mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration

Journal of Cell Science, Oct 2007

Stephanie L. Gupton, Kathryn Eisenmann, Arthur S. Alberts, Clare M. Waterman-Storer

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mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration

Stephanie L. Gupton 2 Kathryn Eisenmann 1 Arthur S. Alberts 1 Clare M. Waterman-Storer 0 0 Department of Cell Biology, The Scripps Research Institute , La Jolla, CA 92037 , and Cell Biology and Physiology Center, National Heart, Lung and Blood Institute , Building 50 South Drive, Bethesda, MD 20892-8019 , USA 1 Van Andel Research Institute , 333 Bostwick Avenue, Grand Rapids, MI 49503 , USA 2 Department of Cell Biology, The Scripps Research Institute , La Jolla, CA 92037 , USA Summary Cell migration requires spatial and temporal regulation of filamentous actin (F-actin) dynamics. This regulation is achieved by distinct actin-associated proteins, which mediate polymerization, depolymerization, severing, contraction, bundling or engagement to the membrane. ce Mammalian Diaphanous-related (mDia) formins, which ne nucleate, processively elongate, and in some cases bundle ic actin filaments, have been extensively studied in vitro, but S their function in the cell has been less well characterized. lle Here we study the role of mDia2 activity in the dynamic C organization of F-actin in migrating epithelial cells. We find fo that mDia2 localizes in the lamella of migrating epithelial l cells, where it is involved in the formation of a stable pool an of cortical actin and in maintenance of polymerizationr u o J - Introduction Cell migration consists of a polarized four step cycle of leading edge protrusion, adhesion to the extracellular matrix (ECM), forward contraction of the cell body, and release of adhesions in the cell rear (Lauffenburger and Horwitz, 1996). Each step of this cycle is mediated by distinct dynamic filamentous actin (F-actin) based structures. At the leading edge of migrating epithelial cells, F-actin is organized into two partially overlapping networks, the lamellipodium network and the lamella network (Gupton et al., 2005; Ponti et al., 2004). The distinct properties of F-actin in the two networks are probably dictated by the distribution and activities of actin-associated proteins that differentially associate with the networks (DesMarais et al., 2002; Ponti et al., 2004), conferring on each distinct functions for migration. In the lamellipodium, which extends from the cell edge to ~2-4 m into the cell, rapid polymerization and subsequent depolymerization of an F-actin array mediated by the Arp2/3 complex and cofilin (Arnaout, 2002; Bailly et al., 1999; Ichetovkin et al., 2002) produces a branched, treadmilling Factin network. This network is pushed rapidly away from the cell edge by plasma-membrane-associated filament elongation (Pollard and Borisy, 2003; Ponti et al., 2004; Svitkina and Borisy, 1999) in a process termed retrograde flow. Cycles of filament polymerization and depolymerization within the competent free filament barbed ends at focal adhesions. Specific inhibition of mDia2 alters focal adhesion turnover and reduces migration velocity. We suggest that the regulation of filament assembly dynamics at focal adhesions may be necessary for the formation of a stable pool of cortical lamella actin and the proper assembly and disassembly dynamics of focal adhesions, making mDia2 an important factor in epithelial cell migration. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/19/3475/DC1 treadmilling lamellipodium network correlate with cycles of leading edge protrusion and retraction (Machacek and Danuser, 2006; Ponti et al., 2004). A few micrometers behind the leading edge, F-actin in the lamella is organized into a network interspersed with bundles, both of which contain myosin II and tropomyosin (DesMarais et al., 2002; Gupton et al., 2005; Ponti et al., 2004; Verkhovsky et al., 1995). F-actin in the lamella undergoes myosin II-dependent retrograde Factin flow that is slower than the polymerization-dependent flow of the lamellipodium (Ponti et al., 2004). Local sites of filament assembly and disassembly are intermingled throughout the lamella. Unlike the lamellipodium, it is unknown what proteins regulate the F-actin assembly and disassembly kinetics of the lamella. Focal adhesions form at the interface between the lamellipodium and lamella F-actin networks (Ponti et al., 2004). Specific actin-binding proteins within focal adhesions may link F-actin in the lamella to transmembrane integrins (Critchley et al., 1999) that bind the ECM, stabilizing leading edge protrusions in the second step of migration. Myosin II contraction in the lamella, pulling on F-actin attached via focal adhesions to the ECM, generates tension for moving the cell body forward in the third step of migration (Lauffenburger and Horowitz, 1996). Actomyosin contraction is also implicated in the disassembly of trailing adhesions in the cell rear in the final step of migration (Kaverina et al., 2002; Webb et al., 2004). Recent studies have brought into question the role of the lamellipodium in cell migration (Pankov et al., 2005). Blocking lamellipodium formation by inhi (...truncated)


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Stephanie L. Gupton, Kathryn Eisenmann, Arthur S. Alberts, Clare M. Waterman-Storer. mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration, Journal of Cell Science, 2007, pp. 3475-3487, 120/19, DOI: 10.1242/jcs.006049