- Email: [email protected]
3 pathway: genetic insights
The WASP–Arp2/3 pathway: genetic insights Maria K Vartiainen1 and Laura M Machesky2 Arp2/3 complex nucleates the formation of dendritic actin filament arrays, which are especially prominent at the leading edges of motile cells. Recent genetic and other loss-of-function studies have highlighted the importance of the Arp2/3 complex for normal cell functions, and especially for cell motility. WASP/ Scar family proteins regulate the activity of the Arp2/3 complex, and also link it to several signaling pathways. Recent studies suggest that Scar is a more important regulator of Arp2/3 activity in actin-dependent morphological processes than WASP, which may have a more restricted role in specialized cellular events. It has also become clear that precise regulation of both Scar and WASP activity is of the utmost importance for their physiological functions. Addresses 1 Cancer Research UK, London Research Institute, Lincoln’s Inn Fields Laboratories, Transcription Laboratory, London, WC2A 3PX, UK 2 School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK e-mail: [email protected]
Current Opinion in Cell Biology 2004, 16:174–181 This review comes from a themed issue on Cell regulation Edited by Craig Montell and Peter Devreotes 0955-0674/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2004.02.004
Abbreviations Abi Abl interactor GBD GTPase binding domain RNAi RNA interference Scar suppressor of cAMP receptor VCA Verprolin-homology-connecting acidic domain WAS Wiskott-Aldrich syndrome WASP Wiskott-Aldrich syndrome protein WAVE WASP family verproline-homologous protein WH1 WASP homology domain 1
Introduction The coordinated assembly and disassembly of actin filaments drives several essential processes that are of crucial importance at both the single-cell and whole-organism level. For example, the chemotactic responses of an amoeba or neutrophil as well as the complicated cell rearrangements during embryogenesis are all based on cell motility and driven, at least in part, by polymerization of actin filaments at the leading edges of the cells [1]. Nucleation of new actin filaments is a key event in any cellular process relying on the dynamic assembly of actin filaments. Since the discovery of the Arp2/3 complex Current Opinion in Cell Biology 2004, 16:174–181
almost a decade ago [2], a broad range of biochemical and cell-biological studies have pointed to the pivotal role of this complex as a regulator of actin filament formation [1,3]. This review focuses on new insights revealed by recent genetic and other loss-of-function studies on the Arp2/3 complex and members of the Wiskott-Aldrich Syndrome protein (WASP)/suppressor of cAMP-receptor (Scar, also known as WASP family verprolin-homologous protein [WAVE]) families. The Arp2/3 complex is composed of two actin-related proteins Arp2 and Arp3, which presumably act as a template for new actin filaments, and five additional subunits, which in humans are called ARPC1–5 (the human nomenclature is used throughout this review) and which serve various structural and regulatory functions. Analysis of homology models from diverse species shows that the folds of the seven subunits, their core residues and the residues at subunit interfaces are well conserved during evolution. Solvent-exposed surfaces are less conserved, but conserved patches of surface residues are candidate sites for binding to nucleation-promoting factors and actin filaments [4], which allow the Arp2/3 complex to nucleate the formation of branched actin filament arrays in response to several signaling pathways. WASP/Scar family proteins are the most potent cellular nucleation promoting factors, and they share a common C-terminal Verprolin-homology-connecting acidic (VCA) domain that is responsible for Arp2/3 complex activation [3]. However, WASP and Scar/WAVE proteins differ both in the signaling inputs that they receive and in their modes of regulation (Figure 1). WASP proteins are autoinhibited: intramolecular interactions mask the VCAdomain in the absence of signaling molecules, such as PtdIns(4,5)P2 and Cdc42 [3]. On the other hand, mammalian Scar/WAVE1 has been shown to be kept inactive in a complex with four other proteins, PIR121/Sra-1, Nap125, HSPC300 and Abl interactor (Abi) 2. This complex can then be disassembled in response to certain signals to initiate Arp2/3-complex-mediated actin polymerization [5].
Arp2/3 complex is important for normal cell functions Genetic studies in yeast, Caenorhabditis elegans and Drosophila all point to the pivotal role of Arp2/3 complex for cell survival (Table 1) [6,7,8,9,10]. Concomitantly, depletion of the ARPC3 subunit by small interfering RNAs in mammalian cells has shown that this protein is essential for cell growth [11]. Consistent with the role of www.sciencedirect.com
The WASP–Arp2/3 pathway: genetic insights Vartiainen and Machesky 175
Figure 1
WASP WH1
B
GBD
ACV
Scar/WAVE P R O
Signals PI(4,5)P2 Rac1 Cdc42 Nck
Abi O Nap125 SHD B PIR121 HSPC 300
Abi PIR121 Nap125
WH1
B
GBD PRO
VCA
SHD
B
PRO
VCA
HSPC 300
Arp2/3-complex-mediated actin polymerization
Membrane trafficking (endocytosis, endosome motility) Specialized dendritic actin arrays (immunological synapses, podosomes)
Arp2/3-complex-mediated actin polymerization
Degradation of uncomplexed Scar
Lamellipodia
Filopodia
Current Opinion in Cell Biology
In the absence of stimulus, WASP is autoinhibited, whereas Scar/WAVE is kept inactive in a regulatory complex with four other proteins. WASP is activated by signaling molecules such as PI(4,5)P2 and Cdc42, which unmask the VCA-domain of WASP, which can then stimulate actin polymerization by the Arp2/3 complex. Scar/WAVE is activated by Rac and Nck, which disassemble the regulatory complex and allow Scar/WAVE to activate the Arp2/3 complex. Uncomplexed Scar/WAVE is also rapidly degraded. Actin polymerization stimulated by WASPs play an important role in membrane trafficking and in the formation of specialized dendritic actin arrays. On the other hand, Scar/WAVEs are important for actin polymerization in the lamellipodia and filopodia, a process which Cdc42 has also been implicated in. B, basic region; PRO, proline rich region; SHD, Scar homology domain.
Arp2/3 as a nucleator of actin filaments, the Arp2/3 mutants often have lower levels of actin filaments [7,9]. Some mutants also show formation of large actin filament aggregates in the absence of Arp2/3 [6,12]. Importantly, the Arp2/3 complex appears to be required for the assembly of only a subset of actin filament structures. In Drosophila, loss-of-function mutations in ARPC1 and Arp3 have shown that Arp2/3 complex is required for regulation of the dynamic actin structures in the blastoderm embryo [9] and for the expansion of ring canals, which allow the flow of cytoplasm from the nurse cell to the developing oocyte. However, the Arp2/3 complex is not required for the initial recruitment of actin filaments at ring canals, nor for the formation of parallel, hexagonally packed actin bundles in the nurse cell or bristles, although it contributes to their organization [10]. In C. elegans, depletion of Arp2/3 complex subunits by RNA interference (RNAi) suggests that the Arp2/3 complex is dispensable for the assembly of cortical actin in the early embryo and for the establishment of anterior–posterior polarity [7,8]. These results suggest that although the Arp2/3 complex does not participate in the nucleation of all actin filament structures in the cell, it may still influwww.sciencedirect.com
ence their organization, indicating that the balance between distinct actin filament assemblies is important for normal cell functions. Branched actin filament networks nucleated by the Arp2/3 are a hallmark of the lamellipodium at the leading edge of motile cells [13]. Accordingly, the Arp2/3 complex appears to be especially important for cell motility. In C. elegans, the Arp2/3 complex is crucial for cell movements after cell-fate decisions during morphogenesis. In Arp2/3-depleted embryos, certain precursor cells fail to ingress into the blastocoel, and therefore gastrulation fails. The Arp2/3 complex is also essential for ventral enclosure, which requires both cell migration and cell adhesion. Arp2/3 subunits appear to be among those few genes that specifically affect the migration part of this process [7,8]. In Drosophila, Arp2/3 complex is required for axon development, a process requiring extensive migration of the growth cones [9]. The role of the Arp2/3 complex in cytokinesis is controversial. Deletion of genes encoding Arp2/3 subunits in Saccharomyces cerevisiae [6] or depletion by RNAi in Current Opinion in Cell Biology 2004, 16:174–181
176 Cell regulation
Table 1 Phenotypes of Arp2/3 complex and WASP/Scar loss-of-function mutants in model organisms.
Saccharomyces cerevisiae, Schizosaccharomyces pombe
Arp2/3
WASP
Scar
References
Some subunits essential, conditional mutations result in defects in the cortical actin cytoskeleton.
One protein Las17p/Bee1p/ Wsp1; required for normal yeast growth, actin organization and endocytosis.
[6,34,51,52]
[12,19,20,21]
Deletion of other subunits causes variable defects in cell viability, growth and actin patches. Arabidopsis thaliana
Mutations in Arp2, Arp3 and ARPC5 result in aberrant cell shapes due to misdirected cell expansion.
Not characterized.
Dictyostelium discoideum
No mutants available.
No mutants available.
Disruption of the gene results in reduced actin levels, abnormal cell morphology during chemotaxis and defects in membrane trafficking.
[38,53]
Caenorhabditis elegans
RNAi results in embryonic arrest during morphogenesis, defects in cell migration during gastrulation and reduced F-actin levels.
RNAi results in several phenotypes including embryonic lethality, larval arrest and sterility, defects in hypodermal cell migration and reduced F-actin levels.
RNAi: no phenotype.
[7,8]
Drosophila melanogaster
All loss-of-function mutations lethal before adult age. Specifically required for cortical division, cytoplasmic organization in the blastoderm, ring canal expansion, axon development, egg chamber structure and adult eye morphology.
Loss-of-function mutations result in defects in Notch-mediated cell fate decisions.
Deletion in Scar locus or P-element insertion results in zygotic lethality, required for same functions as Arp2/3.
[9,10,14, 18,27,31]
RNAi of ARPC4 in cultured cells inhibit lamellipodium formation.
RNAi in cultured cells does not result in any significant phenotype.
RNAi in cells inhibit cell spreading, lamellipodia and filopodia formation.
RNAi of ARPC3 is lethal.
Knockout of WASP leads to various proliferative, morphological, and functional defects in hematopoietic cells.
Knockout of Scar1leads to post natal lethality and various problems in the nervous system; neurons have normal morphology.
Knockout of N-WASP is embryonically lethal at E12 and causes developmental delay, cardiac and neural tube defects, although cells have normal filopodia and lamellipodia
Knockout of Scar2 is lethal at E10-12.5 and causes growth retardation and several morphological defects especially in the cardiovascular system.
Mus musculus
[11,29,30,32, 54,46,47,48]
Cells are deficient in lamellipodia formation and motility in response to growth factors.
C. elegans [8] and Drosophila S2 cells [14] does not interfere with cytokinesis. However, recent studies in the fission yeast S. pombe have shown that the contractile ring is an active site of actin assembly, and that both the Current Opinion in Cell Biology 2004, 16:174–181
formin Cdc12 and the Arp2/3 complex are required for this actin polymerization activity [15]. In fact, Cdc15p, which interacts with both Cdc12 and Arp2/3 activator Myo1p, directly links these actin filament nucleators [16]. www.sciencedirect.com
The WASP–Arp2/3 pathway: genetic insights Vartiainen and Machesky 177
In Drosophila, Arp2/3 mutants have defects in the cortical division cycles in the blastoderm embryo, [9,17] and asymmetric cell divisions in the neuronal lineage [18]. Therefore, the requirement for the Arp2/3 complex in cytokinesis could be cell-type/organism-specific, and clearly requires further study. Rigid cell walls and large vacuoles dominate plant cells, and therefore the cytoskeleton plays different roles in these cells than in most animal cell types. Recent studies have isolated several mutants in actin-binding proteins that have defects in plant cell growth. Among these are the Arabidopsis thaliana CROOKED [19], WURM and DISTORTED [12,20], which encode Arp2/3 subunits ARPC5, Arp2 and Arp3, respectively. Mutations in these genes result in aberrant cell shapes in various cell types, including trichomes, hypocotyl cells and root hairs, due to misdirected cell expansion. Specifically, the generation of fine actin filaments that are often associated with regions of active growth and expansion appears to be impaired in Arp2/3 complex mutants. At the cellular level, the mutant cells often contain large actin filament aggregates, which may prevent vesicle fusion at the expanding sites [12,19,21]. The Arp2/3 complex mutant phenotype can be rescued with the human ortholog [12], and Distorted1 rescues an arp3 mutation in S. cerevisiae [20], indicating a functional conservation of Arp2/3-mediated actin polymerization in very different cellular contexts. Regulation of the Arp2/3 complex activity in plant cells remains to be established, as no plant protein containing a VCA region has been identified. Two uncharacterized Arabidopsis proteins (At4 g18600 and At1 g29170) are, however, predicted to contain a Scar homology domain (SHD) [22]. It is curious that the maize Brk1 gene, which has been shown to play a role in cell expansion, encodes a HSPC300 homolog [23]. Brk1 functions together with Brk2 and Brk3 in a common pathway required for epidermal cell morphogenesis and division [24]. Future studies will reveal the molecular nature of these additional Brk genes. It is tempting to speculate that they may encode a Scar/WAVE ortholog, a plant-specific Arp2/3 complex activator, or other members of the Scarregulatory complex.
WASP/Scar proteins: similar or distinct functions? Yeasts S. cerevisiae and S. pombe have only one WASP/Scar protein and Dictyostelium, C. elegans and Drosophila contain one WASP and one Scar, whereas mammals have five members: WASP, N-WASP, and Scar/WAVE 1–3 (Table 1). The S. cerevisiae WASP/Scar protein Las17p resembles WASP in its overall domain organization, having a Wasp homology 1 domain (WH1, see Figure 1) at its N-terminus. However, it has no obvious GTPase-binding domain (GBD) and does not interact directly with Cdc42. Moreover, recent studies have shown that the purified www.sciencedirect.com
Las17p is not autoinhibited, but two SH3-domain containing proteins, Sla1p and Bbc1p, can inhibit its activity [25]. Therefore, with respect to regulation, Las17p behaves more like Scar/Wave than WASP. The relative importance of the WASP/Scar family members in Arp2/3-dependent cellular functions appears to depend at least partly on the organism. In C. elegans, WASP is required for essentially the same processes as the Arp2/3 complex, including ventral enclosure [7]. Unfortunately, preliminary RNAi studies with Scar in this organism have yielded no phenotype [7]. However, the C. elegans gut on exterior (GEX) genes GEX-2 and GEX-3 encode homologues of PIR121 and Nap125, respectively. RNAi of Gex-2 or transposon insertion in Gex-3 gene results in embryonic lethality, because cells fail to become organized, and tissues are found in abnormal positions with aberrant morphology. Like the Arp2/3 complex, these genes are required for hypodermal cell migration, during both dorsal intercalation and ventral migration [26], suggesting that Scar may also play a role in these processes. In Drosophila, Scar and WASP have clearly distinct functions. Scar appears to be the main regulator of actindependent morphological processes mediated by Arp2/3, such as axon development, egg chamber structure and adult eye morphology [9]. On the other hand, WASP, but not Scar, is required specifically for cell-fate decisions mediated by Notch signaling [9,27]. In the absence of WASP there is excessive differentiation of sensory organ neurons at the expense of non-neuronal cell types, which results from a failure in the execution of asymmetric cell divisions in neural lineages [27]. This activity is Arp2/3-dependent, but interactions with Cdc42 and PtdIns(4,5)P2 are not required [18]. These studies also suggest that members of the WASP/Scar family are the most important physiological activators of the Arp2/3 complex at least in Drosophila, as the defects in the Arp2/3 mutants are a sum of the defects seen in WASP and Scar mutants. At the cellular level, the Arp2/3 complex is responsible for the nucleation of the lamellipodial actin network, which may be subsequently reorganized into filopodia [28]. It has been generally thought that two parallel pathways drive the formation of these structures: from Rac through Scar/WAVE to lamellipodia, and from Cdc42 through WASP to filopodia. There are, however, results that contradict this view. First, Scar is the main regulator of Arp2/3-dependent cellular processes in Drosophila as discussed above [9]. Second, NWASP-deficient mouse fibroblasts have normal filopodia [29,30]. Third, RNAi studies in cultured Drosophila cells have shown that depletion of WASP does not result in any significant phenotype, whereas depletion of Scar inhibits formation of both lamellipodia and Current Opinion in Cell Biology 2004, 16:174–181
178 Cell regulation
filopodia [14,31]. In mammalian cells this situation may be a bit more complicated, as suggested by the fact that in initial studies, Scar1/WAVE1- and Scar2/ WAVE2-deficient fibroblasts have normal filopodia [32,33].
Role of WASP in specialized cellular events If WASPs are not required for the formation of the lamellipodia and filopodia, what is their role in the cell? Several studies provide evidence of the involvement of WASPs in membrane dynamics. The yeast Las17p is required for efficient endocytic internalization [34], and inhibition of N-WASP function with specific antibodies reduces transferrin uptake in mammalian cells [35]. Moreover, in budding yeast, endosome motility requires actin polymerization stimulated by Las17p [36], and N-WASP-deficient ES cells are defective in endomembrane vesicle motility stimulated by PtdIns(4)P5-kinase overexpression [37]. However, Scar has also been implicated in membrane dynamics: SCAR-null Dictyostelium are defective in macropinocytosis, phagocytosis and endosomal membrane flow [38]. WASP was originally identified as the protein mutated in Wiskott-Aldrich syndrome (WAS), which is an X-linked primary immunodeficiency disease characterized by congenital thrombocytopenia, eczema and immune deficiency [39,40]. WASP is specifically expressed in hematopoietic cells, and may therefore be important for the assembly of specialized dendritic actin-filament arrays in these cells. WASP-deficient T cells are abrogated in antigen-induced formation of conjugates between T cells and antigen-presenting cells as a result of defects in immunological synapse formation [41]. WASP is also required for the assembly of natural-killer-cellactivating immunological synapses [42]. T cells from WAS patients are impaired in their ability to cluster the GM1 glycosphingolipid raft marker during T-cell activation, which suggests a critical role for WASP in the movement and subsequent aggregation and clustering of lipid rafts, which are known to be important for immunological synapse formation [43]. WASP also participates in the formation of podosomes, which are specialized adhesion structures of the monocytic lineage that are possibly required for dynamic cell adhesions during cell movement [44]. The role of WASP in some hematopoietic cells may be restricted to certain specialized events, as, for example, WASP-deficient platelets appear to activate the Arp2/3 complex and assemble actin normally [45].
WASP/Scar proteins are required for mammalian development In mice, the severity of phenotypes resulting from deletion of WASP/Scar proteins closely mirrors their expression patterns (Table 1). Defects in WAS knock-out mice as well as in patients with WAS are restricted to the Current Opinion in Cell Biology 2004, 16:174–181
hematopoietic system, where they include several proliferative, morphological and functional defects [39,40]. Deletion of the ubiquitously expressed N-WASP results in death before embryonic day (E) 12 as a result of developmental delay and neural tube and cardiac defects [29,30]. Expression of Scar/WAVE1 is mainly restricted to brain, and, accordingly, the Scar/WAVE1-null mice display several CNS-related problems, such as limb weakness, neuroanatomical malformations and behavioral abnormalities, which presumably lead to postnatal lethality [46,47]. Importantly, the neuronal morphology of Scar1 null cells in vivo as well as their ability to form processes in vitro appear to be normal, indicating that Scar1 is somehow required for the development of the CNS, but not for the formation or extension of neuronal processes [46]. Like N-WASP, Scar/WAVE2 is relatively widely expressed, and Scar/WAVE2-null mice die at E10–E12.5 suffering from haemorrhages, cardiovascular defects due to impaired angiogenesis, developmental delay and growth retardation [32,48]. Scar/WAVE2deficient endothelial cells have morphological defects and do not respond normally to vascular endothelial growth factor, leading to defects in migration [48]. Similarly, Scar/WAVE2-deficient fibroblasts are defective in lamellipodia formation and cell motility in response to platelet-derived growth factor [32]. This indicates that Scar/WAVE2 has an essential role in the active migration of particular cell types. It also seems that Scar/WAVE1 and Scar/WAVE2 have distinct functions during cell migration, at least in mouse embryonic fibroblasts. In these cells, Scar/WAVE2 appears to be required for leading edge extension during directed migration in general, whereas Scar/WAVE1 is essential for matrix-metalloproteinasedependent migration through the extracellular matrix [33]. Taken together, it appears that Scar/WAVE1, Scar/ WAVE2, WASP and N-WASP have specific functions during mammalian development that cannot be compensated by the other family members.
Regulation of WASP/Scar function Despite their different modes of regulation, precise control of both Scar and WASP activity is important for their physiological functions. Regulation of WASP activity is well characterized and has been recently reviewed elsewhere [3]. Regulation of Scar/WAVE proteins by an inhibitory complex [5] appears to be conserved throughout evolution, possibly also in plants [23,24]. In Dictyostelium, deletion of PIR121 results in a phenotype resembling overactivation of Scar, with excessive actin filament formation. Intriguingly, the PIR121-null cells contain very little intact Scar protein, suggesting a novel mechanism to regulate uncomplexed Scar by proteolysis [49]. Similarly, loss of Kette, which is the Drosophila homologue of human Nap125, results in accumulation of F-actin [50]. However, RNAi-mediated depletion of PIR121/Sra-1, Abi or Kette in Drosophila S2 cells results in a Scar-like phenotype rather than in excessive actin www.sciencedirect.com
The WASP–Arp2/3 pathway: genetic insights Vartiainen and Machesky 179
polymerization. Also, these cells display considerable reduction in Scar protein levels [14], indicating that degradation of uncomplexed Scar may be an evolutionarily conserved phenomenon. However, as deletion of PIR121 appears to result in varying phenotypes depending on the system under study, the dynamics of this process may differ. Interestingly, expression of a membrane-tethered Kette induces formation of large actin bundles in a process that is independent of Scar but dependent on WASP. Abi, another component of the Scar complex, may act as a link between Kette and WASP [50], providing a means to integrate these two signaling pathways. It remains to be established whether this kind of crosstalk also operates in other in vivo systems.
Conclusions We can conclude from a survey of the recent literature that genetic and loss-of-function studies have contributed significantly to our understanding of how actin assembly is regulated by the WASP/Scar family proteins and the Arp2/3 complex. In the late 1990s, biochemical and cell biological studies pointed to a crucial role for these proteins in signaling to actin dynamics. More recently, genetic studies have confirmed that the function of Arp2/3 complex as an actin nucleator is likely to be conserved in all eukaryotes and to be essential for life in most cases. However, Arp2/3 does not appear to be the only actin nucleator, but rather it specifically induces branched actin structures as opposed to long parallel bundles. Although many proteins can regulate Arp2/3 function in vitro, WASP/Scar family members appear to be the most important regulators in vivo. Multicellular animals have multiple WASP/Scar proteins with unique roles in specific cell types and contexts, reflecting the numerous and varied processes regulated by these proteins. Genetic studies have also poked some rather large holes in models that had previously been based on biochemical and cellular studies. Mainly, the idea of parallel pathways between the small GTPases, the different WASP/Scar family proteins and specific structures such as lamellipodia and filopodia has proven to be oversimplified. Hopefully future studies will lead to a better understanding of issues such as the relative contribution of the Arp2/3 complex and specific WASP/Scar proteins to various cell structures that use parallel and branched actin filaments. Clearly, the future holds much room for exciting discoveries in the regulation of actin assembly, as actin is one of the most abundant and versatile cellmotility and architectural proteins in eukaryotes.
Update While the review was going to press, a new study from the lab of Baum [55] showed that the Scar complex members Abi, Sra1 and Kette regulate both the stability and localization of Scar in Drosophila cells. The authors suggest www.sciencedirect.com
that the Scar complex is not only inhibitory, but regulatory, and that some components may also play a positive role in actin-based protrusion.
Acknowledgements We wish to thank colleagues for sharing their unpublished data, and regret that space limitations prevented us from citing all of the literature relevant for this review. We also thank G Bompard for the critical reading of this manuscript. MKV is funded by EMBO Long Term Fellowship, and LMM is an MRC Senior Research Fellow.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Pollard TD, Borisy GG: Cellular motility driven by assembly and disassembly of actin filaments. Cell 2003, 112:453-465. This excellent review provides a comprehensive view of the current knowledge of how actin dynamics drive cell motility. 2.
Machesky LM, Atkinson SJ, Ampe C, Vandekerckhove J, Pollard TD: Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin–agarose. J Cell Biol 1994, 127:107-115.
3.
Weaver AM, Young ME, Lee WL, Cooper JA: Integration of signals to the Arp2/3 complex. Curr Opin Cell Biol 2003, 15:23-30.
4.
Beltzner CC, Pollard TD: Identification of functionally important residues of Arp2/3 complex by analysis of homology models from diverse species. J Molec Biol 2004, 336:551-565.
5.
Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW: Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 2002, 418:790-793. This article is the first to identify the protein complex that inhibits the activity of Scar/WAVE1 in cells. It also provides the basic mechanism of Scar/WAVE activation through signal-dependent dissociation of this complex. 6.
Winter DC, Choe EY, Li R: Genetic dissection of the budding yeast Arp2/3 complex: a comparison of the in vivo and structural roles of individual subunits. Proc Natl Acad Sci U S A 1999, 96:7288-7293.
7.
Sawa M, Suetsugu S, Sugimoto A, Miki H, Yamamoto M, Takenawa T: Essential role of the C. elegans Arp2/3 complex in cell migration during ventral enclosure. J Cell Sci 2003, 116:1505-1518. This article provides in vivo evidence for the requirement for Arp2/3complex-mediated actin polymerization during cell motility. By using RNAi-mediated depletion of Arp2/3 complex subunits and WASP the authors show that these proteins play a key role in ventral enclosure in C. elegans. 8.
Severson AF, Baillie DL, Bowerman B: A Formin homology protein and a profilin are required for cytokinesis and Arp2/3-independent assembly of cortical microfilaments in C. elegans. Curr Biol 2002, 12:2066-2075.
9.
Zallen JA, Cohen Y, Hudson AM, Cooley L, Wieschaus E, Schejter ED: SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila. J Cell Biol 2002, 156:689-701. Using a genetic approach, SCAR is shown to be a more important regulator of the Arp2/3 complex during morphological processes in Drosophila than WASP. This article highlights the distinct roles of WASP and Scar/WAVE proteins in different cellular processes. 10. Hudson AM, Cooley L: A subset of dynamic actin rearrangements in Drosophila requires the Arp2/3 complex. J Cell Biol 2002, 156:677-687. 11. Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K: Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci 2001, 114:4557-4565. Current Opinion in Cell Biology 2004, 16:174–181
180 Cell regulation
12. Mathur J, Mathur N, Kernebeck B, Hulskamp M: Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis. Plant Cell 2003, 15:1632-1645. 13. Svitkina TM, Verkhovsky AB, Mcquade KM, Borisy GG: Analysis of the actin–myosin II system in fish epidermal keratocytes: mechanism of cell body translocation. J Cell Biol 1997, 139:397-415. 14. Rogers SL, Weidemann U, Stuurman N, Vale RD: Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J Cell Biol 2003, 162:1079-1088. Using a RNAi-based approach in a new, popular Drosophila cell-culture model, authors study the role of a large number of proteins in lamella formation. This article highlights the role of Scar as a regulator of Arp2/3 activity in the formation of lamellipodia, and shows that the function of the Scar regulatory complex is to inhibit Scar degradation. 15. Pelham RJ, Chang F: Actin dynamics in the contractile ring during cytokinesis in fission yeast. Nature 2002, 419:82-86. Using fission yeast as a model system, the authors show that, during cell division, the contractile ring is a dynamic structure in which actin and other ring components assemble and disassemble continuously from the ring. Actin polymerization by both a formin and the Arp2/3 complex appears to play a key role in the cleavage process.
reorganization of a dendritic network. J Cell Biol 2003, 160:409-421. How filopodia are initiated has remained a mystery for a long time. By using a combination of microscopical techniques, the authors show that dendritic actin arrays of the lamellipodia can be rearranged to form filopodia. 29. Lommel S, Benesch S, Rottner K, Franz T, Wehland J, Kuhn R: Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells. EMBO Rep 2001, 2:850-857. 30. Snapper SB, Takeshima F, Anton I, Liu C-H, Thomas SM, Nguyen D, Dudley D, Fraser H, Purich D, Lopez-Ilasaca M et al.: N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility. Nat Cell Biol 2001, 3:897-904. 31. Biyasheva A, Svitkina T, Baum B, Borisy G: Cascade pathway of filopodia formation downstream of SCAR. J Cell Sci, in press. 32. Yan C, Martinez-Quiles N, Eden S, Shibata T, Takeshima F, Shinkura R, Fujiwara Y, Bronson R, Snapper SB, Kirschner MW et al.: WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility. EMBO J 2003, 22:3602-3612.
16. Carnahan RH, Gould KL: The PCH family protein, Cdc15p, recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe. J Cell Biol 2003, 162:851-862.
33. Suetsugu S, Yamazaki D, Kurisu S, Takenawa T: Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration. Dev Cell 2003, 5:595-609.
17. Stevenson V, Hudson A, Cooley L, Theurkauf WE: Arp2/3-dependent pseudocleavage furrow assembly in syncytial Drosophila embryos. Curr Biol 2002, 12:705-711.
34. Naqvi SN, Zahn R, Mitchell DA, Stevenson BJ, Munn AL: The WASP homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast. Curr Biol 1998, 8:959-962.
18. Tal T, Vaizel-Ohayon D, Schejter ED: Conserved interactions with cytoskeletal but not signaling elements are an essential aspect of Drosophila WASP function. Dev Biol 2002, 243:260-271. 19. Mathur J, Mathur N, Kirik V, Kernebeck B, Purushottam B, Hulskamp M: Arabidopsis CROOKED encodes for the smallest subunit of the Arp2/3 complex and controls cell shape by region specific fine F-actin formation. Development 2003, 130:3137-3146. This is the first article to describe a plant homolog of Arp2/3 complex subunits, and therefore provides evidence for the evolutionarily conserved role of Arp2/3-mediated actin polymerization in very distinct cellular contexts. 20. Le J, El-Assal SED, Basu D, Saad ME, Szymanski DB: Requirements for Arabidopsis ATARP2 and ATARP3 during epidermal development. Curr Biol 2003, 13:1341-1347. 21. Li S, Blanchoin L, Yang Z, Lord EM: The putative Arabidopsis Arp2/3 complex controls leaf cell morphogenesis. Plant Physiol 2003, 132:2034-2044. 22. Deeks MJ, Hussey PJ: Arp2/3 and ‘The shape of things to come’. Curr Opin Plant Biol 2003, 6:561-567. 23. Frank MJ, Smith LG: A small, novel protein highly conserved in plants and animals promotes the polarized growth and division of maize leaf epidermal cells. Curr Biol 2002, 12:849-853. 24. Frank MJ, Cartwright HN, Smith LG: Three Brick genes have distinct functions in a common pathway promoting polarized cell division and cell morphogenesis in the maize leaf epidermis. Development 2003, 130:753-762. 25. Rodal AA, Manning AL, Goode BL, Drubin DG: Negative regulation of yeast WASP by two SH3-domain-containing proteins. Curr Biol 2003, 13:1000-1008. 26. Soto MC, Qadata H, Kasuya H, Inoue M, Tsuboi D, Mello CC, Kaibuchi K: The GEX-2 and GEX-3 proteins are required for tissue morphogenesis and cell migrations in C. elegans. Genes Dev 2002, 16:620-632. 27. Ben-Yaacov S, Le Borgne R, Abrahamson I, Scheweisguth F, Schejter ED: WASP, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling. J Cell Biol 2001, 152:1-13. 28. Svitkina TM, Bulanova EA, Chaga OY, Vignjevic DM, Kojima S, Vasiliev JM, Borisy GG: Mechanism of filopodia initiation by Current Opinion in Cell Biology 2004, 16:174–181
35. Kessels MM, Qualmann B: Syndapins integrate N-WASP in receptor mediated endocytosis. EMBO J 2002, 21:6083-6094. 36. Chang FS, Stefan CJ, Blumer KJ: A WASP homolog powers actin polymerization-dependent motility of endosomes in vivo. Curr Biol 2003, 13:455-463. 37. Benesch S, Lommel S, Steffen A, Stradal TEB, Scaplehorn N, Way M, Wehland J, Rottner K: Phosphatidylinositol 4,5biphosphate (PIP2)-induced vesicle movement depends on N-WASP and involves Nck, WIP, and Grb2. J Biol Chem 2002, 277:37771-37776. 38. Seastone DJ, Harris E, Temesvari LA, Bear JE, Saxe CL, Cardelli J: The WASP-like protein Scar regulates macropinocytosis, phagocytosis and endosomal membrane flow in Dictyostelium. J Cell Sci 2001, 114:2673-2683. 39. Notarangelo LD, Ochs HD: Wiskott-Aldrich syndrome: a model for defective actin reorganization, cell trafficking and synapse formation. Curr Opin Immunol 2003, 15:585-591. 40. Thrasher AJ: WASP in immune-system organization and function. Nat Rev Immunol 2002, 2:635-646. 41. Badour K, Zhang J, Shi F, McGavin MK, Ramperstad V, Hardy LA, Field D, Siminovitch KA: The Wiskott-Aldrich syndrome protein acts downstream of CD2 and the CD2AP and PSTPIP1 adaptors to promote formation of the immunological synapse. Immunity 2003, 18:141-154. 42. Orange JS, Ramesh N, Remold-O’Donnell E, Sasahara Y, Koopman L, Byrne M, Bonilla FA, Rosen FS, Geha RS, Strominger JL: Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunological synapses. Proc Natl Acad Sci U S A 2002, 99:11351-11356. 43. Dupre L, Aiuti A, Trifari S, Martino S, Saracco P, Bordignon C, Roncarolo MG: Wiskott-Aldrich syndrome protein regulates lipid raft dynamics during immunological synapse formation. Immunity 2002, 17:157-166. 44. Burns S, Thrasher AJ, Blundell MP, Machesky L, Jones GE: Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS protein, and differentiation. Blood 2001, 98:1142-1149. 45. Falet H, Hoffmeister KM, Neujahr R, Hartwig JH: Normal Arp2/3 complex activation in platelets lacking WASP. Blood 2002, 100:2113-2122. www.sciencedirect.com
The WASP–Arp2/3 pathway: genetic insights Vartiainen and Machesky 181
46. Dahl JP, Wang-Dunlop J, Gonzales C, Goad MEP, Mark RJ, Kwak SP: Characterization of the WAVE-1 knock-out mouse: implications for CNS development. J Neurosci 2003, 23:3343-3352.
51. Morrell JL, Morphew M, Gould KL: A mutant of Arp2p causes partial disassembly of the Arp2/3 complex and loss of cortical actin function in fission yeast. Mol Biol Cell 1999, 10:4201-4215.
47. Soderling SH, Langeberg LK, Soderling JA, Davee SM, Simerly R, Raber J, Scott JD: Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Proc Natl Acad Sci U S A 2003, 100:1723-1728.
52. Li R: Bee1, a yeast protein with homology to Wiskott-Aldrich syndrome protein, is critical for the assembly of cortical actin cytoskeleton. J Cell Biol 1997, 136:649-658.
48. Yamazaki D, Suetsugu S, Miki H, Kataoka Y, Nishikawa SI, Fujiwara T, Yoshida N, Takenawa T: WAVE2 is required for directed cell migration and cardiovascular development. Nature 2003, 424:452-456. 49. Blagg SL, Stewart M, Sambles C, Insall RH: PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium. Curr Biol 2003, 13:1480-1487. Using PIR121-null Dictyostelium, the authors demonstrate that PIR121 also regulates Scar activity in this organism. This article also contains the first observation of degradation of the uncomplexed Scar. 50. Bogdan S, Klambt C: Kette regulates actin dynamics and genetically interacts with Wave and WASP. Development 2003, 130:4427-4437.
www.sciencedirect.com
53. Bear JE, Rawls JF, Saxe CL III: SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development. J Cell Biol 1998, 142:1325-1335. 54. Zhang J, Shehabeldin A, da Cruz LAG, Butler J, Somani AK, McGavin M, Kozieradzki I, dos Santos AO, Nagy A, Grinstein S et al.: Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes. J Exp Med 1999, 190:1329-1341. 55. Kunda P, Craig G, Dominguez D, Baum B: Abi, Sra1 and Kette control the stability and localization of Scar/WAVE to regulate the formation of actin-based protrusions. Curr Biol 2003, 13:1867-1875.
Current Opinion in Cell Biology 2004, 16:174–181
Current Opinion in Cell Biology 2004, 16:174–181 This review comes from a themed issue on Cell regulation Edited by Craig Montell and Peter Devreotes 0955-0674/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2004.02.004
Abbreviations Abi Abl interactor GBD GTPase binding domain RNAi RNA interference Scar suppressor of cAMP receptor VCA Verprolin-homology-connecting acidic domain WAS Wiskott-Aldrich syndrome WASP Wiskott-Aldrich syndrome protein WAVE WASP family verproline-homologous protein WH1 WASP homology domain 1
Introduction The coordinated assembly and disassembly of actin filaments drives several essential processes that are of crucial importance at both the single-cell and whole-organism level. For example, the chemotactic responses of an amoeba or neutrophil as well as the complicated cell rearrangements during embryogenesis are all based on cell motility and driven, at least in part, by polymerization of actin filaments at the leading edges of the cells [1]. Nucleation of new actin filaments is a key event in any cellular process relying on the dynamic assembly of actin filaments. Since the discovery of the Arp2/3 complex Current Opinion in Cell Biology 2004, 16:174–181
almost a decade ago [2], a broad range of biochemical and cell-biological studies have pointed to the pivotal role of this complex as a regulator of actin filament formation [1,3]. This review focuses on new insights revealed by recent genetic and other loss-of-function studies on the Arp2/3 complex and members of the Wiskott-Aldrich Syndrome protein (WASP)/suppressor of cAMP-receptor (Scar, also known as WASP family verprolin-homologous protein [WAVE]) families. The Arp2/3 complex is composed of two actin-related proteins Arp2 and Arp3, which presumably act as a template for new actin filaments, and five additional subunits, which in humans are called ARPC1–5 (the human nomenclature is used throughout this review) and which serve various structural and regulatory functions. Analysis of homology models from diverse species shows that the folds of the seven subunits, their core residues and the residues at subunit interfaces are well conserved during evolution. Solvent-exposed surfaces are less conserved, but conserved patches of surface residues are candidate sites for binding to nucleation-promoting factors and actin filaments [4], which allow the Arp2/3 complex to nucleate the formation of branched actin filament arrays in response to several signaling pathways. WASP/Scar family proteins are the most potent cellular nucleation promoting factors, and they share a common C-terminal Verprolin-homology-connecting acidic (VCA) domain that is responsible for Arp2/3 complex activation [3]. However, WASP and Scar/WAVE proteins differ both in the signaling inputs that they receive and in their modes of regulation (Figure 1). WASP proteins are autoinhibited: intramolecular interactions mask the VCAdomain in the absence of signaling molecules, such as PtdIns(4,5)P2 and Cdc42 [3]. On the other hand, mammalian Scar/WAVE1 has been shown to be kept inactive in a complex with four other proteins, PIR121/Sra-1, Nap125, HSPC300 and Abl interactor (Abi) 2. This complex can then be disassembled in response to certain signals to initiate Arp2/3-complex-mediated actin polymerization [5].
Arp2/3 complex is important for normal cell functions Genetic studies in yeast, Caenorhabditis elegans and Drosophila all point to the pivotal role of Arp2/3 complex for cell survival (Table 1) [6,7,8,9,10]. Concomitantly, depletion of the ARPC3 subunit by small interfering RNAs in mammalian cells has shown that this protein is essential for cell growth [11]. Consistent with the role of www.sciencedirect.com
The WASP–Arp2/3 pathway: genetic insights Vartiainen and Machesky 175
Figure 1
WASP WH1
B
GBD
ACV
Scar/WAVE P R O
Signals PI(4,5)P2 Rac1 Cdc42 Nck
Abi O Nap125 SHD B PIR121 HSPC 300
Abi PIR121 Nap125
WH1
B
GBD PRO
VCA
SHD
B
PRO
VCA
HSPC 300
Arp2/3-complex-mediated actin polymerization
Membrane trafficking (endocytosis, endosome motility) Specialized dendritic actin arrays (immunological synapses, podosomes)
Arp2/3-complex-mediated actin polymerization
Degradation of uncomplexed Scar
Lamellipodia
Filopodia
Current Opinion in Cell Biology
In the absence of stimulus, WASP is autoinhibited, whereas Scar/WAVE is kept inactive in a regulatory complex with four other proteins. WASP is activated by signaling molecules such as PI(4,5)P2 and Cdc42, which unmask the VCA-domain of WASP, which can then stimulate actin polymerization by the Arp2/3 complex. Scar/WAVE is activated by Rac and Nck, which disassemble the regulatory complex and allow Scar/WAVE to activate the Arp2/3 complex. Uncomplexed Scar/WAVE is also rapidly degraded. Actin polymerization stimulated by WASPs play an important role in membrane trafficking and in the formation of specialized dendritic actin arrays. On the other hand, Scar/WAVEs are important for actin polymerization in the lamellipodia and filopodia, a process which Cdc42 has also been implicated in. B, basic region; PRO, proline rich region; SHD, Scar homology domain.
Arp2/3 as a nucleator of actin filaments, the Arp2/3 mutants often have lower levels of actin filaments [7,9]. Some mutants also show formation of large actin filament aggregates in the absence of Arp2/3 [6,12]. Importantly, the Arp2/3 complex appears to be required for the assembly of only a subset of actin filament structures. In Drosophila, loss-of-function mutations in ARPC1 and Arp3 have shown that Arp2/3 complex is required for regulation of the dynamic actin structures in the blastoderm embryo [9] and for the expansion of ring canals, which allow the flow of cytoplasm from the nurse cell to the developing oocyte. However, the Arp2/3 complex is not required for the initial recruitment of actin filaments at ring canals, nor for the formation of parallel, hexagonally packed actin bundles in the nurse cell or bristles, although it contributes to their organization [10]. In C. elegans, depletion of Arp2/3 complex subunits by RNA interference (RNAi) suggests that the Arp2/3 complex is dispensable for the assembly of cortical actin in the early embryo and for the establishment of anterior–posterior polarity [7,8]. These results suggest that although the Arp2/3 complex does not participate in the nucleation of all actin filament structures in the cell, it may still influwww.sciencedirect.com
ence their organization, indicating that the balance between distinct actin filament assemblies is important for normal cell functions. Branched actin filament networks nucleated by the Arp2/3 are a hallmark of the lamellipodium at the leading edge of motile cells [13]. Accordingly, the Arp2/3 complex appears to be especially important for cell motility. In C. elegans, the Arp2/3 complex is crucial for cell movements after cell-fate decisions during morphogenesis. In Arp2/3-depleted embryos, certain precursor cells fail to ingress into the blastocoel, and therefore gastrulation fails. The Arp2/3 complex is also essential for ventral enclosure, which requires both cell migration and cell adhesion. Arp2/3 subunits appear to be among those few genes that specifically affect the migration part of this process [7,8]. In Drosophila, Arp2/3 complex is required for axon development, a process requiring extensive migration of the growth cones [9]. The role of the Arp2/3 complex in cytokinesis is controversial. Deletion of genes encoding Arp2/3 subunits in Saccharomyces cerevisiae [6] or depletion by RNAi in Current Opinion in Cell Biology 2004, 16:174–181
176 Cell regulation
Table 1 Phenotypes of Arp2/3 complex and WASP/Scar loss-of-function mutants in model organisms.
Saccharomyces cerevisiae, Schizosaccharomyces pombe
Arp2/3
WASP
Scar
References
Some subunits essential, conditional mutations result in defects in the cortical actin cytoskeleton.
One protein Las17p/Bee1p/ Wsp1; required for normal yeast growth, actin organization and endocytosis.
[6,34,51,52]
[12,19,20,21]
Deletion of other subunits causes variable defects in cell viability, growth and actin patches. Arabidopsis thaliana
Mutations in Arp2, Arp3 and ARPC5 result in aberrant cell shapes due to misdirected cell expansion.
Not characterized.
Dictyostelium discoideum
No mutants available.
No mutants available.
Disruption of the gene results in reduced actin levels, abnormal cell morphology during chemotaxis and defects in membrane trafficking.
[38,53]
Caenorhabditis elegans
RNAi results in embryonic arrest during morphogenesis, defects in cell migration during gastrulation and reduced F-actin levels.
RNAi results in several phenotypes including embryonic lethality, larval arrest and sterility, defects in hypodermal cell migration and reduced F-actin levels.
RNAi: no phenotype.
[7,8]
Drosophila melanogaster
All loss-of-function mutations lethal before adult age. Specifically required for cortical division, cytoplasmic organization in the blastoderm, ring canal expansion, axon development, egg chamber structure and adult eye morphology.
Loss-of-function mutations result in defects in Notch-mediated cell fate decisions.
Deletion in Scar locus or P-element insertion results in zygotic lethality, required for same functions as Arp2/3.
[9,10,14, 18,27,31]
RNAi of ARPC4 in cultured cells inhibit lamellipodium formation.
RNAi in cultured cells does not result in any significant phenotype.
RNAi in cells inhibit cell spreading, lamellipodia and filopodia formation.
RNAi of ARPC3 is lethal.
Knockout of WASP leads to various proliferative, morphological, and functional defects in hematopoietic cells.
Knockout of Scar1leads to post natal lethality and various problems in the nervous system; neurons have normal morphology.
Knockout of N-WASP is embryonically lethal at E12 and causes developmental delay, cardiac and neural tube defects, although cells have normal filopodia and lamellipodia
Knockout of Scar2 is lethal at E10-12.5 and causes growth retardation and several morphological defects especially in the cardiovascular system.
Mus musculus
[11,29,30,32, 54,46,47,48]
Cells are deficient in lamellipodia formation and motility in response to growth factors.
C. elegans [8] and Drosophila S2 cells [14] does not interfere with cytokinesis. However, recent studies in the fission yeast S. pombe have shown that the contractile ring is an active site of actin assembly, and that both the Current Opinion in Cell Biology 2004, 16:174–181
formin Cdc12 and the Arp2/3 complex are required for this actin polymerization activity [15]. In fact, Cdc15p, which interacts with both Cdc12 and Arp2/3 activator Myo1p, directly links these actin filament nucleators [16]. www.sciencedirect.com
The WASP–Arp2/3 pathway: genetic insights Vartiainen and Machesky 177
In Drosophila, Arp2/3 mutants have defects in the cortical division cycles in the blastoderm embryo, [9,17] and asymmetric cell divisions in the neuronal lineage [18]. Therefore, the requirement for the Arp2/3 complex in cytokinesis could be cell-type/organism-specific, and clearly requires further study. Rigid cell walls and large vacuoles dominate plant cells, and therefore the cytoskeleton plays different roles in these cells than in most animal cell types. Recent studies have isolated several mutants in actin-binding proteins that have defects in plant cell growth. Among these are the Arabidopsis thaliana CROOKED [19], WURM and DISTORTED [12,20], which encode Arp2/3 subunits ARPC5, Arp2 and Arp3, respectively. Mutations in these genes result in aberrant cell shapes in various cell types, including trichomes, hypocotyl cells and root hairs, due to misdirected cell expansion. Specifically, the generation of fine actin filaments that are often associated with regions of active growth and expansion appears to be impaired in Arp2/3 complex mutants. At the cellular level, the mutant cells often contain large actin filament aggregates, which may prevent vesicle fusion at the expanding sites [12,19,21]. The Arp2/3 complex mutant phenotype can be rescued with the human ortholog [12], and Distorted1 rescues an arp3 mutation in S. cerevisiae [20], indicating a functional conservation of Arp2/3-mediated actin polymerization in very different cellular contexts. Regulation of the Arp2/3 complex activity in plant cells remains to be established, as no plant protein containing a VCA region has been identified. Two uncharacterized Arabidopsis proteins (At4 g18600 and At1 g29170) are, however, predicted to contain a Scar homology domain (SHD) [22]. It is curious that the maize Brk1 gene, which has been shown to play a role in cell expansion, encodes a HSPC300 homolog [23]. Brk1 functions together with Brk2 and Brk3 in a common pathway required for epidermal cell morphogenesis and division [24]. Future studies will reveal the molecular nature of these additional Brk genes. It is tempting to speculate that they may encode a Scar/WAVE ortholog, a plant-specific Arp2/3 complex activator, or other members of the Scarregulatory complex.
WASP/Scar proteins: similar or distinct functions? Yeasts S. cerevisiae and S. pombe have only one WASP/Scar protein and Dictyostelium, C. elegans and Drosophila contain one WASP and one Scar, whereas mammals have five members: WASP, N-WASP, and Scar/WAVE 1–3 (Table 1). The S. cerevisiae WASP/Scar protein Las17p resembles WASP in its overall domain organization, having a Wasp homology 1 domain (WH1, see Figure 1) at its N-terminus. However, it has no obvious GTPase-binding domain (GBD) and does not interact directly with Cdc42. Moreover, recent studies have shown that the purified www.sciencedirect.com
Las17p is not autoinhibited, but two SH3-domain containing proteins, Sla1p and Bbc1p, can inhibit its activity [25]. Therefore, with respect to regulation, Las17p behaves more like Scar/Wave than WASP. The relative importance of the WASP/Scar family members in Arp2/3-dependent cellular functions appears to depend at least partly on the organism. In C. elegans, WASP is required for essentially the same processes as the Arp2/3 complex, including ventral enclosure [7]. Unfortunately, preliminary RNAi studies with Scar in this organism have yielded no phenotype [7]. However, the C. elegans gut on exterior (GEX) genes GEX-2 and GEX-3 encode homologues of PIR121 and Nap125, respectively. RNAi of Gex-2 or transposon insertion in Gex-3 gene results in embryonic lethality, because cells fail to become organized, and tissues are found in abnormal positions with aberrant morphology. Like the Arp2/3 complex, these genes are required for hypodermal cell migration, during both dorsal intercalation and ventral migration [26], suggesting that Scar may also play a role in these processes. In Drosophila, Scar and WASP have clearly distinct functions. Scar appears to be the main regulator of actindependent morphological processes mediated by Arp2/3, such as axon development, egg chamber structure and adult eye morphology [9]. On the other hand, WASP, but not Scar, is required specifically for cell-fate decisions mediated by Notch signaling [9,27]. In the absence of WASP there is excessive differentiation of sensory organ neurons at the expense of non-neuronal cell types, which results from a failure in the execution of asymmetric cell divisions in neural lineages [27]. This activity is Arp2/3-dependent, but interactions with Cdc42 and PtdIns(4,5)P2 are not required [18]. These studies also suggest that members of the WASP/Scar family are the most important physiological activators of the Arp2/3 complex at least in Drosophila, as the defects in the Arp2/3 mutants are a sum of the defects seen in WASP and Scar mutants. At the cellular level, the Arp2/3 complex is responsible for the nucleation of the lamellipodial actin network, which may be subsequently reorganized into filopodia [28]. It has been generally thought that two parallel pathways drive the formation of these structures: from Rac through Scar/WAVE to lamellipodia, and from Cdc42 through WASP to filopodia. There are, however, results that contradict this view. First, Scar is the main regulator of Arp2/3-dependent cellular processes in Drosophila as discussed above [9]. Second, NWASP-deficient mouse fibroblasts have normal filopodia [29,30]. Third, RNAi studies in cultured Drosophila cells have shown that depletion of WASP does not result in any significant phenotype, whereas depletion of Scar inhibits formation of both lamellipodia and Current Opinion in Cell Biology 2004, 16:174–181
178 Cell regulation
filopodia [14,31]. In mammalian cells this situation may be a bit more complicated, as suggested by the fact that in initial studies, Scar1/WAVE1- and Scar2/ WAVE2-deficient fibroblasts have normal filopodia [32,33].
Role of WASP in specialized cellular events If WASPs are not required for the formation of the lamellipodia and filopodia, what is their role in the cell? Several studies provide evidence of the involvement of WASPs in membrane dynamics. The yeast Las17p is required for efficient endocytic internalization [34], and inhibition of N-WASP function with specific antibodies reduces transferrin uptake in mammalian cells [35]. Moreover, in budding yeast, endosome motility requires actin polymerization stimulated by Las17p [36], and N-WASP-deficient ES cells are defective in endomembrane vesicle motility stimulated by PtdIns(4)P5-kinase overexpression [37]. However, Scar has also been implicated in membrane dynamics: SCAR-null Dictyostelium are defective in macropinocytosis, phagocytosis and endosomal membrane flow [38]. WASP was originally identified as the protein mutated in Wiskott-Aldrich syndrome (WAS), which is an X-linked primary immunodeficiency disease characterized by congenital thrombocytopenia, eczema and immune deficiency [39,40]. WASP is specifically expressed in hematopoietic cells, and may therefore be important for the assembly of specialized dendritic actin-filament arrays in these cells. WASP-deficient T cells are abrogated in antigen-induced formation of conjugates between T cells and antigen-presenting cells as a result of defects in immunological synapse formation [41]. WASP is also required for the assembly of natural-killer-cellactivating immunological synapses [42]. T cells from WAS patients are impaired in their ability to cluster the GM1 glycosphingolipid raft marker during T-cell activation, which suggests a critical role for WASP in the movement and subsequent aggregation and clustering of lipid rafts, which are known to be important for immunological synapse formation [43]. WASP also participates in the formation of podosomes, which are specialized adhesion structures of the monocytic lineage that are possibly required for dynamic cell adhesions during cell movement [44]. The role of WASP in some hematopoietic cells may be restricted to certain specialized events, as, for example, WASP-deficient platelets appear to activate the Arp2/3 complex and assemble actin normally [45].
WASP/Scar proteins are required for mammalian development In mice, the severity of phenotypes resulting from deletion of WASP/Scar proteins closely mirrors their expression patterns (Table 1). Defects in WAS knock-out mice as well as in patients with WAS are restricted to the Current Opinion in Cell Biology 2004, 16:174–181
hematopoietic system, where they include several proliferative, morphological and functional defects [39,40]. Deletion of the ubiquitously expressed N-WASP results in death before embryonic day (E) 12 as a result of developmental delay and neural tube and cardiac defects [29,30]. Expression of Scar/WAVE1 is mainly restricted to brain, and, accordingly, the Scar/WAVE1-null mice display several CNS-related problems, such as limb weakness, neuroanatomical malformations and behavioral abnormalities, which presumably lead to postnatal lethality [46,47]. Importantly, the neuronal morphology of Scar1 null cells in vivo as well as their ability to form processes in vitro appear to be normal, indicating that Scar1 is somehow required for the development of the CNS, but not for the formation or extension of neuronal processes [46]. Like N-WASP, Scar/WAVE2 is relatively widely expressed, and Scar/WAVE2-null mice die at E10–E12.5 suffering from haemorrhages, cardiovascular defects due to impaired angiogenesis, developmental delay and growth retardation [32,48]. Scar/WAVE2deficient endothelial cells have morphological defects and do not respond normally to vascular endothelial growth factor, leading to defects in migration [48]. Similarly, Scar/WAVE2-deficient fibroblasts are defective in lamellipodia formation and cell motility in response to platelet-derived growth factor [32]. This indicates that Scar/WAVE2 has an essential role in the active migration of particular cell types. It also seems that Scar/WAVE1 and Scar/WAVE2 have distinct functions during cell migration, at least in mouse embryonic fibroblasts. In these cells, Scar/WAVE2 appears to be required for leading edge extension during directed migration in general, whereas Scar/WAVE1 is essential for matrix-metalloproteinasedependent migration through the extracellular matrix [33]. Taken together, it appears that Scar/WAVE1, Scar/ WAVE2, WASP and N-WASP have specific functions during mammalian development that cannot be compensated by the other family members.
Regulation of WASP/Scar function Despite their different modes of regulation, precise control of both Scar and WASP activity is important for their physiological functions. Regulation of WASP activity is well characterized and has been recently reviewed elsewhere [3]. Regulation of Scar/WAVE proteins by an inhibitory complex [5] appears to be conserved throughout evolution, possibly also in plants [23,24]. In Dictyostelium, deletion of PIR121 results in a phenotype resembling overactivation of Scar, with excessive actin filament formation. Intriguingly, the PIR121-null cells contain very little intact Scar protein, suggesting a novel mechanism to regulate uncomplexed Scar by proteolysis [49]. Similarly, loss of Kette, which is the Drosophila homologue of human Nap125, results in accumulation of F-actin [50]. However, RNAi-mediated depletion of PIR121/Sra-1, Abi or Kette in Drosophila S2 cells results in a Scar-like phenotype rather than in excessive actin www.sciencedirect.com
The WASP–Arp2/3 pathway: genetic insights Vartiainen and Machesky 179
polymerization. Also, these cells display considerable reduction in Scar protein levels [14], indicating that degradation of uncomplexed Scar may be an evolutionarily conserved phenomenon. However, as deletion of PIR121 appears to result in varying phenotypes depending on the system under study, the dynamics of this process may differ. Interestingly, expression of a membrane-tethered Kette induces formation of large actin bundles in a process that is independent of Scar but dependent on WASP. Abi, another component of the Scar complex, may act as a link between Kette and WASP [50], providing a means to integrate these two signaling pathways. It remains to be established whether this kind of crosstalk also operates in other in vivo systems.
Conclusions We can conclude from a survey of the recent literature that genetic and loss-of-function studies have contributed significantly to our understanding of how actin assembly is regulated by the WASP/Scar family proteins and the Arp2/3 complex. In the late 1990s, biochemical and cell biological studies pointed to a crucial role for these proteins in signaling to actin dynamics. More recently, genetic studies have confirmed that the function of Arp2/3 complex as an actin nucleator is likely to be conserved in all eukaryotes and to be essential for life in most cases. However, Arp2/3 does not appear to be the only actin nucleator, but rather it specifically induces branched actin structures as opposed to long parallel bundles. Although many proteins can regulate Arp2/3 function in vitro, WASP/Scar family members appear to be the most important regulators in vivo. Multicellular animals have multiple WASP/Scar proteins with unique roles in specific cell types and contexts, reflecting the numerous and varied processes regulated by these proteins. Genetic studies have also poked some rather large holes in models that had previously been based on biochemical and cellular studies. Mainly, the idea of parallel pathways between the small GTPases, the different WASP/Scar family proteins and specific structures such as lamellipodia and filopodia has proven to be oversimplified. Hopefully future studies will lead to a better understanding of issues such as the relative contribution of the Arp2/3 complex and specific WASP/Scar proteins to various cell structures that use parallel and branched actin filaments. Clearly, the future holds much room for exciting discoveries in the regulation of actin assembly, as actin is one of the most abundant and versatile cellmotility and architectural proteins in eukaryotes.
Update While the review was going to press, a new study from the lab of Baum [55] showed that the Scar complex members Abi, Sra1 and Kette regulate both the stability and localization of Scar in Drosophila cells. The authors suggest www.sciencedirect.com
that the Scar complex is not only inhibitory, but regulatory, and that some components may also play a positive role in actin-based protrusion.
Acknowledgements We wish to thank colleagues for sharing their unpublished data, and regret that space limitations prevented us from citing all of the literature relevant for this review. We also thank G Bompard for the critical reading of this manuscript. MKV is funded by EMBO Long Term Fellowship, and LMM is an MRC Senior Research Fellow.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Pollard TD, Borisy GG: Cellular motility driven by assembly and disassembly of actin filaments. Cell 2003, 112:453-465. This excellent review provides a comprehensive view of the current knowledge of how actin dynamics drive cell motility. 2.
Machesky LM, Atkinson SJ, Ampe C, Vandekerckhove J, Pollard TD: Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin–agarose. J Cell Biol 1994, 127:107-115.
3.
Weaver AM, Young ME, Lee WL, Cooper JA: Integration of signals to the Arp2/3 complex. Curr Opin Cell Biol 2003, 15:23-30.
4.
Beltzner CC, Pollard TD: Identification of functionally important residues of Arp2/3 complex by analysis of homology models from diverse species. J Molec Biol 2004, 336:551-565.
5.
Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW: Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 2002, 418:790-793. This article is the first to identify the protein complex that inhibits the activity of Scar/WAVE1 in cells. It also provides the basic mechanism of Scar/WAVE activation through signal-dependent dissociation of this complex. 6.
Winter DC, Choe EY, Li R: Genetic dissection of the budding yeast Arp2/3 complex: a comparison of the in vivo and structural roles of individual subunits. Proc Natl Acad Sci U S A 1999, 96:7288-7293.
7.
Sawa M, Suetsugu S, Sugimoto A, Miki H, Yamamoto M, Takenawa T: Essential role of the C. elegans Arp2/3 complex in cell migration during ventral enclosure. J Cell Sci 2003, 116:1505-1518. This article provides in vivo evidence for the requirement for Arp2/3complex-mediated actin polymerization during cell motility. By using RNAi-mediated depletion of Arp2/3 complex subunits and WASP the authors show that these proteins play a key role in ventral enclosure in C. elegans. 8.
Severson AF, Baillie DL, Bowerman B: A Formin homology protein and a profilin are required for cytokinesis and Arp2/3-independent assembly of cortical microfilaments in C. elegans. Curr Biol 2002, 12:2066-2075.
9.
Zallen JA, Cohen Y, Hudson AM, Cooley L, Wieschaus E, Schejter ED: SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila. J Cell Biol 2002, 156:689-701. Using a genetic approach, SCAR is shown to be a more important regulator of the Arp2/3 complex during morphological processes in Drosophila than WASP. This article highlights the distinct roles of WASP and Scar/WAVE proteins in different cellular processes. 10. Hudson AM, Cooley L: A subset of dynamic actin rearrangements in Drosophila requires the Arp2/3 complex. J Cell Biol 2002, 156:677-687. 11. Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K: Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci 2001, 114:4557-4565. Current Opinion in Cell Biology 2004, 16:174–181
180 Cell regulation
12. Mathur J, Mathur N, Kernebeck B, Hulskamp M: Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis. Plant Cell 2003, 15:1632-1645. 13. Svitkina TM, Verkhovsky AB, Mcquade KM, Borisy GG: Analysis of the actin–myosin II system in fish epidermal keratocytes: mechanism of cell body translocation. J Cell Biol 1997, 139:397-415. 14. Rogers SL, Weidemann U, Stuurman N, Vale RD: Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J Cell Biol 2003, 162:1079-1088. Using a RNAi-based approach in a new, popular Drosophila cell-culture model, authors study the role of a large number of proteins in lamella formation. This article highlights the role of Scar as a regulator of Arp2/3 activity in the formation of lamellipodia, and shows that the function of the Scar regulatory complex is to inhibit Scar degradation. 15. Pelham RJ, Chang F: Actin dynamics in the contractile ring during cytokinesis in fission yeast. Nature 2002, 419:82-86. Using fission yeast as a model system, the authors show that, during cell division, the contractile ring is a dynamic structure in which actin and other ring components assemble and disassemble continuously from the ring. Actin polymerization by both a formin and the Arp2/3 complex appears to play a key role in the cleavage process.
reorganization of a dendritic network. J Cell Biol 2003, 160:409-421. How filopodia are initiated has remained a mystery for a long time. By using a combination of microscopical techniques, the authors show that dendritic actin arrays of the lamellipodia can be rearranged to form filopodia. 29. Lommel S, Benesch S, Rottner K, Franz T, Wehland J, Kuhn R: Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells. EMBO Rep 2001, 2:850-857. 30. Snapper SB, Takeshima F, Anton I, Liu C-H, Thomas SM, Nguyen D, Dudley D, Fraser H, Purich D, Lopez-Ilasaca M et al.: N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility. Nat Cell Biol 2001, 3:897-904. 31. Biyasheva A, Svitkina T, Baum B, Borisy G: Cascade pathway of filopodia formation downstream of SCAR. J Cell Sci, in press. 32. Yan C, Martinez-Quiles N, Eden S, Shibata T, Takeshima F, Shinkura R, Fujiwara Y, Bronson R, Snapper SB, Kirschner MW et al.: WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility. EMBO J 2003, 22:3602-3612.
16. Carnahan RH, Gould KL: The PCH family protein, Cdc15p, recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe. J Cell Biol 2003, 162:851-862.
33. Suetsugu S, Yamazaki D, Kurisu S, Takenawa T: Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration. Dev Cell 2003, 5:595-609.
17. Stevenson V, Hudson A, Cooley L, Theurkauf WE: Arp2/3-dependent pseudocleavage furrow assembly in syncytial Drosophila embryos. Curr Biol 2002, 12:705-711.
34. Naqvi SN, Zahn R, Mitchell DA, Stevenson BJ, Munn AL: The WASP homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast. Curr Biol 1998, 8:959-962.
18. Tal T, Vaizel-Ohayon D, Schejter ED: Conserved interactions with cytoskeletal but not signaling elements are an essential aspect of Drosophila WASP function. Dev Biol 2002, 243:260-271. 19. Mathur J, Mathur N, Kirik V, Kernebeck B, Purushottam B, Hulskamp M: Arabidopsis CROOKED encodes for the smallest subunit of the Arp2/3 complex and controls cell shape by region specific fine F-actin formation. Development 2003, 130:3137-3146. This is the first article to describe a plant homolog of Arp2/3 complex subunits, and therefore provides evidence for the evolutionarily conserved role of Arp2/3-mediated actin polymerization in very distinct cellular contexts. 20. Le J, El-Assal SED, Basu D, Saad ME, Szymanski DB: Requirements for Arabidopsis ATARP2 and ATARP3 during epidermal development. Curr Biol 2003, 13:1341-1347. 21. Li S, Blanchoin L, Yang Z, Lord EM: The putative Arabidopsis Arp2/3 complex controls leaf cell morphogenesis. Plant Physiol 2003, 132:2034-2044. 22. Deeks MJ, Hussey PJ: Arp2/3 and ‘The shape of things to come’. Curr Opin Plant Biol 2003, 6:561-567. 23. Frank MJ, Smith LG: A small, novel protein highly conserved in plants and animals promotes the polarized growth and division of maize leaf epidermal cells. Curr Biol 2002, 12:849-853. 24. Frank MJ, Cartwright HN, Smith LG: Three Brick genes have distinct functions in a common pathway promoting polarized cell division and cell morphogenesis in the maize leaf epidermis. Development 2003, 130:753-762. 25. Rodal AA, Manning AL, Goode BL, Drubin DG: Negative regulation of yeast WASP by two SH3-domain-containing proteins. Curr Biol 2003, 13:1000-1008. 26. Soto MC, Qadata H, Kasuya H, Inoue M, Tsuboi D, Mello CC, Kaibuchi K: The GEX-2 and GEX-3 proteins are required for tissue morphogenesis and cell migrations in C. elegans. Genes Dev 2002, 16:620-632. 27. Ben-Yaacov S, Le Borgne R, Abrahamson I, Scheweisguth F, Schejter ED: WASP, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling. J Cell Biol 2001, 152:1-13. 28. Svitkina TM, Bulanova EA, Chaga OY, Vignjevic DM, Kojima S, Vasiliev JM, Borisy GG: Mechanism of filopodia initiation by Current Opinion in Cell Biology 2004, 16:174–181
35. Kessels MM, Qualmann B: Syndapins integrate N-WASP in receptor mediated endocytosis. EMBO J 2002, 21:6083-6094. 36. Chang FS, Stefan CJ, Blumer KJ: A WASP homolog powers actin polymerization-dependent motility of endosomes in vivo. Curr Biol 2003, 13:455-463. 37. Benesch S, Lommel S, Steffen A, Stradal TEB, Scaplehorn N, Way M, Wehland J, Rottner K: Phosphatidylinositol 4,5biphosphate (PIP2)-induced vesicle movement depends on N-WASP and involves Nck, WIP, and Grb2. J Biol Chem 2002, 277:37771-37776. 38. Seastone DJ, Harris E, Temesvari LA, Bear JE, Saxe CL, Cardelli J: The WASP-like protein Scar regulates macropinocytosis, phagocytosis and endosomal membrane flow in Dictyostelium. J Cell Sci 2001, 114:2673-2683. 39. Notarangelo LD, Ochs HD: Wiskott-Aldrich syndrome: a model for defective actin reorganization, cell trafficking and synapse formation. Curr Opin Immunol 2003, 15:585-591. 40. Thrasher AJ: WASP in immune-system organization and function. Nat Rev Immunol 2002, 2:635-646. 41. Badour K, Zhang J, Shi F, McGavin MK, Ramperstad V, Hardy LA, Field D, Siminovitch KA: The Wiskott-Aldrich syndrome protein acts downstream of CD2 and the CD2AP and PSTPIP1 adaptors to promote formation of the immunological synapse. Immunity 2003, 18:141-154. 42. Orange JS, Ramesh N, Remold-O’Donnell E, Sasahara Y, Koopman L, Byrne M, Bonilla FA, Rosen FS, Geha RS, Strominger JL: Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunological synapses. Proc Natl Acad Sci U S A 2002, 99:11351-11356. 43. Dupre L, Aiuti A, Trifari S, Martino S, Saracco P, Bordignon C, Roncarolo MG: Wiskott-Aldrich syndrome protein regulates lipid raft dynamics during immunological synapse formation. Immunity 2002, 17:157-166. 44. Burns S, Thrasher AJ, Blundell MP, Machesky L, Jones GE: Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS protein, and differentiation. Blood 2001, 98:1142-1149. 45. Falet H, Hoffmeister KM, Neujahr R, Hartwig JH: Normal Arp2/3 complex activation in platelets lacking WASP. Blood 2002, 100:2113-2122. www.sciencedirect.com
The WASP–Arp2/3 pathway: genetic insights Vartiainen and Machesky 181
46. Dahl JP, Wang-Dunlop J, Gonzales C, Goad MEP, Mark RJ, Kwak SP: Characterization of the WAVE-1 knock-out mouse: implications for CNS development. J Neurosci 2003, 23:3343-3352.
51. Morrell JL, Morphew M, Gould KL: A mutant of Arp2p causes partial disassembly of the Arp2/3 complex and loss of cortical actin function in fission yeast. Mol Biol Cell 1999, 10:4201-4215.
47. Soderling SH, Langeberg LK, Soderling JA, Davee SM, Simerly R, Raber J, Scott JD: Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Proc Natl Acad Sci U S A 2003, 100:1723-1728.
52. Li R: Bee1, a yeast protein with homology to Wiskott-Aldrich syndrome protein, is critical for the assembly of cortical actin cytoskeleton. J Cell Biol 1997, 136:649-658.
48. Yamazaki D, Suetsugu S, Miki H, Kataoka Y, Nishikawa SI, Fujiwara T, Yoshida N, Takenawa T: WAVE2 is required for directed cell migration and cardiovascular development. Nature 2003, 424:452-456. 49. Blagg SL, Stewart M, Sambles C, Insall RH: PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium. Curr Biol 2003, 13:1480-1487. Using PIR121-null Dictyostelium, the authors demonstrate that PIR121 also regulates Scar activity in this organism. This article also contains the first observation of degradation of the uncomplexed Scar. 50. Bogdan S, Klambt C: Kette regulates actin dynamics and genetically interacts with Wave and WASP. Development 2003, 130:4427-4437.
www.sciencedirect.com
53. Bear JE, Rawls JF, Saxe CL III: SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development. J Cell Biol 1998, 142:1325-1335. 54. Zhang J, Shehabeldin A, da Cruz LAG, Butler J, Somani AK, McGavin M, Kozieradzki I, dos Santos AO, Nagy A, Grinstein S et al.: Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes. J Exp Med 1999, 190:1329-1341. 55. Kunda P, Craig G, Dominguez D, Baum B: Abi, Sra1 and Kette control the stability and localization of Scar/WAVE to regulate the formation of actin-based protrusions. Curr Biol 2003, 13:1867-1875.
Current Opinion in Cell Biology 2004, 16:174–181