Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hairs cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its 2-fold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely driven by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ('teeth') that appear to be interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, supporting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings present a phagocytic cup-shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

Fascin-induced actin protrusions are suppressed by dendritic networks in GUVs

The interactions between actin networks and cell membrane are immensely important for eukaryotic cell functions including cell shape changes, motility, polarity establishment, and adhesion. Actin binding proteins are known to compete and cooperate, using finite amount of actin monomers, to form distinct actin networks. How actin bundling protein fascin and actin branching protein Arp2/3 complex compete to remodel membranes is not entirely clear. To investigate fascin- and Arp2/3-mediated actin network remodeling, we applied a reconstitution approach encapsulating bundled and dendritic actin networks inside giant unilamellar vesicles (GUVs). Independently reconstituted, membrane-bound Arp2/3 nucleation forms an actin cortex in GUVs whereas fascin mediates formation of actin bundles that protrude out of GUVs. Co-encapsulating both fascin and Arp2/3 complex leads to polarized dendritic aggregates and significantly reduces membrane protrusions, irrespective of whether the dendritic network is membrane-bound or not. However, reducing Arp2/3 complex while increasing fascin restores membrane protrusion. Such changes in network assembly and the subsequent interplay with membrane can be attributed to competition between fascin and Arp2/3 complex to utilize a finite pool of actin. [Media: see text] [Media: see text]

Cadherin puncta are interdigitated dynamic actin protrusions necessary for stable cadherin adhesion

Cadherins harness the actin cytoskeleton to build cohesive sheets of cells using paradoxically weak bonds, but the molecular mechanisms are poorly understood. In one popular model, actin organizes cadherins into large, micrometer-sized clusters known as puncta. Myosin is thought to pull on these puncta to generate strong adhesion. Here, however, we show that cadherin puncta are actually interdigitated actin microspikes generated by actin polymerization mediated by three factors (Arp2/3, EVL, and CRMP-1). The convoluted membranes in these regions give the impression of cadherin clustering by fluorescence microscopy, but the ratio of cadherin to membrane is constant. Nevertheless, these interlocking fingers of membrane are important for adhesion because perturbing their formation disrupts cell adhesion. In contrast, blocking myosin-dependent contractility does not disrupt either the interdigitated microspikes or lateral membrane adhesion. “Puncta” are zones of strong cell–cell adhesion not due to cadherin clustering but that occur because the interdigitated microspikes expand the surface area available for adhesive bond formation and increase the asperity of the cell surface to promote friction between cells.

Drosophila USP22/nonstop polarizes the actin cytoskeleton during collective border cell migration

Polarization of the actin cytoskeleton is vital for the collective migration of cells in vivo. During invasive border cell migration in Drosophila, actin polarization is directly controlled by the Hippo signaling complex, which resides at contacts between border cells in the cluster. Here, we identify, in a genetic screen for deubiquitinating enzymes involved in border cell migration, an essential role for nonstop/USP22 in the expression of Hippo pathway components expanded and merlin. Loss of nonstop function consequently leads to a redistribution of F-actin and the polarity determinant Crumbs, loss of polarized actin protrusions, and tumbling of the border cell cluster. Nonstop is a component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex, but SAGA’s histone acetyltransferase module, which does not bind to expanded or merlin, is dispensable for migration. Taken together, our results uncover novel roles for SAGA-independent nonstop/USP22 in collective cell migration, which may help guide studies in other systems where USP22 is necessary for cell motility and invasion.