Scar/WAVE drives actin protrusions independently of its VCA domain using proline-rich domains.

Cell migration requires the constant modification of cellular shape by reorganization of the actin cytoskeleton. Fine-tuning of this process is critical to ensure new actin filaments are formed only at specific times and in defined regions of the cell. The Scar/WAVE complex is the main catalyst of pseudopod and lamellipodium formation during cell migration. It is a pentameric complex highly conserved through eukaryotic evolution and composed of Scar/WAVE, Abi, Nap1/NCKAP1, Pir121/CYFIP, and HSPC300/Brk1. Its function is usually attributed to activation of the Arp2/3 complex through Scar/WAVE's VCA domain, while other parts of the complex are expected to mediate spatial-temporal regulation and have no direct role in actin polymerization. Here, we show in both B16-F1 mouse melanoma and Dictyostelium discoideum cells that Scar/WAVE without its VCA domain still induces the formation of morphologically normal, actin-rich protrusions, extending at comparable speeds despite a drastic reduction of Arp2/3 recruitment. However, the proline-rich regions in Scar/WAVE and Abi subunits are essential, though either is sufficient for the generation of actin protrusions in B16-F1 cells. We further demonstrate that N-WASP can compensate for the absence of Scar/WAVE's VCA domain and induce lamellipodia formation, but it still requires an intact WAVE complex, even if without its VCA domain. We conclude that the Scar/WAVE complex does more than directly activating Arp2/3, with proline-rich domains playing a central role in promoting actin protrusions. This implies a broader function for the Scar/WAVE complex, concentrating and simultaneously activating many actin-regulating proteins as a lamellipodium-producing core.

Formation and closure of macropinocytic cups in Dictyostelium.

Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment.

An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome.

Cellular condensates can comprise membrane-less ribonucleoprotein assemblies with liquid-like properties. These cellular condensates influence various biological outcomes, but their liquidity hampers their isolation and characterization. Here, we investigated the composition of the condensates known as processing bodies (PBs) in the model plant Arabidopsis thaliana through a proximity-biotinylation proteomics approach. Using in situ protein-protein interaction approaches, genetics and high-resolution dynamic imaging, we show that processing bodies comprise networks that interface with membranes. Surprisingly, the conserved component of PBs, DECAPPING PROTEIN 1 (DCP1), can localize to unique plasma membrane subdomains including cell edges and vertices. We characterized these plasma membrane interfaces and discovered a developmental module that can control cell shape. This module is regulated by DCP1, independently from its role in decapping, and the actin-nucleating SCAR-WAVE complex, whereby the DCP1-SCAR-WAVE interaction confines and enhances actin nucleation. This study reveals an unexpected function for a conserved condensate at unique membrane interfaces.

AKT and SGK kinases regulate cell migration by altering Scar/WAVE complex activation and Arp2/3 complex recruitment.

Cell polarity and cell migration both depend on pseudopodia and lamellipodia formation. These are regulated by coordinated signaling acting through G-protein coupled receptors and kinases such as PKB/AKT and SGK, as well as the actin cytoskeletal machinery. Here we show that both Dictyostelium PKB and SGK kinases (encoded by pkbA and pkgB) are dispensable for chemotaxis towards folate. However, both are involved in the regulation of pseudopod formation and thus cell motility. Cells lacking pkbA and pkgB showed a substantial drop in cell speed. Actin polymerization is perturbed in pkbA- and reduced in pkgB- and pkbA-/pkgB- mutants. The Scar/WAVE complex, key catalyst of pseudopod formation, is recruited normally to the fronts of all mutant cells (pkbA-, pkgB- and pkbA-/pkgB-), but is unexpectedly unable to recruit the Arp2/3 complex in cells lacking SGK. Consequently, loss of SGK causes a near-complete loss of normal actin pseudopodia, though this can be rescued by overexpression of PKB. Hence both PKB and SGK are required for correct assembly of F-actin and recruitment of the Arp2/3 complex by the Scar/WAVE complex during pseudopodia formation.

The Amoebal Model for Macropinocytosis.

Macropinocytosis is a relatively unexplored form of large-scale endocytosis driven by the actin cytoskeleton. Dictyostelium amoebae form macropinosomes from cups extended from the plasma membrane, then digest their contents and absorb the nutrients in the endo-lysosomal system. They use macropinocytosis for feeding, maintaining a high rate of fluid uptake that makes assay and experimentation easy. Mutants collected over the years identify cytoskeletal and signalling proteins required for macropinocytosis. Cups are organized around plasma membrane domains of intense PIP3, Ras and Rac signalling, proper formation of which also depends on the RasGAPs NF1 and RGBARG, PTEN, the PIP3-regulated protein kinases Akt and SGK and their activators PDK1 and TORC2, Rho proteins, plus other components yet to be identified. This PIP3 domain directs dendritic actin polymerization to the extending lip of macropinocytic cups by recruiting a ring of the SCAR/WAVE complex around itself and thus activating the Arp2/3 complex. The dynamics of PIP3 domains are proposed to shape macropinocytic cups from start to finish. The role of the Ras-PI3-kinase module in organizing feeding structures in unicellular organisms most likely predates its adoption into growth factor signalling, suggesting an evolutionary origin for growth factor signalling.

Extracellular Signalling Modulates Scar/WAVE Complex Activity through Abi Phosphorylation.

The lamellipodia and pseudopodia of migrating cells are produced and maintained by the Scar/WAVE complex. Thus, actin-based cell migration is largely controlled through regulation of Scar/WAVE. Here, we report that the Abi subunit-but not Scar-is phosphorylated in response to extracellular signalling in Dictyostelium cells. Like Scar, Abi is phosphorylated after the complex has been activated, implying that Abi phosphorylation modulates pseudopodia, rather than causing new ones to be made. Consistent with this, Scar complex mutants that cannot bind Rac are also not phosphorylated. Several environmental cues also affect Abi phosphorylation-cell-substrate adhesion promotes it and increased extracellular osmolarity diminishes it. Both unphosphorylatable and phosphomimetic Abi efficiently rescue the chemotaxis of Abi KO cells and pseudopodia formation, confirming that Abi phosphorylation is not required for activation or inactivation of the Scar/WAVE complex. However, pseudopodia and Scar patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Dictyostelium, in which Scar and Abi are both unphosphorylatable, can still form pseudopods, but migrate substantially faster. We conclude that extracellular signals and environmental responses modulate cell migration by tuning the behaviour of the Scar/WAVE complex after it has been activated.

Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1.

Rac1 is a major regulator of actin dynamics, with GTP-bound Rac1 promoting actin assembly via the Scar/WAVE complex. CYRI competes with Scar/WAVE for interaction with Rac1 in a feedback loop regulating actin dynamics. Here, we reveal the nature of the CYRI-Rac1 interaction, through crystal structures of CYRI-B lacking the N-terminal helix (CYRI-BΔN) and the CYRI-BΔN:Rac1Q61L complex, providing the molecular basis for CYRI-B regulation of the Scar/WAVE complex. We reveal CYRI-B as having two subdomains - an N-terminal Rac1 binding subdomain with a unique Rac1-effector interface and a C-terminal Ratchet subdomain that undergoes conformational changes induced by Rac1 binding. Finally, we show that the CYRI protein family, CYRI-A and CYRI-B can produce an autoinhibited hetero- or homodimers, adding an additional layer of regulation to Rac1 signaling.

Developmental Modulation of Root Cell Wall Architecture Confers Resistance to an Oomycete Pathogen.

The cell wall is the primary interface between plant cells and their immediate environment and must balance multiple functionalities, including the regulation of growth, the entry of beneficial microbes, and protection against pathogens. Here, we demonstrate how API, a SCAR2 protein component of the SCAR/WAVE complex, controls the root cell wall architecture important for pathogenic oomycete and symbiotic bacterial interactions in legumes. A mutation in API results in root resistance to the pathogen Phytophthora palmivora and colonization defects by symbiotic rhizobia. Although api mutant plants do not exhibit significant overall growth and development defects, their root cells display delayed actin and endomembrane trafficking dynamics and selectively secrete less of the cell wall polysaccharide xyloglucan. Changes associated with a loss of API establish a cell wall architecture with altered biochemical properties that hinder P. palmivora infection progress. Thus, developmental stage-dependent modifications of the cell wall, driven by SCAR/WAVE, are important in balancing cell wall developmental functions and microbial invasion.

Adhesion stimulates Scar/WAVE phosphorylation in mammalian cells.

The Scar/WAVE complex catalyzes the protrusion of pseudopods and lamellipods, and is therefore a principal regulator of cell migration. However, it is unclear how its activity is regulated, beyond a dependence on Rac. Phosphorylation of the proline-rich region, by kinases such as Erk2, has been suggested as an upstream activator. We have recently reported that phosphorylation is not required for complex activation. Rather, it occurs after Scar/WAVE has been activated, and acts as a modulator. Neither chemoattractant signaling nor Erk2 affects the amount of phosphorylation, though in Dictyostelium it is promoted by cell-substrate adhesion. We now report that cell-substrate adhesion also promotes Scar/WAVE2 phosphorylation in mammalian cells, suggesting that the process is evolutionarily conserved.

Wash exhibits context-dependent phenotypes and, along with the WASH regulatory complex, regulates Drosophila oogenesis.

WASH, a Wiskott-Aldrich syndrome (WAS) family protein, has many cell and developmental roles related to its function as a branched actin nucleation factor. Similar to mammalian WASHC1, which is embryonic lethal, Drosophila Wash was found to be essential for oogenesis and larval development. Recently, however, Drosophila wash was reported to be homozygous viable. Here, we verify that the original wash null allele harbors an unrelated lethal background mutation; however, this unrelated lethal mutation does not contribute to any Wash oogenesis phenotypes. Significantly, we find that: (1) the homozygous wash null allele retains partial lethality, leading to non-Mendelian inheritance; (2) the allele's functions are subject to its specific genetic background; and (3) the homozygous stock rapidly accumulates modifications that allow it to become robust. Together, these results suggest that Wash plays an important role in oogenesis via the WASH regulatory complex. Finally, we show that another WAS family protein, SCAR/WAVE, plays a similar role in oogenesis and that it is upregulated as one of the modifications that allows the wash allele to survive in the homozygous state.

Actin filament reorganisation controlled by the SCAR/WAVE complex mediates stomatal response to darkness.

Stomata respond to darkness by closing to prevent excessive water loss during the night. Although the reorganisation of actin filaments during stomatal closure is documented, the underlying mechanisms responsible for dark-induced cytoskeletal arrangement remain largely unknown. We used genetic, physiological and cell biological approaches to show that reorganisation of the actin cytoskeleton is required for dark-induced stomatal closure. The opal5 mutant does not close in response to darkness but exhibits wild-type (WT) behaviour when exposed to abscisic acid (ABA) or CaCl2 . The mutation was mapped to At5g18410, encoding the PIR/SRA1/KLK subunit of the ArabidopsisSCAR/WAVE complex. Stomata of an independent allele of the PIR gene (Atpir-1) showed reduced sensitivity to darkness and F1 progenies of the cross between opal5 and Atpir-1 displayed distorted leaf trichomes, suggesting that the two mutants are allelic. Darkness induced changes in the extent of actin filament bundling in WT. These were abolished in opal5. Disruption of filamentous actin using latrunculin B or cytochalasin D restored wild-type stomatal sensitivity to darkness in opal5. Our findings suggest that the stomatal response to darkness is mediated by reorganisation of guard cell actin filaments, a process that is finely tuned by the conserved SCAR/WAVE-Arp2/3 actin regulatory module.

A plasma membrane template for macropinocytic cups.

Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery.

Homologs of SCAR/WAVE complex components are required for epidermal cell morphogenesis in rice.

Filamentous actins (F-actins) play a vital role in epidermal cell morphogenesis. However, a limited number of studies have examined actin-dependent leaf epidermal cell morphogenesis events in rice. In this study, two recessive mutants were isolated: less pronounced lobe epidermal cell2-1 (lpl2-1) and lpl3-1, whose leaf and stem epidermis developed a smooth surface, with fewer serrated pavement cell (PC) lobes, and decreased papillae. The lpl2-1 also exhibited irregular stomata patterns, reduced plant height, and short panicles and roots. Molecular genetic studies demonstrated that LPL2 and LPL3 encode the PIROGI/Specifically Rac1-associated protein 1 (PIR/SRA1)-like and NCK-associated protein 1 (NAP1)-like proteins, respectively, two components of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (SCAR/WAVE) regulatory complex involved in actin nucleation and function. Epidermal cells exhibited abnormal arrangement of F-actins in both lpl2 and lpl3 expanding leaves. Moreover, the distorted trichomes of Arabidopsis pir could be partially restored by an overexpression of LPL2 A yeast two-hybrid assay revealed that LPL2 can directly interact with LPL3 in vitro Collectively, the results indicate that LPL2 and LPL3 are two functionally conserved homologs of the SCAR/WAVE complex components, and that they play an important role in controlling epidermal cell morphogenesis in rice by organising F-actin.

Diaphanous regulates SCAR complex localization during Drosophila myoblast fusion.

From Drosophila to man, multinucleated muscle cells form through cell-cell fusion. Using Drosophila as a model system, researchers first identified, and then demonstrated, the importance of actin cytoskeletal rearrangements at the site of fusion. These actin rearrangements at the fusion site are regulated by SCAR and WASp mediated Arp2/3 activation, which nucleates branched actin networks. Loss of SCAR, WASp or both leads to defects in myoblast fusion. Recently, we have found that the actin regulator Diaphanous (Dia) also plays a role both in organizing actin and in regulating Arp2/3 activity at the fusion site. In this Extra View article, we provide additional data showing that the Abi-SCAR complex accumulates at the fusion site and that excessive SCAR activity impairs myoblast fusion. Using constitutively active Dia constructs, we provide additional evidence that Dia functions upstream of SCAR activity to regulate actin dynamics at the fusion site and to localize the Abi-SCAR complex.

A dual role model for active Rac1 in cell migration.

Over time we have come to appreciate that the complex regulation of Rho GTPases involves additional mechanisms beyond the activating role of RhoGEFs, the inactivating function of RhoGAPs and the sequestering activity of RhoGDIs. One class of regulatory mechanisms includes direct modifications of Rho proteins such as isoprenylation, phosphorylation and SUMOylation. Rho GTPases can also regulate each other by means of crosstalk signaling, which is again mostly mediated by GEFs, GAPs and GDIs. More complex mutual regulation ensues when and where two or more Rho proteins activate a common molecular target, i.e., share a common effector. We have recently unraveled a reciprocal mechanism wherein spatiotemporal dynamics of Rac1 activity during migration of Dictyostelium cells is apparently regulated by antagonizing interactions of Rac1-GTP with two distinct effectors. By monitoring specific fluorescent probes, activated Rac1 is simultaneously present at the leading edge, where it participates in Scar/WAVE-mediated actin polymerization, and at the trailing edge, where it induces formation of a DGAP1/cortexillin actin-bundling complex. Strikingly, in addition to their opposed localization, the two populations of activated Rac1 also display opposite kinetics of recruitment to the plasma membrane upon stimulation by chemoattractants. These findings with respect to Rac1 in Dictyostelium suggest a novel principle for regulation of Rho GTPase activity that might also play a role in other cell types and for other Rho family members.

Rac1 function during fucoid development.

Morphogenesis in fucoid algae begins with adhesive secretion and rhizoid germination, developmental events that secure the alga within the intertidal zone. The importance of the actin cytoskeleton during these processes has been well established; but in general, little is known about actin regulation within the stramenopile lineage. Based on conserved strategies for regulation of actin in other lineages, co-localization of the Arp2/3 complex with actin structures that are essential for rhizoid formation may implicate members of the Rho family of small GTPases in the signaling pathway(s) regulating actin polymerization during fucoid development. Our lab recently demonstrated Rac1 dependent regulation of endomembrane polarization, polarization of adhesive secretion, germination and tip growth in the fucoid brown alga Silvetia compressa. We also present new evidence revealing Rac1 localization during germination in S. compressa, and show that membrane localization is essential for proper Rac1 function.