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Methods in Cell Biology 2018Productive chromosome movements require that a large multiprotein complex called the kinetochore assemble on sister centromeres. The kinetochore fulfills two critical...
Productive chromosome movements require that a large multiprotein complex called the kinetochore assemble on sister centromeres. The kinetochore fulfills two critical functions as (1) the physical linkage between chromosomes and spindle microtubules and (2) a mechanomolecular sensor that relays a spindle assembly checkpoint signal delaying anaphase onset until chromosomes are attached to spindle microtubules and bioriented. Given its central roles in such a vital process, the kinetochore is one of the most important force-transducing structures in cells; yet it has been technically challenging to measure kinetochore forces. Barriers to measuring cellular forces have begun to be broken by the development of fluorescence-based tension sensors. In this chapter, two methods will be described for measuring kinetochore forces in living cells and strategies for applying these sensors to other force-transducing processes and molecules will be discussed.
Topics: Animals; Biomechanical Phenomena; Biosensing Techniques; Cytological Techniques; Drosophila; Fluorescence Resonance Energy Transfer; Mitosis; Photobleaching; Talin; Vinculin
PubMed: 29804669
DOI: 10.1016/bs.mcb.2018.03.007 -
Scientific Reports Jul 2019Focal adhesions (FAs) are multiprotein structures that link the intracellular cytoskeleton to the extracellular matrix. They mediate cell adhesion and migration, crucial...
Focal adhesions (FAs) are multiprotein structures that link the intracellular cytoskeleton to the extracellular matrix. They mediate cell adhesion and migration, crucial to many (patho-) physiological processes. We examined in two cell types from different species the binding dynamics of functionally related FA protein pairs: paxillin and vinculin versus zyxin and VASP. In photobleaching experiments ~40% of paxillin and vinculin remained stably associated with a FA for over half an hour. Zyxin and VASP predominantly displayed more transient interactions. We show protein binding dynamics are influenced by FA location and orientation. In FAs located close to the edge of the adherent membrane paxillin, zyxin and VASP were more dynamic and had larger bound fractions. Zyxin and VASP were also more dynamic and had larger bound fractions at FAs perpendicular compared to parallel to this edge. Finally, we developed a photoconversion assay to specifically visualise stably bound proteins within subcellular structures and organelles. This revealed that while paxillin and vinculin are distributed evenly throughout FAs, their stably bound fractions form small clusters within the FA-complex. These clusters are more concentrated for paxillin than for vinculin and are mostly found at the proximal half of the FA where actin also enters.
Topics: Animals; Bone Neoplasms; Cell Adhesion Molecules; Cytoskeleton; Dogs; Extracellular Matrix; Focal Adhesions; Humans; Madin Darby Canine Kidney Cells; Microfilament Proteins; Osteosarcoma; Paxillin; Phosphoproteins; Tumor Cells, Cultured; Vinculin; Zyxin
PubMed: 31320676
DOI: 10.1038/s41598-019-46905-2 -
Experimental Cell Research Apr 2016External forces play a key role in shaping development and normal physiology. Aberrant responses to forces, or changes in the nature of such forces, are implicated in a... (Review)
Review
External forces play a key role in shaping development and normal physiology. Aberrant responses to forces, or changes in the nature of such forces, are implicated in a variety of diseases. Cells contain several types of adhesions, linking them to their external environment. It is through these adhesions that forces are both sensed (from the outside inwards) and applied (from inside to out). Furthermore, several adhesion-based proteins are sensitive to changes in intracellular forces, utilising them for activation and regulation. Here, we outline how vinculin, a key component of integrin-mediated adhesions linking the actin cytoskeleton to the extracellular matrix (ECM), is regulated by force and acts as force transducing protein. We discuss the role of vinculin in vivo and its place in health and disease; summarise the proposed mechanisms by which vinculin is recruited to and activated at integrin-ECM adhesions; and discuss recent findings that place vinculin as the major force sensing and transmitting component of cell-matrix adhesion complexes. Finally, we discuss the role of vinculin in regulating the cellular responses to both the physical properties of the external environment and to externally applied physical stimuli.
Topics: Cell Adhesion; Extracellular Matrix; Humans; Integrins; Models, Biological; Stress, Mechanical; Vinculin
PubMed: 26607713
DOI: 10.1016/j.yexcr.2015.11.017 -
Medical Sciences (Basel, Switzerland) Aug 2022Vinculin and talin-1, which are cytoskeletal proteins affecting focal adhesions, were reported to be down-expressed in atherosclerotic lesions. Recently, we reported...
Vinculin and talin-1, which are cytoskeletal proteins affecting focal adhesions, were reported to be down-expressed in atherosclerotic lesions. Recently, we reported high concentrations of plasma talin-1 in patients with coronary artery disease (CAD). However, blood vinculin concentrations in CAD patients have not been clarified. Plasma vinculin concentrations as well as talin-1 were studied in 327 patients in whom coronary angiography was performed. CAD was proven in 177 patients (1-vessel, = 79; 2-vessel, = 57; 3-vessel disease, = 41). However, vinculin concentrations were not markedly different between the CAD(-) and CAD groups (median 122.5 vs. 119.6 pg/mL, = 0.325) or among patients with CAD(-), 1-, 2-, and 3-vessel diseases (122.5, 112.8, 107.9, and 137.2 pg/mL, = 0.202). In contrast, talin-1 concentrations were higher in CAD than the CAD(-) group (0.29 vs. 0.23 ng/mL, = 0.006) and increased stepwise in the number of stenotic vessels: 0.23 in CAD(-), 0.28 in 1-vessel, 0.29 in 2-vessel, and 0.33 ng/mL in 3-vessel disease ( = 0.043). No correlation was observed between vinculin and talin-1 concentrations. In multivariate analysis, vinculin concentrations were not a factor for CAD. In conclusion, plasma vinculin concentrations in patients with CAD were not high and were not associated with the presence or severity of CAD.
Topics: Coronary Angiography; Coronary Artery Disease; Focal Adhesions; Humans; Talin; Vinculin
PubMed: 36135831
DOI: 10.3390/medsci10030046 -
Oncotarget Oct 2015
Topics: Animals; Cell Communication; Cell Membrane; Cell Movement; Focal Adhesions; Humans; Mice; Vinculin
PubMed: 26431280
DOI: 10.18632/oncotarget.5868 -
The Journal of International Medical... Jan 2020This study aimed to explore the expression of vinculin in non-small cell lung cancer (NSCLC) and to analyze its correlation with clinical features and prognosis of NSCLC.
OBJECTIVE
This study aimed to explore the expression of vinculin in non-small cell lung cancer (NSCLC) and to analyze its correlation with clinical features and prognosis of NSCLC.
METHODS
The expression of vinculin in cancer tissues and paracancer tissues was detected by real-time PCR, western blotting, and immunohistochemistry. Correlations between vinculin-positive expression and clinical features were analyzed by Pearson correlation analysis, and those between vinculin expression and prognosis were analyzed by Cox multivariate analysis.
RESULTS
Vinculin expression was significantly lower in cancer tissues than in paracancer tissues. Pearson correlation analysis showed that vinculin expression was significantly correlated with tumor–node–metastasis (TNM) stage and lymph node metastasis in NSCLC. Cox multivariate analysis showed that vinculin-negative expression and TNM stage were independent risk factors for NSCLC prognosis. Kaplan–Meier analysis showed that the 5-year overall survival (OS) rate was 20.51% for all NSCLC patients, and was significantly higher for vinculin-positive patients with NSCLC than vinculin-negative patients.
CONCLUSIONS
Vinculin gene transcription is inhibited in NSCLC, and low vinculin expression promotes malignancy in NSCLC. Therefore, vinculin could be used as a prognostic marker for NSCLC and a potential target for its treatment.
Topics: Carcinoma, Non-Small-Cell Lung; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Prognosis; Proportional Hazards Models; RNA, Messenger; Regression Analysis; Survival Analysis; Vinculin
PubMed: 30947597
DOI: 10.1177/0300060519839523 -
Journal of the American Society of... Jun 2018Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex...
Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein. We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin. Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and -actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well. Palladin has an important role in podocyte function and .
Topics: Actins; Animals; Cytoskeletal Proteins; Cytoskeleton; Female; Focal Adhesions; Gene Expression; Gene Silencing; Humans; Kidney Glomerulus; Male; Mice, Knockout; Microfilament Proteins; Morpholinos; Phosphoproteins; Podocytes; RNA, Messenger; Vinculin; Zebrafish; Zebrafish Proteins
PubMed: 29720549
DOI: 10.1681/ASN.2017091039 -
ELife Mar 2021Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated...
Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated NA formation, we find these molecules concurrently recruited to the subclass of NAs maturing to focal adhesions. After the initial recruitment under minimal load, vinculin accumulates in maturing NAs at a ~ fivefold higher rate than in non-maturing NAs, and is accompanied by a faster traction force increase. We identify the R8 domain in talin, which exposes a vinculin-binding-site (VBS) in the absence of load, as required for NA maturation. Disruption of R8 domain function reduces load-free vinculin binding to talin, and reduces the rate of additional vinculin recruitment. Taken together, these data show that the concurrent recruitment of talin and vinculin prior to mechanical engagement with integrins is essential for the traction-mediated unfolding of talin, exposure of additional VBSs, further recruitment of vinculin, and ultimately, NA maturation.
Topics: Animals; Binding Sites; CHO Cells; Cricetinae; Cricetulus; Focal Adhesions; Mice; Protein Binding; Talin; Vinculin
PubMed: 33783351
DOI: 10.7554/eLife.66151 -
Matrix Biology : Journal of the... Sep 2011Cells in a three-dimensional (3D) extracellular matrix environment often display different properties and behavior compared to cells cultured on a two-dimensional (2D)... (Comparative Study)
Comparative Study Review
Cells in a three-dimensional (3D) extracellular matrix environment often display different properties and behavior compared to cells cultured on a two-dimensional (2D) substrate. Recent studies characterizing the cell-matrix adhesions formed by cells within a 3D matrix have arrived at contradictory conclusions regarding the presence and composition of adhesions. Here we review this literature, and provide a comparative compilation of information found in published studies from the 3D cell-matrix adhesion field in order to identify shared and divergent conclusions and conceptually important areas that require further research. Although there is a general consensus that discrete cell-matrix adhesions exist in various 3D matrix environments, there are specific exceptions, particularly in cells undergoing amoeboid migration. There are also technical issues to consider when imaging adhesions in 3D matrix; for example, over-expression of a cytoskeletal cell adhesion component can potentially cloud the visualization of adhesions and even alter the mode of cell migration. Properties such as stiffness and local matrix topography may also affect the composition of cell-matrix adhesions. For example, even though cells contain integrin-based 3D adhesions, there can be substantial variability within these adhesions in the presence of force-dependent cytoskeletal components such as vinculin. These new findings and ideas provide promising new leads for understanding the regulation and function of cell-matrix adhesions in 3D matrix.
Topics: Animals; Biophysical Phenomena; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell-Matrix Junctions; Collagen; Cytoskeleton; Elasticity; Extracellular Matrix; Gels; Humans; Imaging, Three-Dimensional; Integrins; Vinculin
PubMed: 21723391
DOI: 10.1016/j.matbio.2011.06.001 -
ELife Jul 2020Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting...
Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.
Topics: Actin Cytoskeleton; Actins; Membranes, Artificial; Phosphatidylinositols; Talin; Vinculin
PubMed: 32657269
DOI: 10.7554/eLife.56110