scholarly journals pH sensing by FAK-His58 regulates focal adhesion remodeling

2013 ◽  
Vol 202 (6) ◽  
pp. 849-859 ◽  
Author(s):  
Chang-Hoon Choi ◽  
Bradley A. Webb ◽  
Michael S. Chimenti ◽  
Matthew P. Jacobson ◽  
Diane L. Barber
Keyword(s):  
Focal Adhesion ◽  
Conformational Changes ◽  
Cancer Metastasis ◽  
Ph Sensor ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Initial Step ◽  
Ph Sensing ◽  
Cellular Processes ◽  
Dynamics Simulations

Intracellular pH (pHi) dynamics regulates diverse cellular processes, including remodeling of focal adhesions. We now report that focal adhesion kinase (FAK), a key regulator of focal adhesion remodeling, is a pH sensor responding to physiological changes in pH. The initial step in FAK activation is autophosphorylation of Tyr397, which increased with higher pHi. We used a genetically encoded biosensor to show increased pH at focal adhesions as they mature during cell spreading. We also show that cells with reduced pHi had attenuated FAK-pY397 as well as defective cell spreading and focal adhesions. Mutagenesis studies indicated FAK-His58 is critical for pH sensing and molecular dynamics simulations suggested a model in which His58 deprotonation drives conformational changes that may modulate accessibility of Tyr397 for autophosphorylation. Expression of FAK-H58A in fibroblasts was sufficient to restore defective autophosphorylation and cell spreading at low pHi. These data are relevant to understanding cancer metastasis, which is dependent on increased pHi and FAK activity.

scholarly journals Structural and mechanistic insights into mechanoactivation of focal adhesion kinase

2019 ◽  
Vol 116 (14) ◽  
pp. 6766-6774 ◽  
Author(s):  
Magnus Sebastian Bauer ◽  
Fabian Baumann ◽  
Csaba Daday ◽  
Pilar Redondo ◽  
Ellis Durner ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Single Molecule ◽  
Conformational Changes ◽  
Cell Attachment ◽  
Focal Adhesions ◽  
Kinase Domain ◽  
Initial Rupture ◽  
Dynamics Simulations ◽  
Force Range

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.

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 Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization

2021 ◽  
Author(s):  
Hugo Arellano-Santoyo ◽  
Rogelio A Hernandez-Lopez ◽  
Emma Stokasimov ◽  
Ray YR Wang ◽  
David Pellman ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Single Molecule ◽  
Conformational Changes ◽  
Intracellular Transport ◽  
Structural Features ◽  
Cellular Processes ◽  
Tubulin Binding ◽  
Dynamics Simulations ◽  
Spatiotemporal Control ◽  
Unique Ability

The microtubule (MT) cytoskeleton is central to cellular processes including axonal growth, intracellular transport, and cell division, all of which rely on precise spatiotemporal control of MT organization. Kinesin-8s play a key role in regulating MT length by combining highly processive directional motility with MT-end disassembly. However, how kinesin-8 switches between these two apparently opposing activities remains unclear. Here, we define the structural features underlying this molecular switch through cryo-EM analysis of the yeast kinesin-8, Kip3 bound to MTs, and molecular dynamics simulations to approximate the complex of Kip3 with the curved tubulin state found at the MT plus-end. By integrating biochemical and single-molecule biophysical assays, we identified specific intra- and intermolecular interactions that modulate processive motility and MT disassembly. Our findings suggest that Kip3 undergoes conformational changes in response to tubulin curvature that underlie its unique ability to interact differently with the MT lattice than with the MT-end.

scholarly journals Focal adhesions are sites of integrin extension

2010 ◽  
Vol 188 (6) ◽  
pp. 891-903 ◽  
Author(s):  
Janet A. Askari ◽  
Christopher J. Tynan ◽  
Stephen E.D. Webb ◽  
Marisa L. Martin-Fernandez ◽  
Christoph Ballestrem ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Cell Spreading ◽  
Integrin Subunit ◽  
Adherent Cells ◽  
Extended Form ◽  
Integrin Α5β1 ◽  
Mediate Cell

Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)–based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin α5β1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled α5β1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of β1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.

A Method for Identification of the Constitutive Law of Biological Filaments From Their Dynamic Equilibria

2015 ◽  
Author(s):  
Soheil Fatehiboroujeni ◽  
Sachin Goyal ◽  
Apostol Gramada
Keyword(s):  
Conformational Changes ◽  
Inverse Method ◽  
Vital Role ◽  
Constitutive Law ◽  
Cellular Processes ◽  
Inverse Approach ◽  
Double Stranded Dna ◽  
Dynamics Simulations ◽  
Dynamic Equilibria ◽  
Atomistic Structure

There are several biological filaments that play vital role in cellular processes via twisting and bending deformations. From the double-stranded DNA molecule containing genetic information to the cytoskeletal fibers that provide shape to the cell, biological filaments undergo conformational changes as they perform their biological tasks. Therefore the ability of a filament to deform, which depends on their atomistic structure, is a characteristic property that governs its biological functions. Since there is no direct analytic method to derive the deformability or constitutive law of such filaments from their atomistic structure, the constitutive law has to be identified from their actual deformations. An inverse approach based on a continuum rod model was developed recently that uses deformations in static equilibrium to estimate the constitutive law in bending and torsion. We extend the inverse method to use dynamic states of deformations, and consequently expand its scope to leverage a wide variety of choices in molecular dynamics simulations for identifying the constitutive law. This paper presents and validates the technique applying it to filaments with artificial atomistic structure.

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

2007 ◽  
Vol 293 (3) ◽  
pp. H1978-H1986 ◽  
Author(s):  
Charles S. Wallace ◽  
Sophie A. Strike ◽  
George A. Truskey
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Tissue Engineered Blood Vessels ◽  
Focal Adhesion Formation ◽  
Fibrillar Adhesion

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the α5β1-integrin complex, whereas ECs used either α5β1- or αvβ3-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC α5β1-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.

scholarly journals Identification of integrin-stimulated sites of serine phosphorylation in FRNK, the separately expressed C-terminal domain of focal adhesion kinase: a potential role for protein kinase A

1997 ◽  
Vol 324 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Alan RICHARDSON ◽  
John D. SHANNON ◽  
Reid B. ADAMS ◽  
Michael D. SCHALLER ◽  
J. Thomas PARSONS
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Serine Phosphorylation ◽  
Early Event ◽  
Kinase A

Focal adhesion kinase (pp125FAK) is a protein tyrosine kinase that is localized to focal adhesions in many cell types and which undergoes tyrosine phosphorylation after integrin binding to extracellular matrix. In some cells the C-terminal non-catalytic domain of pp125FAK is expressed as a separate protein referred to as FRNK (FAK-related, non-kinase). We have previously shown that overexpression of FRNK inhibits tyrosine phosphorylation of pp125FAK and its substrates as well as inhibiting cell spreading on fibronectin. In this report we identify Ser148 and Ser151 as residues in FRNK that are phosphorylated after tyrosine phosphorylation of pp125FAK and in response to integrin binding to fibronectin. Tyrosine phosphorylation of pp125FAK appears to be an early event after integrin occupancy, and serine phosphorylation of FRNK occurs significantly later. Treatment of fibroblasts with a series of protein kinase A inhibitors delayed serine phosphorylation of FRNK as well as cell spreading on fibronectin and tyrosine phosphorylation of pp125FAK. However, these PKA inhibitors are unlikely to delay cell spreading simply by preventing serine phosphorylation of FRNK, as overexpression of FRNK containing mutations of Ser148 and Ser151 either singly or jointly to either alanine or glutamate residues did not significantly alter the ability of FRNK to act as an inhibitor of pp125FAK.

scholarly journals Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation.

1997 ◽  
Vol 17 (12) ◽  
pp. 6906-6914 ◽  
Author(s):  
A Richardson ◽  
R K Malik ◽  
J D Hildebrand ◽  
J T Parsons
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Sh2 Domain ◽  
Inhibitory Effects ◽  
C Terminus ◽  
Separate Protein ◽  
Additional Support ◽  
Autophosphorylation Site

pp125FAK is a tyrosine kinase that appears to regulate the assembly of focal adhesions and thereby promotes cell spreading on the extracellular matrix. In some cells, the C terminus of pp125FAK is expressed as a separate protein, pp41/43FRNK. We have previously shown that overexpression of pp41/43FRNK inhibits tyrosine phosphorylation of pp125FAK and paxillin and, in addition, delays cell spreading and focal adhesion assembly. Thus, pp41/43FRNK functions as a negative inhibitor of adhesion signaling and provides a tool to dissect the mechanism by which pp125FAK promotes cell spreading. We report here that the inhibitory effects of pp41/43FRNK expression can be rescued by the co-overexpression of wild-type pp125FAK and partially rescued by catalytically inactive variants of pp125FAK. However, coexpression of an autophosphorylation site mutant of pp125FAK, which fails to bind the SH2 domain of pp60c-Src, or a mutant that fails to bind paxillin did not promote cell spreading. In contrast, expression of pp41/43FRNK and pp60c-Src reconstituted cell spreading and tyrosine phosphorylation of paxillin but did so without inducing tyrosine phosphorylation of pp125FAK. These data provide additional support for a model whereby pp125FAK acts as a "switchable adaptor" that recruits pp60c-Src to phosphorylate paxillin, promoting cell spreading. In addition, these data point to tyrosine phosphorylation of paxillin as being a critical step in focal adhesion assembly.

scholarly journals STIM1 Controls the Focal Adhesion Dynamics and Cell Migration by Regulating SOCE in Osteosarcoma

2021 ◽  
Vol 23 (1) ◽  
pp. 162
Author(s):  
Yu-Shan Lin ◽  
Yi-Hsin Lin ◽  
MyHang Nguyen Thi ◽  
Shih-Chuan Hsiao ◽  
Wen-Tai Chiu
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Cancer Metastasis ◽  
Focal Adhesions ◽  
Dominant Negative ◽  
Osteosarcoma Cell ◽  
Wild Type ◽  
Calpain Activity ◽  
The Impact ◽  
Constitutively Active

The dysregulation of store-operated Ca2+ entry (SOCE) promotes cancer progression by changing Ca2+ levels in the cytosol or endoplasmic reticulum. Stromal interaction molecule 1 (STIM1), a component of SOCE, is upregulated in several types of cancer and responsible for cancer cell migration, invasion, and metastasis. To explore the impact of STIM1-mediated SOCE on the turnover of focal adhesion (FA) and cell migration, we overexpressed the wild-type and constitutively active or dominant negative variants of STIM1 in an osteosarcoma cell line. In this study, we hypothesized that STIM1-mediated Ca2+ elevation may increase cell migration. We found that constitutively active STIM1 dramatically increased the Ca2+ influx, calpain activity, and turnover of FA proteins, such as the focal adhesion kinase (FAK), paxillin, and vinculin, which impede the cell migration ability. In contrast, dominant negative STIM1 decreased the turnover of FA proteins as its wild-type variant compared to the cells without STIM1 overexpression while promoting cell migration. These unexpected results suggest that cancer cells need an appropriate amount of Ca2+ to control the assembly and disassembly of focal adhesions by regulating calpain activity. On the other hand, overloaded Ca2+ results in excessive calpain activity, which is not beneficial for cancer metastasis.

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