Adhesion Disassembly Research Articles

EGF‐induced ERK‐activation downstream of FAK requires rac1‐NADPH oxidase

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox-sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1-NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1-NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAK's ability to act as a scaffold for EGF-mediated signaling, including activation of ERK. Accordingly, we found that EGF-induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK-regulated S910-site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading.

A direct interaction between the large GTPase dynamin-2 and FAK regulates focal adhesion dynamics in response to active Src

Tumor cell migration is supported in part by the cyclic formation and disassembly of focal adhesions (FAs); however, the mechanisms that regulate this process are not fully defined. The large guanosine 5'-triphosphatase dynamin (Dyn) plays an important role in FA dynamics and is activated by tyrosine phosphorylation. Using a novel antibody specific to phospho-dynamin (pDyn-Tyr-231), we found that Dyn2 is phosphorylated at FAs by Src kinase and is recruited to FAs by a direct interaction with the 4.1/ezrin/radizin/moesin domain of focal adhesion kinase (FAK), which functions as an adaptor between Src and Dyn2 to facilitate Dyn2 phosphorylation. This Src-FAK-Dyn2 trimeric complex is essential for FA turnover, as mutants disrupting the formation of this complex inhibit FA disassembly. Importantly, phosphoactivated Dyn2 promotes FA turnover by mediating the endocytosis of integrins in a clathrin-dependent manner. This study defines a novel mechanism of how Dyn2 functions as a downstream effector of FAK-Src signaling in turning over FAs.

Calpain-mediated Proteolysis of Paxillin Negatively Regulates Focal Adhesion Dynamics and Cell Migration

The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.

Steroid Receptor Coactivator-1 Upregulates Integrin α5 Expression to Promote Breast Cancer Cell Adhesion and Migration

Metastatic breast cancer remains a lethal disease with poorly understood molecular mechanisms. Steroid receptor coactivator-1 (SRC-1 or NCOA1) is overexpressed in a subset of breast cancers with poor prognosis. It potentiates gene expression by serving as a coactivator for nuclear receptors and other transcription factors. We previously reported that SRC-1 promotes breast cancer metastasis without affecting primary mammary tumor formation. Herein, we found that SRC-1 deficiency in mouse and human breast cancer cells substantially reduced cell adhesion and migration capabilities on fibronectin and significantly extended the time of focal adhesion disassembly and reassembly. In agreement with this phenotype, SRC-1 expression positively correlated with integrin α(5) (ITGA5) expression in estrogen receptor-negative breast tumors whereas SRC-1 deficiency decreased ITGA5 expression. Furthermore, ITGA5 reduction in SRC-1-deficient/insufficient breast cancer cells or knockdown of ITGA5 in SRC-1-expressing breast cancer cells was associated with a disturbed integrin-mediated signaling. Critical downstream changes included reduced phosphorylation and/or dampened activation of focal adhesion kinase, paxillin, Rac1, and Erk1/2 during cell adhesion. Finally, we found that SRC-1 enhanced ITGA5 promoter activity through an AP-1 (activator protein)-binding site proximal to the transcriptional initiation site; both SRC-1 and c-Jun were recruited to this promoter region in breast cancer cells. These results show that SRC-1 can promote breast cancer metastasis by directly enhancing ITGA5 expression and thus promoting ITGA5-mediated cell adhesion and migration. Therefore, targeting ITGA5 in SRC-1-positive breast cancers may result in inhibition of SRC-1-promoted breast cancer metastasis.

ERK-MAPK Drives Lamellipodia Protrusion by Activating the WAVE2 Regulatory Complex

Cell movement begins with a leading edge protrusion, which is stabilized by nascent adhesions and retracted by mature adhesions. The ERK-MAPK (extracellular signal-regulated kinase-mitogen-activated protein kinase) localizes to protrusions and adhesions, but how it regulates motility is not understood. We demonstrate that ERK controls protrusion initiation and protrusion speed. Lamellipodial protrusions are generated via the WRC (WAVE2 regulatory complex), which activates the Arp2/3 actin nucleator for actin assembly. The WRC must be phosphorylated to be activated, but the sites and kinases that regulate its intermolecular changes and membrane recruitment are unknown. We show that ERK colocalizes with the WRC at lamellipodial leading edges and directly phosphorylates two WRC components: WAVE2 and Abi1. The phosphorylations are required for functional WRC interaction with Arp2/3 and actin during cell protrusion. Thus, ERK coordinates adhesion disassembly with WRC activation and actin polymerization to promote productive leading edge advancement during cell migration.

Stiffness and Load-Dependent Spreading of Cell-Surface Adhesions are Emergent Properties of a Molecular Model

Cells plated on a 2-D substrate form adhesions with that surface. These adhesions, consisting of aggregates of various proteins, including integrins, paxillin, vinculin, talin and others, are thought to be important in mechanosensation, the process by which the cell senses and adapts to mechanical properties of the surface, such as stiffness. At a molecular level, integrins in the adhesion bind to the extracellular matrix, while the various other proteins are involved in, among other things, binding to actin filaments that, in turn, apply a load to the adhesion. Several of these proteins (e.g. talin) undergo load-dependent state transitions that are thought to be important in both the load-dependent and the surface stiffness-dependent stability of the adhesions. Based on these molecular-level observations, we consider a grossly simplified version of an adhesion, made up of “molecules” that can bind to the surface in a strain-dependent manner and can undergo a load-dependent state transition. Remarkably, we find that, in Monte-Carlo simulations of these molecules, molecular aggregates are formed in a load- and stiffness-dependent fashion that closely mimics that seen in experiment. Furthermore, we find that these adhesions exhibit three phases of growth: 1) nucleation, where small, transient molecular aggregates form; 2) maturation, where adhesions grow quadratically in time; and 3) decay, where a short steady-state is followed by adhesion disassembly. These three phases of adhesion growth are also experimentally observed. These various properties of the Monte-Carlo simulation may be simply understood by analytic calculations. We therefore conclude that many experimental observations of stiffness- and load-dependent adhesion growth are emergent properties of molecular-mechanical systems with strain-dependent surface binding and a load-dependent state transition.

High Resolution Microscopy in Live-Cell Imaging

We have used PALM (photoactivation localization microscopy) imaging to study the formation and disassembly of focal adhesions of live HeLa cells in a high resolution pulse chase experiment using a monomeric variant of the photoactivatable protein IrisFP [1]. mIrisFP is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green- to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms [2]. A subpopulation of mIrisFP molecules is photoconverted to the red form by irradiating a specific region of the cell with a pulse of violet light. Migration of the tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by PALM imaging, exploiting the photoswitching capability of the red species. Online image analysis was performed by using our recently software [3]. [1] Adam et al., Proc. Natl. Acad. Sci. USA. 105 (2008) 18343. [2] Fuchs et al., Nat. Methods 7 (2010) 627. [3] Hedde et al., Nat. Methods 6 (2009) 689.

Mechano-Stimulation of Fibroblasts by Adjusting Viscous Drag of Mobile Cell Linkers in Biomembrane-Mimicking Substrates

Recent advancements in the design of polymeric substrates of tunable rigidity have shown that cells probe the viscoelasticity of their environment through an adaptive process of focal contact assembly/disassembly that critically affects cell adhesion and morphology. However, the specific mechanisms of this process of mechano-sensitivity have not yet been fully uncovered, in part due to the limitations of existing engineered cell substrates, which are characterized by immobilized cell linkers. To overcome this limitation, we here present a biomembrane-mimicking cell substrate based on polymer-tethered lipid muli-bilayers, in which laterally mobile cell linkers enable the free assembly and disassembly of focal adhesions. In this experimental platform, the mechano-stimulation of plated cells is accomplished by altering the viscous drag of cell linkers through the number of lipid bilayers in the solid-supported multi-bilayer stack. Results from microscopy experiments are discussed, which illustrate that the number of bilayers in the multi-bilayer stack has a profound impact on various cellular properties of plated 3T3 fibroblasts, including adhesion, morphology, shape fluctuations, migration, and cytoskeletal organization. Furthermore, this biomembrane-mimicking substrate is integrated into a force traction microscopy assay, which confirms that the presence of the fluid multi-bilayer system leads to a notable reduction in cellular traction forces. Our experiments illustrate that the described biomembrane-mimicking cell substrate is particularly well suited to monitor plated cells under conditions of weak force transduction conditions between cells and underlying substrate.

Molecular and Structural Insight for the Role of Key Residues of Thrombospondin-1 and Calreticulin in Thrombospondin-1- Calreticulin Binding

Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on cell surface signals focal adhesion disassembly. Residues of Lys 24 and 32 in TSP1 and amino acids 24-26 and 32-34 in CRT are critical for TSP1-CRT binding (Faseb J, 22:3968, 2008; J Biol Chem, 268:26784, 1993). This study investigated molecular and structural basis for the effect of these key residues in TSP1 and CRT on TSP1-CRT binding. Based on a validated TSP1-CRT complex structure (Biochemistry, 47:3685, 2010), we adopted steered molecular dynamics simulations to determine the effect of the mutations of these key residues on TSP1-CRT binding and validated the simulation results with experimental observation. We further performed 30 ns molecular dynamics simulations for wild type TSP1, CRT, TSP1 K24A&K32A mutant and mutant CRT (residues 24-26 & 32-34 mutated to Ala) and studied the conformational and structural changes of TSP1 and CRT by the mutations of the critical residues. Results showed that mutations of residues 24 and 32 to Ala in TSP1 and of amino acids 24-26 and 32-34 in CRT to Ala result in a shortened β strand in the binding site, decreased hydrogen bond occupancy for β strand pairs that are located within or near the binding site, increased conformational flexibility of the binding site, a changed degree of dynamic correlated motion between the residues in the binding site and the other residues in protein, and a changed degree of overall correlated motions between the residues in protein. These changes could directly contribute to the loss or reduced binding of TSP1-CRT complex and further affect TSP1-CRT binding induced cellular activities. Results from this study provide molecular and structural insight for the role of the critical residues in TSP1 and CRT in TSP1-CRT binding.

Molecular and Structural Insight into the Role of Key Residues of Thrombospondin-1 and Calreticulin in Thrombospondin-1−Calreticulin Binding

Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface signals focal adhesion disassembly, leading to the intermediate adhesive phenotype, cell migration, anoikis resistance, and collagen stimulation. Residues Lys 24 and 32 in TSP1 and amino acids 24-26 and 32-34 in CRT have been shown through biochemical and cell-based approaches to be critical for TSP1-CRT binding and signaling. This study investigated the molecular and structural basis for these key TSP1 and CRT residues in TSP1-CRT binding. On the basis of a validated TSP1-CRT complex structure, we adopted steered molecular dynamics simulations to determine the effect of mutation of these key residues on TSP1-CRT binding and validated the simulation results with experimental observations. We further performed 30 ns molecular dynamics simulations for wild-type TSP1, CRT, K24A/K32A mutant TSP1, and mutant CRT (residues 24-26 and 32-34 mutated to Ala) and studied the conformational and structural changes in TSP1 and CRT as the result of mutation of these critical residues. Results showed that mutation of residues 24 and 32 to Ala in TSP1 and of amino acids 24-26 and 32-34 to Ala in CRT results in a shortened β-strand in the binding site, decreased hydrogen bond occupancy for β-strand pairs that are located within or near the binding site, increased conformational flexibility of the binding site, a changed degree of dynamically correlated motion between the residues in the binding site and the other residues in protein, and a changed degree of overall correlated motion between the residues in the protein. These changes could directly contribute to the loss or weakened binding between TSP1 and CRT and the resultant effects on TSP1-CRT binding-induced cellular activities. Results from this study provide a molecular and structural insight into the role of these critical residues of TSP1 and CRT in TSP1-CRT binding.

Alveolar Type II Cells Escape Stress Failure Caused by Tonic Stretch through Transient Focal Adhesion Disassembly

Mechanical ventilation-induced excessive stretch of alveoli is reported to induce cellular stress failure and subsequent lung injury, and is therefore an injurious factor to the lung. Avoiding cellular stress failure is crucial to ventilator-induced lung injury (VILI) treatment. In the present study, primary rat alveolar type II (ATII) cells were isolated to evaluate their viability and the mechanism of their survival under tonic stretch. By the annexin V/ PI staining and flow cytometry assay, we demonstrated that tonic stretch-induced cell death is an immediate injury of mechanical stress. In addition, immunofluorescence and immunoblots assay showed that the cells experienced an expansion-contraction-reexpansion process, accompanied by partial focal adhesion (FA) disassembly during contraction. Manipulation of integrin adherent affinity by altering bivalent cation levels in the culture medium and applying an integrin neutralizing antibody showed that facilitated adhesion affinity promoted cell death under tonic stretch, while lower level of adhesion protected the cells from stretch-induced stress failure. Finally, a simplified numerical model was established to reveal that adequate disassembly of FAs reduced the forces transmitting throughout the cell. Taken together, these results indicate that ATII cells escape stress failure caused by tonic stretch via active cell morphological remodeling, during which cells transiently disassemble FAs to unload mechanical forces.

Distinct roles for paxillin and Hic-5 in regulating breast cancer cell morphology, invasion, and metastasis

Individual metastatic tumor cells exhibit two interconvertible modes of cell motility during tissue invasion that are classified as either mesenchymal or amoeboid. The molecular mechanisms by which invasive breast cancer cells regulate this migratory plasticity have yet to be fully elucidated. Herein we show that the focal adhesion adaptor protein, paxillin , and the closely related Hic-5 have distinct and unique roles in the regulation of breast cancer cell lung metastasis by modulating cell morphology and cell invasion through three-dimensional extracellular matrices (3D ECMs). Cells depleted of paxillin by RNA interference displayed a highly elongated mesenchymal morphology, whereas Hic-5 knockdown induced an amoeboid phenotype with both cell populations exhibiting reduced plasticity, migration persistence, and velocity through 3D ECM environments. In evaluating associated signaling pathways, we determined that Rac1 activity was increased in cells devoid of paxillin whereas Hic-5 silencing resulted in elevated RhoA activity and associated Rho kinase–induced nonmuscle myosin II activity. Hic-5 was essential for adhesion formation in 3D ECMs, and analysis of adhesion dynamics and lifetime identified paxillin as a key regulator of 3D adhesion assembly, stabilization, and disassembly.

Calpain 2 Controls Turnover of LFA-1 Adhesions on Migrating T Lymphocytes

The immune cells named T lymphocytes circulate around the body fulfilling their role in immunosurveillance by monitoring the tissues for injury or infection. To migrate from the blood into the tissues, they make use of the integrin LFA-1 which is exclusively expressed by immune cells. These highly motile cells attach and migrate on substrates expressing the LFA-1 ligand ICAM-1. The molecular events signaling LFA-1 activation and adhesion are now reasonably well identified, but the process of detaching LFA-1 adhesions is less understood. The cysteine protease calpain is involved in turnover of integrin-mediated adhesions in less motile cell types. In this study we have explored the involvement of calpain in turnover of LFA-1-mediated adhesions of T lymphocytes. Using live cell imaging and immunohistochemistry, we demonstrate that turnover of adhesions depends on the Ca2+-dependent enzyme, calpain 2. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition results in inefficient disassembly of LFA-1 adhesions causing T lymphocyte elongation and shedding of LFA-1 clusters behind the migrating T lymphocytes. We show that calpain 2 is distributed throughout the T lymphocyte, but is most active at the trailing edge as detected by expression of its fluorescent substrate CMAC,t-BOC-Leu-Met. Extracellular Ca2+ entry is essential for the activity of calpain 2 that is constantly maintained as the T lymphocytes migrate. Use of T cells from a patient with mutation in ORAI1 revealed that the major calcium-release-activated-calcium channel is not the ion channel delivering the Ca2+. We propose a model whereby Ca2+ influx, potentially through stretch activated channels, is sufficient to activate calpain 2 at the trailing edge of a migrating T cell and this activity is essential for the turnover of LFA-1 adhesions.

Genetic Variants In Platelet Supervillin but Not Archvillin Are Associated with PFA-100 Closure Times In African Americans

AbstractAbstract 4311Myocardial infarction typically occurs after atherosclerotic plaque rupture that exposes collagen under high shear stress, favoring platelet adhesion, activation with ADP release, aggregation and platelet thrombus formation. There is a well-established genetic contribution to myocardial infarction, and we have previously shown a shear-dependent assay of platelet function is heritable (JTH 2007;5:1617). Collagen-induced platelet aggregation is highly heritable in African Americans (AAs) but not in European Americans, which may be relevant since AAs have the highest overall coronary heart disease mortality rate of all ethnic groups in the US. The mechanisms for these ethnic differences in platelet function are not clear. The goal of the current study was to identify genetic variants in AAs affecting the Platelet Function Analyzer (PFA)-100 (ADP cartridge) that is both collagen- and shear-dependent. We recruited healthy AA subjects and measured closure times in the PFA-100. Subjects were excluded if there was evidence of exposure to anti-platelet therapy (absent response to arachidonic acid). Closure times showed a normal distribution (mean CT = 96 sec +/− 20 sec [SD]). Genome-wide genotyping with the Infinium II Assay using the Illumina BeadStation 500 system was performed. After controlling for population admixture and excluding markers with a less than 95% call rate, 131 subjects passed criteria for ethnicity and successful genotyping of 956,604 tagSNPs. Statistical analysis identified a locus on chromosome 10 that appeared to be associated with PFA-100 closure times. Of the 7 most significantly associated SNPs, 5 were in SVIL, the gene encoding supervillin. The closest flanking genes to the SNP with the best P value were >77 kb upstream (2 pseudogenes) and >214 kb downstream (LTZ1; lysozyme-like 1). After adjusting for VWF and fibrinogen levels, rs10826650 in SVIL was associated with PFA-100 closure times (P = 5.92E–07). We pursued SVIL (chr 10p11.23) because it is known to form a high-affinity link between the actin cytoskeleton and the plasma membrane during cell spreading and disassembly of focal adhesions. Supervillin slows the rate of cell spreading and increases myosin contractility by linking the myosin II heavy chain with myosin light chain kinase. Supervillin also decreases the stability of focal adhesions and co-localizes with signaling molecules in cholesterol-rich lipid rafts. Prior work has identified 2 transcript variants encoding different isoforms of supervillin. Isoform 1 (supervillin) is reported to be most abundantly expressed in muscle, bone marrow, thyroid gland and salivary gland; isoform 2 (archvillin) is reported as muscle specific. Because neither isoform has been characterized in platelets, we performed gene expression analysis with 23 leukocyte depleted platelet (LDP) RNA samples (<1 leukocyte per 5 million platelets) using the Sentrix BeadChip and BeadStation system from Illumina, Inc. A probe recognizing both isoforms was highly expressed in these samples, whereas a probe specific for the archvillin isoform was barely detectable. The level of SVIL mRNA expression was higher than PECAM1 but less than GPIbα. These microarray data were confirmed by RT-PCR using LDP RNA and PCR primers specific for supervillin. Notably, SVIL was not detected in megakaryocyte lines, HEL or Meg-01. Anti-supervillin antisera recognized the appropriate sized ∼205 kD polypeptide by immunoblotting. PFA-100 closure times were found to directly correlate with supervillin (r = 0.481, P = 0.02) but not with archvillin (r = 0.11, P=n.s.) mRNA levels. In summary, we have demonstrated for the first time that platelets contain supervillin but not the alternately spliced isoform archvillin, and that expression is associated with PFA-100 closure times in AAs. Because higher supervillin expression was associated with longer closure times, this finding is consistent with an inhibitory role for supervillin in regulating the rapid actin cytoskeletal rearrangement required to form platelet thrombi on collagen under shear stress. Disclosures:No relevant conflicts of interest to declare.

Mechanical Integration of Actin and Adhesion Dynamics in Cell Migration

Directed cell migration is a physical process that requires dramatic changes in cell shape and adhesion to the extracellular matrix. For efficient movement, these processes must be spatiotemporally coordinated. To a large degree, the morphological changes and physical forces that occur during migration are generated by a dynamic filamentous actin (F-actin) cytoskeleton. Adhesion is regulated by dynamic assemblies of structural and signaling proteins that couple the F-actin cytoskeleton to the extracellular matrix. Here, we review current knowledge of the dynamic organization of the F-actin cytoskeleton in cell migration and the regulation of focal adhesion assembly and disassembly with an emphasis on how mechanical and biochemical signaling between these two systems regulate the coordination of physical processes in cell migration.

Kinectin-mediated endoplasmic reticulum dynamics supports focal adhesion growth in the cellular lamella

Focal adhesions (FAs) control cell shape and motility, which are important processes that underlie a wide range of physiological functions. FA dynamics is regulated by cytoskeleton, motor proteins and small GTPases. Kinectin is an integral endoplasmic reticulum (ER) membrane protein that extends the ER along microtubules. Here, we investigated the influence of the ER on FA dynamics within the cellular lamella by disrupting the kinectin-kinesin interaction by overexpressing the minimal kinectin-kinesin interaction domain on kinectin in cells. This perturbation resulted in a morphological change to a rounded cell shape and reduced cell spreading and migration. Immunofluorescence and live-cell imaging demonstrated a kinectin-dependent ER extension into the cellular lamella and ER colocalisation with FAs within the cellular lamella. FRAP experiments showed that ER contact with FAs was accompanied with an increase in FA protein recruitment to FAs. Disruption of the kinectin-kinesin interaction caused a reduction in FA protein recruitment to FAs. This suggests that the ER supports FA growth within the cellular lamella. Microtubule targeting to FAs is known to promote adhesion disassembly; however, ER contact increased FA size even in the presence of microtubules. Our results suggest a scenario whereby kinectin-kinesin interaction facilitates ER transport along microtubules to support FA growth.

Filamin A regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion

Filamin A regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion

P130Cas Src-Binding and Substrate Domains Have Distinct Roles in Sustaining Focal Adhesion Disassembly and Promoting Cell Migration

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas −/− mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.

Filamin A regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion

The actin cross-linking protein filamin A (FLNa) functions as a scaffolding protein and couples cell cytoskeleton to extracellular matrix and integrin receptor signaling. In this study, we report that FLNa suppresses invasion of breast cancer cells and regulates focal adhesion (FA) turnover. Two large progression tissue microarrays from breast cancer patients revealed a significant decrease of FLNa levels in tissues from invasive breast cancer compared with benign disease and in lymph node-positive compared with lymph node-negative breast cancer. In breast cancer cells and orthotopic mouse breast cancer models, down-regulation of FLNa stimulated cancer cell migration, invasion, and metastasis formation. Time-lapse microscopy and biochemical assays after FLNa silencing and rescue with wild-type or mutant protein resistant to calpain cleavage revealed that FLNa regulates FA disassembly at the leading edge of motile cells. Moreover, FLNa down-regulation enhanced calpain activity through the mitogen-activated protein kinase-extracellular signal-regulated kinase cascade and stimulated the cleavage of FA proteins. These results document a regulation of FA dynamics by FLNa in breast cancer cells.

Maspin Regulates Endothelial Cell Adhesion and Migration through an Integrin Signaling Pathway

Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the activation of integrin β(1), which subsequently led to distribution pattern changes of vinculin and F-actin. These results indicated that maspin affects cell adhesion and cytoskeleton reorganization through an integrin signal transduction pathway. Analysis of HUVECs following maspin treatment revealed increased integrin-linked kinase activities and phosphorylated FAK levels, consistent with increased cell adhesion. Interestingly, when HUVECs were induced to migrate by migration stimulatory factor bFGF, active Rac1 and cdc42 small GTPase levels were decreased dramatically at 30 min following maspin treatment. Using phosphorylated FAK at Tyr(397) as an indicator of focal adhesion disassembly, maspin-treated HUVECs had elevated FAK phosphorylation compared with the mock treated control. The results were a reduction in focal adhesion disassembly and the retardation in EC migration. This study uncovers a mechanism by which maspin exerts its effect on EC adhesion and migration through an integrin signal transduction pathway.