scholarly journals The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL)

2001 ◽  
Vol 154 (1) ◽  
pp. 161-176 ◽  
Author(s):  
Kip A. West ◽  
Huaye Zhang ◽  
Michael C. Brown ◽  
Sotiris N. Nikolopoulos ◽  
M.C. Riedy ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Deletion Mutant ◽  
Morphological Changes ◽  
Cell Spreading ◽  
Small Gtpases ◽  
Random Motility ◽  
Phenotypic Changes ◽  
Rho Family ◽  
Mutant Cells

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinΔLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin–PKL interaction. In cells overexpressing paxillinΔLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLΔPBS2) induced phenotypic changes reminiscent of paxillinΔLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

scholarly journals Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton

2000 ◽  
Vol 355 (1399) ◽  
pp. 965-970 ◽  
Author(s):  
Alan Hall ◽  
Catherine D. Nobes
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Fundamental Property ◽  
Small Gtpases ◽  
Membrane Receptors ◽  
Embryonic Cells ◽  
Filamentous Actin ◽  
Rho Family ◽  
Plasma Membrane Receptors

The actin cytoskeleton plays a fundamental role in all eukaryotic cells—it is a major determinant of cell morphology and polarity and the assembly and disassembly of filamentous actin structures provides a driving force for dynamic processes such as cell motility, phagocytosis, growth cone guidance and cytokinesis. The ability to reorganize actin filaments is a fundamental property of embryonic cells during development; the shape changes accompanying gastrulation and dorsal closure, for example, are dependent on the plasticity of the actin cytoskeleton, while the ability of cells or cell extensions, such as axons, to migrate within the developing embryo requires rapid and spatially organized changes to the actin cytoskeleton in response to the external environment. W ork in mammalian cells over the last decade has demonstrated the central role played by the highly conserved Rho family of small GTPases in signal transduction pathways that link plasma membrane receptors to the organization of the actin cytoskeleton.

Sequential activation of individual PKC isozymes in integrin-mediated muscle cell spreading: a role for MARCKS in an integrin signaling pathway

2002 ◽  
Vol 115 (10) ◽  
pp. 2151-2163 ◽  
Author(s):  
Marie-Hélène Disatnik ◽  
Stéphane C. Boutet ◽  
Christine H. Lee ◽  
Daria Mochly-Rosen ◽  
Thomas A. Rando
Keyword(s):  
Actin Cytoskeleton ◽  
Muscle Cell ◽  
Focal Adhesion ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Muscle Cells ◽  
Integrin Signaling ◽  
Pkc Isozymes ◽  
Pkc Activation

To understand how muscle cell spreading and survival are mediated by integrins, we studied the signaling events initiated by the attachment of muscle cells to fibronectin (FN). We have previously demonstrated that muscle cell spreading on FN is mediated by α5β1 integrin, is associated with rapid phosphorylation of focal adhesion kinase and is dependent on activation of protein kinase C (PKC). Here we investigated the role of individual PKC isozymes in these cellular processes. We show that α,δ and ϵPKC are expressed in muscle cells and are activated upon integrin engagement with different kinetics — ϵPKC was activated early, whereas α and δPKC were activated later. Using isozyme-specific inhibitors, we found that the activation of ϵPKC was necessary for cell attachment to FN. However, using isozyme-specific activators, we found that activation of each of three isozymes was sufficient to promote the spreading of α5-integrin-deficient cells on FN. To investigate further the mechanism by which integrin signaling and PKC activation mediate cell spreading, we studied the effects of these processes on MARCKS, a substrate of PKC and a protein known to regulate actin dynamics. We found that MARCKS was localized to focal adhesion sites soon after cell adhesion and that MARCKS translocated from the membrane to the cytosol during the process of cell spreading. This translocation correlated with different phases of PKC activation and with reorganization of the actin cytoskeleton. Using MARCKS-antisense cDNA, we show that α5-expressing cells in which MARCKS expression is inhibited fail to spread on FN, providing evidence for the crucial role of MARCKS in muscle cell spreading. Together, the data suggest a model in which early activation of ϵPKC is necessary for cell attachment; the later activation of α or δPKC may be necessary for the progression from attachment to spreading. The mechanism of PKC-mediated cell spreading may be via the phosphorylation of signaling proteins, such as MARCKS, that are involved in the reorganization of the actin cytoskeleton.

scholarly journals Cupidin, an Isoform of Homer/Vesl, Interacts with the Actin Cytoskeleton and Activated Rho Family Small GTPases and Is Expressed in Developing Mouse Cerebellar Granule Cells

1999 ◽  
Vol 19 (19) ◽  
pp. 8389-8400 ◽  
Author(s):  
Yoko Shiraishi ◽  
Akihiro Mizutani ◽  
Haruhiko Bito ◽  
Kazuko Fujisawa ◽  
Shuh Narumiya ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Granule Cells ◽  
Cerebellar Granule Cells ◽  
Small Gtpases ◽  
Cerebellar Granule ◽  
Rho Family

scholarly journals Small GTPase RhoG Is a Key Regulator for Neurite Outgrowth in PC12 Cells

2000 ◽  
Vol 20 (19) ◽  
pp. 7378-7387 ◽  
Author(s):  
Hironori Katoh ◽  
Hidekazu Yasui ◽  
Yoshiaki Yamaguchi ◽  
Junko Aoki ◽  
Hirotada Fujita ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Morphological Changes ◽  
Cell Types ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Wild Type ◽  
Rho Family ◽  
Constitutively Active

ABSTRACT The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.

scholarly journals Socius Is a Novel Rnd GTPase-Interacting Protein Involved in Disassembly of Actin Stress Fibers

2002 ◽  
Vol 22 (9) ◽  
pp. 2952-2964 ◽  
Author(s):  
Hironori Katoh ◽  
Amane Harada ◽  
Kazutoshi Mori ◽  
Manabu Negishi
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Body ◽  
Inhibitory Effect ◽  
Small Gtpases ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Cell Periphery ◽  
Cellular Processes ◽  
Rho Family ◽  
Actin Stress Fibers

ABSTRACT Rho family small GTPases are key regulators of the actin cytoskeleton in various cell types. The Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been recently identified as new members of the Rho family of GTPases, and expression of Rnd1 or Rnd3 in fibroblasts causes the disassembly of actin stress fibers and the retraction of the cell body to produce extensively branching cellular processes. Here we have performed a yeast two-hybrid screening by using Rnd1 as bait and identified a novel protein that specifically binds to Rnd GTPases. We named this protein Socius. Socius directly binds to Rnd GTPases through its COOH-terminal region. When transfected into COS-7 cells, Socius is translocated to the cell periphery in response to Rnd1 and Rnd3 and colocalized with the GTPases. While expression of wild-type Socius in Swiss 3T3 fibroblasts has little effect on the actin cytoskeleton, the expression of a membrane-targeted form of Socius, containing a COOH-terminal farnesylation motif (Socius-CAAX), induces a dramatic loss of stress fibers. The inhibitory effect of Socius-CAAX on stress fiber formation is enhanced by truncation of its NH2 terminus. On the other hand, the expression of Socius-CAAX or its NH2 terminus-truncated form suppresses the Rnd-induced retraction of the cell body and the production of extensively branching cellular processes, although the disassembly of stress fibers is observed. We propose that Socius participates in the Rnd GTPase-induced signal transduction pathways, leading to reorganization of the actin cytoskeleton.

scholarly journals Angiopoietin-Like 3 Induces Podocyte F-Actin Rearrangement through IntegrinαVβ3/FAK/PI3K Pathway-Mediated Rac1 Activation

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Yi Lin ◽  
Jia Rao ◽  
Xi-liang Zha ◽  
Hong Xu
Keyword(s):  
Actin Cytoskeleton ◽  
Pi3k Pathway ◽  
Small Gtpases ◽  
Glomerular Diseases ◽  
Differentiated Cells ◽  
Foot Process ◽  
Rho Family ◽  
Actin Rearrangement ◽  
Cytoskeleton Rearrangement

Glomerular podocytes are highly differentiated cells whose foot processes, which are mainly maintained by the architecture of actin filaments, have a unique morphology. A rearrangement of F-actin in podocytes causes changes in their motility that involve foot process effacement and proteinuria in glomerular diseases. Members of the Rho family small GTPases, especially RhoA, Rac1, and Cdc42, are key molecules in the regulation of actin cytoskeleton rearrangement. Our previous study showed that angiopoietin-like 3 (Angptl3) can increase the motility of podocytesin vitro. In this study, we found that recombinant Angptl3 treatment, together with the activation of Rac1, could cause F-actin rearrangement in podocytes. We also found that these effects could be blocked by an integrinαVβ3inhibitor, implicating integrinαVβ3as the Angptl3 receptor in its effects on actin cytoskeleton rearrangement. In addition, we studied the molecular pathway for this process. Our results showed that in podocytes, Angptl3 could induce actin filament rearrangement, mainly in lamellipodia formation, and that this process was mediated by integrinαVβ3-mediated FAK and PI3K phosphorylation and Rac1 activation. Our results might provide a new explanation for the effect of Angptl3 on increasing podocyte motility.

scholarly journals Overexpression of a Novel Rho Family GTPase, RacC, Induces Unusual Actin-based Structures and Positively Affects Phagocytosis inDictyostelium discoideum

1998 ◽  
Vol 9 (10) ◽  
pp. 2891-2904 ◽  
Author(s):  
David J. Seastone ◽  
Eunkyung Lee ◽  
John Bush ◽  
David Knecht ◽  
James Cardelli
Keyword(s):  
Actin Cytoskeleton ◽  
Kinase Inhibitors ◽  
Morphological Changes ◽  
Fluid Phase ◽  
Binding Motif ◽  
Cytoskeleton Organization ◽  
Cellular Processes ◽  
Actin Cytoskeleton Organization ◽  
A Cell ◽  
Rho Family

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin–rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif–containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis inDictyostelium.

Simulation of Stress Fiber Remodeling and Mixed-Mode Focal Adhesion Assembly During Cell Spreading and for Cells Adhered to Elastic Substrates

2011 ◽  
Author(s):  
William Ronan ◽  
Vikram S. Deshpande ◽  
Robert M. McMeeking ◽  
J. Patrick McGarry
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Physical Environment ◽  
Mixed Mode ◽  
Computational Models ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Stress Fiber ◽  
Cellular Processes ◽  
Elastic Substrates

Cell spreading is governed by two cooperative cellular processes: the association of binding proteins to form focal adhesions, and the active remodeling of the actin cytoskeleton as the cell spreads [1]. The interaction between these two processes is poorly understood, and previous computational models have only examined each process in isolation. Previous studies have established that cells possess the ability to sense and react to their physical environment, for example cells seeded on substrates of varying stiffness exhibit a different cytoskeletal response [2,3].

scholarly journals The actin cytoskeleton and plasma membrane connection: PtdIns(4,5)P(2) influences cytoskeletal protein activity at the plasma membrane

2000 ◽  
Vol 113 (21) ◽  
pp. 3685-3695 ◽  
Author(s):  
A.S. Sechi ◽  
J. Wehland
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Adhesion Molecules ◽  
Focal Adhesion ◽  
Small Gtpases ◽  
Cytoskeletal Protein ◽  
Protein Activity ◽  
Associated Proteins ◽  
Tight Connection

The co-ordination of rearrangements of the actin cytoskeleton depends on its tight connection to the plasma membrane. Phosphatidylinositol 4,5-bisphosphate is thought to transmit signals originating at the plasma membrane to the underlying actin cytoskeleton. This lipid binds to, and influences the activity of, several actin-associated proteins in vitro that regulate the architecture of the actin cytoskeleton. Signalling intermediates in this process include focal adhesion molecules such as vinculin and members of two families of proteins, ERM and WASP. These proteins interact with phosphatidylinositol 4,5-bisphosphate and appear to be regulated by interplay between small GTPases and phosphatidylinositol 4,5-bisphosphate metabolism, and thus link the plasma membrane with cytoskeletal remodelling.

scholarly journals Hic-5-Reduced Cell Spreading on Fibronectin: Competitive Effects between Paxillin and Hic-5 through Interaction with Focal Adhesion Kinase

2001 ◽  
Vol 21 (16) ◽  
pp. 5332-5345 ◽  
Author(s):  
Naoyuki Nishiya ◽  
Kouichi Tachibana ◽  
Motoko Shibanuma ◽  
Jun-Ichi Mashimo ◽  
Kiyoshi Nose
Keyword(s):  
Signal Transduction ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cell Spreading ◽  
3T3 Cells ◽  
Competitive Effects ◽  
Mouse Embryo Fibroblasts ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
Nih 3T3

ABSTRACT Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKβ/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK−/− mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.

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