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Andrés J. García - One of the best experts on this subject based on the ideXlab platform.
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contractility modulates cell Adhesion strengthening through Focal Adhesion kinase and assembly of vinculin containing Focal Adhesions
Journal of Cellular Physiology, 2010Co-Authors: David W Dumbauld, Nathan D Gallant, Kristin E Michael, Heungsoo Shin, Harish Radhakrishna, Andrés J. GarcíaAbstract:Actin-myosin contractility modulates Focal Adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast Adhesion strengthening using a hydrodynamic Adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in Adhesion strengthening to levels 30-40% higher than serum-free cultures. Inhibition of myosin light chain kinase or Rho-kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum-induced enhancements in strengthening. Blebbistatin-induced inhibition of myosin II reduced serum-induced Adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in Adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from Focal Adhesions without changes in integrin binding. In vinculin-null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in Adhesion strength, indicating that the effects of contractility on adhesive force are vinculin-dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum-induced Adhesion strengthening as well as eliminated Focal Adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter Adhesion strength or Focal Adhesion assembly. These results indicate that contractility modulates Adhesion strengthening via FAK-dependent, vinculin-containing Focal Adhesion assembly.
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cell Adhesion strengthening contributions of adhesive area integrin binding and Focal Adhesion assembly
Molecular Biology of the Cell, 2005Co-Authors: Nathan D Gallant, Kristin E Michael, Andrés J. GarcíaAbstract:Mechanical interactions between a cell and its environment regulate migration, contractility, gene expression, and cell fate. We integrated micropatterned substrates to engineer adhesive area and a hydrodynamic assay to analyze fibroblast Adhesion strengthening on fibronectin. Independently of cell spreading, integrin binding and Focal Adhesion assembly resulted in rapid sevenfold increases in Adhesion strength to steady-state levels. Adhesive area strongly modulated Adhesion strength, integrin binding, and vinculin and talin recruitment, exhibiting linear increases for small areas. However, above a threshold area, Adhesion strength and Focal Adhesion assembly reached a saturation limit, whereas integrin binding transitioned from a uniform distribution to discrete complexes. Adhesion strength exhibited exponential increases with bound integrin numbers as well as vinculin and talin recruitment, and the relationship between Adhesion strength and these biochemical events was accurately described by a simple mechanical model. Furthermore, Adhesion strength was regulated by the position of an adhesive patch, comprised of bound integrins and cytoskeletal elements, which generated a constant 200-nN adhesive force. Unexpectedly, Focal Adhesion assembly, in particular vinculin recruitment, contributed only 30% of the Adhesion strength. This work elucidates the roles of adhesive complex size and position in the generation of cell-extracellular matrix forces.
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surface chemistry modulates Focal Adhesion composition and signaling through changes in integrin binding
Biomaterials, 2004Co-Authors: Benjamin G Keselowsky, David M Collard, Andrés J. GarcíaAbstract:Abstract Biomaterial surface properties influence protein adsorption and elicit diverse cellular responses in biomedical and biotechnological applications. However, the molecular mechanisms directing cellular activities remain poorly understood. Using a model system with well-defined chemistries (CH 3 , OH, COOH, NH 2 ) and a fixed density of the single adhesive ligand fibronectin, we investigated the effects of surface chemistry on Focal Adhesion assembly and signaling. Surface chemistry strongly modulated integrin binding and specificity— α 5 β 1 integrin binding affinity followed the pattern OH>NH 2 COOH>CH 3 , while integrin α V β 3 displayed the relationship COOH>NH 2 ≫OHCH 3 . Immunostaining and biochemical analyses revealed that surface chemistry modulates the structure and molecular composition of cell-matrix Adhesions as well as Focal Adhesion kinase (FAK) signaling. The neutral hydrophilic OH functionality supported the highest levels of recruitment of talin, α-actinin, paxillin, and tyrosine-phosphorylated proteins to adhesive structures. The positively charged NH 2 and negatively charged COOH surfaces exhibited intermediate levels of recruitment of Focal Adhesion components, while the hydrophobic CH 3 substrate displayed the lowest levels. These patterns in Focal Adhesion assembly correlated well with integrin α 5 β 1 binding. Phosphorylation of specific tyrosine residues in FAK also showed differential sensitivity to surface chemistry. Finally, surface chemistry-dependent differences in adhesive interactions modulated osteoblastic differentiation. These differences in Focal Adhesion assembly and signaling provide a potential mechanism for the diverse cellular responses elicited by different material properties.
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surface chemistry modulates Focal Adhesion composition and signaling through changes in integrin binding
Biomaterials, 2004Co-Authors: Benjamin G Keselowsky, David M Collard, Andrés J. GarcíaAbstract:Biomaterial surface properties influence protein adsorption and elicit diverse cellular responses in biomedical and biotechnological applications. However, the molecular mechanisms directing cellular activities remain poorly understood. Using a model system with well-defined chemistries (CH3, OH, COOH, NH2) and a fixed density of the single adhesive ligand fibronectin, we investigated the effects of surface chemistry on Focal Adhesion assembly and signaling. Surface chemistry strongly modulated integrin binding and specificity--alpha5beta1 integrin binding affinity followed the pattern OH>NH2=COOH>CH3, while integrin alphaVbeta3 displayed the relationship COOH>NH2>>OH=CH3. Immunostaining and biochemical analyses revealed that surface chemistry modulates the structure and molecular composition of cell-matrix Adhesions as well as Focal Adhesion kinase (FAK) signaling. The neutral hydrophilic OH functionality supported the highest levels of recruitment of talin, alpha-actinin, paxillin, and tyrosine-phosphorylated proteins to adhesive structures. The positively charged NH2 and negatively charged COOH surfaces exhibited intermediate levels of recruitment of Focal Adhesion components, while the hydrophobic CH3 substrate displayed the lowest levels. These patterns in Focal Adhesion assembly correlated well with integrin alpha5beta1 binding. Phosphorylation of specific tyrosine residues in FAK also showed differential sensitivity to surface chemistry. Finally, surface chemistry-dependent differences in adhesive interactions modulated osteoblastic differentiation. These differences in Focal Adhesion assembly and signaling provide a potential mechanism for the diverse cellular responses elicited by different material properties.
Michael D. Schaller - One of the best experts on this subject based on the ideXlab platform.
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the Focal Adhesion targeting domain of Focal Adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation
Structure, 2004Co-Authors: Kirk C Prutzman, Michael D. Schaller, Guanghua Gao, Michelle L King, Vidhya V Iyer, Geoffrey A Mueller, Sharon L CampbellAbstract:Abstract The Focal Adhesion targeting (FAT) domain of Focal Adhesion kinase (FAK) is critical for recruitment of FAK to Focal Adhesions and contains tyrosine 926, which, when phosphorylated, binds the SH2 domain of Grb2. Structural studies have shown that the FAT domain is a four-helix bundle that exists as a monomer and a dimer due to domain swapping of helix 1. Here, we report the NMR solution structure of the avian FAT domain, which is similar in overall structure to the X-ray crystal structures of monomeric forms of the FAT domain, except that loop 1 is longer and less structured in solution. Residues in this region undergo temperature-dependent exchange broadening and sample aberrant phi and psi angles, which suggests that this region samples multiple conformations. We have also identified a mutant that dimerizes ∼8 fold more than WT FAT domain and exhibits increased phosphorylation of tyrosine 926 both in vitro and in vivo.
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nmr solution structure of the Focal Adhesion targeting domain of Focal Adhesion kinase in complex with a paxillin ld peptide evidence for a two site binding model
Journal of Biological Chemistry, 2004Co-Authors: Guanghua Gao, Michael D. Schaller, Kirk C Prutzman, Michelle L King, Danielle M Scheswohl, Eugene F Derose, Robert E London, Sharon L CampbellAbstract:Abstract Focal Adhesion kinase (FAK) is a non-receptor tyrosine kinase that is regulated by integrins. Upon activation, FAK generates signals that modulate crucial cell functions, including cell proliferation, migration, and survival. The C-terminal Focal Adhesion targeting (FAT) sequence mediates localization of FAK to discrete regions in the cell called Focal Adhesions. Several binding partners for the FAT domain of FAK have been identified, including paxillin. We have determined the solution structure of the avian FAT domain in complex with a peptide mimicking the LD2 motif of paxillin by NMR spectroscopy. The FAT domain retains a similar fold to that found in the unliganded form when complexed to the paxillin-derived LD2 peptide, an antiparallel four-helix bundle. However, noticeable conformational changes were observed upon the LD2 peptide binding, especially the position of helix 4. Multiple lines of evidence, including the results obtained from isothermal titration calorimetry, intermolecular nuclear Overhauser effects, mutagenesis, and protection from paramagnetic line broadening, support the existence of two distinct paxillin-binding sites on the opposite faces of the FAT domain. The structure of the FAT domain-LD2 complex was modeled using the program HADDOCK based on our solution structure of the LD2-bound FAT domain and mutagenesis data. Our model of the FAT domain-LD2 complex provides insight into the molecular basis of FAK-paxillin binding interactions, which will aid in understanding the role of paxillin in FAK targeting and signaling.
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paxillin a Focal Adhesion associated adaptor protein
Oncogene, 2001Co-Authors: Michael D. SchallerAbstract:Paxillin is a Focal Adhesion-associated, phosphotyrosine-containing protein that may play a role in several signaling pathways. Paxillin contains a number of motifs that mediate protein-protein interactions, including LD motifs, LIM domains, an SH3 domain-binding site and SH2 domain-binding sites. These motifs serve as docking sites for cytoskeletal proteins, tyrosine kinases, serine/threonine kinases, GTPase activating proteins and other adaptor proteins that recruit additional enzymes into complex with paxillin. Thus paxillin itself serves as a docking protein to recruit signaling molecules to a specific cellular compartment, the Focal Adhesions, and/or to recruit specific combinations of signaling molecules into a complex to coordinate downstream signaling. The biological function of paxillin coordinated signaling is likely to regulate cell spreading and motility.
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biochemical signals and biological responses elicited by the Focal Adhesion kinase
Biochimica et Biophysica Acta, 2001Co-Authors: Michael D. SchallerAbstract:Abstract The Focal Adhesion kinase, FAK, is an important component of an integrin-dependent signaling pathway, which functions to transmit signals from the extracellular matrix into the cytoplasm. FAK is an essential gene product, since the fak −/− mouse exhibits embryonic lethality. A number of important biological processes, including cell motility and cell survival, are controlled by integrin-dependent signals and FAK has been implicated in regulating these processes. This review will focus upon recent findings providing insight into the mechanisms by which FAK transmits biochemical signals and elicits biological effects.
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paxillin a tyrosine phosphorylated Focal Adhesion associated protein binds to the carboxyl terminal domain of Focal Adhesion kinase
Molecular Biology of the Cell, 1995Co-Authors: Jeffrey D Hildebrand, Michael D. Schaller, J T ParsonsAbstract:Focal Adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called Focal Adhesions. pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals. Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk. In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin. The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified Focal Adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK. The interaction of paxillin and pp125FAK is independent of the Adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.
David D. Schlaepfer - One of the best experts on this subject based on the ideXlab platform.
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Focal Adhesion kinase signaling in unexpected places.
Current opinion in cell biology, 2017Co-Authors: Elizabeth G. Kleinschmidt, David D. SchlaepferAbstract:Focal Adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase first identified at extracellular matrix and integrin receptor cell Adhesion sites and is a key regulator of cell movement. FAK is activated by a variety of stimuli. Herein, we discuss advances in conformational-associated FAK activation and dimerization mechanisms. Additionally, new roles have emerged for FAK signaling at cell Adhesions, adherens junctions, endosomes, and the nucleus. In light of these new findings, we review how FAK activation at these sites is connected to the regulation of integrin recycling-activation, vascular permeability, cell survival, and transcriptional regulation, respectively. Studies uncovering FAK signaling connections in unexpected places within cells have yielded important new regulatory insights in cell biology.
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cortactin as a target for fak in the regulation of Focal Adhesion dynamics
PLOS ONE, 2012Co-Authors: Alok Tomar, Christine Lawson, Majid Ghassemian, David D. SchlaepferAbstract:Background Efficient cell movement requires the dynamic regulation of Focal Adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without Focal Adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.
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Detection of Focal Adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer
Nature Communications, 2011Co-Authors: Jihye Seong, Mingxing Ouyang, Shaoying Lu, Yue Zhuo, Nicholas M. Llewellyn, David D. Schlaepfer, Jun-lin GuanAbstract:The Focal Adhesion kinase has a role in cell Adhesion and migration. In this study, a fluorescent resonance energy transfer biosensor is designed to monitor Focal Adhesion kinase activity at membrane microdomains, revealing that the mechanisms that activate Focal Adhesion kinase are stimulus dependent.
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Focal Adhesion kinase in command and control of cell motility
Nature Reviews Molecular Cell Biology, 2005Co-Authors: Satyajit Sujit Kumar Mitra, Daniel Hanson, David D. SchlaepferAbstract:A central question in cell biology is how membrane-spanning receptors transmit extracellular signals inside cells to modulate cell Adhesion and motility. Focal Adhesion kinase (FAK) is a crucial signalling component that is activated by numerous stimuli and functions as a biosensor or integrator to control cell motility. Through multifaceted and diverse molecular connections, FAK can influence the cytoskeleton, structures of cell Adhesion sites and membrane protrusions to regulate cell movement.
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Focal Adhesion kinase suppresses rho activity to promote Focal Adhesion turnover
Journal of Cell Science, 2000Co-Authors: Xiangdong Ren, David D. Schlaepfer, David J Sieg, William B Kiosses, Carol A Otey, Martin A SchwartzAbstract:Focal Adhesion kinase (FAK) is activated and localized at Focal Adhesions upon cell Adhesion to extracellular matrices. Cells lacking FAK show increased Focal Adhesion number and decreased cell migration, functions that are regulated by the small GTPase Rho. We now report that fibroblasts from FAK-/- mice failed to transiently inhibit Rho activity when plated on fibronectin. Re-expression of FAK restored normal Rho regulation. Turnover of Focal Adhesions correlated inversely with Rho activity. The presence or absence of FAK was mimicked by inhibiting or activating Rho, respectively. These data suggest that loss of FAK resulting in constitutive activation of Rho and inhibition of Focal Adhesion turnover can account for deficiencies in cell migration and embryonic lethality of the FAK knockout.
Sharon L Campbell - One of the best experts on this subject based on the ideXlab platform.
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the Focal Adhesion targeting domain of Focal Adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation
Structure, 2004Co-Authors: Kirk C Prutzman, Michael D. Schaller, Guanghua Gao, Michelle L King, Vidhya V Iyer, Geoffrey A Mueller, Sharon L CampbellAbstract:Abstract The Focal Adhesion targeting (FAT) domain of Focal Adhesion kinase (FAK) is critical for recruitment of FAK to Focal Adhesions and contains tyrosine 926, which, when phosphorylated, binds the SH2 domain of Grb2. Structural studies have shown that the FAT domain is a four-helix bundle that exists as a monomer and a dimer due to domain swapping of helix 1. Here, we report the NMR solution structure of the avian FAT domain, which is similar in overall structure to the X-ray crystal structures of monomeric forms of the FAT domain, except that loop 1 is longer and less structured in solution. Residues in this region undergo temperature-dependent exchange broadening and sample aberrant phi and psi angles, which suggests that this region samples multiple conformations. We have also identified a mutant that dimerizes ∼8 fold more than WT FAT domain and exhibits increased phosphorylation of tyrosine 926 both in vitro and in vivo.
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nmr solution structure of the Focal Adhesion targeting domain of Focal Adhesion kinase in complex with a paxillin ld peptide evidence for a two site binding model
Journal of Biological Chemistry, 2004Co-Authors: Guanghua Gao, Michael D. Schaller, Kirk C Prutzman, Michelle L King, Danielle M Scheswohl, Eugene F Derose, Robert E London, Sharon L CampbellAbstract:Abstract Focal Adhesion kinase (FAK) is a non-receptor tyrosine kinase that is regulated by integrins. Upon activation, FAK generates signals that modulate crucial cell functions, including cell proliferation, migration, and survival. The C-terminal Focal Adhesion targeting (FAT) sequence mediates localization of FAK to discrete regions in the cell called Focal Adhesions. Several binding partners for the FAT domain of FAK have been identified, including paxillin. We have determined the solution structure of the avian FAT domain in complex with a peptide mimicking the LD2 motif of paxillin by NMR spectroscopy. The FAT domain retains a similar fold to that found in the unliganded form when complexed to the paxillin-derived LD2 peptide, an antiparallel four-helix bundle. However, noticeable conformational changes were observed upon the LD2 peptide binding, especially the position of helix 4. Multiple lines of evidence, including the results obtained from isothermal titration calorimetry, intermolecular nuclear Overhauser effects, mutagenesis, and protection from paramagnetic line broadening, support the existence of two distinct paxillin-binding sites on the opposite faces of the FAT domain. The structure of the FAT domain-LD2 complex was modeled using the program HADDOCK based on our solution structure of the LD2-bound FAT domain and mutagenesis data. Our model of the FAT domain-LD2 complex provides insight into the molecular basis of FAK-paxillin binding interactions, which will aid in understanding the role of paxillin in FAK targeting and signaling.
Pascal Schneider - One of the best experts on this subject based on the ideXlab platform.
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syndecan 4 par 3 signaling regulates Focal Adhesion dynamics in mesenchymal cells
Cell Communication and Signaling, 2020Co-Authors: Alejandra Valdivia, Areli Cardenas, Marianne Brenet, Horacio Maldonado, Milene Kong, Jorge Diaz, Keith Burridge, Pascal SchneiderAbstract:BACKGROUND Syndecans regulate cell migration thus having key roles in scarring and wound healing processes. Our previous results have shown that Thy-1/CD90 can engage both αvβ3 integrin and Syndecan-4 expressed on the surface of astrocytes to induce cell migration. Despite a well-described role of Syndecan-4 during cell movement, information is scarce regarding specific Syndecan-4 partners involved in Thy-1/CD90-stimulated cell migration. METHODS Mass spectrometry (MS) analysis of complexes precipitated with the Syndecan-4 cytoplasmic tail peptide was used to identify potential Syndecan-4-binding partners. The interactions found by MS were validated by immunoprecipitation and proximity ligation assays. The conducted research employed an array of genetic, biochemical and pharmacological approaches, including: PAR-3, Syndecan-4 and Tiam1 silencing, active Rac1 GEFs affinity precipitation, and video microscopy. RESULTS We identified PAR-3 as a Syndecan-4-binding protein. Its interaction depended on the carboxy-terminal EFYA sequence present on Syndecan-4. In astrocytes where PAR-3 expression was reduced, Thy-1-induced cell migration and Focal Adhesion disassembly was impaired. This effect was associated with a sustained Focal Adhesion Kinase activation in the siRNA-PAR-3 treated cells. Our data also show that Thy-1/CD90 activates Tiam1, a PAR-3 effector. Additionally, we found that after Syndecan-4 silencing, Tiam1 activation was decreased and it was no longer recruited to the membrane. Syndecan-4/PAR-3 interaction and the alteration in Focal Adhesion dynamics were validated in mouse embryonic fibroblast (MEF) cells, thereby identifying this novel Syndecan-4/PAR-3 signaling complex as a general mechanism for mesenchymal cell migration involved in Thy-1/CD90 stimulation. CONCLUSIONS The newly identified Syndecan-4/PAR-3 signaling complex participates in Thy-1/CD90-induced Focal Adhesion disassembly in mesenchymal cells. The mechanism involves Focal Adhesion kinase dephosphorylation and Tiam1 activation downstream of Syndecan-4/PAR-3 signaling complex formation. Additionally, PAR-3 is defined here as a novel adhesome-associated component with an essential role in Focal Adhesion disassembly during polarized cell migration. These novel findings uncover signaling mechanisms regulating cell migration, thereby opening up new avenues for future research on Syndecan-4/PAR-3 signaling in processes such as wound healing and scarring.