Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.

• MeSH
• Amino Acid Motifs MeSH
• Biomechanical Phenomena MeSH
• Cell Adhesion MeSH
• Cell Line MeSH
• Fibroblasts cytology   metabolism   MeSH
• Focal Adhesions metabolism   ultrastructure   MeSH
• Focal Adhesion Protein-Tyrosine Kinases metabolism   MeSH
• Phosphorylation MeSH
• Protein Interaction Maps MeSH
• Mice MeSH
• Peptides chemistry   metabolism   MeSH
• src Homology Domains MeSH
• Crk-Associated Substrate Protein analysis   metabolism   MeSH
• Protein Binding MeSH
• Vinculin analysis   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

CAS is a docking protein, which was shown to act as a mechanosensor in focal adhesions. The unique assembly of structural domains in CAS is important for its function as a mechanosensor. The tension within focal adhesions is transmitted to a stretchable substrate domain of CAS by focal adhesion-targeting of SH3 and CCH domain of CAS, which anchor the CAS protein in focal adhesions. Mechanistic models of the stretching biosensor propose equal roles for both anchoring domains. Using deletion mutants and domain replacements, we have analyzed the relative importance of the focal adhesion anchoring domains on CAS localization and dynamics in focal adhesions as well as on CAS-mediated mechanotransduction. We confirmed the predicted prerequisite of the focal adhesion targeting for CAS-dependent mechanosensing and unraveled the critical importance of CAS SH3 domain in mechanosensing. We further show that CAS localizes to the force transduction layer of focal adhesions and that mechanical stress stabilizes CAS in focal adhesions.

• MeSH
• Cell Adhesion MeSH
• Mechanotransduction, Cellular * MeSH
• Fibroblasts cytology   metabolism   MeSH
• Focal Adhesions metabolism   MeSH
• Stress, Mechanical MeSH
• Mutant Proteins chemistry   MeSH
• Mice MeSH
• Protein Domains MeSH
• Recombinant Fusion Proteins metabolism   MeSH
• Signal Transduction MeSH
• Protein Stability MeSH
• Crk-Associated Substrate Protein chemistry   metabolism   MeSH
• Structure-Activity Relationship MeSH
• Green Fluorescent Proteins metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Hippo effectors YAP/TAZ act as on-off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described by a hierarchical model in which elements of Hippo pathway are under the control of focal adhesions (FAs). Here we unveil the molecular mechanism by which cell spreading and RhoA GTPase activity control FA formation through YAP to stabilize the anchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAP co-transcriptional function and involves the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity leads to the modification of cell mechanics, force development and adhesion strength, and determines cell shape, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify this Hippo effector as the key determinant of cell mechanics in response to ECM cues.

• MeSH
• Cell Differentiation genetics   physiology   MeSH
• Cell Membrane metabolism   MeSH
• Cell Line MeSH
• Mechanotransduction, Cellular genetics   physiology   MeSH
• Extracellular Matrix metabolism   MeSH
• Focal Adhesions genetics   metabolism   physiology   MeSH
• HEK293 Cells MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Actin Cytoskeleton metabolism   MeSH
• Cell Line, Tumor MeSH
• Cell Movement genetics   physiology   MeSH
• rhoA GTP-Binding Protein genetics   metabolism   MeSH
• Gene Expression Profiling MeSH
• Transcription Factors genetics   metabolism   MeSH
• Cell Shape MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Sunitinib malate is a small molecule that targets multiple receptor tyrosine kinases and blocks their activity. Receptors targeted by sunitinib are implicated in tumor vascularization and are overexpressed by vascular tumors encountered in infants, namely, hemangiomas. Of note is that there is still no definitive treatment for these commonly occurring tumors of infancy. The purpose of this study was to investigate the effects of sunitinib malate on hemangioma using endothelial cells isolated from a murine model of the neoplasm (sEnd.2). The effects of the drug on cell growth were evaluated using the crystal violet assay and flow cytometry, while the scratch assay was employed to measure cell migration. Proteins associated with cell migration and angiogenesis were detected using western blotting. Sunitinib was investigated further to determine its effects on the production of reactive oxygen species, a parameter associated with the promotion of neovascularization in tumors. The results showed that sunitinib significantly reduced the growth of sEnd.2 cells by causing the cells to accumulate in the sub-G1 phase of the cell cycle, and also induced a significant decrease in the migration of these hemangioma cells (P < 0.05). The western blot assay showed a decrease in the expression of adhesion proteins, focal adhesion kinase and paxillin at IC50 doses, although the expression of cadherin did not change significantly (P < 0.05). In addition, transforming growth factor-β1 (TGF-β1) expression was decreased in sunitinib-treated cells at the same dose. The adhesion proteins as well as TGF-β1 regulate cell movement and have been implicated in tumor progression. Thus, sunitinib malate may have potential in the treatment of hemangiomas.

• MeSH
• Cell Migration Assays statistics & numerical data   MeSH
• Cell Cycle MeSH
• Cell Growth Processes drug effects   MeSH
• Endothelial Cells drug effects   MeSH
• Focal Adhesion Protein-Tyrosine Kinases MeSH
• Hemangioma * drug therapy   MeSH
• Models, Animal MeSH
• Mice MeSH
• Flow Cytometry MeSH
• Statistics as Topic MeSH
• Sunitinib * pharmacology   therapeutic use   MeSH
• Vascular Endothelial Growth Factors MeSH
• Cell Survival MeSH
• Treatment Outcome MeSH
• Blotting, Western MeSH
• Check Tag
• Mice MeSH
• Publication type
• Clinical Study MeSH
• Research Support, Non-U.S. Gov't MeSH

Cells attaching to the extracellular matrix spontaneously acquire front-rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front-rear polarity in spreading cells.

• MeSH
• Cell Adhesion genetics   physiology   MeSH
• Cell Line MeSH
• Fibroblasts cytology   metabolism   MeSH
• Focal Adhesion Protein-Tyrosine Kinases genetics   metabolism   MeSH
• Rats MeSH
• Microscopy, Phase-Contrast MeSH
• Cell Movement genetics   physiology   MeSH
• Cell Polarity genetics   physiology   MeSH
• Receptors for Activated C Kinase genetics   metabolism   MeSH
• RNA Interference MeSH
• Cell Shape genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Beyond its structural role in the skeleton, the extracellular matrix (ECM), particularly basement membrane proteins, facilitates communication with intracellular signaling pathways and cell to cell interactions to control differentiation, proliferation, migration and survival. Alterations in extracellular proteins cause a number of skeletal disorders, yet the consequences of an abnormal ECM on cellular communication remains less well understood METHODS: Clinical and radiographic examinations defined the phenotype in this unappreciated bent bone skeletal disorder. Exome analysis identified the genetic alteration, confirmed by Sanger sequencing. Quantitative PCR, western blot analyses, immunohistochemistry, luciferase assay for WNT signaling were employed to determine RNA, proteins levels and localization, and dissect out the underlying cell signaling abnormalities.  Migration and wound healing assays examined cell migration properties. FINDINGS: This bent bone dysplasia resulted from biallelic mutations in LAMA5, the gene encoding the alpha-5 laminin basement membrane protein. This finding uncovered a mechanism of disease driven by ECM-cell interactions between alpha-5-containing laminins, and integrin-mediated focal adhesion signaling, particularly in cartilage. Loss of LAMA5 altered β1 integrin signaling through the non-canonical kinase PYK2 and the skeletal enriched SRC kinase, FYN. Loss of LAMA5 negatively impacted the actin cytoskeleton, vinculin localization, and WNT signaling. INTERPRETATION: This newly described mechanism revealed a LAMA5-β1 Integrin-PYK2-FYN focal adhesion complex that regulates skeletogenesis, impacted WNT signaling and, when dysregulated, produced a distinct skeletal disorder. FUNDING: Supported by NIH awards R01 AR066124, R01 DE019567, R01 HD070394, and U54HG006493, and Czech Republic grants INTER-ACTION LTAUSA19030, V18-08-00567 and GA19-20123S.

• MeSH
• Alleles * MeSH
• Cell Adhesion genetics   MeSH
• Chondrocytes metabolism   MeSH
• Phenotype MeSH
• Focal Adhesion Kinase 2 genetics   metabolism   MeSH
• Genetic Predisposition to Disease MeSH
• Genetic Association Studies MeSH
• Bone and Bones abnormalities   diagnostic imaging   MeSH
• Laminin genetics   metabolism   MeSH
• Humans MeSH
• Mutation * MeSH
• DNA Mutational Analysis MeSH
• Wnt Signaling Pathway MeSH
• Signal Transduction * MeSH
• src-Family Kinases metabolism   MeSH
• Bone Diseases, Developmental diagnosis   etiology   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Interaction of leukemia blasts with the bone marrow extracellular matrix often results in protection of leukemia cells from chemotherapy and in persistence of the residual disease which is on the basis of subsequent relapses. The adhesion signaling pathways have been extensively studied in adherent cells as well as in mature haematopoietic cells, but the adhesion structures and signaling in haematopoietic stem and progenitor cells, either normal or malignant, are much less explored. We analyzed the interaction of leukemia cells with fibronectin (FN) using interference reflection microscopy, immunofluorescence, measurement of adherent cell fraction, real-time microimpedance measurement and live cell imaging. We found that leukemia cells form very dynamic adhesion structures similar to early stages of focal adhesions. In contrast to adherent cells, where Src family kinases (SFK) belong to important regulators of focal adhesion dynamics, we observed only minor effects of SFK inhibitor dasatinib on leukemia cell binding to FN. The relatively weak involvement of SFK in adhesion structure regulation might be associated with the lack of cytoskeletal mechanical tension in leukemia cells. On the other hand, active Lyn kinase was found to specifically localize to leukemia cell adhesion structures and a less firm cell attachment to FN was often associated with higher Lyn activity (this unexpectedly occurred also after cell treatment with the inhibitor SKI-1). Lyn thus may be important for signaling from integrin-associated complexes to other processes in leukemia cells.

• MeSH
• Cell Adhesion drug effects   physiology   MeSH
• Dasatinib pharmacology   MeSH
• Fibronectins metabolism   MeSH
• Focal Adhesions drug effects   metabolism   MeSH
• Focal Adhesion Protein-Tyrosine Kinases metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Leukemia drug therapy   MeSH
• Humans MeSH
• src-Family Kinases drug effects   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Lithium is the gold standard treatment for bipolar disorder (BD). However, its mechanism of action is incompletely understood, and prediction of treatment outcomes is limited. In our previous multi-omics study of the Pharmacogenomics of Bipolar Disorder (PGBD) sample combining transcriptomic and genomic data, we found that focal adhesion, the extracellular matrix (ECM), and PI3K-Akt signaling networks were associated with response to lithium. In this study, we replicated the results of our previous study using network propagation methods in a genome-wide association study of an independent sample of 2039 patients from the International Consortium on Lithium Genetics (ConLiGen) study. We identified functional enrichment in focal adhesion and PI3K-Akt pathways, but we did not find an association with the ECM pathway. Our results suggest that deficits in the neuronal growth cone and PI3K-Akt signaling, but not in ECM proteins, may influence response to lithium in BD.

• MeSH
• Bipolar Disorder * drug therapy   genetics   MeSH
• Genome-Wide Association Study MeSH
• Focal Adhesions MeSH
• Phosphatidylinositol 3-Kinases genetics   MeSH
• Humans MeSH
• Lithium * pharmacology   therapeutic use   MeSH
• Multiomics MeSH
• Proto-Oncogene Proteins c-akt genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Lithium (Li) is one of the most effective drugs for treating bipolar disorder (BD), however, there is presently no way to predict response to guide treatment. The aim of this study is to identify functional genes and pathways that distinguish BD Li responders (LR) from BD Li non-responders (NR). An initial Pharmacogenomics of Bipolar Disorder study (PGBD) GWAS of lithium response did not provide any significant results. As a result, we then employed network-based integrative analysis of transcriptomic and genomic data. In transcriptomic study of iPSC-derived neurons, 41 significantly differentially expressed (DE) genes were identified in LR vs NR regardless of lithium exposure. In the PGBD, post-GWAS gene prioritization using the GWA-boosting (GWAB) approach identified 1119 candidate genes. Following DE-derived network propagation, there was a highly significant overlap of genes between the top 500- and top 2000-proximal gene networks and the GWAB gene list (Phypergeometric = 1.28E-09 and 4.10E-18, respectively). Functional enrichment analyses of the top 500 proximal network genes identified focal adhesion and the extracellular matrix (ECM) as the most significant functions. Our findings suggest that the difference between LR and NR was a much greater effect than that of lithium. The direct impact of dysregulation of focal adhesion on axon guidance and neuronal circuits could underpin mechanisms of response to lithium, as well as underlying BD. It also highlights the power of integrative multi-omics analysis of transcriptomic and genomic profiling to gain molecular insights into lithium response in BD.

• MeSH
• Antimanic Agents pharmacology   therapeutic use   MeSH
• Bipolar Disorder * drug therapy   genetics   MeSH
• Genome-Wide Association Study * methods   MeSH
• Pharmacogenetics methods   MeSH
• Focal Adhesions * drug effects   genetics   MeSH
• Genomics methods   MeSH
• Gene Regulatory Networks * drug effects   genetics   MeSH
• Induced Pluripotent Stem Cells drug effects   metabolism   MeSH
• Humans MeSH
• Lithium * pharmacology   therapeutic use   MeSH
• Multiomics MeSH
• Neurons metabolism   drug effects   MeSH
• Lithium Compounds pharmacology   therapeutic use   MeSH
• Gene Expression Profiling methods   MeSH
• Transcriptome * genetics   drug effects   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Focal adhesions are specific types of cellular adhesion structures through which both mechanical force and regulatory signals are transmitted. Recently, the existence of focal adhesions in 3D environment has been questioned. Using a unique life-like model of dermis-based matrix we analysed the presence of focal adhesions in a complex 3D environment. Although the dermis-based matrix constitutes a 3D environment, the interface of cell-to-matrix contacts on thick bundled fibres within this matrix resembles 2D conditions. We call this a quasi-2D situation. We suggest that the quasi-2D interface of cell-to-matrix contacts constituted in the dermis-based matrix is much closer to in tissue conditions than the meshed structure of mostly uniform thin fibres in the gel-based matrices. In agreement with our assumption, we found that the cell adhesion structures are formed by cells that invade the dermis-based matrix and that these structures are of similar size as focal adhesions formed on fibronectin-coated coverslips (2D). In both 2D situation and the dermis-based matrix, we observed comparable vinculin dynamics in focal adhesions and comparable enlargement of the focal adhesions in response to a MEK inhibitor. We conclude that focal adhesions that are formed in the 3D environment are similar in size and dynamics as those seen in the 2D setting.

• MeSH
• Cell Culture Techniques * MeSH
• Butadienes pharmacology   MeSH
• Focal Adhesions drug effects   metabolism   ultrastructure   MeSH
• Fluorescence Recovery After Photobleaching MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors   metabolism   MeSH
• Cell Line, Tumor MeSH
• Nitriles pharmacology   MeSH
• Dermis drug effects   metabolism   MeSH
• Sus scrofa MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH