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BRAFV600E is the most common driver mutation in human cutaneous melanoma and is frequently accompanied by loss of the tumor-suppressing phosphatase PTEN. Recent evidence suggests a co-operative role for RAC1 activity in BRAFV600E-driven melanoma progression and drug resistance. However, the underlying molecular mechanisms and the role of RAC1 downstream targets are not well-explored. In this study, we examine the role of the NCKAP1 subunit of the pentameric cytoskeletal SCAR/WAVE complex, a major downstream target of RAC1, in a mouse model of melanoma driven by BRAFV600E;PTEN loss. The SCAR/WAVE complex is the major driver of lamellipodia formation and cell migration downstream of RAC1 and depends on NCKAP1 for its integrity. Targeted deletion of Nckap1 in the melanocyte lineage delayed tumor onset and progression of a mutant Braf;Pten loss‒driven melanoma mouse model. Nckap1-depleted tumors displayed fibrotic stroma with increased collagen deposition concomitant with enhanced immune infiltration. Nckap1 loss slowed proliferation and tumor growth, highlighting a role in cell-cycle progression. Altogether, we propose that NCKAP1-orchestrated actin polymerization is essential for tumor progression and maintenance of tumor tissue integrity in a mutant Braf/Pten loss‒driven mouse model for melanoma.

Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. Here we show that N-WASP is required for the metastatic process, with roles in both chemotaxis, steering cells out of the tumour, and matrix remodelling, allowing them to escape. Lysophosphatidic acid, a signalling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for PDAC cells. We find they efficiently break LPA down as they respond to it, setting up a self-generated gradient that directs cells out of the tumour. N-WASP depleted cells are unable to respond to LPA gradients and show altered RhoA activation, leading to a loss of cell contractility and traction forces, and reduced metastasis in vitro and in vivo. N-WASP couples LPA receptor signalling to RhoA via the endocytic adapter SNX18, and promotes sensitivity by recycling the receptor back to the surface. Coordinated by N-WASP, the LPA-LPAR signalling loop promotes RhoA-mediated contractility and force generation. Perturbing this pathway chemically or by CRISPR deletion causes PDAC cells to lose their ability to invade through complex 3D environments or peritoneal explants, and remodel fibrillar collagen. We thus reveal N-WASP as a central controller of a chemotactic loop between PDAC cells and microenvironmental conditions that drives metastasis.

How cells breach basement membrane barriers remains an area of active research. In this issue of Developmental Cell, Kelley et al. (2019) reveal that the C. elegans anchor cell uses physical force to breach basement membrane in the absence of matrix metalloproteases during its developmental invasion.

Cell polarity arises out of asymmetry of the distribution and organisation of cell contents. Polarity is an important feature of all living organisms and much energy is devoted to breaking symmetry and establishing polarity. Recent developments in our understanding of how the budding yeast Saccharomyces cerevisiae establishes and maintains polarity for cell division shed light on universal mechanisms that may be relevant to both asymmetric cell division and polarised cell migration in other organisms. Here, we summarise some of the recent developments in our understanding of polarity of the cytoskeleton and associated signalling molecules as it relates to cell migration. Parallels are drawn between planar cell polarity and apical-basal polarity in epithelial tissues and front-back polarity in migrating cells.

The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P2. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P2 or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD–lipid interaction falls into the 78–170 μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD–lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD–lipid interaction.

Around a fifth of melanomas exhibit an activating mutation in the oncogene NRas that confers constitutive signaling to proliferation and promotes tumor initiation. NRas signals downstream of the major melanocyte tyrosine kinase receptor c-kit and activated NRas results in increased signaling via the extracellular signal-regulated kinase (ERK)/MAPK/ERK kinase/mitogen-activated protein kinase (MAPK) pathways to enhance proliferation. The Ras oncogene also activates signaling via the related Rho GTPase Rac1, which can mediate growth, survival, and motility signaling. We tested the effects of activated NRas(Q61K) on the proliferation, motility, and invasiveness of melanoblasts and melanocytes in the developing mouse and ex vivo explant culture as well as in a melanoma transplant model. We find an important role for Rac1 downstream of NRas(Q61K) in mediating dermal melanocyte survival in vivo in mouse, but surprisingly NRas(Q61K) does not appear to affect melanoblast motility or proliferation during mouse embryogenesis. We also show that genetic deletion or pharmacological inhibition of Rac1 in NRas(Q61K) induced melanoma suppresses tumor growth, lymph node spread, and tumor cell invasiveness, suggesting a potential value for Rac1 as a therapeutic target for activated NRas-driven tumor growth and invasiveness.Journal of Investigative Dermatology advance online publication, 21 June 2012; doi:10.1038/jid.2012.186.

Summary Background SCAR/WAVE is a principal regulator of pseudopod growth in crawling cells. It exists in a stable pentameric complex, which is regulated at multiple levels that are only beginning to be understood. SCAR/WAVE is phosphorylated at multiple sites, but how this affects its biological activity is unclear. Here we show that dephosphorylation of Dictyostelium SCAR controls normal pseudopod dynamics. Results We demonstrate that the C-terminal acidic domain of most Dictyostelium SCAR is basally phosphorylated at four serine residues. A small amount of singly phosphorylated SCAR is also found. SCAR phosphorylation site mutants cannot replace SCAR's role in the pseudopod cycle, though they rescue cell size and growth. Unphosphorylatable SCAR is hyperactive—excessive recruitment to the front results in large pseudopods that fail to bifurcate because they continually grow forward. Conversely, phosphomimetic SCAR is weakly active, causing frequent small, disorganized pseudopods. Even in its regulatory complex, SCAR is normally held inactive by an interaction between the phosphorylated acidic and basic domains. Loss of basic residues complementary to the acidic phosphosites yields a hyperactive protein similar to unphosphorylatable SCAR. Conclusions Regulated dephosphorylation of a fraction of the cellular SCAR pool is a key step in SCAR activation during pseudopod growth. Phosphorylation increases autoinhibition of the intact complex. Dephosphorylation weakens this interaction and facilitates SCAR activation but also destabilizes the protein. We show that SCAR is specifically dephosphorylated in pseudopods, increasing activation by Rac and lipids and supporting positive feedback of pseudopod growth.

SummaryDuring embryogenesis, melanoblasts proliferate and migrate ventrally through the developing dermis and epidermis as single cells. Targeted deletion of Rac1 in melanoblasts during embryogenesis causes defects in migration, cell-cycle progression, and cytokinesis. Rac1 null cells migrate markedly less efficiently, but surprisingly, global steering, crossing the dermal/epidermal junction, and homing to hair follicles occur normally. Melanoblasts navigate in the epidermis using two classes of protrusion: short stubs and long pseudopods. Short stubs are distinct from blebs and are driven by actin assembly but are independent of Rac1, Arp2/3 complex, myosin, or microtubules. Rac1 positively regulates the frequency of initiation of long pseudopods, which promote migration speed and directional plasticity. Scar/WAVE and Arp2/3 complex drive actin assembly for long pseudopod extension, which also depends on microtubule dynamics. Myosin contractility balances the extension of long pseudopods by effecting retraction and allowing force generation for movement through the complex 3D epidermal environment.

Cell migration is an essential feature of eukaryotic life, required for processes ranging from feeding and phagoctyosis to development, healing, and immunity. Migration requires the actin cytoskeleton, specifically the localized polymerization of actin filaments underneath the plasma membrane. Here we summarize recent developments in actin biology that particularly affect structures at the leading edge of the cell, including the structure of actin branches, the multiple pathways that lead to cytoskeleton assembly and disassembly, and the role of blebs. Future progress depends on connecting these processes and components to the dynamic behavior of the whole cell in three dimensions.

Summary. Background: During platelet spreading, the actin cytoskeleton undergoes marked changes, forming filopodia, lamellipodia and stress fibres. In the present study, we report the identification of a novel actin-rich structure, termed an actin nodule, which appears prior to lamellipodia and stress fibre formation. Methods: Platelet spreading was monitored using human platelets and mouse GFP-actin platelets using real-time and end-point DIC, and fluorescent and electron microscopy (EM). Results: We identified a small, novel actin structure, the actin nodule, in the early stages of adhesion and spreading, which we hypothesize to be a precursor of lamellipodia and stress fibres. Nodule formation shows an inverse correlation to Rho kinase and myosin-II activity, is independent of PI3kinase, but dependent on Src kinase activity. Actin nodules contain multiple proteins, including Arp2/3, Fyn, Rac, and b1and b3- integrins, but not Src. EM analysis revealed that actin filaments extend in all directions from the nodules. Actin nodules are present on multiple matrices, including fibrinogen, laminin and VWF + botrocetin. Conclusion: This work identifies a novel platelet actin structure, which we propose is a precursor to both lamellipodia and stress fibres and acts to drive platelet spreading.

The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.

Endocytosis is an important way for cells to take up liquids and particles from their environment. It requires membrane bending to be coupled with membrane fission, and the actin cytoskeleton has an active role in membrane remodelling. Here, we review recent research into the function of Bin-Amphiphysin-Rvs (BAR) domain proteins, which can sense membrane curvature and recruit actin to membranes. BAR proteins interact with the endocytic and cytoskeletal machinery, including the GTPase dynamin (which mediates vesicle fission), N-WASP (an Arp2/3 complex regulator) and synaptojanin (a phosphoinositide phosphatase). We describe three classes of BAR domains, BAR, N-BAR and F-BAR, providing examples of each discussing and how they function in linking membranes to the actin cytoskeleton in endocytosis.

Recent studies have shown that platelet adhesion and subsequent aggregation can occur in vivo in the absence of the two principal platelets adhesive ligands, von Willebrand factor and fibrinogen. These results highlight a possible role for fibronectin in supporting thrombus formation.To evaluate the platelet integrins and subsequent activation pathways associated with fibronectin-dependent platelet adhesion utilizing both human and murine platelets.Platelets can adhere to fibronectin via the integrin alpha(IIb)beta(3), leading to formation of lamellipodia. This is mediated through an interaction with the tenth type III domain in fibronectin. Spreading on fibronectin promotes alpha(IIb)beta(3)-mediated Ca(2+) mobilization and tyrosine phosphorylation of focal adhesion kinase and phospholipase C gamma2. In contrast, studies with blocking antibodies and mice demonstrate that alpha(5)beta(1) and alpha(v)beta(3) support adhesion and promote formation of filopodia but not lamellipodia or tyrosine phosphorylation of these proteins. Further, neither alpha(5)beta(1) nor alpha(v)beta(3) is able to induce formation of lamellipodia in the presence of platelets agonists, such as collagen-related-peptide (CRP).These observations demonstrate that integrins alpha(5)beta(1) and alpha(v)beta(3) support platelet adhesion and the generation of filopodia but that, in contrast to the integrin alpha(IIb)beta(3), are unable to promote formation of lamellipodia.

Background/Aims : Previously, trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to exhibit strong antifibrotic characteristics in hepatic stellate cells (HSC), which are known to play a central role in chronic liver diseases. TSA retained a more quiescent phenotype in spite of culture conditions that favor transdifferentiation into activated HSC. Methods : To identify TSA-sensitive genes, differential mRNA display, Northern and Western blot analysis were used and genes were functionally validated by using contraction and motility assays. Results : TSA prevented new actin filament formation by down-regulation of two nucleating proteins, actin related protein 2 (Arp2) and Arp3, and by up-regulation of adducin like protein 70 (ADDL70) and gelsolin, two capping proteins. RhoA, a key mediator in the development of the actin cytoskeleton, decreased following TSA exposure. Expression of proteins of Class III intermediate filaments was affected by TSA. Furthermore, F-actin and G-actin were expressed heterogeneously under influence of TSA. Functionally, TSA treatment abrogated migration of quiescent HSC, while migration was reduced in transitional HSC. The endothelin-1-induced contractility properties of HSC was not affected by TSA. Conclusions : These data indicate that TSA affects the development of the actin cytoskeleton in quiescent HSC and thereby abrogates the process of HSC transdifferentiation.

Phagocytosis through Fcγ receptor (FcγR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway [1]. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcγR phagocytosis, while Rho controls actin assembly at CR phagosomes [2, 3]. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho → ROK → myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcγR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcγR phagocytosis [4–6], to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.

Background APC loss is a common initiating event in colorectal cancer but additional mutations are required for progression along the adenoma–carcinoma pathway. The Neural Wiskott–Aldrich syndrome protein (N-WASP) is crucial for invasion in a variety of cancers. Its role in colorectal cancer has not been investigated; however, it is implicated in Wnt signalling (the pathway affected when APC function is lost). The aim of this project was to investigate the role of N-WASP in intestinal tumorigenesis and its potential as a prognostic biomarker. Methods The effect of N-Wasp knockout (N-Waspfl/fl) on survival and tumour burden was investigated using two established murine models of intestinal tumorigenesis (Apcfl/+ and Apcfl/+ KrasG12D/+). Immunohistochemistry for N-WASP was performed on two human tissue microarrays—namely, colorectal cancers with linked clinicopathological data and long-term follow-up, and early cancers diagnosed via bowel screening. N-WASP expression was assessed by weighted histoscore, and correlation with survival was tested. Findings N-Wasp knockout decreased survival in the Apcfl/+ model (median survival 143 days [IQR 127·3–167·0] vs 269 [228·8–304·5], p vs 8 [4·34], p 2 [SD 0·238] vs 1·547 [0·609], p=0·0317), there was no difference in mean size of the largest tumour. There was no effect of N-Wasp knockout on survival or small intestinal tumour burden in the Apcfl/+ KrasG12D/+ model. However, they developed fewer colonic tumours (mean 16·8 [SD 10·2] vs 35·4 [11·5], p=0·0018). In the human tissue microarray studies, there was no difference in overall or cancer-specific survival between tumours with high and low N-WASP expression. Interpretation We found that loss of N-Wasp promotes early tumorigenesis in Apc-deficient intestinal epithelium in mice. N-WASP stabilises epithelial cell–cell junctions and might be a regulator of Wnt signalling. Further research is required to ascertain whether either of these roles explains the effects seen here and whether it promotes invasion and metastasis in more advanced intestinal cancers, as seen in other tissues. Funding Funded by a Medical Research Council grant via the Scottish Clinical Pharmacology and Pathology Programme (SCP3).

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5–7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.

Although recent years have yielded many insights into the molecular basis for cell motility, this complex process still holds many secrets. Recent models have suggested that a major active signal to nucleate new networks of actin filaments comes from a pathway known as the WASP-Arp2/3 pathway (reviewed in Machesky and Insall 1999xMachesky, L.M. and Insall, R.H. J. Cell Biol. 1999; 146: 267–272Crossref | PubMed | Scopus (199)See all ReferencesMachesky and Insall 1999). The Ena/VASP proteins facilitate the formation of new networks through enhancement of filament elongation and filament capture (Laurent et al. 1999xLaurent, V., Loisel, T.P., Harbeck, B., Wehman, A., Groebe, L., Jockusch, B.M., Wehland, J., Gertler, F.B., and Carlier, M.F. J. Cell Biol. 1999; 144: 1245–1258Crossref | PubMed | Scopus (237)See all ReferencesLaurent et al. 1999). Now we must incorporate the idea that Ena/VASP proteins may actually slow eukaryotic cell translocation although they speed up Listeria translocation (1xBashaw, G.J., Kidd, T., Murray, D., Pawson, T., and Goodman, C.S. Cell. 2000; 101: 703–715Abstract | Full Text | Full Text PDF | PubMedSee all References, 2xBear, J.E., Loureiro, J.J., Libova, I., Fassler, R., Wehland, J., and Gertler, F.B. Cell. 2000; 101: 717–728Abstract | Full Text | Full Text PDF | PubMedSee all References). How can we reconcile these apparently contradictory findings?First, it is probably much too simplistic to assume that increased actin polymerization correlates directly with increased translocation. For example, Dictyostelium cells show a maximum of assembled actin in response to a pulse of extracellular cAMP at a point when the cells are completely rounded up prior to polarization and increased translocation. This cringe response is not a migratory state, but the cells have up to 3-fold more F-actin than resting cells (Hall et al. 1988xHall, A.L., Schlein, A., and Condeelis, J. J. Cell. Biochem. 1988; 37: 285–299Crossref | PubMedSee all ReferencesHall et al. 1988). Furthermore, many of the signaling molecules generally thought to trigger actin polymerization, such as the small GTPases Rac1 and RhoA, can trigger neurite retraction or even growth cone collapse. It is unclear whether a rise in F-actin accompanies a pause, retraction or even collapse, but this should be testable. Small local amounts of the actin polymerization blocking drug cytochalasin D can actually increase neurite extension, perhaps by allowing the microtubule cytoskeleton to penetrate the cell cortex and advance the leading edge (Lanier and Gertler 2000xLanier, L.M. and Gertler, F.B. Curr. Opin. Neurobiol. 2000; 10: 80–87Crossref | PubMed | Scopus (161)See all ReferencesLanier and Gertler 2000). Clearly, the role of F-actin in cell translocation is complex, so simply equating F-actin amount or even turnover with rate of translocation is oversimplistic.Listeria cells appear to have a simple built-in polarity—so any increase in actin polymerization would be expected to enhance the translocation speed—but eukaryotic motile cells exhibit much more complex types of movement. Perhaps the shape and polarity of a eukaryotic cell may be a better indicator of its speed than the amount of filamentous actin or the rate of actin filament turnover. Rapidly moving fibroblasts or Dictyostelium cells most often show a very polarized phenotype—the cell is elongated with a clear uropod at the rear and a small but consistent lamellipodium at the front. Stationary cells or cells that are changing direction often show multiple pseudopodia and lamellipodia in all directions. This could be analogous to the pause and turn state of an axon during pathfinding. If the axon needs to make a decision, it may use Ena, whether downstream of the repulsive receptor Robo or other attractive/repulsive receptors, to enhance the production of actin-based filopodia and lamellipodia while it pauses and explores the environment. Furthermore, neurons could require actin assembly for both protrusion and retraction. Contractility, as induced by Rho GTPases, requires large actin-myosin networks, which are probably assembled in response to cues such as repulsion (Kozma et al. 1997xKozma, R., Sarner, S., Ahmed, S., and Lim, L. Mol. Cell Biol. 1997; 17: 1201–1211Crossref | PubMedSee all ReferencesKozma et al. 1997). Figure 3Figure 3 shows a cartoon sketch of some different shapes of motile cells in what could be termed pause and explore versus rapid polarized translocation. It is easy to imagine that the actin polymerization requirements for pause and explore might be quite high and that a cell might concentrate its efforts into only a small leading edge area when it translocates in a single direction. If this model holds any truth, we might expect to see both repulsive and attractive receptors coupling to Ena and other parameters, such as the location of activation, to be variable.Figure 3Examples of Cells Translocating Unidirectionally toward a Target versus Cells Migrating Randomly or Pausing to Sample the EnvironmentRed indicates areas of rapid actin filament assembly.View Large Image | View Hi-Res Image | Download PowerPoint SlideAnother plausible way to think of Ena/VASP proteins as translocation restrictors is that the kinds of effects that Ena/VASP have on actin filaments may be to promote a stable network that enhances adhesion and/or pauses the leading edge. Both cell–cell and cell–substratum adhesion have the effect of slowing down cell translocation and both are likely to require actin polymerization. The effects of Ena/VASP proteins at the leading edges of cells might be to promote adhesion or alternatively to modify the structure of actin networks nucleated by the Arp2/3 complex. Lanier et al. 1999xLanier, L.M., Gates, M.A., Witke, W., Menzies, A.S., Wehman, A.M., Macklis, J.D., Kwiatkowski, D., Soriano, P., and Gertler, F.B. Neuron. 1999; 22: 313–325Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesLanier et al. 1999 found Mena at the tips of growth cone filopodia, so perhaps Ena/VASP reshape actin into parallel bundles, allowing structures such as filopodia to protrude from branched lamellipodial networks. These filopodia could then form the basis for cell–cell junctions in keratinocytes or cell–substratum exploration in neurites or fibroblasts. It would be interesting to use fluorescence microscopy to examine directly the effects of Ena/VASP proteins on dendritic networks produced by the Arp2/3 complex in vitro (Blanchoin et al. 2000xBlanchoin, L., Amann, K.J., Higgs, H.N., Marchand, J.B., Kaiser, D.A., and Pollard, T.D. Nature. 2000; 404: 1007–1011Crossref | PubMed | Scopus (356)See all ReferencesBlanchoin et al. 2000).Clearly, many questions arise as to the reconciliation of the role of Ena/VASP proteins in Listeria translocation, in vitro and in eukaryotic cells. Some of the questions will probably be answered by resolution of the differences between genetic systems, where expression levels and cellular context within a whole organism are directing the results and biochemical systems that are oversimplified at times but allow direct access to molecular mechanisms. Other questions will be of great biological interest, such as the real function of filopodia and lamellipodia in an advancing neurite, the different contexts in which actin assembly is triggered, and the fine-tuning of the cytoskeleton by numerous signaling and actin binding proteins that results in all of the complex behaviors of motile cells.*E-mail: l.m.machesky@bham.ac.uk

Background: Phosphatidylinositol 4,5-bisphosphate (PIP2) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol–sphingolipid-rich membrane microdomains called ‘rafts’, in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. Results: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP2, promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP2 levels, suggesting that it induces comets by changing PIP2 homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-β-cyclodextrin reduced comets, establishing that rafts promote comet formation. Conclusions: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP2 synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.

Identification of bona fide tumor suppressors is often challenging because of the large number of alterations present in most human cancers. To evaluate candidates present within regions recurrently deleted in human cancers we coupled high-resolution genomic analysis with a two-stage genetic study using RNA interference (RNAi). We found that Cyfip1, a subunit of the WAVE complex, which regulates cytoskeletal dynamics, is commonly deleted in human epithelial cancers. Reduced expression of Cyfip1 is commonly observed during invasion of epithelial tumors and it associated with poor prognosis in same tumor types. Silencing of Cyfip1 disturbed normal epithelial morphogenesis in vitro and cooperated with oncogenic Ras to produce invasive carcinomas in vivo. Mechanistically, we have linked alterations in WAVE-regulated actin dynamics with impaired cell-cell adhesion and cell-ECM interactions. Thus, we propose Cyfip1 as an invasion suppressor gene.

Molecules involved in the interaction between the extracellular matrix, cell membrane and cytoskeleton are of central importance in morphogenesis. Talin is a large cytoskeletal protein with a modular structure consisting of an amino-terminal membrane-interacting domain, with sequence similarities to members of the band 4.1 family, and a carboxy-terminal region containing F-actin-binding and vinculin-binding domains [1,2]. It also interacts with the cytoplasmic tail of β integrins which, on the external face of the membrane, bind to extracellular matrix proteins [3]. The possible roles of talin in multicellular morphogenesis in development remain largely unexplored. In Dictyostelium , a eukaryotic microorganism capable of multicellular morphogenesis, a talin homologue (TALA) has previously been identified and shown to play an important role in cell-to-substrate adhesion and maintenance of normal elastic properties of the cell [4–6]. Here, we describe a second talin homologue (TALB) that is required for multicellular morphogenesis in the development of Dictyostelium . Unlike any other talin characterised to date, it contains an additional carboxy-terminal domain homologous to the villin headpiece.

The actin-related proteins (Arps) constitute a recently characterized family of proteins, many of which function as members of multiprotein complexes. The discovery that two family members, Arp2 and Arp3, act as multifunctional organizers of actin filaments in all eukaryotes has generated much excitement. Over the past two years, newly discovered properties of the Arp2/3 complex have suggested a central role in the control of actin polymerization. First, it promotes actin assembly on the surface of the motile intracellular pathogen Listeria monocytogenes. Second, it can nucleate and cross-link actin filaments in vitro. Third, it localizes with dynamic actin-rich spots of mammalian cells suggesting a role in protrusion; it is found in cortical actin patches in the budding and fission yeasts where it may control patch movement and cortical actin function. Clearly, the complex has a central role in actin cytoskeletal function and will be the subject of much research in the coming years.

Cell motility requires actin assembly mediated by Rac, a Rho family GTPase. In this issue, Palamidessi et al. (2008) show that Rab5-directed trafficking of Rac to the cell membrane is required for Rac-mediated actin assembly. Trafficking of Rac may influence the mode of cell migration during morphogenesis and cancer metastasis.

Recent studies suggest that a family of proteins known as ‘IQGAPs’, which contain several previously characterized sequence domains that could connect cell-signalling pathways to the actin cytoskeleton, are involved in forming actin rings during cytokinesis.

The Rho family of GTP-binding proteins has yielded fresh insights into cell signalling in relation to motility, shape and the control of the actin cytoskeleton. Rho itself is probably near the top of several diverse signalling cascades and has been implicated in cell adhesion, actin filament organization, control of mitogen-activated protein kinase pathways and phospholipid synthesis and turnover. As a member of the Ras superfamily, Rho is regulated by GDP-GTP exchange factors (GEFs) that have homology to the dbl oncogene, and by GTPase-activating proteins (GAPs). These proteins regulate the nucleotide (GDP or GTP) bound to Rho, thus determining the activity of Rho and the interactions of Rho with many of its downstream targets. In the past year, many new targets of Rho have been identified, which hopefully will uncover molecular connections among the diverse downstream effects of Rho activation.

Cdc42 is a key regulator of cell polarity and actin dynamics. One of its effectors, WASP, initiates the assembly of new actin filaments. In this issue of Cell, Ho et al. (2004) show that a previously unknown regulator named Toca-1 is required for Cdc42 to activate WASP. This discovery changes our picture of how small GTPases and multiple other signals converge to stimulate actin polymerization and cell motility.

The involvement of Nck in pedestal formation by EPEC highlights the similar strategies adopted by this bacterium and the Vaccinia virus to hijack the host cell's cytoskeleton.

DEAD-box RNA helicases, defined by the sequence Asp–Glu–Ala–Asp (DEAD, in single-letter amino-acid code), regulate RNA unwinding and secondary structure in an ATP-dependent manner in vitro [1] and control mRNA stability and protein translation. Both yeast and mammals have large families of DEAD-box proteins, many of unknown function. We have disrupted a Dictyostelium discoideum gene, helC , which encodes helicase C, a member of the DEAD-box family of RNA helicases that shows strong homology to the product of the essential Saccharomyces cerevisiae gene dbp5 [2] and to related helicases in mouse and Schizosaccharomyces pombe . The HelC protein also shows weaker homology to the translation initiation factor elF-4a. Other DEAD-box-containing proteins, which are less closely related to HelC, have been implicated in developmental roles in Drosophila [3] and Xenopus laevis; one example is the Xenopus Vasa-like protein (XVLP) [4–6]. In Drosophila and Xenopus , Vasa and XVLP, respectively, are required for the establishment of tissue polarity during development. In yeast, DEAD-box helicases such as Prp8 [7] are components of the spliceosome and connect pre-mRNA splicing with the cell cycle. Disruption of the helC gene in D. discoideum led to developmental asynchrony, failure to differentiate and aberrant morphogenesis. We postulate that one reason for the existence of large families of homologous DEAD-box proteins in yeast, mammals and Dictyostelium could be that some DEAD-box proteins have developmentally specific roles regulating protein translation or mRNA stability.