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
• buněčné kultury * MeSH
• butadieny farmakologie   MeSH
• fokální adheze účinky léků   metabolismus   ultrastruktura   MeSH
• FRAP MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy kinas antagonisté a inhibitory   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nitrily farmakologie   MeSH
• škára účinky léků   metabolismus   MeSH
• Sus scrofa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Src kinase plays an important role in a multitude of fundamental cellular processes and is often found deregulated in tumors. Active Src adopts an open conformation, whereas inactive Src is characterized by a very compact structure stabilized by inhibitory intramolecular interactions. Taking advantage of this spatial regulation, we constructed a fluorescence resonance energy transfer (FRET)-based Src biosensor and analyzed conformational changes of Src following Src activation and the spatiotemporal dynamics of Src activity in cells. We found that activatory mutations either in regulatory or kinase domains induce opening of the Src structure. Surprisingly, we discovered that Src inhibitors differ in their effect on the Src structure, some counterintuitively inducing an open conformation. Finally, we analyzed the dynamics of Src activity in focal adhesions by FRET imaging and found that Src is rapidly activated during focal adhesion assembly, and its activity remains steady and high throughout the life cycle of focal adhesion and decreases during focal adhesion disassembly.

• MeSH
• biosenzitivní techniky metody   MeSH
• fokální adheze metabolismus   MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mutageneze MeSH
• rezonanční přenos fluorescenční energie MeSH
• skupina kinas odvozených od src-genu antagonisté a inhibitory   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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
• aminokyselinové motivy MeSH
• biomechanika MeSH
• buněčná adheze MeSH
• buněčné linie MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fokální adheze metabolismus   ultrastruktura   MeSH
• fokální adhezní tyrosinkinasy metabolismus   MeSH
• fosforylace MeSH
• mapy interakcí proteinů MeSH
• myši MeSH
• peptidy chemie   metabolismus   MeSH
• src homologní domény MeSH
• substrátový protein asociovaný s Crk analýza   metabolismus   MeSH
• vazba proteinů MeSH
• vinkulin analýza   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

• MeSH
• barvení a značení MeSH
• buněčná adheze fyziologie   MeSH
• časové faktory MeSH
• fibroblasty fyziologie   MeSH
• krysa rodu rattus MeSH
• mikroskopie metody   MeSH
• myši MeSH
• paxilin chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• buněčná adheze MeSH
• buněčný převod mechanických signálů * MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fokální adheze metabolismus   MeSH
• mechanický stres MeSH
• mutantní proteiny chemie   MeSH
• myši MeSH
• proteinové domény MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• signální transdukce MeSH
• stabilita proteinů MeSH
• substrátový protein asociovaný s Crk chemie   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• buněčná diferenciace genetika   fyziologie   MeSH
• buněčná membrána metabolismus   MeSH
• buněčné linie MeSH
• buněčný převod mechanických signálů genetika   fyziologie   MeSH
• extracelulární matrix metabolismus   MeSH
• fokální adheze genetika   metabolismus   fyziologie   MeSH
• HEK293 buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mikrofilamenta metabolismus   MeSH
• nádorové buněčné linie MeSH
• pohyb buněk genetika   fyziologie   MeSH
• rhoA protein vázající GTP genetika   metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• transkripční faktory genetika   metabolismus   MeSH
• tvar buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• buněčná adheze genetika   fyziologie   MeSH
• buněčné linie MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fokální adhezní tyrosinkinasy genetika   metabolismus   MeSH
• krysa rodu rattus MeSH
• mikroskopie fázově kontrastní MeSH
• pohyb buněk genetika   fyziologie   MeSH
• polarita buněk genetika   fyziologie   MeSH
• receptory pro aktivovanou kinasu C genetika   metabolismus   MeSH
• RNA interference MeSH
• tvar buňky genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• analýza buněčné migrace statistika a číselné údaje   MeSH
• buněčný cyklus MeSH
• buňky - růstové procesy účinky léků   MeSH
• endoteliální buňky účinky léků   MeSH
• fokální adhezní tyrosinkinasy MeSH
• hemangiom * farmakoterapie   MeSH
• modely u zvířat MeSH
• myši MeSH
• průtoková cytometrie MeSH
• statistika jako téma MeSH
• sunitinib * farmakologie   terapeutické užití   MeSH
• vaskulární endoteliální růstové faktory MeSH
• viabilita buněk MeSH
• výsledek terapie MeSH
• western blotting MeSH
• Check Tag
• myši MeSH
• Publikační typ
• klinická studie MeSH
• práce podpořená grantem 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
• alely * MeSH
• buněčná adheze genetika   MeSH
• chondrocyty metabolismus   MeSH
• fenotyp MeSH
• fokální adhezní kinasa 2 genetika   metabolismus   MeSH
• genetická predispozice k nemoci MeSH
• genetické asociační studie MeSH
• kosti a kostní tkáň abnormality   diagnostické zobrazování   MeSH
• laminin genetika   metabolismus   MeSH
• lidé MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• signální dráha Wnt MeSH
• signální transdukce * MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• vývojové onemocnění kostí diagnóza   etiologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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
• bipolární porucha * farmakoterapie   genetika   MeSH
• celogenomová asociační studie MeSH
• fokální adheze MeSH
• fosfatidylinositol-3-kinasy genetika   MeSH
• lidé MeSH
• lithium * farmakologie   terapeutické užití   MeSH
• multiomika MeSH
• protoonkogenní proteiny c-akt genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH