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51
Rho-stimulated contractility drives the formation of stress fibers and focal adhesions
- J. Cell Biol
, 1996
"... Abstract. Activated rhoA, a ras-related GTP-binding protein, stimulates the appearance of stress fibers, focal adhesions, and tyrosine phosphorylation in quiescent cells (Ridley, A.J., and A. Hall, 1992. Cell. 70:389-399). The pathway by which rho triggers these events has not been elucidated. Many ..."
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Cited by 309 (5 self)
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Abstract. Activated rhoA, a ras-related GTP-binding protein, stimulates the appearance of stress fibers, focal adhesions, and tyrosine phosphorylation in quiescent cells (Ridley, A.J., and A. Hall, 1992. Cell. 70:389-399). The pathway by which rho triggers these events has not been elucidated. Many of the agents that activate rho (e.g., vasopressin, endothelin, lysophosphatidic acid) stimulate the contractility of smooth muscle and other cells. We have investigated whether rho's induction of stress fibers, focal adhesions, and tyrosine phosphorylation is the result of its stimulation of contractility. We demonstrate that stimulation of fibroblasts with lysophosphatidic acid, which activates rho, induces myosin light chain phosphorylation. This precedes the forma-tion of stress fibers and focal adhesions and is accompanied by increased contractility. Inhibition of contractility by several different mechanisms leads to inhibition of rho-induced stress fibers, focal adhesions, and tyrosine phosphorylation. In addition, when contractility is inhibited, integrins disperse from focal adhesions as stress fibers and focal adhesions disassemble. Conversely, upon stimulation of contractility, diffusely distributed integrins are aggregated into focal adhesions. These results suggest that activated rho stimulates contractility, driving the formation of stress fibers and focal adhesions and elevating tyrosine phosphorylation. A model is proposed to account for how contractility could promote these events. F OCAL adhesions are sites where cells in culture adhere strongly to the underlying extracellular matrix (ECM) 1 via specific members of the integrin family of ECM receptors (Burridge et al., 1988; Jockusch et al., 1995). At their cytoplasmic face, focal adhesions provide attachment for bundles of actin filaments (stress fibers). More than just sites of structural linkage between the ECM on the outside and the cytoskeleton on the inside, focal adhesions are regions of signal transduction. Components involved in multiple signal transduction pathways have been identified in focal adhesions (Hynes, 1992;
Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism
, 2001
"... Abstract. The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was sho ..."
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Cited by 134 (6 self)
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Abstract. The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy.
Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling
- J. Cell Biol
, 1997
"... Abstract. We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus e ..."
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Cited by 84 (15 self)
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Abstract. We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally “pioneering ” MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated
Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly
- J. Cell Biol
, 1998
"... Abstract. Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mo ..."
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Cited by 62 (0 self)
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Abstract. Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTPbinding protein Rho, blocks the binding of fibronectin and the 70-kD NH 2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH 2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin’s assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov,
The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly
- J. Cell
, 1997
"... Abstract. The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Arp complex). ..."
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Cited by 58 (0 self)
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Abstract. The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Arp complex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat–containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.
p21-Activated Kinase 1 (Pak1) Regulates Cell Motility in Mammalian Fibroblasts
"... Abstract. The p21 (Cdc42/Rac) activated kinase Pak1 regulates cell morphology and polarity in most, if not all, eukaryotic cells. We and others have established that Pak’s effects on these parameters are mediated by changes in the organization of cortical actin. Because cell motility requires polari ..."
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Cited by 53 (5 self)
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Abstract. The p21 (Cdc42/Rac) activated kinase Pak1 regulates cell morphology and polarity in most, if not all, eukaryotic cells. We and others have established that Pak’s effects on these parameters are mediated by changes in the organization of cortical actin. Because cell motility requires polarized rearrangements of the actin/myosin cytoskeleton, we examined the role of Pak1 in regulating cell movement. We established clonal tetracycline-regulated NIH-3T3 cell lines that inducibly express either wild-type Pak1, a kinase-dead, or constitutively-active forms of this enzyme, and examined the morphology, F-actin organization, and motility of these cells. Expression of any of these forms of Pak1 induced dramatic changes in actin organization which were not inhibited by coexpression of a dominant-negative
Identification of novel graded polarity actin filament bundles in locomoting heart fibroblasts: implications for the generation of motile force
- J. Cell Biol
, 1997
"... We have determined the structural organiza-tion and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with ..."
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Cited by 36 (3 self)
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We have determined the structural organiza-tion and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with barbed ends fac-ing forward. In the lamella, cell body, and tail there are two observable types of actin filament organization. A less abundant type is located on the inner surface of the plasma membrane and is composed of short, overlap-ping actin bundles (0.25–2.5 m m) that repeatedly alter-nate in polarity from uniform barbed ends forward to uniform pointed ends forward. This type of organiza-tion is similar to the organization we show for actin fila-ment bundles (stress fibers) in nonlocomoting cells (PtK2 cells) and to the known organization of muscle sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly ventrally located and is composed of long, overlapping bundles (average 13 m
Regulation of Cell Contraction and Membrane Ruffling by Distinct Signals in Migratory Cells
"... Abstract. Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are r ..."
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Cited by 35 (1 self)
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Abstract. Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively. Key words: adaptor proteins • cell migration • mitogen-activated protein kinase • myosin • signal transduction
Small-molecule inhibitors of actin dynamics and cell motility
- Curr. Topics Med. Chem
, 2003
"... Abstract: Cell motility is a central feature of a range of normal and pathological processes, including embryonic development, tissue repair, immune cell function, angiogenesis, and cancer metastasis. The dynamics of the actin cytoskeleton power cell migration. A large number of proteins are known o ..."
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Cited by 34 (6 self)
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Abstract: Cell motility is a central feature of a range of normal and pathological processes, including embryonic development, tissue repair, immune cell function, angiogenesis, and cancer metastasis. The dynamics of the actin cytoskeleton power cell migration. A large number of proteins are known or suspected to play roles in regulating actin dynamics. While there are now many available small molecules that target the actin cytoskeleton directly, there is a paucity of specific inhibitors of actin-binding proteins and other immediate regulators of actin dynamics and cell movement. This makes the field of exceptional interest as a meeting place between the goals of chemical biology and the needs of cell biology. Furthermore, while regulators of the cell cycle have been recognized for some time as targets for anti-cancer drug development, controlling actin dynamics and cell motility as a therapeutic approach has received scant attention in comparison until recently. This review deals with small-molecule inhibitors of actin dynamics as they relate to cell shape change and motility, from compounds targeting actin directly to those targeting proteins involved in the fundamental control of the actin cytoskeleton.
Visualization and molecular analysis of actin assembly in living cells
- J. Cell
, 1998
"... Abstract. Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibrobl ..."
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Cited by 29 (1 self)
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Abstract. Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place. Key words: actin assembly • Arp2/3 complex • capping protein • cell motility • Rho family GTPase
