Role of Actin Polymerization and Adhesion to Extracellular Matrix in Rac- and Rho-induced Cytoskeletal Reorganization

Most animal cells use a combination of actin-myosin–based contraction and actin polymerization– based protrusion to control their shape and motility. The small GTPase Rho triggers the formation of contractile stress fibers and focal adhesion complexes (Ridley, A.J., and A. Hall. 1992. Cell. 70:389–399) while a close relative, Rac, induces lamellipodial protrusions and focal complexes in the lamellipodium (Nobes, C.D., and A. Hall. 1995. Cell. 81:53–62; Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. Cell. 70:401–410); the Rho family of small GTPases may thus play an important role in regulating cell movement. Here we explore the roles of actin polymerization and extracellular matrix in Rho- and Rac-stimulated cytoskeletal changes. To examine the underlying mechanisms through which these GTPases control F-actin assembly, fluorescently labeled monomeric actin, Cy3-actin, was introduced into serum-starved Swiss 3T3 fibroblasts. Incorporation of Cy3- actin into lamellipodial protrusions is concomitant with F-actin assembly after activation of Rac, but Cy3-actin is not incorporated into stress fibers formed immediately after Rho activation. We conclude that Rac induces rapid actin polymerization in ruffles near the plasma membrane, whereas Rho induces stress fiber assembly primarily by the bundling of actin filaments. Activation of Rho or Rac also leads to the formation of integrin adhesion complexes. Integrin clustering is not required for the Rho-induced assembly of actin-myosin filament bundles, or for vinculin association with actin bundles, but is required for stress fiber formation. Integrin-dependent focal complex assembly is not required for the Rac-induced formation of lamellipodia or membrane ruffles. It appears, therefore, that the assembly of large integrin complexes is not required for most of the actin reorganization or cell morphology changes induced by Rac or Rho activation in Swiss 3T3 fibroblasts.

Download Full-text

ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts

The Journal of Cell Biology ◽

10.1083/jcb.143.7.1981 ◽

1998 ◽

Vol 143 (7) ◽

pp. 1981-1995 ◽

Cited By ~ 108

Author(s):

J.C. Norman ◽

D. Jones ◽

S.T. Barry ◽

M.R. Holt ◽

S. Cockcroft ◽

...

Keyword(s):

Focal Adhesion ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

3T3 Fibroblasts ◽

Stress Fiber Formation ◽

Swiss 3T3 ◽

Swiss 3T3 Fibroblasts ◽

Actin Stress Fibers

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPγS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPγS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion–like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated Δ17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPγS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Δ17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.

Download Full-text

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

The Journal of Cell Biology ◽

10.1083/jcb.200112107 ◽

2002 ◽

Vol 157 (5) ◽

pp. 819-830 ◽

Cited By ~ 146

Author(s):

Takahiro Tsuji ◽

Toshimasa Ishizaki ◽

Muneo Okamoto ◽

Chiharu Higashida ◽

Kazuhiro Kimura ◽

...

Keyword(s):

Focal Adhesions ◽

Small Gtpase ◽

Dominant Negative ◽

Stress Fibers ◽

Induced Membrane ◽

3T3 Fibroblasts ◽

C3 Exoenzyme ◽

Swiss 3T3 ◽

Swiss 3T3 Fibroblasts ◽

Membrane Ruffle

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632–induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632–induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.

Download Full-text

Regulation of reactive oxygen species and stress fiber formation by calpeptin in Swiss 3T3 fibroblasts

Cellular Signalling ◽

10.1016/s0898-6568(01)00244-3 ◽

2002 ◽

Vol 14 (3) ◽

pp. 205-210 ◽

Cited By ~ 3

Author(s):

Soo-Jung Kim ◽

Zee-Won Lee ◽

Soo-Mi Kweon ◽

Soohyun Kim ◽

Kwon-Soo Ha

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Stress Fiber ◽

Fiber Formation ◽

Oxygen Species ◽

3T3 Fibroblasts ◽

Stress Fiber Formation ◽

Swiss 3T3 ◽

Swiss 3T3 Fibroblasts

Download Full-text

Dynamics of Actin Stress Fibers and Focal Adhesions during Slow Migration in Swiss 3T3 Fibroblasts: Intracellular Mechanism of Cell Turning

BioMed Research International ◽

10.1155/2016/5749749 ◽

2016 ◽

Vol 2016 ◽

pp. 1-16 ◽

Cited By ~ 3

Author(s):

Michiko Sugawara ◽

Hiromi Miyoshi ◽

Takuya Miura ◽

Hiroto Tanaka ◽

Ken-ichi Tsubota ◽

...

Keyword(s):

Focal Adhesions ◽

Leading Edge ◽

Stress Fibers ◽

Lateral Region ◽

3T3 Fibroblasts ◽

Self Assembling ◽

Swiss 3T3 ◽

Cell Retraction ◽

Swiss 3T3 Fibroblasts ◽

Actin Stress Fibers

To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs) coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs) were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts.

Download Full-text