scholarly journals Arrestins regulate cell spreading and motility via focal adhesion dynamics

2015 ◽  
Vol 26 (4) ◽  
pp. 622-635 ◽  
Author(s):  
Whitney M. Cleghorn ◽  
Kevin M. Branch ◽  
Seunghyi Kook ◽  
Christopher Arnette ◽  
Nada Bulus ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion ◽  
Cell Attachment ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Wild Type

Focal adhesions (FAs) play a key role in cell attachment, and their timely disassembly is required for cell motility. Both microtubule-dependent targeting and recruitment of clathrin are critical for FA disassembly. Here we identify nonvisual arrestins as molecular links between microtubules and clathrin. Cells lacking both nonvisual arrestins showed excessive spreading on fibronectin and poly-d-lysine, increased adhesion, and reduced motility. The absence of arrestins greatly increases the size and lifespan of FAs, indicating that arrestins are necessary for rapid FA turnover. In nocodazole washout assays, FAs in arrestin-deficient cells were unresponsive to disassociation or regrowth of microtubules, suggesting that arrestins are necessary for microtubule targeting–dependent FA disassembly. Clathrin exhibited decreased dynamics near FA in arrestin-deficient cells. In contrast to wild-type arrestins, mutants deficient in clathrin binding did not rescue the phenotype. Collectively the data indicate that arrestins are key regulators of FA disassembly linking microtubules and clathrin.

scholarly journals Src SH2 Arginine 175 Is Required for Cell Motility: Specific Focal Adhesion Kinase Targeting and Focal Adhesion Assembly Function

2006 ◽  
Vol 26 (12) ◽  
pp. 4399-4409 ◽  
Author(s):  
Myeong Gu Yeo ◽  
Michael A. Partridge ◽  
Ellen J. Ezratty ◽  
Qiong Shen ◽  
Gregg G. Gundersen ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Src Kinase ◽  
Focal Adhesions ◽  
Sh2 Domain ◽  
Wild Type ◽  
Binding Function ◽  
Generation Capacity ◽  
Substrate Phosphorylation

ABSTRACT Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.

scholarly journals The Cytoplasmic Tyrosines of Integrin Subunit β1 Are Involved in Focal Adhesion Kinase Activation

2000 ◽  
Vol 20 (15) ◽  
pp. 5758-5765 ◽  
Author(s):  
Krister Wennerberg ◽  
Annika Armulik ◽  
Takao Sakai ◽  
Marjam Karlsson ◽  
Reinhard Fässler ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Phosphorylation Site ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Integrin Subunit ◽  
Wild Type ◽  
Directed Cell Migration ◽  
Dependent Cell

ABSTRACT We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit β1 (Y783 and Y795) to phenylalanines markedly reduces the capability of β1A integrins to mediate directed cell migration. In this study, β1-dependent cell spreading was found to be delayed in GD25 cells expressing β1AY783/795F compared to that in wild-type GD25-β1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to β1-dependent adhesion in GD25-β1AY783/795F cells compared to that in wild-type GD25-β1A or mutants in which only a single tyrosine was altered (β1AY783F or β1AY795F). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via β1AY783/795F lies at the level of the initial autophosphorylation step. Indeed, β1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the β1AY783/795F cells, consistent with the impairment in FAK activation. In contrast, p130CAS overall tyrosine phosphorylation was unaffected by the β1 mutations. Despite the defect in β1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-β1AY783/795F cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit β1A are critical mediators of FAK activation and cell spreading in GD25 cells.

scholarly journals The Association of ASAP1, an ADP Ribosylation Factor-GTPase Activating Protein, with Focal Adhesion Kinase Contributes to the Process of Focal Adhesion Assembly

2002 ◽  
Vol 13 (6) ◽  
pp. 2147-2156 ◽  
Author(s):  
Yunhao Liu ◽  
Joost C. Loijens ◽  
Karen H. Martin ◽  
Andrei V. Karginov ◽  
J. Thomas Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Ph Domain ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Transient Overexpression ◽  
Adp Ribosylation Factor

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60Src. Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). GlutathioneS-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.

scholarly journals FAK Potentiates Rac1 Activation and Localization to Matrix Adhesion Sites: A Role for βPIX

2007 ◽  
Vol 18 (1) ◽  
pp. 253-264 ◽  
Author(s):  
Fumin Chang ◽  
Christopher A. Lemmon ◽  
Dongeun Park ◽  
Lewis H. Romer
Keyword(s):  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Cell Attachment ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Cell Spreading ◽  
Subcellular Location ◽  
Dominant Negative ◽  
Membrane Ruffling ◽  
Adhesion Sites

FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon cell attachment to extracellular matrix. FAK null cells spread poorly and exhibit altered focal adhesion turnover. Rac1 is a member of the Rho-family GTPases that promotes membrane ruffling, leading edge extension, and cell spreading. We investigated the activation and subcellular location of Rac1 in FAK null and FAK reexpressing fibroblasts. FAK reexpressers had a more robust pattern of Rac1 activation after cell adhesion to fibronectin than the FAK null cells. Translocation of Rac1 to focal adhesions was observed in FAK reexpressers, but seldom in FAK null cells. Experiments with constitutively active L61Rac1 and dominant negative N17Rac1 indicated that the activation state of Rac1 regulated its localization to focal adhesions. We demonstrated that FAK tyrosine-phosphorylated βPIX and thereby increased its binding to Rac1. In addition, βPIX facilitated the targeting of activated Rac1 to focal adhesions and the efficiency of cell spreading. These data indicate that FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of βPIX.

EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway

1998 ◽  
Vol 111 (5) ◽  
pp. 615-624 ◽  
Author(s):  
H. Xie ◽  
M.A. Pallero ◽  
K. Gupta ◽  
P. Chang ◽  
M.F. Ware ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Motility ◽  
Map Kinase ◽  
Signaling Pathway ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Focal Adhesions ◽  
Short Term ◽  
Egfr Kinase

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

scholarly journals Campylobacter jejuni Triggers Signaling through Host Cell Focal Adhesions To Inhibit Cell Motility

mBio ◽  
2021 ◽  
Author(s):  
Courtney M. Klappenbach ◽  
Nicholas M. Negretti ◽  
Jesse Aaron ◽  
Teng-Leong Chew ◽  
Michael E. Konkel
Keyword(s):  
Epithelial Cells ◽  
Cell Motility ◽  
Host Cell ◽  
Campylobacter Jejuni ◽  
Focal Adhesion ◽  
Foodborne Pathogen ◽  
Focal Adhesions ◽  
Structure And Function ◽  
Content Type ◽  
And Function

Campylobacter jejuni is a major foodborne pathogen that causes severe gastritis. We investigated the dynamics of focal adhesion structure and function in C. jejuni -infected epithelial cells.

Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2417-2426 ◽  
Author(s):  
Özlem Yilmaz ◽  
Patrick A. Young ◽  
Richard J. Lamont ◽  
George E. Kenny
Keyword(s):  
Porphyromonas Gingivalis ◽  
Focal Adhesion ◽  
Cell Signalling ◽  
Focal Adhesions ◽  
Wild Type ◽  
Cortical Actin ◽  
Membrane Ruffling ◽  
Invasion Mechanism ◽  
Signalling Molecules ◽  
Gingival Epithelial Cell

Porphyromonas gingivalis, an oral pathogen, can internalize within primary gingival epithelial cells (GECs) through an invasion mechanism mediated by interactions between P. gingivalis fimbriae and integrins on the surface of the GECs. Fimbriae–integrin-based signalling events were studied by fluorescence microscopy, and the subcellular localization of integrin-associated signalling molecules paxillin and focal adhesion kinase (FAK), and the architecture of the actin and microtubule cytoskeleton were examined. GECs infected with P. gingivalis for 30 min demonstrated significant redistribution of paxillin and FAK from the cytosol to cell peripheries and assembly into focal adhesion complexes. In contrast, a fimbriae-deficient mutant of P. gingivalis did not contribute substantially to activation of paxillin or FAK. After 24 h, the majority of paxillin and FAK had returned to the cytoplasm with significant co-localization with P. gingivalis in the perinuclear region. Wild-type P. gingivalis induced nucleation of actin filaments forming microspike-like protrusions and long stable microfilaments distributed throughout the cells. Fimbriae mutants promoted a rich cortical actin meshwork accompanied by membrane ruffling dispersed along the cell membrane. Remarkable disassembly and nucleation of the actin and microtubule filamentous network was observed following 24 h infection with either wild-type or fimbriae-deficient mutants of P. gingivalis. The results show that fimbriated P. gingivalis cells induce formation of integrin-associated focal adhesions with subsequent remodelling of the actin and tubulin cytoskeleton.

scholarly journals Interleukin 1-induced calcium signalling in chondrocytes requires focal adhesions

1997 ◽  
Vol 324 (2) ◽  
pp. 653-658 ◽  
Author(s):  
Laura LUO ◽  
Tony CRUZ ◽  
Christopher McCULLOCH
Keyword(s):  
Signal Transduction ◽  
Focal Adhesion ◽  
Cell Attachment ◽  
Interleukin 1 ◽  
Primary Cultures ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Ion Concentration ◽  
Significant Difference ◽  
In Cells

The cytokine interleukin 1 (IL-1) is an important mediator of connective-tissue destruction in arthritic joints but the mechanisms by which IL-1 mediates signal transduction in chondrocytes is poorly understood. Previous results have indicated that IL-1 receptors co-localize with focal adhesions [Qwarnstrom, Page, Gillis and Dower (1988) J. Biol. Chem. 263, 8261–8269], discrete adhesive domains of cells that function in cell attachment and possibly in signal transduction. We have determined whether focal adhesions restrict IL-1-induced Ca2+ signalling in primary cultures of bovine chondrocytes. In cells grown for 24 h on fibronectin, the basal intracellular Ca2+ ion concentration ([Ca2+]i) was 100±3 nM. Optimal increases of [Ca2+]i above baseline were induced by 10 nM IL-1 (183±30 nM above baseline). There was no significant difference between cells plated on fibronectin or type II collagen (P > 0.2; 233±90 nM above baseline). Ca2+ transients were significantly decreased by the inclusion of 0.5 mM EGTA in the bathing buffer (74±11 nM above baseline), and 1 μM thapsigargin completely blocked Ca2+ transients. Cells plated on poly-(l-lysine) or suspended cells showed no Ca2+ increases, whereas cells grown on fibronectin exhibited IL-1-induced Ca2+ responses that corresponded temporally to the time-dependent cell spreading after plating on fibronectin. Cells plated on poly-(l-lysine) and incubated with fibronectin-coated beads exhibited vinculin staining in association with the beads. In identical cell preparations, IL-1 induced a 136±39 nM increase of [Ca2+]i above baseline in response to 10 nM IL-1β. There were no IL-1-induced Ca2+ increases when cells on poly-(l-lysine) were incubated with fibronectin-coated beads for only 15 min at 37 °C, in cells maintained for 3 h at 4 °C, in cells incubated with BSA beads for 3 h at 37 °C, or in cells pretreated with cytochalasin D. Labelling of IL-1 receptors with 125I-IL-1β showed 3-fold more specific labelling of focal adhesion complexes in cells incubated with fibronectin-coated beads compared with cells incubated with BSA-coated beads, indicating that IL-1 receptor binding or the number of IL-1 receptors was increased in focal adhesions. These results indicate that, in chondrocytes, IL-1-induced Ca2+ signalling is dependent on focal adhesion formation and that focal adhesions recruit IL-1 receptors by redistribution in the cell membrane.

scholarly journals Concerted Activity of Tyrosine Phosphatase SHP-2 and Focal Adhesion Kinase in Regulation of Cell Motility

1999 ◽  
Vol 19 (4) ◽  
pp. 3125-3135 ◽  
Author(s):  
Santos Mañes ◽  
Emilia Mira ◽  
Concepción Gómez-Mouton ◽  
Zhizuang Joe Zhao ◽  
Rosa Ana Lacalle ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Cell Motility ◽  
Focal Adhesion ◽  
Tyrosine Phosphatase ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Igf I ◽  
Mitogen Activated Protein ◽  
Mcf 7

ABSTRACT The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Using MCF-7 cells, we found that insulin-like growth factor I (IGF-I) induces the adhesion of MCF-7 to vitronectin and collagen in a dose- and time-dependent manner, suggesting that IGF-I triggers the activation of different integrins. On the other hand, IGF-I promotes the association of insulin receptor substrate 1 with the focal adhesion kinase (FAK), paxillin, and the tyrosine phosphatase SHP-2, resulting in FAK and paxillin dephosphorylation. Abrogation of SHP-2 catalytic activity with a dominant-negative mutant (SHP2-C>S) abolishes IGF-I-induced FAK dephosphorylation, and cells expressing SHP2-C>S show reduced IGF-I-stimulated chemotaxis compared with either mock- or SHP-2 wild-type-transfected cells. This impairment of cell migration is recovered by reintroduction of a catalytically active SHP-2. Interestingly, SHP-2-C>S cells show a larger number of focal adhesion contacts than wild-type cells, suggesting that SHP-2 activity participates in the integrin deactivation process. Although SHP-2 regulates mitogen-activated protein kinase activity, the mitogen-activated protein kinase kinase inhibitor PD-98059 has only a marginal effect on MCF-7 cell migration. The role of SHP-2 as a general regulator of cell chemotaxis induced by other chemotactic agents and integrins is discussed.

scholarly journals Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

1994 ◽  
Vol 5 (9) ◽  
pp. 977-988 ◽  
Author(s):  
S Kawaguchi ◽  
J M Bergelson ◽  
R W Finberg ◽  
M E Hemler
Keyword(s):  
Amino Acids ◽  
Cell Adhesion ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Inverse Correlation ◽  
Cytoplasmic Domain ◽  
Negative Regulator ◽  
Wild Type

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.

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