In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. The gene kette (correctly termed Hem-protein, or simply Hem) is required for the normal projection of the VUM axons and interference with kette function disrupts glial migration. In spite of the fact that glial migration is disrupted in kette mutants, both the axon guidance and glial migration phenotypes have their origin in midline neuron expression and not in midline glial expression. Axonal projection defects are found for many moto- and interneurons in kette mutants. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The Hem/Kette protein is homologous to the transmembrane protein HEM-2/NAP1 (Nck-associated protein) evolutionary conserved from worms to vertebrates. In the CNS, the membrane protein Kette could be participating directly in the neuron-glial interaction at the midline, where it could act as a signal to direct glial migration. Alternatively, Kette could serve as a receptor of possibly glial-derived signals during VUM growth cone guidance. The experimental data suggest that Kette transduces information to the neuronal cytoskeleton, which is in agreement with a receptor function (Hummel, 2000).
In vitro analysis has shown a specific interaction of the vertebrate HEM-2/NAP1 with the SH2-SH3 adapter protein NCK and the small GTPase RAC1, which both have been implicated in regulating cytoskeleton organization and axonal growth. Hypomorphic kette mutations lead to axonal defects similar to mutations in the Drosophila NCK homolog dreadlocks. Furthermore, kette and dock mutants genetically interact. NCK is thought to interact with the small G proteins Rac1 and Cdc42, which play a role in axonal growth. In line with these observations, a kette phenocopy can be obtained following directed expression of mutant Cdc42 or RAC1 in the CNS midline. In addition, the kette mutant phenotype can be partially rescued by expression of an activated Rac1 transgene. These data suggest an important role of the HEM-2 protein in cytoskeletal organization during axonal pathfinding (Hummel, 2000).
Most of the Kette protein is found in the cytoplasm where it colocalizes with F-actin to which it can bind via its N-terminal domain. Some Kette protein is localized at the membrane and accumulates at focal contact sites. Loss of Kette protein results in the accumulation of cytosolic F-actin. Actin dynamics crucially depend on the ability of the protein to switch from a monomeric (G-actin) to a filamentous form (F-actin). Polymerization of F-actin starts with the de novo nucleation of an actin trimer, a process that occurs relatively slowly and requires the action of the Arp2/3 complex (see Drosophila Arp2/3 component Suppressor of profilin 2). Subsequent elongation is fast and cells have to prevent spontaneous actin polymerization by expressing a variety of actin-binding proteins such as profilin. The nucleation activity of the Arp2/3 complex in turn is regulated by a set of activators, such as the members of the Wasp (Wiskott-Aldrich syndrome protein) and Wave (Scar - FlyBase) families. Wave, which does not bind Cdc42, is trans-inhibited through its association with members of the Kette family: Sra1 (specifically Rac associated 1) and Abi (Abelson-interactor). Rac1 binding, presumably to Sra1, relieves the inhibitory function of this complex (Bogdan, 2003 and references therein).
To date it is still unclear how Kette could regulate the organization of the actin cytoskeleton in vivo. Since Kette has been predicted to be an integral membrane protein with six transmembrane domains, it might serve as a receptor recruiting Nck/Dock or Rho-GTPases to the membrane. Biochemical and genetic evidence reveals that Kette is found predominantly in the cytosol. Only a small amount of Kette is recruited to the plasma membrane. In vivo as well as in tissue culture models, Kette protein colocalizes with F-actin, and co-sedimentation assays reveal a direct interaction with F-actin. Within the membrane, Kette accumulates at the insertion sites of large F-actin bundles, suggesting that targeted localization of Kette may be required for its function. Loss of Kette protein leads to a Scar/Wave-dependent accumulation of F-actin within the cell. Ectopic expression of wild-type or different truncated Kette proteins in tissue culture cells or during Drosophila development does not affect F-actin formation or viability. However, expression of membrane-tethered Kette efficiently induces ectopic bundles of F-actin in a process depending on Wasp but not on Scar/Wave. These data indicate that Kette fulfils a novel role in regulating F-actin organization by antagonizing Wave and activating Wasp-dependent actin polymerization (Bogdan, 2003).
Thus Kette is able to modulate the activity of both Wasp and Scar/Wave and thus contribute to the regulation of F-actin dynamics. Wasp and Wave together with Cdc42 and Rac1 control different aspects of cortical actin dynamics. Cdc42 and Wasp are required for filopodia formation, whereas dominant-negative Wave disrupts the Rac1-dependent formation of branched network of F-actin bundles required to form lamellipodia. Wave localizes to membrane ruffles induced by activated Rac1 and Wasp accumulates in microspikes containing bundled F-actin (Bogdan, 2003 and references therein).
A large 500 kDa complex comprising Nap1/Kette, PIR121/Sra1, Abi, HSPC300 and Wave silences the otherwise constitutive activity of Wave in stimulating actin polymerization (Eden, 2002). The 500 kDa complex is stabilized by direct protein-protein interactions that have been demonstrated between Nap1/Kette and Abi, and Nap1/Kette and PIR121/Sra1 (also called Gex2). The exact binding partners of Wave are presently unknown (Bogdan, 2003 and references therein).
Two modes of Wave activation have been demonstrated in vitro: (1) activated Rac1 is able to bind to PIR121/Sra1 and can relieve Wave inhibition by dissociating the Nap1/Kette, PIR121/Sra1 and Abi sub-complex (Eden, 2002); (2) PIR121/Sra1 is able to bind to the first SH3 domain of SH2SH3 adapter Nck, which is sufficient to activate the Wave complex (Eden, 2002). In vivo these two mechanisms may work at the same time to fully activate Wave (Bogdan, 2003 and references therein).
In agreement with the work of Eden (2002) disruption of Kette function leads to an excess formation of cytoplasmic F-actin. However, expression of even very high levels of wild-type Kette protein do not evoke any mutant phenotype. Thus, in wild type cells the inactive Wave complexes are already formed and Kette overexpression does not result in an additional sequestering of Wave into the silencing complexes and/or an incorporation of additional Kette into these complexes. In addition, since Kette requires Sra1 function to bind to membrane-associated adapters such as Nck, excess cytosolic Kette will not be able to reorganize subcellular Wave distribution (Bogdan, 2003).
Further support for the notion that Kette mediates repression of Scar/Wave activity stems from genetic analyses. Embryos lacking zygotic kette function display a characteristic CNS phenotype, whereas loss of zygotic Scar/Wave expression does not affect embryonic nervous system development. The kette mutant phenotype, which is due to defects in neurite outgrowth (Hummel, 2000), is significantly suppressed by reducing the dose of Scar/Wave expression. This demonstrates that in wild-type embryos, Kette acts as a negative regulator of Scar/Wave. Similar results are obtained when Kette and or Scar/Wave expression is reduced in Drosophila S2 cells. These experiments also reveal that Scar/Wave is required for the normal subcellular distribution of Kette, which may, however, be an indirect effect caused by the disruption of the F-actin actin cytoskeleton (Bogdan, 2003).
Kette protein localizes to the plasma membrane where it accumulates in focal adhesion contacts. A prime candidate that may mediate recruitment of Kette to the membrane is the SH2 SH3 adapter protein Nck which, besides binding of the Sra1/Kette/Abi complex, also recruits numerous other proteins to focal contact sites. Among these is Wasp, which binds to the third SH3 domain of Nck (Bogdan, 2003 and references therein).
kette interacts with dock, which encodes the Drosophila Nck homolog (Hummel, 2000). Membrane recruitment of Kette is sufficient to activate actin polymerization in the cell cortex mediated by Wasp. How is this brought about? One explanation might be that recruitment of Kette to the membrane disintegrates the inhibitory Wave complex, independent of the Nck/Sra1 association. This would then lead to an excess of Wave activity and subsequently to an excess of actin polymerization. However, the genetic data clearly show that membrane bound Kette functions independent of Scar/Wave but depends on Wasp (Bogdan, 2003).
Wasp usually adopts an auto-inhibited conformation and is activated after Cdc42, Nck binding or phosphorylation. A structure-function analysis of the Drosophila Wasp has demonstrated that the Cdc42-binding domain is not necessary for function, suggesting that alternative pathways, such as phosphorylation can activate Wasp. Kette might be a part of such an alternative pathway; a genetic interaction between kette and wasp in the regulation of actin dynamics has been demonstrated. Regulation of Wasp by Kette is not mediated by direct protein-protein interaction, but probably involves Abi that is able to link Kette and Wasp. The Abl interactor (Abi) protein localizes to sites of actin polymerization at the tips of lamellipodia and filopodia and has been implicated in the cytoskeletal reorganization in response to growth factor stimulation. As a positive regulator of the non-receptor tyrosine-kinase Abelson (Abl) Abi may bring Abl into position to phosphorylate and thus activate Wasp. Abl is known to phosphorylate many proteins regulating focal adhesion and F-actin dynamics and overexpression of activated Abl induces F-Actin formation in Cdc42-independent manner. Some tyrosine kinases are activate by phosphorylation of Wasp; however, direct phosphorylation of Wasp by Abl remains to be demonstrated (Bogdan, 2003 and references therein).
Further support of the model that suggests in vivo Kette activates Wasp but suppresses Wave comes from the phenotypic analyses of Drosophila kette, wasp and scar/wave mutants. Mutations in kette have been isolated due to defects in commissure formation in the embryonic CNS (Hummel, 2000). If Kette acts via activating Wasp, similar phenotypes are expected following disruption of either gene. This is indeed the case and loss of zygotic and maternal Wasp function results in a kette-like embryonic CNS phenotype (Bogdan, 2003 and references therein).
In agreement with the proposed function of Kette in regulating both, Wasp and Wave, is its subcellular distribution. Whereas the majority of Kette is present in the cytoplasm to keep Wave in its inactive state (Eden, 2002) some is present in the leading edge of lamellipodia-like structures. However, the highest amounts of Kette are present at the insertion points of large F-actin bundles where N-Wasp is also present. Kette might be recruited to these focal adhesion sites via Sra1/Nck; via Wasp, it could enhance the formation of F-actin bundles (Bogdan, 2003 and references therein).
It is presently unclear just how Wasp activity results in straight F-actin bundles, whereas Wave stimulates the formation of a meshed F-actin network. In the cytosol, Kette may act as a scaffold protein that keeps Wave close to F-actin and recruits additional factors to F-actin such as Profilin, which not only binds to Kette but also enhances actin nucleation. Thus, Kette could promote the formation of a meshed F-actin network characteristic for lamellipodia. At the membrane other proteins may interact with Kette and in this respect it is interesting to note that the F-actin crosslinking protein Filamin, which plays an important role in filopodia formation, also binds to Kette. This suggests that Kette, in addition to regulating Wasp and Wave, may also contribute to the decision whether filopodia or lamellipodia are formed (Bogdan, 2003).
In animal cells, GTPase signaling pathways are thought to generate cellular protrusions by modulating the activity of downstream actin-regulatory proteins. Although the molecular events linking activation of a GTPase to the formation of an actin-based process with a characteristic morphology are incompletely understood, Rac-GTP is thought to promote the activation of SCAR/WAVE, whereas Cdc42 is thought to initiate the formation of filopodia through WASP. SCAR and WASP then activate the Arp2/3 complex to nucleate the formation of new actin filaments, which through polymerization exert a protrusive force on the membrane. Using RNAi to screen for genes regulating cell form in an adherent Drosophila cell line, a set of genes, including Abi/E3B1 (an SH3 domain-containing Abl substrate), was identified that is absolutely required for the formation of dynamic protrusions. These genes delineate a pathway from Cdc42 and Rac to SCAR and the Arp2/3 complex. Efforts to place Abi in this signaling hierarchy reveal that Abi and two components of a recently identified SCAR complex, Sra1 (p140/PIR121/CYFIP) and Kette (Nap1/Hem), protect SCAR from proteasome-mediated degradation and are critical for SCAR localization and for the generation of Arp2/3-dependent protrusions. It is concluded that in Drosophila cells SCAR is regulated by Abi, Kette, and Sra1, components of a conserved regulatory SCAR complex. By controlling the stability, localization, and function of SCAR, these proteins may help to ensure that Arp2/3 activation and the generation of actin-based protrusions remain strictly dependant on local GTPase signaling (Kunda, 2003).
With the advent of RNAi it is possible to use Drosophila cells in culture as a model system to test the cell-biological function of genes identified by genomic sequencing; such genes include those involved in the generation of actin-based protrusions. S2R+ cells are particularly amenable to this type of loss-of-function analysis because a large number of distinct actin-related phenotypes can be readily distinguished in this cell type. Using such an approach, several genes were identified from a set of putative actin regulators that are absolutely required for the maintenance of S2R+ cell shape and for the formation of lamellipodia. In each case, the gene-specific dsRNA identified causes cells to assume a starfish-like morphology with multiple slender cell extensions. This change in form is accompanied by the loss of actin filaments from the cell periphery, resulting in a more diffuse, non-cortical F-actin distribution. Genes with this characteristic RNAi phenotype included a known activator of the Arp2/3 complex, the sole Drosophila SCAR/WAVE homolog, and Drosophila Abi, an SH3 domain-containing Abl substrate. In contrast, Drosophila WASP, another Arp2/3 complex activator, had no discernable RNAi phenotype in this assay and did not visibly accentuate the SCARRNAi phenotype. RNAi targeting of Arc-p34 and Arc-p20, two components of the Drosophila Arp2/3 complex, led to a similar change in S2R+ cell shape, implying that this spiky phenotype reflects the inability to nucleate new cortical actin filaments (Kunda, 2003).
Although this analysis delineated a putative pathway (Cdc42>Rac>SCAR>Arp2/3 complex) that promotes the nucleation of actin filaments, it was not clear where to place Abi within this signaling hierarchy. A recent biochemical study, however, noted that Abi copurifies with mammalian homologs of Kette, Sra1, and HSPC300 (see Medline: 12181570 ) as part of a regulatory SCAR complex in extracts from mammalian brains. By using RNAi to test the functions of the equivalent Drosophila proteins, it was found that Abi, Sra1, and Kette are essential for the generation of protrusions and for the stability of SCAR protein. Similarly, in parallel studies in Drosophila and in Dictyostelium, reduced levels of SCAR protein were observed in cells lacking individual components of the complex. These data suggest that the presence of SCAR in a regulatory complex and its sensitivity to degradation have been highly conserved during evolution. Furthermore, the idea that these proteins form a physical complex in Drosophila is supported by the colocalization of Kette and SCAR at the tips of protrusions. The data concerning the function of the fifth component of the putative SCAR complex, HSPC300, are more equivocal. Although treatment of S2R+ cells with HSPC300 dsRNA compromised their ability to form lamellipodia and caused a reproducible, if partial, reduction in SCAR protein levels, it was not possible to measure the extent of RNAi-mediated HSPC300 silencing. Therefore, although the data support a role for HSPC300 in the regulation of SCAR, it has not yet been determined whether HSPC300 is absolutely required for the generation of SCAR-dependent protrusions, as are Abi, Kette, and Sra1 (Kunda, 2003).
Given the apparent sensitivity of SCAR to proteolysis, changes in local or global SCAR stability could modulate the rate of actin filament formation. Furthermore, if SCAR is released from the complex after the binding of Rac-GTP, as predicted, SCAR degradation could also act as a brake to limit SCAR-dependent actin filament nucleation. In either case, one would expect SCAR protein to exhibit a relatively short half-life in vivo. In actively ruffling S2R+ cells, however, SCAR appears to be relatively stable because proteasome inhibitors or inhibitors of transcription or translation have little effect on SCAR protein levels. These findings lead to the conclusion that most SCAR is present in stable complexes in wild-type cells. For this reason, the conserved instability of SCAR protein may simply provide cells with a mechanism to rapidly eliminate free, nascent, or mislocalized SCAR, protecting cells from the potentially adverse effects of this potent, constitutively active protein. Nevertheless, under special circumstances or in other cell types, proteasome-mediated degradation of SCAR may help to limit the extent of actin filament nucleation induced after a burst of Rac-GTP (see below) (Kunda, 2003).
Although Abi, Kette, and Sra1 are required for preventing SCAR degradation, the data clearly point to their having additional functions. Most importantly, the morphological changes observed in AbiRNAi cells precede the loss of SCAR protein. In addition, increasing the SCAR protein levels in AbiRNAi cells fails to rescue their morphological defects; it also fails to do so in KetteRNAi and Sra1RNAi cells. This might seem unexpected given that SCAR is able to activate the Arp2/3 complex on its own, both in vitro and in Drosophila cells. In the absence of complex components, however, SCAR fails to become properly localized at the cell cortex. So, by localizing SCAR at the cortex, the complex may play a critical role in harnessing its activity for the generation of protrusive force (Kunda, 2003).
Three recent genetic studies reported observations that conflict with data presented here. In particular, data presented in these studies show that Sra1/Kette and SCAR display an antagonistic relationship in the Drosophila nervous system. Although more work will have to be done to unravel such apparently contradictory data, some of these discrepancies may reflect differences in the relative levels of SCAR and components of the inhibitory complex in the model system under investigation. If the total cellular pool of Abi, Sra1, and Kette is bound up in stable, Rac-responsive SCAR complexes, a reduction in the level of any one component will lead to a reduction in Arp2/3-dependent actin nucleation (as observed in this study). In contrast, if Abi, Kette, and Sra1 are present in excess of SCAR, they will limit the ability of free, active SCAR to nucleate actin filaments (Kunda, 2003).
A speculative model is presented for the regulation of SCAR. This model attempts to reconcile the current findings with data from recent in vitro and genetic studies. It is proposed that nascent SCAR is rapidly incorporated into an inhibitory complex that contains Abi, Sra1, and Kette and protects the protein from proteolysis. The complex localizes at the cell cortex , where it is responsive to Rac signaling. The binding of Sra1 to Rac-GTP may induce a transient change in the makeup or conformation of the complex, which may free the SCAR VCA domain to interact with the Arp2/3 complex, whose activation triggers a burst of new actin filament formation. Finally, the extent of actin filament formation in response to a pulse of Rac-GTP may be limited by the presence of the inhibitory complex and to a lesser extent by proteasome-mediated degradation. In summary, it is proposed that cells regulate SCAR stability, localization, and activity to ensure that actin nucleation and the formation of cellular protrusions are precisely regulated in time and space (Kunda, 2003).
Although it is well established that the WAVE/SCAR complex transduces Rac1 signaling to trigger Arp2/3-dependent actin nucleation, regulatory mechanisms of this complex and its versatile function in the nervous system are poorly understood. The Drosophila proteins SCAR, CYFIP and Kette, orthologs of WAVE/SCAR complex components, all show strong accumulation in axons of the central nervous system and indeed form a complex in vivo. Neuronal defects of SCAR, CYFIP and Kette mutants are, despite the initially proposed function of CYFIP and Kette as SCAR silencers, indistinguishable and are as diverse as ectopic midline crossing and nerve branching as well as synapse undergrowth at the larval neuromuscular junction. The common phenotypes of the single mutants are readily explained by the finding that loss of any one of the three proteins leads to degradation of its partners. As a consequence, each mutant is unambiguously to be judged as defective in multiple components of the complex even though each component affects different signaling pathways. Indeed, SCAR-Arp2/3 signaling is known to control axonogenesis whereas CYFIP signaling to the Fragile X Mental Retardation Protein fly ortholog contributes to synapse morphology. Thus, these results identify the WAVE/SCAR complex as a multifunctional unit orchestrating different pathways and aspects of neuronal connectivity (Schenck, 2004).
Formal evidence is provided that SCAR, CYFIP and Kette proteins form a complex. Co-immunoprecipitation experiments were performed from cytoplasmic extracts of Drosophila Schneider (S2) cells using antibodies raised against CYFIP. Extract and co-immunoprecipitated material were subjected to Western blot analysis using antibodies against SCAR and Kette, the fly orthologs of WAVE and Hem-2/NAP125, respectively, which both associate with the human CYFIP2 protein. Anti-Kette antibody reveals a band of 112 kDa, whereas anti-SCAR reveals a doublet of about 66 and 70 kDa, one band of which may represent a post-translationally modified SCAR protein. The two Drosophila proteins are found to specifically co-immunoprecipitate with CYFIP. Using the same antibodies, it was found that, in wild-type embryos, Kette is present in longitudinal connectives as well as commissures during establishment of the axonal network, like SCAR and CYFIP. By late stages, all three proteins accumulate in longitudinal connectives, strongly suggesting that they act as a physical and functional unit during embryogenesis. In summary, the WAVE/SCAR complex is conserved in Drosophila and its members accumulate in axons of the nervous system (Schenck, 2004).
The mammalian WAVE/SCAR complex has been shown to be an integral part of Rac1 GTPase signaling pathways that coordinate actin cytoskeleton remodeling. Although mutations in single components call for a role of these proteins in construction of the nervous system, little is known about regulation and function of the WAVE/SCAR complex in this tissue. Evidence is provided that SCAR, CYFIP and Kette, which co-localise during embryogenesis, are submitted to interdependent, posttranscriptional, control. Moreover, the WAVE/SCAR complex acts as a functional unit coordinating different aspects of axonal and synapse development, revealing its role in core signaling pathways underlying neuronal connectivity (Schenck, 2004).
The analysis of CYFIP, SCAR and Kette mutant phenotypes and their genetic interaction with dFMR1 call for distinct pathways being triggered by the WAVE/SCAR unit. Better understanding of specific contribution requires a more complete knowledge on these signaling pathways. First conclusions, however, can be drawn. Guidance of embryonic central axons is, for example, affected in WAVE/SCAR complex but not in dFMR1 mutants and is hence controlled by dFMR1-independent pathways downstream of the WAVE/SCAR complex. In fact, central axons may be under control of the SCAR-Arp2/3 pathway, because mutations in different subunits of the Arp2/3 complex result in disruption of these axon tracts (Schenck, 2004).
In contrast, dFMR1 as well as WAVE/SCAR complex mutants affect NMJ morphology, suggesting a role of one or more complex components in this process. Indeed, overexpressed CYFIP rescues the dFMR1 gain of function phenotype, while overexpressed Kette and SCAR do not. This indicates that only CYFIP can signal to dFMR1 and suggests that the Kette and SCAR synaptic phenotypes are indirect consequences of CYFIP protein degradation. The fact that nevertheless, CYFIP, Kette or SCAR mutations compensate for the dFMR1 loss of function phenotype further supports the view that the WAVE/SCAR complex acts as an integral unit. While these studies do not exclude a direct role of WAVE/SCAR-mediated Arp2/3-dependent actin nucleation in synapse morphology, they clearly highlight the importance of CYFIP signaling to dFMR1. Interestingly, it has been recently shown that WASP, the second actin nucleation promoting factor, as well as its interacting protein Nervous wreck, control NMJ morphology. WASP is also directly linked to the WAVE/SCAR complex by its interaction with the Abi protein, indicating that proper synapse morphology requires integration of several related signaling pathways. Understanding the molecular basis of neuronal connectivity clearly implies evaluation of the specific contribution and integration of Arp2/3 and Fragile X Mental Retardation Protein mediated pathways at the synapse (Schenck, 2004).
Recent studies on fly and vertebrate cell cultures have shown that overexpressed SCAR or WAVE2 in cells that are knocked down for other components of the complex fail to be recruited to the cell periphery and do not rescue cytoskeletal defects. Loss and gain of function data show that WAVE/SCAR complex function relies on the integrity of all its components and that not only SCAR, but also its partners require proper control of protein stability and localisation. Surprisingly, the overexpressed SCAR protein can still accumulate, at least in part, at central axons, whereas excess CYFIP and Kette proteins cannot, suggesting the possibility that SCAR is directly connected to the translocation machinery responsible for axonal recruitment of the WAVE/SCAR complex (Schenck, 2004).
The observation that even upon simultaneous overexpression in pairwise or triple combinations SCAR is found in axons whereas excess CYFIP and Kette are not, is explained by the recent finding that CYFIP and Kette do not bind SCAR directly and must hence fail to travel piggybaggy with SCAR (Schenck, 2004).
Even properly localised excess of SCAR, however, is not capable of inducing an aberrant phenotype. Localisation of SCAR is hence a prerequisite but not sufficient to activate Arp2/3-dependent changes in the actin cytoskeleton, calling for an additional level of SCAR activity control. Whether this control occurs through phosphorylation, as in the case of the WAVE/SCAR related protein WASP and as suggested by the doublet revealed by anti-SCAR in immunoblotting, remains to be determined (Schenck, 2004).
Direct comparison of axonal and synaptic phenotypes displayed by CYFIP, Kette and SCAR mutant alleles has revealed that they are undistinguishable, a finding that suggested a common pathogenic mechanism. Indeed, in the developing nervous system, not only SCAR is subjected to protein turnover if either CYFIP or Kette are missing, as predictable from studies in cellular systems, but also CYFIP and Kette are lost if one of their partners is absent. These results demonstrate for the first time that not only SCAR levels are regulated by CYFIP and Kette dose, but also CYFIP and Kette levels depend on the dose of their protein partners. Thus, instead of being considered as single mutants, CYFIP, Kette and SCAR mutants have unambiguously to be judged as defective in multiple components of the WAVE/SCAR complex. This common biochemical basis (i.e., lack of all three proteins) clearly accounts for the identical observed phenotypes in the loss of function conditions, regardless of any effect these proteins may exert on each other in this tissue (Schenck, 2004).
An important question that has remained so far unanswered by studies on the WAVE/SCAR complex is why WAVE/SCAR requires four associated proteins to transduce Rac1 signaling to the Arp2/3 complex, whereas the WAVE/SCAR-related protein WASP is capable of doing this job on its own. It is speculated that the hetreopentameric WAVE/SCAR complex constitutes a checkpoint for a multitude of signaling pathways, which ensures their simultaneous activation. Several hints exist now in the literature indicating additional functions of Kette, Abi and CYFIP proteins. Whereas the functional significance of Kette interaction with signaling proteins like dynamin and Eps8 and Abi interaction with the Abl nonreceptor tyrosin kinase remain to be validated, this work has delineated a first pathway specific to one of the WAVE/SCAR-associated proteins, CYFIP signaling to dFMR1 (Schenck, 2004).
These data show that integrity of the WAVE/SCAR complex plays a pivotal function in nervous system development and that CYFIP and Kette do not simply function as SCAR silencers or proteins merely stabilising/localizing SCAR. This is of particular interest if one considers that a series of genes connected to the WAVE/SCAR complex and its associated signaling pathways are implicated in human mental retardation. First, several mutations directly affecting Rho/Rac regulatory or effector proteins cause X-linked mental retardation. Moreover, the most frequent cause of hereditary mental retardation is due to mutations in the Fragile X Mental Retardation gene, which is connected to Rac1 via CYFIP and thereby to the WAVE/SCAR complex. Finally, MEGAP (mental disorder-associated GAP protein), also known as WRP or srGAP3, encoded by one of the few so far identified autosomal mental retardation genes, is directly linked to the WAVE/SCAR complex. Indeed, MEGAP/WRP/srGAP3 is a negative regulator of the Rac1 GTPase and binds directly to WAVE1, suggesting that the protein terminates Rac1 signaling to the complex. The WAVE/SCAR complex is thus central to signaling pathways mutated in impaired conditions of neuronal functioning (Schenck, 2004).
In light of the data obtained in fly, one can speculate that also (some of) the different human genetic conditions mentioned above may have a common biochemical basis. If it can be formally proven that, analogous to flies, also the recently reported WAVE1 knockout mouse, notably characterised by cognitive deficits, is devoid of CYFIP and Kette proteins, this would provide the first direct evidence for the implication of this complex not only in neuronal connectivity but also in cognitive function (Schenck, 2004).
To examine SCAR protein expression and subcellular localization, a polyclonal antibody to the unique SCAR NH2-terminal domain was generated. SCAR protein was found to be present in early blastoderm embryos and in the embryonic CNS consistent with its mRNA expression. In the blastoderm, SCAR protein colocalizes with filamentous actin structures that are dynamically regulated during the cell cycle. In the CNS, SCAR protein is specifically localized to axons. This pattern of SCAR protein expression in the embryo provides an initial indication of the potential sites of SCAR gene activity (Zallen, 2002).
To investigate SCAR function, two mutant alleles of SCAR were identified and characterized. The recessive lethal P-element insertion mutation l(2)k13811 lies within the 5' UTR of the SCAR transcript, 208 nucleotides upstream of the translation start codon. Chromosomes bearing precise excisions of this insertion complement the lethality of l(2)k13811 and are homozygous viable. In addition, ubiquitous expression of a full-length SCAR cDNA rescues the lethality of the l(2)k13811 insertion. These data demonstrate that the zygotic recessive lethality is due to disruption of the SCAR gene by the l(2)k13811 insertion, and this allele is referred as SCARk13811. Moreover, embryos that are maternally and zygotically mutant for the SCARk13811 allele display a strong reduction in staining with the anti-SCAR antibody, confirming that this insertion disrupts SCAR expression (Zallen, 2002).
To obtain deletions in the SCAR locus, imprecise excision alleles of the SCARk13811 insertion were generated, all of which are homozygous lethal and fail to complement the lethality of SCARk13811. The homozygous lethality of the Delta37 excision allele was rescued by ubiquitous expression of the full-length SCAR cDNA, and this allele is referred to as SCARDelta37. SCARDelta37 was molecularly characterized and was found to remove all SCAR sequences downstream of the insertion site. This excision event also removed portions of the neighboring piwi transcription unit. Since piwi function is restricted to maintenance and proliferation of germline stem cells, the SCARDelta37 phenotypes described in this study, in distinct tissues, are likely to represent consequences of disrupting SCAR function alone. Developmental defects were considerably weaker in SCARk13811, indicating that the insertion allele retains partial SCAR activity (Zallen, 2002).
In addition to SCAR and WASp (Ben-Yaacov, 2001), the sequenced Drosophila genome contains predicted homologs of the seven members of the Arp2/3 complex. Mutations have been recovered in two Arp2/3 complex components, Arp3 and Arpc1. This set of mutations provides an opportunity to analyze the role of Arp2/3-based signaling in different contexts within a multicellular organism and to ascertain the physiological contributions of the SCAR and Wasp activators (Zallen, 2002 and references therein).
Homozygous mutations in either SCAR allele result in zygotic lethality, which can occur during late embryogenesis, larval, and early pupal stages. However, maternally provided SCAR gene products may compensate for loss of zygotic gene function and mask an essential requirement during embryogenesis. To interfere with the maternal gene contribution, FLP-mediated recombination was used to produce homozygous mutant clones within the germline of heterozygous females. Strong disruption of maternal SCAR or Arpc1 in this manner results in developmental arrest during oogenesis. However, germline clones homozygous for the weaker SCARk13811 or Arpc1R337st alleles give rise to fertilizable eggs, enabling study of functional requirements for SCAR and the Arp2/3 complex during embryogenesis. These embryos are designated SCARmat and Arpc1mat, respectively. To compare the roles of SCAR and Wasp, embryos derived from germline clones were examined for the strong loss of function WASp3 allele (WASpmat embryos). The WASp3 frameshift mutation truncates the protein before the highly conserved WA domain that is required for Arp2/3 activation and is a probable null allele (Ben-Yaacov, 2001; Zallen, 2002).
The early blastoderm embryo undergoes 13 nuclear divisions without accompanying cytokinesis, producing a multinucleate syncytium. The majority of nuclei migrate to the surface by cycle 10, where they undergo four synchronous rounds of division before their compartmentalization into individual cells during interphase of cycle 14. Surface nuclei maintain a uniform distribution throughout these final syncytial divisions. Examination of the spatial distribution of nuclei revealed a requirement for SCAR and Arpc1, but not Wasp, during these cortical division cycles. SCARmat and Arpc1mat mutants exhibit defects in the uniform spacing of interphase nuclei beginning in cycle 11, whereas WASpmat mutants displayed wild-type nuclear organization. By cycles 12 and 13, increased defects in nuclear spacing in SCAR and Arpc1 were accompanied by the appearance of abnormal nuclear morphologies, including large or elongate DNA masses that are likely to represent the fusion of adjacent nuclei (Zallen, 2002).
In wild-type syncytial embryos, nuclei are maintained in two separate populations: a uniform layer of surface nuclei and an internal mass of yolk nuclei. In SCARmat and Arpc1mat embryos, displacement of surface nuclei into the interior was first observed at cycle 12 and increased in severity by cycle 14. The severity of these defects is strongly correlated with increased division cycles, demonstrating a late onset progressive defect. Nuclear displacement is rarely observed in wild-type and WASpmat embryos (Zallen, 2002).
The syncytial blastoderm contains well-defined filamentous actin structures that exhibit dynamic cell cycle regulation. Actin is organized into caps overlying individual nuclei during interphase of cortical cycles 10-14. During mitosis, actin is redistributed into a network of metaphase furrows that separate adjacent spindles. Genetic and drug interference studies demonstrate that organization of the actin cytoskeleton is crucial for the uniform arrangement of blastoderm nuclei. The nuclear defects in SCAR mutants, and SCAR protein colocalization with filamentous actin, raise the possibility that SCAR may function in the regulation of actin structures in the blastoderm embryo (Zallen, 2002).
In SCARmat embryos, actin caps appeared largely intact during interphase, although some defects were observed. In particular, SCARmat actin caps are consistently smaller and less rounded than in wild type. In a subset of embryos, primarily in later syncytial divisions 12 and 13, actin caps appear less discrete and gaps are observed in regions where nuclei are abnormally clustered. In contrast to the relatively mild defects in interphase caps, metaphase furrows are completely absent in the majority of SCARmat embryos undergoing mitosis, and actin accumulates in aberrant structures positioned above rather than between individual spindles. A partial and discontinuous metaphase furrow network is observed in a minority of SCARmat embryos. These two mitotic phenotypes are mutually exclusive and consistent across the entire embryo surface. These observations suggest an abnormal reorganization of interphase actin as SCARmat embryos enter mitosis, indicating that the transition between interphase caps and metaphase furrows requires SCAR function (Zallen, 2002).
Major defects in cortical actin structures were also observed in Arpc1mat embryos, where interphase actin caps are abnormal and actin appears to be depleted from the regions above individual nuclei. This depletion is apparent most readily in cross-section. As in SCAR, metaphase furrows fail to form in Arpc1mat embryos. However, unlike SCAR, metaphase actin exhibits a diffuse localization to the broad region between spindles. These results demonstrate that the Arp2/3 complex component Arpc1 is required for the formation of both interphase actin caps and metaphase actin furrows. The greater severity of the Arpc1 phenotype compared with SCAR could reflect a difference in residual gene activity of these partial loss of function alleles (Zallen, 2002).
SCAR and Arpc1, therefore, provide functions that are critical for proper formation of cortical actin structures. In contrast, actin caps and furrows formed normally in WASpmat embryos. It is worth emphasizing in this context that both the SCAR and Arpc1 phenotypes result from a partial loss of gene function, whereas early embryogenesis can proceed normally despite complete lack of WASp gene activity (Zallen, 2002).
In addition to defects in organization of microfilament structures, overall actin levels in Arpc1mat and SCARmat embryos appear consistently lower than in wild type. To rigorously assess differences in actin levels, surface filamentous actin was quantitated in syncytial embryos using a phalloidin fluorescence assay. Both Arpc1mat and SCARmat mutants were found to exhibit significantly reduced levels of surface actin compared with control embryos. The more severe loss of actin in Arpc1mat correlates with the greater disruption of actin structures in this mutant. These results indicate that Arpc1 and SCAR are both required for the generation of bulk filamentous actin in the blastoderm and suggest a common basis for the defects in cortical actin structures of Arpc1 and SCAR mutant embryos (Zallen, 2002).
The striking enrichment of SCAR expression in the CNS prompted an examination of CNS morphology in SCAR mutants using the axon-specific BP102 monoclonal antibody. In wild-type embryos, CNS axons travel in two longitudinal bundles that flank the midline and two commissural bundles that cross the midline in each segment. Although no apparent defects were observed in homozygous SCAR embryos, maternal contribution of SCAR transcript or protein may provide sufficient wild-type SCAR activity to mask a functional requirement in the CNS. Partial maternal SCAR function provided by the weak SCARk13811 allele at lower temperature (20–22°C rather than 25°C) is sufficient to produce embryos that develop normally through the blastoderm stages, allowing an examination of later CNS development (Zallen, 2002).
Reduction of SCAR function achieved in this manner indeed causes dramatic CNS defects. The phenotypes observed in these mutants (designated SCARmat/zyg embryos) require disruption of zygotic SCAR function. In SCARmat/zyg embryos, frequent breaks occur in longitudinal and commissural bundles. In extreme cases, a severe depletion of all axon bundles is observed. At a lower frequency, SCARmat/zyg embryos exhibit defects in commissure fasciculation and separation (18% of segments), and medial (13%) or lateral (9%) displacement of axons (Zallen, 2002).
Since SCAR is essential for normal CNS axon morphology, the zygotic effect of mutations in two members of the Arp2/3 complex, Arp3 and Arpc1, was also examined. Arp3 zygotic mutant embryos exhibit a partially penetrant defect in CNS axon morphology with a range of phenotypes that strongly resemble SCARmat/zyg mutants. In particular, the majority of Arp3 mutants display breaks in the longitudinal and commissural axon bundles. A subset of Arp3 mutants exhibit defects such as commissure defasciculation or fusion and medially or laterally displaced axons. Arp3 heterozygotes also exhibit a low penetrance of axon defects. The CNS morphology of zygotic Arpc1 single mutants appear normal, perhaps due to the continued presence of maternal gene products. However, combining zygotic Arpc1 mutations with an Arp3 heterozygous background produces defects that are significantly more severe than in Arp3 heterozygotes alone. These phenotypes demonstrate a similar functional requirement for SCAR and Arp2/3 complex components during CNS development (Zallen, 2002).
The contribution of Wasp function to CNS axon morphology is more difficult to assess, since complete removal of maternal and zygotic WASp using the strong WASp3 allele (Waspmat/zyg embryos) produces cell fate defects in CNS lineages (Ben-Yaacov, 2001). An apparent thickening of commissural bundles suggestive of an increase in neuronal number is observed in a majority of WASpmat/zyg embryos. In addition, most WASpmat/zyg embryos contain one to two segments with axon bundles collapsed at the midline. Despite these phenotypes, WASpmat/zyg embryos does not exhibit the severe defects in axon morphology present in SCAR and Arp3 mutants. Although removal of zygotic SCAR or WASp function alone does not disrupt CNS morphology, zygotic reduction of SCAR and WASp together produces significant defects that resemble the strong SCARmat/zyg phenotype. Therefore, although loss of WASp function alone does not cause the significant axon defects produced by loss of SCAR, WASp can influence axon morphology in situations where SCAR function is compromised (Zallen, 2002).
Although the partial reduction of SCAR function associated with the SCARk13811 insertion allele is sufficient for normal egg production, the more severe SCARDelta37 excision allele produces small and abnormally shaped eggs indicative of a defect in oogenesis. Drosophila ovaries house a series of egg chambers that each contain 16 cells (the oocyte and a 15-cell nurse cell complex) interconnected by cytoplasmic bridges (ring canals) that arise from incomplete cytokinesis during mitosis. Morphological defects are apparent in SCARDelta37 germline clones during the final phases of oogenesis. In particular, nurse cells become multinucleate, since many of the actin-lined nurse cell membranes are absent. The morphological abnormalities extend to additional structures, including the actin-rich ring canals, which are significantly smaller than in wild-type and aberrantly shaped (Zallen, 2002).
The defects observed in SCAR mutant egg chambers closely resemble phenotypes described in mutants for the Arp2/3 complex subunits Arpc1 and Arp3 (Hudson, 2002), resulting in late stage deterioration of the nurse cell complex. In marked contrast to the Arpc1, Arp3, and SCAR phenotypes, oogenesis in germline clones for the strong loss of function WASp3 allele appears wild type. No apparent morphological abnormalities were observed in WASp3 late stage egg chambers, which can support embryonic development after fertilization (Ben-Yaacov, 2001). This phenotypic analysis indicates that SCAR, rather than WASp, is the major mediator of Arp2/3 function during Drosophila oogenesis, much as was observed in the blastoderm and the embryonic CNS (Zallen, 2002).
The above phenotypic analyses identify several Arp2/3-dependent morphological processes that rely on SCAR but are largely independent of Wasp. Does the reciprocal situation exist -- namely, are there Arp2/3-mediated events that rely on Wasp but are independent of SCAR? WASp provides an essential contribution to cell fate decisions in several neural lineages in the Drosophila embryo and adult (Ben-Yaacov, 2001). Furthermore, the Arp2/3 complex component Arpc1 is required for Wasp-dependent cell fate changes during sensory organ development, and association with Arp2/3 is essential for WASp function in this context. This requirement provides an opportunity to examine whether developmental events dependent on Wasp also require SCAR function (Zallen, 2002).
In the adult peripheral nervous system, a primary consequence of mutations in WASp is the excessive differentiation of sensory organ neurons at the expense of nonneuronal cell types, resulting in a marked absence of mechanosensory bristles. The ey-FLP-FRT system to generate mosaic SCAR and Arpc1 heterozygous flies in which head capsule structures and cuticle are derived from homozygous mutant clones induced in the eye imaginal disc. Arpc1 mosaic heads, like WASp mosaic heads, display a pronounced bristle loss phenotype, which results from cell fate defects similar to those present in WASp mutants. In contrast, the sensory organ pattern in mosaic heads of strong SCAR alleles appears wild type, suggesting that SCAR does not play an essential role in lineage decisions mediated by Wasp and the Arp2/3 complex (Zallen, 2002).
In addition to loss of sensory organ structures, Arpc1 mosaics display abnormalities in eye structure, including a reduction in the overall size of the eye, irregularly shaped ommatidia, and a distinct loss of lens material in most eye facets. Mosaics for the SCARDelta37 excision allele present a very similar eye phenotype, with the exception that interommatidial bristles are largely present. As noted previously (Ben-Yaacov, 2001), mutations in WASp have no discernible effect on eye morphology apart from the bristle loss phenotype. This analysis provides a striking example of the distinct requirements for SCAR and WASp, which mediate separate aspects of Arp2/3 complex function during adult development (Zallen, 2002).
The protrusion of two distinct actin-containing organelles, lamellipodia and filopodia, is thought to be regulated by two parallel pathways: from Rac1 through Scar/WAVEs to lamellipodia, and from Cdc42 through N-WASP to filopodia. This hypothesis was tested in Drosophila, which contains a single gene for each of the WASP subfamilies, SCAR and WASp. Targeted depletion of SCAR or WASp by dsRNA-mediated interference was performed in two Drosophila cultured cell lines expressing lamellipodial and filopodial protrusion. Knockdown was verified by laser capture microdissection and RT-PCR, as well as Western blotting. Morphometrical, kinetic and electron microscopy analyses of the SCAR-depleted phenotype in both cell types revealed strong inhibition of lamellipodial formation and cell spreading, as expected. More importantly, filopodia formation was also strongly inhibited; this is not consistent with the parallel pathway hypothesis. By contrast, depletion of WASp did not produce any significant phenotype, except for a slight inhibition of spreading, showing that both lamellipodia and filopodia in Drosophila cells are regulated predominantly by SCAR. A new, cascade pathway model of filopodia regulation is proposed in which SCAR signals to lamellipodia and then filopodia arise from lamellipodia in response to additional signal(s) (Biyasheva, 2004).
Depletion of SCAR from S2R+ cells is correlated with the development of a strong, penetrant and highly reproducible phenotype, which reflected the declining ability of cells to form lamellipodia and spread over the substratum. Although some heterogeneity inevitably accompanies the development of the phenotype (probably due to uneven dsRNA uptake), the progression clearly occurs from the appearance of slight irregularity of the lamellipodial outline to the formation of long narrow processes to virtual failure of a cell to spread (Biyasheva, 2004).
The formation of linear processes reminiscent of filopodia during SCAR depletion could have resulted from the undepleted activity of WASp leading to the induction of filopodia, as is predicted by the model of two parallel signaling pathways. However, a detailed analysis of these aberrant processes showed that they are more like extremely narrowed residual lamellipodia than filopodia. The curved shape, uneven thickness and kinetic behavior of these processes are distinct from those seen during normal filopodial protrusion. Structurally, the processes of SCAR-depleted cells do not contain bundles of long parallel filaments that are characteristic of filopodia. Instead, they are filled with a sparse dendritic network like that seen in lamellipodia. The weak presence of the lamelipodial marker Arp3 and absence of the filopodial marker Ena in these processes also suggests that they are not filopodia. Thus, the depletion of SCAR in S2R+ cells does not induce filopodia, but produces narrow aberrant processes, probably by a combination of residual SCAR activity and retraction elsewhere along the cell perimeter. These findings in Drosophila cells are in agreement with the previous work in Dictyostelium, where SCAR-deficient cells manifested abnormal cell size and morphology, and instead of normal broad lamellipodia formed 'pseudopodia-like extensions' depleted of F-actin (Biyasheva, 2004).
The formation of narrow lamellipodia following depletion of SCAR provides an additional insight into the function of SCAR. Scar/WAVEs in mammalian and Drosophila cells are localized to the extreme edge of protruding lamellipodia. Possibly, SCAR acts cooperatively in the process of protrusion. That is, protrusion at the leading edge may be more probable at sites of a pre-existing protrusion. A consequence of SCAR depletion would be that protrusion would become progressively restricted to smaller domains (Biyasheva, 2004). In contrast to the striking effect of SCAR depletion, WASp depletion did not generate a phenotype in S2R+ cells aside from a minor, albeit statistically significant, reduction in the cell projected area. One possible explanation for the lack of a significant phenotype might be the incomplete knockdown of WASp, only 75% depletion of WASp was achieved, as compared with greater than 95% depletion of SCAR. However, considering that several diverse assays were used for phenotypic analysis, it is believed that if WASp made a significant contribution to protrusive behavior, a phenotype would have been detectable (Biyasheva, 2004).
Taken together, the results using S2R+ cells indicate that SCAR is the major regulator of protrusive activity in these cells. It is necessary for the assembly of the actin dendritic network, formation of lamellipodia and cell spreading onto the substratum. By contrast, WASp makes little, if any, contribution to actin-based protrusion in these cells (Biyasheva, 2004).
The lack of filopodia formation in S2R+ cells after SCAR inhibition may be explained by an intrinsic deficiency of the filopodial machinery in these cells. Therefore, to test the role of SCAR in filopodial formation, BG2 cells were used -- these are, in addition to lamellipodia, very rich in filopodia, similar to other cells of neuronal origin (Biyasheva, 2004).
The parallel signaling hypothesis predicts that depletion of the lamellipodial regulator, SCAR, will not affect filopodia, which, according to the model, would be induced by WASp. By contrast, the cascade model predicts that SCAR depletion will inhibit not only lamellipodia, but also filopodia. The results support the latter prediction. It was confirmed that SCAR in BG2 cells, as in S2R+ cells, regulates lamellipodia. Indeed, depletion of SCAR in BG2 cells inhibits lamellipodia, leading to decreased spreading and altered shape similar to results obtained with S2R+ cells. By EM criteria, the lamellipodial dendritic network in BG2 cells was significantly reduced; only a few small patches of the dendritic network remained at some cell edges (Biyasheva, 2004).
The conclusive evidence helped in distinguishing between two alternative models of regulation expected from effects of SCAR depletion on filopodia formation. The results clearly show that SCAR RNAi in BG2 cells inhibits not only lamellipodia, but also filopodia. Although light microscopic evaluation of static cultures per se gave an impression of filopodia sustaining in SCAR-depleted BG2 cells, more detailed analyses, namely, EM structural assay and a quantitative kinetic analysis of filopodial initiation during cell spreading, revealed that filopodia formation, in fact, was strongly inhibited. These results allowed the parallel pathway model to be ruled out and validate the cascade pathway model for actin-based protrusions in Drosophila cell lines (Biyasheva, 2004).
In contrast to SCAR, depletion of WASp in BG2 cells did not result in any changes in cell morphology, kinetics of protrusion or cytoskeletal architecture except for a slight inhibition of spreading, similar to what was observed in S2R+ cells. BG2 cells after WASp depletion retained not only lamellipodia, but also numerous perfectly looking filopodia indistinguishable from those in control cells. These results show that WASp does not play a distinctive role for filopodia formation, thus further corroborating the priority of a cascade pathway model over the parallel pathway model (Biyasheva, 2004).
Regulation of lamellipodia and filopodia The requirement of SCAR activity in Drosophila cells for both lamellipodial and filopodial protrusion is fully consistent with the idea that filopodial initiation in motile cells occurs downstream of lamellipodial protrusion, as suggested by previous studies. Therefore, a model stipulating a two-step cascade regulation of filopodial protrusion in Drosophila cells is proposed. SCAR activates the Arp2/3 complex which then induces assembly of the dendritic network in lamellipodia. The lamellipodium by itself represents an important part of the protrusive machinery, but it is suggested that the lamellipodium also provides a foundation for subsequent reorganization into filopodial bundles. Such reorganization would be dependent on additional as-yet-unspecified signals. Indirect evidence in support of the cascade regulation model comes from in vivo studies of Drosophila. In mutations removing all three Drosophila isoforms of Rac, both lamellipodia and filopodia are abolished during dorsal closure. Such a result would not be predicted if Rac signaled only to lamellipodia and a parallel pathway existed for filopodia (Biyasheva, 2004).
In general, these results and interpretations of the roles of SCAR and WASp at the cellular level are consistent with conclusions drawn from studies on Drosophila development in vivo: SCAR mutations result in more severe phenotypes and affect many structures, whereas mutations in WASp had very little phenotype. SCAR, rather than WASp, is the major mediator of Arp2/3 function during Drosophila oogenesis and in the development of the central nervous system. SCAR and Arp2/3 are essential for proper spatial distribution of nuclei during cortical divisions, as well as for metaphase furrow formation and ring canal formation. Overall actin levels are lower in SCAR and Arp2/3 mutant cells. By contrast, WASp function seems to be restricted to specific processes in development, such as the proper execution of asymmetrical cell divisions in neural lineages or in bristle formation in ommatidia. Nevertheless, WASp performs its role via the Arp2/3 complex. Although WASp does not seem to contribute to protrusive activity in the Drosophila cell lines tested in these experiments, consideration of mutant developmental phenotypes suggests that WASp can activate the Arp2/3 complex in certain cellular contexts and induce downsteam events that are dependent on dendritic nucleation of actin filaments (Biyasheva, 2004).
If WASP subfamily members are not essential for filopodia formation, then what are the signaling molecules for the second step of the postulated cascade pathway? A key element driving network reorganization into filopodial bundles appears to be a multiprotein complex at filopodial tips. It is proposed that the tip complex is a site at which the second step of the cascade signaling is directed. In favor of this hypothesis, IRSp53, an effector of Cdc42, induces filopodia by recruiting Mena, a protein that localizes to the filopodial tips and promotes the elongation of actin filaments by protecting them from capping. Also, a formin family protein Drf3 has recently been shown to be a downstream effector of Cdc42; it is localized to filopodial tips and plays a role in filopodia formation. The IRSp53 or Drf3 pathway or an as-yet-unidentified pathway, rather than N-WASP, may be responsible for Cdc42 signaling to filopodia. Irrespective of the specific molecules involved in the signaling pathway, the structural-kinetic analyses suggest that they share the property of targeting the filopodial tip complex to drive filopodial formation (Biyasheva, 2004).
During the cell cycle, the Golgi, like other organelles, has to be duplicated in mass and number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. This study shows that in Drosophila S2 cells, the Golgi likely duplicates in mass to form a paired structure during G1/S phase and remains so until G2 when the two stacks separate, ready for entry into mitosis. Pairing requires an intact actin cytoskeleton which in turn depends on Abi/Scar but not WASP. This actin-dependent pairing is not limited to flies but also occurs in mammalian cells. It is further shown that preventing the Golgi stack separation at G2 blocks entry into mitosis, suggesting that this paired organization is part of the mitotic checkpoint, similar to what has been proposed in mammalian cells (Kondylis, 2007).
During the cell cycle, the Golgi, like other organelles, has to duplicate in mass and/or number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. The process of Golgi duplication and inheritance in mammalian cells is still debated, as different modes of Golgi biogenesis have been proposed. One reason why this issue is not yet settled could be due to the elaborate organization of the Golgi stacks, which are interconnected to form a single-copy organelle capping the nucleus, thus impeding clear visualization of organelle duplication and segregation. Therefore, this study has exploited the relatively small number of Golgi stacks in Drosophila tissue-cultured S2 cells to revisit this issue (Kondylis, 2007).
In S2 cells, the Golgi stacks are found in close proximity to transitional endoplasmic reticulum (tER) sites, forming tER-Golgi units (Kondylis, 2003; Herpers, 2004). Their number nearly doubles at G2 phase. In an effort to identify factors mediating this process, focus was placed on cytoskeletal elements that have been involved in the organization of the mammalian Golgi apparatus. Microtubules are involved in mammalian Golgi ribbon maintenance, as their depolymerization leads to its reorganization into individual Golgi stacks in close proximity to ER exit sites (Kondylis, 2007 and references therein).
F-actin has also been implicated in the organization of the mammalian Golgi apparatus; its depolymerization leads to a compact appearance of this organelle without disruption of cisternal stacking. A key regulator of actin polymerization is the Arp2/3 complex. Its F-actin nucleation activity is triggered both by Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous (WAVE/Scar) proteins, which are in turn regulated by Rho small GTPases. WASP exists in an autoinhibited state that is released by the cooperative action of Cdc42, PI(4,5)P, and other SH3-containing proteins. In contrast, WAVE/Scar proteins, together with Sra-1, Kette (Nap1), Abi, and HSPC300, form a stable complex, which is itself regulated by Rac (Kondylis, 2007 and references therein).
Rho GTPases have recently been implicated in maintaining Golgi architecture. Cdc42 has been localized on the Golgi membrane and shown to recruit the Arp2/3 complex around this organelle via ARHGAP10. Furthermore, suppression of the brain-specific Rho-binding protein Citron-N in neurons was shown to lead to fragmentation of the Golgi apparatus, and Rho1 was proposed to exert its local effect on F-actin by regulating ROCK and profilin activity (Kondylis, 2007 and references therin).
This study shows that drug-induced F-actin depolymerization in S2 cells leads to doubling of the number of tER-Golgi units independent of anterograde transport. Using live cell imaging, electron microscopy, and three-dimensional (3D) electron tomography, this study shows that each Golgi is organized as a pair of stacks held together by an actin-based mechanism, both in Drosophila and in human cells. In S2 cells, this is mediated by Abi and Scar, suggesting a novel role for the Rac signaling cascade in Golgi architecture. Last, it was shown that the Golgi stacks undergo separation at G2 through modulation of Abi and Scar, and that blocking this separation prevents cells from entering mitosis, supporting the existence of a G2/M checkpoint related to Golgi structural organization (Kondylis, 2007).
The two Golgi stacks could be physically linked without displaying membrane continuity or being interconnected, for instance through intercisternal tubular connections, either permanent or transient. Tubules connecting cisternae of adjacent stacks are involved in the formation of the Golgi ribbon in mammalian cells and, recently, GM130 and GRASP65 have been proposed to be required for their integrity. However, the putative tubules connecting the two stacks in the pair would have different molecular requirements, at least in Drosophila, since depletion of dGM130 or dGRASP does not lead to their separation (Kondylis, 2003; Kondylis, 2005; Kondylis, 2007 and references therein).
F-actin could provide a physical link holding the paired Golgi stacks together, or it could help in the formation/maintenance of intercisternal tubules. In addition, short actin filaments have been proposed to link spectrin mosaics leading to the formation of a skeleton that surrounds the Golgi complex. One of its functions could be to hold the two Golgi stacks close enough to allow the formation and fusion of the tubules. It could also surround the tubules themselves, thus providing membrane stability. The localization of Abi and Scar at the periphery of the tER-Golgi units and between the two stacks in a pair is consistent with both proposed functions. These tomography studies so far have not revealed clear membrane continuities between Golgi cisternae, though examples have been found of a tubular network which is shared by the paired stacks (Kondylis, 2007).
tER sites behave similarly to the Golgi, as they also separate at G2 and upon F-actin depolymerization. Because little is known about the mechanism regulating the biogenesis of tER sites, it is difficult to envisage how the two parts could be held together. The spectrin-actin mesh described above could be common to Golgi and tER sites, and Golgi and tER site scission could be achieved in a synchronized fashion. Alternatively, either of these organelles could split first and lead to the scission of the other, perhaps by providing positional information. Recently, the centrosome component centrin 2 that is also localized to tER sites in Trypanosoma has been shown to give such positioning information. A more in-depth study combining immunogold labeling and 3D tomography would be required to elucidate such fine details of tER-Golgi structural organization (Kondylis, 2007).
Drosophila Rho1 is unlikely to have a role in holding the two Golgi stacks together. The overexpression of the Rho1 constitutively inactive mutant or treatment of S2 cells with ROCK or myosin light chain inhibitors (Y27632 and blebbistatin) did not affect the Golgi number. Cdc42 is also unlikely to participate as the depletion of its downstream effector WASP did not lead to Golgi separation, although the overexpression of the Cdc42T17N dominant negative did. However, this effect could be due to nonspecific sequestration of the guanine nucleotide exchange factor involved in maintaining the paired Golgi stacks and may be shared with other small GTPases (Kondylis, 2007).
Interestingly, the results are consistent with a role for Rac GTPases in Drosophila Golgi architecture. Expression of the constitutively inactive form of Rac1 led to a near-doubling in the Golgi number, and depletion of Scar/WAVE or Abi, which are regulated by Rac GTPases, led to a similar phenotype. The identical results obtained in Scar and Abi RNAi suggest that this well-established Scar/WAVE pentameric complex is involved in holding the paired Golgi stacks together by promoting F-actin polymerization. These data indicate that the Rac signaling pathway is involved. However, the Scar/Abi complex has recently been shown to also stimulate Arp2/3 and F-actin polymerization independently of Rac. This would need to be investigated further (Kondylis, 2007).
This study shows that the separation of the paired Golgi stacks occurs at G2, prior to mitosis. A similar phenomenon has already been reported during cell division in Toxoplasma gondii, where a single Golgi stack grows as a duplicated organelle and is separated as the cell divides. However, the mechanism underlying this separation is not known (Kondylis, 2007).
The Golgi doubling in number at G2 phase resembles many aspects of this observed upon F-actin depolymerization. In both cases, a similar increase in Golgi number and decrease in their size are observed. Furthermore, this study has shown that it is the modulation of the F-actin cytoskeleton and the activity of Abi/Scar at G2 that lead to Golgi stack separation. (1) It was found that both Scar and Abi localized to the Golgi, strongly arguing for having a role in actin remodeling around this organelle. (2) The Golgi stacks in G2 cells remain insensitive to F-actin depolymerization. (3) Cells depleted of Abi and Scar that exhibit separated Golgi stacks do not split them further at G2. (4) The overexpression of Abi prevents Golgi separation at G2. This strongly suggests that the F-actin/Abi/Scar-mediated link of the two stacks has been severed in a G2-specific manner, perhaps by kinases such as Polo (Kondylis, 2007).
Because tER sites and the Golgi apparatus ultimately disperse later in mitosis, both in mammalian and Drosophila S2 cells, the Golgi stack separation prior to dispersion might be part of the proposed Golgi G2/M checkpoint. Indeed, reagents that interfere with the GRASP65/55 phosphorylation by Polo and ERK/MEK, respectively, arrest or delay the cell cycle at the G2/M transition. This study shows that blocking Golgi separation at G2 by overexpressing Abi also prevents S2 cells from entering mitosis. This strengthens the relationship between Golgi organization and mitotic entry, although it cannot formally be excluded that the mitotic block observed is partly due to additional effects of Abi overexpression, for instance at the plasma membrane (Kondylis, 2007).
It is proposed that at G2, the paired stacks are separated along with the adjacent tER sites. As the cell enters mitosis, the Golgi membrane and the tER sites disperse, and are segregated into the two daughter cells, where the tER-Golgi units are rebuilt. The Golgi could be rebuilt as a very small paired stack in close association with Scar, Abi, and F-actin, or as a single stack that will duplicate by a mechanism that still needs to be unraveled. Since G1 cells are all sensitive to F-actin depolymerization, this suggests that the formation of the paired Golgi stack starts just after the exit from mitosis and persists until S phase, when the Golgi seems to grow significantly. A more detailed understanding will come from EM study of S and G2 cells (Kondylis, 2007).
One of the remaining questions regards the impact of the Abi/Scar role on Golgi organization during development. Using Scar/WAVE, Abi, Kette, and Sra-1 mutants, as well as transgenic flies carrying inducible RNAi constructs, it will be possible to assess whether any of the observed phenotypes (defects in oogenesis, cell and organ morphology, neuroanatomical malformations, and failure in cell migration) is in part due to defects in Golgi organization (Kondylis, 2007).
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date revised: 10 April 2008
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