 complex generates forces at multiple stages of endocytosis. FCH-BIN amphiphysin RVS (F-BAR)/SH3 domain proteins play key roles in this process by coordinating membrane deformation with WASp-dependent actin polymerization. However, it is not known how other WASp ligands, such as the small GTPase Cdc42, coordinate with F-BAR/SH3 proteins to regulate actin polymerization at membranes. Nervous Wreck (Nwk) is a conserved neuronal F-BAR/SH3 protein that localizes to periactive zones at the Drosophila larval neuromuscular junction (NMJ) and is required for regulation of synaptic growth via bone morphogenic protein signaling. This study shows that Nwk interacts with the endocytic proteins dynamin and Dynamin associated protein 160 (Dap160) and functions together with Cdc42 to promote WASp-mediated actin polymerization in vitro and to regulate synaptic growth in vivo. Cdc42 function is associated with Rab11-dependent recycling endosomes, and this study shows that Rab11 colocalizes with Nwk at the NMJ. Together, these results suggest that synaptic growth activated by growth factor signaling is controlled at an endosomal compartment via coordinated Nwk and Cdc42-dependent actin assembly (Rodal, 2008).
Nwk interacts with the endocytic machinery and activates Wsp/Arp2/3 actin polymerization together with Cdc42 to regulate synaptic growth upstream of growth factor signaling. Mapping these interactions and activities provides a critical framework for determining the mechanism by which endocytic accessory proteins and the cytoskeleton control membrane deformation during endocytosis (Rodal, 2008).
Nwk activates Wsp/Arp2/3 actin polymerization via its SH3a domain, and Nwk-SH3b is not required for Wsp binding or activation, but is required for the residual Wsp-inhibitory activity of Nwk when SH3a function is abolished. This activity may be more pronounced on endogenous Wsp, which is more tightly autoinhibited than recombinant WASp, raising the possibility that Nwk-SH3b could potently regulate Nwk-SH3a-dependent activation of Wsp. Thus, ligands of Nwk-SH3b are in a position to serve as activators of Nwk and Wsp/Arp2/3 actin polymerization. Nwk-SH3b is required for interactions between Nwk and Dap160, which is an excellent candidate for acting upstream of Nwk, because dap160 mutants exhibit synaptic overgrowth and temperature-sensitive seizures like those of nwk mutants, and Nwk is mislocalized in dap160 NMJs. Recently, it was reported that the fragment of Dap160 containing its last two SH3 domains is required for interaction with full-length Nwk in Drosophila extracts, leading to the hypothesis that the C terminal proline-rich region of Nwk mediates these interactions (O'Connor-Giles, 2008). The current results show instead that interactions between purified Nwk{Delta}C (i.e., Nwk lacking the C terminus) and both endogenous full-length Dap160 as well as purified Dap160 SH3 domain-containing fragment depend on Nwk SH3b. Two possible interpretations can reconcile these results. Nwk SH3b may interact with a noncanonical SH3 domain-binding site in the intervening sequences between the Dap160 SH3 domains. Alternatively, Nwk SH3b may function in an intramolecular interaction within Nwk that is required to expose one of several proline-rich sequences in the N-terminal region Nwk for interaction with Dap160 SH3 domains. Thus, it is concluded that Nwk SH3b is important for Dap160-Nwk interactions via an indirect or noncanonical mechanism. Further experiments will be needed to identify the Nwk-binding site on Dap160 and to confirm activity of Dap160 on Nwk in vitro (Rodal, 2008).
Nwk-SH3a is required for interactions of Nwk with both dynamin and Wsp. Other F-BAR/SH3 family members have been postulated to link dynamin and Wsp by multimerization via their F-BAR domains (Itoh, 2006; Tsujita, 2006; Shimada, 2007), but endogenous complexes containing Wsp and dynamin have only been demonstrated for the F-BAR/SH3 protein syndapin (Kessels, 2006). Nwk could thus be in a position to bring dynamin and Wsp together. It has not been possible to coimmunoprecipitate endogenous Wsp and Nwk using the available antibodies. However, dynamin immunoprecipitates contain Nwk but not Wsp, suggesting that Nwk-SH3a may switch associations between dynamin and Wsp. Another interpretation is that Wsp and dynamin binding are restricted to separate populations of Nwk molecules, and that the SH3a domain thus acts in two parallel biochemical pathways (Rodal, 2008).
In vivo analysis reflects the complexity of these SH3 domain interactions. SH3a and SH3b of Nwk have both separate and overlapping functions in regulating synaptic growth, perhaps reflecting the multivalent nature of interactions in the Nwk network. [In addition to binding Nwk, Dap160 binds to both dynamin and to Wsp.] Furthermore, the fact that mutation of both SH3 domains together (Nwk-SH3a*b*) produces additional dominant effects suggests that a non-SH3 ligand of Nwk is inappropriately titrated away from its function after mutation of Nwk SH3 domains. An excellent candidate ligand is the membrane itself, because the Nwk F-BAR domain has the potential to bind to and tubulate phospholipid bilayers. Determining the specific order and regulation of F-BAR/SH3 domain protein interactions with competing SH3 domain ligands and with the membrane will be important for uncovering the molecular mechanisms of these proteins during endocytosis (Rodal, 2008).
NMJ overgrowth with an excess of satellite boutons is a hallmark of endocytic mutants. Nwk interacts with the endocytic machinery and cdc42 and nwk mutants exhibit overproliferation of satellite boutons. A prominent function of endocytosis in nerve terminals is the recycling of synaptic vesicles. However, nwk single mutants and cdc42; nwk double mutants show no detectable defect in endocytosis of synaptic vesicles. One interpretation of this result is that receptor endocytosis is more sensitive to perturbation than synaptic vesicle recycling. However, given the documented function of Cdc42 and Wsp in endosomes, it is more likely that Nwk functions in a later step of endocytic traffic. Importantly, although the synaptic vesicle endocytosis defects in shi (dynamin) and dap160 reflect the function of these molecules in the internalization step of endocytosis, synaptic overgrowth in these mutants could arise from defects at later steps of endocytic traffic, because dynamin functions in a variety of membrane-trafficking events, ranging from Golgi traffic to endosome traffic (van Dam and Stoorvogel, 2002Go; Kessels et al., 2006Go) (Rodal, 2008).
The endosomal system is organized into subdomains defined by specific members of the Rab GTPase family and adopts distinct morphology and ultrastructure in different cell types. Thus, functionally conserved Rab subdomains provide a unifying approach to understanding structurally diverse membrane systems. Rab11 controls the function of the recycling endosome in directing traffic to the cell surface and colocalizes with Nwk in periactive zones at the Drosophila NMJ [although it can occasionally be observed in larger puncta]. Like cdc42 and nwk mutants, rab11 mutants have a profound defect in synaptic growth, exhibiting excessive satellite boutons. Cdc42 and WASp have recently been implicated in recycling endosome function. Thus, periactive zones may be the synaptic representation of the recycling endosome, with Cdc42 and Nwk controlling actin polymerization-dependent traffic of signaling complexes at this Rab11-positive compartment. Whether Cdc42 functions as a signal-responsive element in this compartment or forms part of the constitutive machinery for membrane traffic remains uncertain (Rodal, 2008).
The TGF-β/BMP family member Gbb activates downstream signals that may be the critical targets of Nwk/Cdc42-mediated endocytosis in synaptic growth. Indeed, recent work has shown that Gbb signaling is required for synaptic overgrowth in nwk mutants, phosphorylation of the Gbb signaling target Mothers against decapentaplegic (Mad) is upregulated in nwk mutants, and Nwk biochemically interacts with the intracellular domain of the Gbb receptor Tkv. However, other signaling pathways could equally be regulated by Nwk/Cdc42-mediated endocytosis, lead to upregulation of phosphorylated Mad, and contribute to the synaptic overgrowth in cdc42; nwk mutants. One candidate pathway is the presynaptic component of the Wnt/Wg cascade, which may converge on Gbb/Mad regulation in the synapse as observed in other tissues. It has not been possible to detect any change in the steady-state localization of candidate cargoes in synaptic boutons in nwk or cdc42 mutants, suggesting that Nwk and Cdc42 are not required for the gross morphology of endosomes, but instead contribute to the rate of cargo trafficking through this compartment. Determining the specific signaling pathways, receptors, and their activation states in recycling endosomes will require tools to measure the activity and rates of traffic of specific receptors in situ (Rodal, 2008).
Nwk is conserved from insects to higher vertebrates, and the mammalian genome encodes two Nwk homologs, which have not yet been characterized. However, Cdc42 and WASp-induced actin polymerization have been implicated in synapse formation in Aplysia sensory neurons and in mammalian hippocampal cultures. These reports suggest that the direct consequence of activating these proteins was the formation of filopodia that mature into synapses. An alternative hypothesis, consistent with the established function of Cdc42 and WASp family members in generating force for intracellular membrane traffic rather than in filopodial formation, is that synaptic growth regulatory functions of Cdc42 and WASp depend on endosomal traffic of signaling complexes by a similar mechanism to Drosophila Nwk-Wsp-induced synapse formation (Rodal, 2008).
Different actin-filament-based structures co-exist in many cells. This study characterises dynamic actin-based protrusions that form at distinct positions within columnar epithelial cells in the Drosophila pupal notum, focusing on basal filopodia and sheet-like intermediate-level protrusions that extend between surrounding epithelial cells. Using a genetic analysis, it was found that the form and distribution of these actin-filament-based structures depends on the activities of apical polarity determinants, not on basal integrin signalling. Bazooka/Par3 acts upstream of the RacGEF Sif/TIAM1 to limit filopodia to the basal domain, whereas Cdc42, aPKC and Par6 are required for normal protrusion morphology and dynamics. Downstream of these polarity regulators, Sif/TIAM1, Rac, SCAR and Arp2/3 complexes catalyse actin nucleation to generate lamellipodia and filopodia, whose form depends on the level of Rac activation. Taken together, these data reveal a role for Baz/Par3 in the establishment of an intercellular gradient of Rac inhibition, from apical to basal, and an intimate association between different apically concentrated Par proteins and Rho-family GTPases in the regulation of the distribution and structure of the polarised epithelial actin cytoskeleton (Georgiou, 2010).
Although many studies have used the segregation of apical, junctional and basolateral markers as a model of epithelial polarity, and a number of studies have reported the existence of cell protrusions in the notum and other epithelia, these structures and the genes regulating their formation have not been characterised in detail. This study used Neuralized-Gal4 to express GFP-fusion proteins in isolated epithelial cells to reveal the dynamic shape of cells within the dorsal thorax of the fly during pupal development. Using this method, distinct populations of protrusions were characterised based on their form, dynamics and location within the basolateral domain of columnar epithelial cells. The analysis reveals dynamic protrusions at three distinct locations within the epithelial cell: apical microvillus-like structures, intermediate-level sheet-like protrusions and basal-level lamellipodia and filopodia. Importantly, although these are all dependent on continued actin filament dynamics, these populations of protrusions rely on different gene activities for their formation (Georgiou, 2010).
Cdc42, Rac, SCAR/WAVE and the Arp2/3 complex are required for the formation of basal lamellipodia and filopodia, but not for the formation of the apical microvillus-like structures. This analysis also confirms that HSPC300 should be considered to be a functional component of the SCAR complex. Moreover, the SCAR and Arp2/3 complexes are required to induce the formation of both lamellipodia and filopodia in this system. Although many studies have suggested that Rac activates the SCAR complex to induce branched Arp2/3-dependent actin nucleation that underlies lamellipodial formation, whereas Cdc42 is required to induce filopodial formation, this analysis suggests that the macroscopic form of the protrusion in a tissue context is not dictated by the nucleator used. In this, the current results are in line with several recent studies in cell culture. Instead, the macroscopic structure generated depends on the local level of Rac activity, with high levels of Rac driving filopodial formation and low levels leading to lamellipodial formation. Since the forces required to distort the membrane to generate finger-like protrusions are likely to be greater than those required to generate the equivalent section of a sheet-like protrusion, protrusion morphology might be a product of a force balance between membrane tension, extracellular confinement and local actin-filament formation. Since wild-type cells have a graded distribution of protrusions, with lamellipodia predominating apically and filopodia basally, wild-type cell morphology might reflect a gradient in the level of Rac activation, from high basal levels to low apical levels (Georgiou, 2010).
Within this system, Cdc42-Par6-aPKC and Baz/Par3 appear to have antagonistic roles in the formation of basolateral protrusions. Cdc42-Par6-aPKC is required for actin filament formation and protrusion dynamics, whereas Baz/Par3 ensures the separation of basal and intermediate protrusions by limiting the extent of basal filopodia along the apical-basal axis. In this, the current analysis adds to the growing body of evidence that Baz/Par3 and Par6-aPKC have distinct molecular targets. Moreover, the data confirm that Par6-aPKC act together with the Rho-family GTPase Cdc42. Significantly, the loss of Baz/Par3 phenocopies gain-of-function mutations in Rac and the overexpression of the Rac-GEF Sif/TIAM1, a Par3-interacting protein. Baz/Par3 might therefore serve as a cell-intrinsic cue to polarise the dynamic actin cytoskeleton along the epithelial apical-basal axis, giving epithelial cells their characteristic polarised morphology (Georgiou, 2010).
Baz/Par3 has previously been implicated in the restriction of actin polymerisation to specific subcompartments within a cell, allowing for the formation of distinct populations of protrusions. This has been studied most extensively in hippocampal neurons, in which Par3 was shown to interact with TIAM1 to regulate the activation of Rac within distinct domains of the cell during axon specification and dendritic spine morphogenesis. Indeed, it has been suggested that the formation of a Cdc42-Par6-Par3-TIAM1-Rac1 complex is required to establish neuronal polarity. The current study suggests that Baz/Par3 acts in a similar fashion in the morphogenesis and positioning of dynamic protrusions in epithelia. However, this analysis reveals an antagonistic relationship between Sif/TIAM1 and Baz/Par3 in protrusion formation. Baz/Par3 might sequester Sif/TIAM1 to prevent its association with Rac. Furthermore, because the loss of Cdc42, Par6 or aPKC results in the loss of basolateral protrusions and a marked reduction in the GFP:Moe reporter (a phenotype that can be rescued by the coexpression of RacV12 or Sif) Cdc42, Par6 and aPKC are probably required for the basal activation of Rac in epithelial cells in the Drosophila notum. Thus, signals from apically concentrated polarity determinants appear to be communicated and translated into local protrusion formation within the basolateral domain. Whether this occurs through the diffusion of an apically localised regulator or via long-range transmission of polarity information e.g. via microtubules, will be an important area of future research. An intriguing correlation is the largely apical localisation of Baz and its proximity to intermediate-level sheet-like protrusions. This would suggest a possible gradient of Baz/Par3-mediated Rac inhibition, allowing sheet-like protrusions at an intermediate level and restricting filopodial protrusions to the very base of the cell. Since Baz/Par3 has been shown to localise PTEN to apical junctions, it is possible that Baz recruits PTEN, which acts on PtdIns(3,4,5)P3 to generate a PtdIns(3,4,5)P3 gradient from high levels basally to low levels apically. PIP3 could then act to aid in the recruitment and activation of Rac at the membrane (Georgiou, 2010).
Taken together, these data demonstrate that different components of the apical determinants of cell polarity act in conjunction with the Rho-family GTPases Cdc42 and Rac to regulate the positioning of lamellipodial and filopodial protrusions over the entire span of the apical-basal cell axis. Significantly, in this tissue context, Rac, SCAR and Arp2/3 complexes promote the formation of both lamellipodia and filopodia, whose structure appears to depend on the level of Rac activation (Georgiou, 2010).
Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. DC and the follicular epithelium were used to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. It is proposed that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. It is concluded that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion (Bahri, 2010).
Some embryos lacking zygotic Pak function successfully bring the epidermal flanks together at the dorsal midline but fail to restore septate junctions and adherens junctions at the LE in the DME cells. Thus, Pak at the LE membrane of the dorsal-most epithelial cells (DME) is regulating establishment of apicobasal polarity during a mesenchymal-epithelial transition. It is suspected that Pak is acting through different routes in its regulation of adherens junction formation versus septate junction formation. This study has focused on Pak regulation of the Scrib complex in septate junction formation at the LE. The data indicate that Pak is a component of the Scrib complex at the lateral membrane. Although Pak might be associating with the Scrib complex throughout epithelia, it might only be required for recruitment of the Scrib complex in epithelia derived from a mesenchymal-like intermediate such as the follicular epithelium and the LE. With the exception of the LE, apicobasal polarity in the epidermis is determined much earlier in development with formation of the blastoderm by cellularization. The epidermis is therefore a primary epithelium that does not arise from a mesenchymal intermediate, and Pak function does not appear to be required for apicobasal polarity in primary epithelia (Bahri, 2010).
Localization of Pak at the lateral membrane in both the follicular epithelium and in the epidermis is integrin-dependent. Studies using organ culture of embryonic kidney mesenchyme and MDCK cells demonstrate a requirement for integrins in apicobasal polarity of epithelia derived from MET, and this study has shown that βPS-integrin is required for Scrib complex and septate junction protein recruitment at the LE and in the follicular epithelium. Furthermore, previous studies in the follicular epithelium and another Drosophila epithelium derived from MET, the midgut, have demonstrated a requirement for integrins in the maintenance of apicobasal polarity. It is proposed that, at the LE, the absence of the septate junction diffusion barrier allows the accumulation of integrin complexes along the lateral membrane. These lateral integrin complexes recruit Pak, around which the Scrib complex is assembled. Thus, the absence of septate junctions allows the recruitment of proteins needed for the assembly of septate junctions. The model suggests that there might be transient Pak-mediated links between integrin and the Scrib complex. Interestingly, Dlg and βPS-integrin have been shown to co-immunoprecipitate from fly head extracts, consistent with these proteins existing in a complex in the nervous system and/or in epithelia (Bahri, 2010).
The data and recent studies on the amnioserosa support the idea that septate junctions restrict the accumulation of lateral integrins. The amnioserosa is devoid of septate junction proteins such as FasIII, and this might be owing to absence in this tissue of the transcription factor Grainy head, which promotes expression of septate junction proteins. The wild-type amnioserosa has high levels of lateral βPS-integrin, but ectopic expression of Grainy head in the amnioserosa leads to an accumulation of septate junction proteins and an accompanying disruption of βPS-integrin localization. Similarly, at the completion of DC, septate junctions appear at the LE and this is accompanied by downregulation of LE lateral integrins. In pak14pak376A and cora14 embryos where LE septate junctions are deficient, lateral LE βPS-integrin persists (Bahri, 2010).
A recent study in mammalian cell culture indicates that Scrib recruits Pak to the LE (Nola, 2008), and this study has shown that Pak localization in the follicular epithelium is Scrib-dependent. This study of the LE at the end of DC demonstrates that the relationship between Cdc42/Rac signaling complexes and Scrib can act in the opposite direction: membrane-localized Pak recruits the Scrib complex. A bidirectional interaction between the Scrib complex and Cdc42/Rac signaling complexes, including Pak, might be a crucial regulator of events at the LE of closing epithelia during both wound healing and development in diverse systems. Scrib at the newly formed LE can lead to recruitment of the Cdc42/Rac signaling complex, allowing acquisition of mesenchymal characteristics and polarized cell migration. When the opposing epithelial flanks meet up, events can be reversed with Pak recruiting the Scrib complex to the lateral membrane, contributing to restoration of apicobasal polarity and cell adhesion at the LE during MET. The Scrib/Pak complex is believed to be a 'toggle switch', enabling the epithelial membrane to shift back and forth between a migratory state characterized by actin-based extensions and an apicobasal polarized state characterized by cell-cell adhesion (Bahri, 2010).
Cells heal disruptions in their plasma membrane using a sophisticated, efficient, and conserved response involving the formation of a membrane plug and assembly of an actomyosin ring. This paper describes how Rho family GTPases modulate the cytoskeleton machinery during single cell wound repair in the genetically amenable Drosophila embryo model. Rho, Rac, and Cdc42 were found to rapidly accumulate around the wound and segregate into dynamic, partially overlapping zones. Genetic and pharmacological assays show that each GTPase makes specific contributions to the repair process. Rho1 is necessary for myosin II activation, leading to its association with actin. Rho1, along with Cdc42, is necessary for actin filament formation and subsequent actomyosin ring stabilization. Rac is necessary for actin mobilization toward the wound. These GTPase contributions are subject to crosstalk among the GTPases themselves and with the cytoskeleton. Rho1 GTPase was found to use several downstream effectors, including Diaphanous, Rok, and Pkn, simultaneously to mediate its functions. These results reveal that the three Rho GTPases are necessary to control and coordinate actin and myosin dynamics during single-cell wound repair in the Drosophila embryo. Wounding triggers the formation of arrays of Rho GTPases that act as signaling centers that modulate the cytoskeleton. In turn, coordinated crosstalk among the Rho GTPases themselves, as well as with the cytoskeleton, is required for assembly/disassembly and translocation of the actomyosin ring. The cell wound repair response is an example of how specific pathways can be activated locally in response to the cell's needs (Abreu-Blanco, 2013).
Actin and myosin II recruitment and organization at the wound edge are key and conserved elements of the single cell wound repair process in different organisms. In epithelial cells and Xenopus oocytes, wounding induces a strong cytoskeletal response dependent on calcium and Rho family GTPase signaling. In particular, the contractile actomyosin ring formed in Xenopus oocyte wounds is accompanied by the flow of cortical F-actin filaments toward the wound from neighboring regions. Myosin II also accumulates as foci at the wound edge, and its recruitment is independent of F-actin and cortical flow, although its subsequent assembly into a continuous ring does require an intact F-actin network. Interestingly, this study found that actin and myosin II are both actively mobilized toward the wound in the Drosophila syncytial embryo, in a process dependent on cortical flow (Abreu-Blanco, 2013).
This mobilization of myosin II is necessary for proper actomyosin ring assembly and function. In the context of Drosophila cell wound repair, Rac coordinates the actin and microtubule network that influences actin and myosin recruitment, consistent with its known functions in regulating the polymerization of both actin and microtubules. The sharp actomyosin ring formed at the leading edge of the wound, as well as the accompanying halo of dynamic actin, provides the contractile force driving rapid wound closure. This study found that Rho1 is necessary for myosin II activation, leading to its association with actin: actin fails to accumulate as a ring at the wound edge when myosin is disrupted. This is consistent with Rho's known role in increasing the phosphorylation of the myosin II regulatory light chain via its downstream effector Rok. Rho1, along with Cdc42, also functions upstream of the assembly and constriction of the actomyosin ring, where they likely trigger actin filament formation and subsequent actomyosin ring stabilization (Abreu-Blanco, 2013).
In addition to Rho family GTPases regulating actomyosin ring assembly, the actin and myosin II cytoskeleton reciprocally mediates GTPase function. For example, the assembly/ disassembly of the actin network in Xenopus oocyte wounds has been shown to disrupt the local activation and inactivation patterns of Rho and Cdc42, which are required for wound healing progression. In the Drosophila model, actin is required for the translocation, refinement, and maintenance of the Rho family GTPase arrays, while myosin II is required for their recruitment and organization. The actomyosin ring acts as scaffold for signaling molecules that, in turn, are responsible for the polymerization of actin and activation of myosin II. Stabilization of the actin network by Jasplakinolide treatment provides strong evidence that the actomyosin array and the underlying signals translocate together. A striking observation from this model is that Rho1, the first Rho family GTPase to be recruited, accumulates at the wound site even when the actin and myosin II cytoskeleton is severely disrupted, indicating that its recruitment is independent of cytoskeleton integrity. Cdc42 and Rac1, which accumulate after Rho1, are sensitive to disruption of actin and myosin II. Thus, Rho family GTPases are required in Drosophila syncytial embryos for proper wound healing through reciprocal regulation with the dynamic and integrated actin and myosin II networks (Abreu-Blanco, 2013).
Rho family GTPases localize at the wound edge with a precise and characteristic organization pattern. This study shows the recruitment of Rac GTPases to the wound: Rac1 and Rac2 accumulate as graded ring-like arrays at the wound edge. By analyzing the distribution of the endogenous Rho GTPases, it was found that Rho, Cdc42, and Rac (Rac1/2) form partially overlapping concentric arrays that correlate with their specific functions during wound repair. Another interesting observation is that Rho is recruited to the wound before the onset of actin and myosin II recruitment and followed by Cdc42 and Rac (Rac1/2) with a 90 s delay. The localization of active Rho GTPases was analyzed using a combination of activity biosensors and downstream effectors. In the Drosophila model, active Rho1 localizes as a discrete array internal to the actomyosin ring. This is in contrast to that observed in Xenopus oocyte wounds, where active Rho and Cdc42 arrays are formed as discrete concentric rings overlapping with myosin II and actin, respectively. Although the timing and configuration of the GTPase arrays is different in the two cell wound models, they both achieve the same end result: organization of a highly contractile actomyosin ring and a dynamic surrounding zone of actin and myosin assembly/ disassembly, thereby ensuring that a region of highly contractility enriched in myosin II is followed by one of low contractility where actin can assemble. Indeed, the organization of Rho GTPases as local arrays is not only restricted to contractile ring structures such as those observed in wound repair and cytokinesis, but is a common strategy in different biological processes. In the context of cell migration, coordinated zones of Rho family GTPases at the cell's leading edge regulate the waves of cellular protrusion and retraction necessary for migration. In this scenario, Rho activation occurs first, followed by Cdc42 and Rac1 with a 40 s delay. Rho accumulates at the front of the cell concomitant with protrusions and its levels are reduced during retraction, while Rac1 accumulates behind the Rho array with its levels remaining high during the retraction phase. These patterns of activation and organization correlate with their proposed activities: Rho modulates contraction and polymerization, whereas Cdc42 and Rac1 regulate adhesion dynamics (Abreu-Blanco, 2013).
Negative feedback among Rho GTPases is a conserved theme in multiple cellular and developmental processes. In the Xenopus wound model, Rho has been shown to negatively modulate the integrity of the Cdc42 array, while inhibition of Cdc42 activity strongly suppresses local Rho activation. Moreover, Abr, a protein with Rho/Cdc42 GEF and Cdc42 GAP activity has been shown to accumulate at the Rho zone where its GAP activity is required to locally suppress Cdc42 activity, allowing Rho and Cdc42 to segregate into their respective zones. It was not possible to specifically deplete Cdc42 in the Drosophila syncytial embryo wounding assays, so its effects on Rho1 and Rac were not specifically addressed in this model. Nonetheless, the results support the idea of GTPase crosstalk playing a role in the control of GTPase array organization, segregation, and levels. A notable example of crosstalk in this context comes from the expansion of the Rac1 wound-induced array in embryos where Rho1 levels were depleted, suggesting that high levels of Rho1 at the wound edge inhibit Rac1 accumulation at the interior of the actomyosin ring, thereby allowing Rac1 to control the dynamic actin halo surrounding the wound. These intricate and highly regulated interactions among Rho family GTPases allow them to efficiently execute their multiple functions in the cell (Abreu-Blanco, 2013).
A current challenge in the field is to determine how Rho GTPases are maintained locally at high levels that dynamically adjust during wound closure. Recent studies in the Xenopus model propose a mechanism of GTPase treadmilling wherein Rho and Cdc42 are subject to rapid local activation and inactivation, which is different for each GTPase: the Rho activity zone is shaped by trailing edge inactivation, whereas Cdc42 undergoes variable inactivation across the array. The development of biologically active photoactivatable Rho family GTPase reporters will be necessary to determine the relevance of this option in the Drosophila cell wound model. An alternate possibility is that Rho family GTPases and their regulators are anchored at the plasma membrane in an actin-dependent manner. Putative candidates for anchoring proteins are the ERM family proteins, which are known to interact and regulate Rho GTPases in different cellular process (Abreu-Blanco, 2013).
It was surprising to find that Rho1 utilizes multiple downstream effectors simultaneously during wound repair, begging the question of how this specificity is achieved, maintained, and tweaked dynamically. Considering the complexity of wound repair, cytoskeletal responses are likely regulated via several signaling pathways that converge on the contractile ring. These pathways require precise coordination to provide and integrate the different components required for the dynamic assembly and disassembly of contractile ring machinery as the wound is drawn closed. Rho family GTPases, rather than switching the behavior of the entire cell, would need to be capable of locally modulating the dynamics of the cytoskeleton from one part of the cell to another. This specificity associated with simultaneous recruitment and function of downstream effectors is likely to be a key regulatory feature for dynamic orchestration of cell wound repair. Future challenges include defining the molecular composition of these signaling modules and delineating the combinations and specific subcellular localizations of Rho GTPases, GEFs, GAPs, upstream regulators, and downstream effectors that are needed for the proper functioning of these pathways (Abreu-Blanco, 2013).
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 Robos 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).
The role of tip and rear cells in collective migration is still a matter of debate and their differences at the cytoskeletal level are poorly understood. This study analysed these issues in the Drosophila trachea, an organ that develops from the collective migration of clusters of cells that respond to Branchless (Bnl), a FGF homologue expressed in surrounding tissues. Individual cells in the migratory cluster were tracked and their features were characterized; two prototypical types of cytoskeletal organization were unveiled that account for tip and rear cells respectively. Indeed, once the former are specified, they remain as such throughout migration. Furthermore, it was shown that FGF signalling in a single tip cell can trigger the migration of the cells in the branch. Finally, specific Rac activation was found at the tip cells, and how FGF-independent cell features such as adhesion and motility act on coupling the behaviour of trailing and tip cells was analyzed. Thus, the combined effect of FGF promoting leading cell behaviour and the modulation of cell properties in a cluster can account for the wide range of migratory events driven by FGF (Lebreton, 2013).
Among the tracheal branches from each placode, two grow towards the ventral side of the embryo, one in the anterior and the other in the posterior region of the segment, the lateral trunk anterior (LTa) and the lateral trunk posterior (LTp) respectively. By a combination of migration, intercalation and elongation, the tip cell of the LTp migrates towards the central nervous system (CNS), and the resulting ganglionic branch (GB) connects the CNS to the main tracheal tube. Another cell from the LTp migrates towards the LTa of the adjacent posterior metamere and makes a fusion branch that connects the two LT branches. This study focused on this branch (LTp/GB) because its complex morphology and pattern of migration make it particularly appropriate for analysing the morphology and behaviour of the tip and trailing cell during tracheal collective migration (Lebreton, 2013).
The FGF signalling pathway is involved in many morphogenetic events requiring collective migration of cell clusters. However, it is not entirely clear whether in these events FGF signalling is directly involved in triggering cell migration, or alternatively if it is required for other processes such as cell determination which only affect cell migration indirectly. Moreover, while FGF might be required it is not clear either whether all the cells or just a subset of those need to directly receive the signal to sustain the migration of the entire cluster. One well-studied case is the role of FGF in the development of the zebra fish lateral line. In that case, FGF appears to be produced by the leading cells which signal to the trailing cells, the cells where FGF signalling is active. Restriction of FGF signalling is thereafter required for the asymmetric expression of the receptors for the chemokines that guide migration (Lebreton, 2013).
A very different scenario applies in the case of Drosophila tracheal migration. On the one hand, FGF is expressed in groups of cells outside the migrating cluster. On the other hand the results in the LTp/GB indicate that FGF signalling is required and sufficient in the leading cells, and not in the trailing cells, for the migration of the whole cell cluster. Therefore, in spite of its widespread involvement, the mechanisms triggered by FGF signalling in collective migration appear to be quite different (Lebreton, 2013).
Rho inactivation produced breaks and detachment in the LTp/GB cluster while its constitutive activation led these cells to hold together impairing migration. Likewise, upon Cdc42 inactivation LTp/GB cells were associated by thin extensions associated in some cases with breaks, while upon its constitutive activation, the LTp/GB transient pyramidal organisation did not evolve, or evolved much more slowly, towards branch elongation. However, the phenotypes from each RhoGTPase mutants don't look alike and the detailed analysis suggests that Rho impinges primarily on cell adhesion while Cdc42 does so on cell motility (Lebreton, 2013).
These results are consistent with previous findings that show a role for Rho in regulating adherens junctions stability and for Cdc42 as the main mediator of filopodia formation. It is noted, however, that Cdc42 was found to exert in the LTp/GB an opposite effect to the one identified in other systems, as Cdc42DN mutants showed more protrusions and were more actin-enriched basally than wild-type cells and Cdc42ACT mutants showed a reduced the motility of LTp/GB (Lebreton, 2013).
There is an increasing amount of data pointing to the different effect of RhoGTPases in vitro versus in vivo models and also among various cell types. A unidirectional assignment between a specific cellular process in vivo and a single RhoGTPase is probably an oversimplification and this was not the aim of the current study. Rather the study relied on mutant forms of the RhoGTPases to modulate cell features, either individually or collectively, to assess their role in the overall behaviour of the cell cluster. In doing so, the results point to a critical role for a balance between cell adhesion and cell motility for the collective migration of a cell cluster (Lebreton, 2013).
The results support the following model for the specification, features and behaviour of leading cells in the migration of the LTp/GB branch. Upon signalling from the FGF pathway, tip cells reorganise their cytoskeleton features (actin enrichment at the basal membrane, small apical surface and an apicobasal polarity along the proximo-distal axis), thereby enabling them to acquire leading behaviour. Indeed, FGF can induce migratory capacity to the whole cluster by signalling only the tip cells, where a dynamic transition between states of Rac activity is needed to acquire a leading role. How the behaviour of tip cells leads collective migration thereafter depends on the features of the cells in the cluster, which are determined by various regulators (among these, the RhoGTPases) which act, at least in part, in an FGF-independent manner. Ultimately, the balance between individual cell properties such as cell adhesion, motility and apicobasal polarity will (1) determine the net movement of the overall cell bodies or alternatively changes in cell shape in terms of elongation, (2) control the migratory speed and (3) define whether cells will migrate individually or in clusters and whether clusters will bifurcate in different paths. The combined effect of the changes promoting leading cell behaviour and modulation of cell features is likely to be a widely exploited mechanism in collective migration. In particular, the actual balance between these cell features may dictate the specifics of each migratory process and, consequently, the final shape of the tissues and organs they contribute to generate (Lebreton, 2013).
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).
By acting as a dynamic link between adjacent cells in a monolayer, adherens junctions (AJs) maintain the integrity of epithelial tissues while allowing for neighbor exchange. Although it is not currently understood how this combination of AJ stability and plasticity is achieved, junctionally associated actin filaments are likely to play a role, because actin-based structures have been implicated in AJ organization and in the regulation of junctional turnover. Through exploring the role of actin cytoskeletal regulators in the developing Drosophila notum, this study has identified a critical role for Cdc42-aPKC-Par6 in the maintenance of AJ organization. In this system, the loss or inhibition of Cdc42-aPKC-Par6 leads to junctional discontinuities, the formation of ectopic junctional structures, and defects in apical actin cytoskeletal organization. Affected cells also undergo progressive apical constriction and, frequently, delamination. Surprisingly, this Cdc42-aPKC-Par6-dependent regulation of junctional stability was found to be independent of several well-known targets of Cdc42-aPKC-Par6: Baz, Lgl, Rac, and SCAR. However, similar AJ defects are observed in wasp, arp2/3, and dynamin mutant cells, suggesting a requirement for actin-mediated endocytosis in the maintenance of junctional stability downstream of Cdc42. This was confirmed in endocytosis assays, which revealed a requirement for Cdc42, Arp2/3, and Dynamin for normal rates of E-cadherin internalization. In conclusion, by focusing on the molecular mechanisms required to maintain an epithelium, this analysis reveals a novel role for the epithelial polarity machinery, Cdc42-Par6-aPKC, in local AJ remodeling through the control of Arp2/3-dependent endocytosis (Georgiou, 2008).
This analysis of Cdc42 in Drosophila epithelial cells reveals a novel role for Cdc42, both in the regulation of the actin cytoskeleton and in AJ maintenance and stability. In this capacity, Cdc42 appears to function together with junctional Par6 and aPKC. It may appear surprising that Cdc42, aPKC, and Par6 act relatively independently of Baz (Par3) in the notum, given that Baz has been shown to act as a landmark to define the future site of E-cad localization and AJ formation in the embryo and to define neuroblast polarity upstream of Cdc42, aPKC, and Par6. However, the localization and function of Baz are distinct from those of Par6 and aPKC in many tissues. Moreover, the differences go further, because in the context of the notum, Cdc42, Par6, and aPKC are not required to maintain a polarized epithelium, and do not appear to function together with Lgl. A possible explanation for the reduced dependency of epithelial architecture in the notum on these molecules is the structural support gained from overlying apical cuticle and the basal lamina during its relatively slow development. Because of this, junctional defects in cdc42 mutant clones in this tissue are less likely to be an indirect consequence of a primary defect in apical-basal polarity. Moreover, the inherent stability of this epithelium made it possible to identify a novel role for the Cdc42-Par6-aPKC complex in the communication of polarity information across cell-cell junctions within the plane of the epithelium, something that may have been obscured by apical-basal polarity defects in studies in less stable epithelia (Georgiou, 2008).
Several lines of evidence suggest that the AJ defects seen in cdc42, aPKC, or par6 mutant cells result from defects in the internalization of junctional material, rather than from defects in the delivery of E-cadherin to the plasma membrane. First, junctional E-cadherin levels remain high in cdc42 mutant clones, enabling these cells to pull on surrounding cells. Second, in cdc42 clones, tubules and tubule-derived puncta containing E-cadherin, alpha-Catenin, and beta-Catenin remain continuous with the cell surface—as visualized with a probe for extracellular E-cadherin. This is the case, even though the vast majority of the rare, large E-cadherin puncta seen by light microscopy in the wild-type are found associated with an internalized fluid phase marker. Third, similar structures accumulate after a transient block in endocytic vesicle scission, resulting from the inhibition of Dynamin function. These data suggest that the primary defect induced by loss of Cdc42 is a defect in the endocytosis-mediated turnover of junctional material. Nevertheless, a detailed analysis of the molecular dynamics of E-cadherin-GFP at wild-type and mutant AJs is needed to quantify the contribution of Cdc42-mediated endocytosis to the normal rate of E-cadherin turnover (Georgiou, 2008).
How then do Cdc42, aPKC, and Par6 activate endocytosis to drive normal junctional turnover? Having identified a role for Cdc42 (aPKC and Par6) in apical actin organization, attention was focused on actin cytoskeletal regulators known to act downstream of Cdc42 in answering this question. Significantly, both WASp and the Arp2/3 complex (but not Rac or SCAR) were found to be required for the maintenance of a normal apical actin cytoskeleton and AJs. Because WASp is a well-established Cdc42 target, this suggested that the effect of Cdc42 on junctional endocytosis is mediated directly through WASp. The junctional phenotypes observed in WASp, however, appear weaker than those seen after the loss of Arp2/3, Cdc42, or Dynamin. This may be the result of protein perdurance. Alternatively, this observation may point to the existence of other proteins that act in parallel with WASp to stimulate Arp2/3-mediated vesicle scission downstream of Cdc42. In fact, a number of Cdc42 targets have been shown to affect actin nucleation and membrane tubulation. Nevertheless, the striking similarities between the AJ phenotypes induced by loss of Arp2/3, Dynamin, and Cdc42, together with recent data implicating Cdc42, Par6, and aPKC in the regulation of vesicle trafficking and endocytosis, provide strong evidence that the primary defect in each case is a block in actin-mediated endocytosis. On the basis of this analysis, it is suggested that the cdc42, par6, and apkc phenotype arises in the following way. First, a reduction in the activity of Cdc42-aPKC-Par6 on one side of an AJ translates into a reduction of Cdc42-aPKC-Par6 activity on the opposing side, resulting in a concomitant reduction in the activity of WASp and the Arp2/3 complex, leading to defects in Dynamin-dependent endocytosis along the entire cell-cell interface. The resulting failure to remove excess material from the ends of the AJ causes junctional spreading, as observed in electron micrographs. This leads to the formation of the discontinuous junctions, junctional extensions, and punctate surface structures visible in confocal images. Over time, this has the effect of destabilizing AJs, leading to the loss of apical material and, eventually, to cell delamination. In this view, Cdc42-Par6-aPKC regulate local Arp2/3-mediated endocytosis to maintain AJs in a state of dynamic equilibrium. Internalized junctional material can then be recycled back to the cell surface to engage in cell-cell adhesion in a well-regulated fashion. Importantly, this model predicts that the stability of AJs is intimately linked to their turnover -- a feature that makes AJs inherently plastic (Georgiou, 2008).
Polarity of the Drosophila oocyte is essential for correct development of the egg and future embryo. The Par proteins Par-6, aPKC and Bazooka are needed to maintain oocyte polarity and localize to specific domains early in oocyte development. To date, no upstream regulator or mechanism for localization of the Par proteins in the oocyte has been identified. This study analyzed the role of the small GTPase Cdc42 in oocyte polarity. Cdc42 was shown to be required to maintain oocyte fate, which it achieves by mediating localization of Par proteins at distinct sites within this cell. Cdc42 localization itself is polarized to the anterolateral cortex of the oocyte, and Cdc42 is needed for maintenance of oocyte polarity throughout oogenesis. The data show that Cdc42 ensures the integrity of the oocyte actin network and that disruption of this network with Latrunculin A phenocopies loss of Cdc42 or Par protein function in early stages of oogenesis. Finally, it was showm that Cdc42 and Par proteins, as well as Cdc42/Par and Arp3, interact in the context of oocyte polarity, and that loss of Par proteins reciprocally affects Cdc42 localization and the actin network. These results reveal a mutual dependence between Par proteins and Cdc42 for their localization, regulation of the actin cytoskeleton and, consequently, for the establishment of oocyte polarity. This most likely allows for the robustness in symmetry breaking in the cell (Leibfried, 2013).
The findings show that Cdc42 is required for oocyte polarity throughout oogenesis. The following findings were made: (1) Cdc42 localizes to the anterolateral cortex of the young oocyte; (2) Cdc42 interacts with Par proteins in the germline in vivo; (3) mutants for Cdc42, aPKC or Baz display a disrupted actin cytoskeleton at the anterolateral cortex; and (4) disrupting the actin cytoskeleton with Latrunculin A results in loss of anterior-to-posterior movement of the oocyte-specific protein Orb, phenocopying loss of Cdc42 or the Par proteins. Thus, the cortical actin cytoskeleton is crucial for the establishment of oocyte polarity (Leibfried, 2013).
This is in line with previous observations linking the actin cytoskeleton and Par proteins in the generation of cell polarity. Loss of Baz results in an increase in actin protrusions in Drosophila epithelia and a decrease in actin at synapses. In C. elegans, active CDC-42 localizes to the anterior during the polarity maintenance phase, when it is important for PAR-6 localization, and the anterior actin cap is depleted in par-3 mutants during polarity establishment. Similar to the current observations, actin depolymerization does not affect Par protein localization in C. elegans. By contrast, drug-induced actin depolymerization has been shown to disrupt Baz apical localization during cellularization and to interfere with its cortical association during gastrulation in Drosophila (Leibfried, 2013).
Although the molecular relationship between Par proteins and actin has not been clearly delineated, in mammals Par-3 (Pard3) associates with actin regulators, including the RacGEF Tiam1 and LIM kinase 2. In the current study, it was shown that Cdc42 localization depends on the Par complex and that Cdc42, aPKC, Baz and Par-6 interact in vivo in biochemical or genetic assays. This interaction is required for oocyte polarity. Par-6 interacts biochemically with Cdc42 and Baz via its semi-CRIB and PDZ domains and via its PB1 domain with the PB1 domain of aPKC. Baz interacts biochemically with the kinase domain of aPKC. Indeed, a quaternary complex of Myc-Cdc42, HA-Par-6b, PKCτ/λ and Par-3 can be isolated from transfected COS-7 cells. In Drosophila, a Baz mutant lacking its aPKC-interaction domain supports early oogenesis and an aPKC mutant that cannot bind Par-6 also develops late egg chambers. Together, these results and the current data indicate that interaction of Cdc42, Par-6, aPKC and Baz is required for their correct function in the germline, and that the binding of aPKC to either Par-6 or to Baz is sufficient to ensure this interaction, highlighting the role of all three Par proteins in actin regulation via their interaction with Cdc42. This quaternary relationship seems important for the regulation of polarity establishment, whereas studies in mature epithelial cells have delineated separate functions of Par-6/aPKC/Cdc42 and Baz for polarity maintenance (Leibfried, 2013).
Early oocyte polarity and its maintenance were previously linked to the microtubule network. Microtubules play an important role in early oogenesis, as their disruption with Colchicine leads to a 16-nurse-cell phenotype. Indeed, oocyte specification depends on the accumulation of the oocyte-specific protein BicD, which is a component of the microtubule-related dynactin complex (Leibfried, 2013).
The results point to a sequential involvement of actin and microtubules in polarizing the oocyte: in the early stages, after oocyte specification, the Par proteins together with Cdc42 establish cortical domains and a pronounced cortical actin cytoskeleton. The interdependence of these proteins for their localization persists during oogenesis, allowing for robustness of symmetry breaking. At later stages, knockdown of Cdc42 results in reduced amounts of Baz and Par-1 at the anterior and posterior of the oocyte, respectively. As Par-1 is required for microtubule organization, this most likely leads to the observed mislocalization of axis determinants. Similarly, disrupting the actin cytoskeleton with drugs or by knockdown of actin-binding proteins has been shown to result in bundling of microtubules and premature ooplasmic streaming, leading to loss of oocyte polarity. Hence, microtubules act in oocyte specification and late polarity events, whereas Cdc42 and actin dominate in the establishment and maintenance of polarity in the developing oocyte (Leibfried, 2013).
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