Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation.

BACKGROUND: Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed. METHODS: Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number. RESULTS: Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells. CONCLUSIONS: Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation.

Convergence of Ras- and Rac-regulated formin pathways is pivotal for phagosome formation and particle uptake in Dictyostelium.

Macroendocytosis comprising phagocytosis and macropinocytosis is an actin-driven process regulated by small GTPases that depend on the dynamic reorganization of the membrane that protrudes and internalizes extracellular material by cup-shaped structures. To effectively capture, enwrap, and internalize their targets, these cups are arranged into a peripheral ring or ruffle of protruding actin sheets emerging from an actin-rich, nonprotrusive zone at its base. Despite extensive knowledge of the mechanism driving actin assembly of the branched network at the protrusive cup edge, which is initiated by the actin-related protein (Arp) 2/3 complex downstream of Rac signaling, our understanding of actin assembly in the base is still incomplete. In the Dictyostelium model system, the Ras-regulated formin ForG was previously shown to specifically contribute to actin assembly at the cup base. Loss of ForG is associated with a strongly impaired macroendocytosis and a 50% reduction in F-actin content at the base of phagocytic cups, in turn indicating the presence of additional factors that specifically contribute to actin formation at the base. Here, we show that ForG synergizes with the Rac-regulated formin ForB to form the bulk of linear filaments at the cup base. Consistently, combined loss of both formins virtually abolishes cup formation and leads to severe defects of macroendocytosis, emphasizing the relevance of converging Ras- and Rac-regulated formin pathways in assembly of linear filaments in the cup base, which apparently provide mechanical support to the entire structure. Remarkably, we finally show that active ForB, unlike ForG, additionally drives phagosome rocketing to aid particle internalization.

Ena/VASP clustering at microspike tips involves lamellipodin but not I-BAR proteins, and absolutely requires unconventional myosin-X.

Sheet-like membrane protrusions at the leading edge, termed lamellipodia, drive 2D-cell migration using active actin polymerization. Microspikes comprise actin-filament bundles embedded within lamellipodia, but the molecular mechanisms driving their formation and their potential functional relevance have remained elusive. Microspike formation requires the specific activity of clustered Ena/VASP proteins at their tips to enable processive actin assembly in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering are poorly understood. Systematic analyses of B16-F1 melanoma mutants lacking potential candidate proteins revealed that neither inverse BAR-domain proteins, nor lamellipodin or Abi is essential for clustering, although they differentially contribute to lamellipodial VASP accumulation. In contrast, unconventional myosin-X (MyoX) identified here as proximal to VASP was obligatory for Ena/VASP clustering and microspike formation. Interestingly, and despite the invariable distribution of other relevant marker proteins, the width of lamellipodia in MyoX-KO mutants was significantly reduced as compared with B16-F1 control, suggesting that microspikes contribute to lamellipodium stability. Consistently, MyoX removal caused marked defects in protrusion and random 2D-cell migration. Strikingly, Ena/VASP-deficiency also uncoupled MyoX cluster dynamics from actin assembly in lamellipodia, establishing their tight functional association in microspike formation.

VASP boosts protrusive activity of macroendocytic cups and drives phagosome rocketing after internalization.

Ena/VASP proteins are powerful actin polymerases that drive the processive elongation of actin filaments. Members of this protein family have been implicated in a variety of important cellular processes including axon guidance, cell migration and adhesion. However, the specific function of these proteins in macroendocytosis, comprising macropinocytosis and phagocytosis remain rather poorly understood. Here, we used the professional phagocyte Dictyostelium discoideum to address the function and dynamics of its only family member VASP in macroendocytosis. Confocal time-lapse imaging revealed that VASP localized prominently in a circumferential narrow band at the advancing rim of the phagocytic cup followed by its aperture-like convergence upon particle internalization. Loss of VASP resulted in substantial defects in both, macropinocytosis of bulk fluid and phagocytosis of yeast particles. Consistently, VASP-deficiency coincided with diminished speed of the protruding rim and an impaired internalization rate. Most intriguingly, after cup closure, VASP condensed at the distal side of internalized phagosomes and initiated localized de-novo actin assembly to propel the phagosome by an actin-rich comet deeper into the cell, resembling intracellular movement of rocketing Listeria cells. In line with these findings, travelled distance and speed of rocketing phagosomes in VASP-deficient cells were markedly impaired.

Capping protein is dispensable for polarized actin network growth and actin-based motility.

Heterodimeric capping protein (CP) binds the rapidly growing barbed ends of actin filaments and prevents the addition (or loss) of subunits. Capping activity is generally considered to be essential for actin-based motility induced by Arp2/3 complex nucleation. By stopping barbed end growth, CP favors nucleation of daughter filaments at the functionalized surface where the Arp2/3 complex is activated, thus creating polarized network growth, which is necessary for movement. However, here using an in vitro assay where Arp2/3 complex-based actin polymerization is induced on bead surfaces in the absence of CP, we produce robust polarized actin growth and motility. This is achieved either by adding the actin polymerase Ena/VASP or by boosting Arp2/3 complex activity at the surface. Another actin polymerase, the formin FMNL2, cannot substitute for CP, showing that polymerase activity alone is not enough to override the need for CP. Interfering with the polymerase activity of Ena/VASP, its surface recruitment or its bundling activity all reduce Ena/VASP's ability to maintain polarized network growth in the absence of CP. Taken together, our findings show that CP is dispensable for polarized actin growth and motility in situations where surface-directed polymerization is favored by whatever means over the growth of barbed ends in the network.

Formins specify membrane patterns generated by propagating actin waves.

Circular actin waves separate two distinct areas on the substrate-attached cell surface from each other: an external area from an inner territory that is circumscribed by the wave. These areas differ in composition of actin-associated proteins and of phosphoinositides in the membrane. At the propagating wave, one area is converted into the other. By photo-conversion of Eos-actin and analysis of actin network structures we show that both in the inner territory and the external area the actin network is subject to continuous turnover. To address the question of whether areas in the wave pattern are specified by particular actin polymerizing machines, we locate five members of the formin family to specific regions of the wave landscape using TIRF microscopy and constitutively active formin constructs tagged with fluorescent protein. Formin ForB favors the actin wave and ForG the inner territory, whereas ForA, ForE, and ForH are more strongly recruited to the external area. Fluctuations of membrane binding peculiar to ForB indicate transient states in the specification of membrane domains before differentiation into ForB decorated and depleted ones. Annihilation of the patterns by 1 µM of the formin inhibitor SMIFH2 supports the implication of formins in their generation.

Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins.

The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA- /E-/H- and racE- mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.