Vicente-Manzanares, M., Zareno, J., Whitmore, L., Choi, C. K. & Horwitz, A. F. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells. J. Cell Biol. 176, 573-580

Department of Cell Biology, University of Virginia, Charlottesville, VA 22908.
The Journal of Cell Biology (Impact Factor: 9.83). 03/2007; 176(5):573-80. DOI: 10.1083/jcb.200612043
Source: PubMed


We have used isoform-specific RNA interference knockdowns to investigate the roles of myosin IIA (MIIA) and MIIB in the component processes that drive cell migration. Both isoforms reside outside of protrusions and act at a distance to regulate cell protrusion, signaling, and maturation of nascent adhesions. MIIA also controls the dynamics and size of adhesions in central regions of the cell and contributes to retraction and adhesion disassembly at the rear. In contrast, MIIB establishes front-back polarity and centrosome, Golgi, and nuclear orientation. Using ATPase- and contraction-deficient mutants of both MIIA and MIIB, we show a role for MIIB-dependent actin cross-linking in establishing front-back polarity. From these studies, MII emerges as a master regulator and integrator of cell migration. It mediates each of the major component processes that drive migration, e.g., polarization, protrusion, adhesion assembly and turnover, polarity, signaling, and tail retraction, and it integrates spatially separated processes.

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Available from: Miguel vicente-manzanares, Jul 09, 2014
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    • "may alter actomyosin interactions in migrating cells, where tensile forces are tightly controlled and are linked to focal adhesion turnover [24] [25] and cell– extracellular matrix adhesion [25] [26]. Thus, SEPT9 may influence actomyosin activity at stress fibers and focal adhesions. "
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    ABSTRACT: Septins are a highly conserved family of proteins in eukaryotes that is recognized as a novel component of the cytoskeleton. Septin 9 (SEPT9) interacts directly with actin filaments and functions as an actin stress fiber cross-linking protein that promotes the maturation of nascent focal adhesions and cell migration. However, the molecular details of how SEPT9 interacts with F-actin remain unknown. Here, we use electron microscopy and image analysis to show that SEPT9 binds to F-actin in a highly polymorphic fashion. We demonstrate that the basic domain (B-domain) of the N-terminal tail of SEPT9 is responsible for actin cross-linking, while the GTP-binding domain (G-domain) does not bundle F-actin. We show that the B-domain of SEPT9 binds to three sites on F-actin, and the two of these sites overlap with the binding regions of myosin and cofilin. SEPT9 inhibits actin-dependent ATPase activity of myosin and competes with the weakly-bound state of myosin for binding to F-actin. At the same time, SEPT9 significantly reduces the extent of F-actin depolymerization by cofilin. Taken together, these data suggest that SEPT9 protects actin filaments from depolymerization by cofilin and myosin, and indicate a mechanism by which SEPT9 could maintain the integrity of growing and contracting actin filaments. Copyright © 2015. Published by Elsevier Ltd.
    Journal of Molecular Biology 08/2015; 427(20). DOI:10.1016/j.jmb.2015.07.026 · 4.33 Impact Factor
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    • "Inhibition of NM II ATPase activity by a specific inhibitor, blebbistatin [14], makes the leading edge more dynamic by increasing the frequency of protrusions and retractions, induces frequent changes in the growth cone shape and reduces directed motility [13]. In fibroblasts, specific depletion of NM IIA or IIB resulted in a similar (2–3-fold) increase in the rate of protrusion [15]. These results suggest that the loss of NM II contractility by direct inhibition of its ATPase activity inversely correlates with actin dynamics. "
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    ABSTRACT: Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family. NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II-GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II-GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation. Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF-Rho GTPase signaling pathway.
    PLoS ONE 04/2014; 9(4):e95212. DOI:10.1371/journal.pone.0095212 · 3.23 Impact Factor
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    • "Together with the increased lamellar retrograde flow in cells lacking dynamin2, these observations support the idea that dynamin2 promotes engagement of F-actin at maturing focal adhesions to slow lamellar retrograde flow. Although contractile forces also contribute to adhesion maturation [58]–[60], recent evidence highlights the importance of actin filament organization, particularly filament crosslinking, for adhesion assembly and maturation [9], [10], [33], [61]. We suggest that actin filament remodeling by dynamin2 integrates lamellipodial and lamellar actin networks and their interactions at adhesions. "
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    ABSTRACT: Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2's action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.
    PLoS ONE 04/2014; 9(4):e94330. DOI:10.1371/journal.pone.0094330 · 3.23 Impact Factor
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