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
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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)