Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

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Direct Observation of Catch-Bonds in Focal Adhesions of Living Cells

Biophysical Journal ◽

10.1016/j.bpj.2011.11.088 ◽

2012 ◽

Vol 102 (3) ◽

pp. 12a

Author(s):

Navid Bonakdar ◽

Achim Schilling ◽

Claus Metzner ◽

Ben Fabry

Keyword(s):

Direct Observation ◽

Focal Adhesions ◽

Living Cells ◽

Catch Bonds

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Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells

Journal of Cellular Physiology ◽

10.1002/jcp.20550 ◽

2006 ◽

Vol 207 (1) ◽

pp. 187-194 ◽

Cited By ~ 149

Author(s):

Tanmay P. Lele ◽

Jay Pendse ◽

Sanjay Kumar ◽

Matthew Salanga ◽

John Karavitis ◽

...

Keyword(s):

Focal Adhesions ◽

Living Cells ◽

Mechanical Forces

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Focal adhesions are hotspots for keratin filament precursor formation

The Journal of Cell Biology ◽

10.1083/jcb.200511124 ◽

2006 ◽

Vol 173 (3) ◽

pp. 341-348 ◽

Cited By ~ 63

Author(s):

Reinhard Windoffer ◽

Anne Kölsch ◽

Stefan Wöll ◽

Rudolf E. Leube

Keyword(s):

Focal Adhesions ◽

Living Cells ◽

Stress Fibers ◽

Regulatory Function ◽

Cell Cortex ◽

Hairpin Rna ◽

Actin Turnover ◽

Keratin Filament ◽

Short Hairpin ◽

Multicolor Fluorescence

Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia. Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement.

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Total internal reflection energy transfer (TIRET) microscopy for analysis of focal adhesions in living cells

10.1117/12.662073 ◽

2006 ◽

Cited By ~ 1

Author(s):

Brigitte Angres ◽

Heiko Steuer ◽

Michael Wagner ◽

Petra Weber ◽

Herbert Schneckenburger

Keyword(s):

Energy Transfer ◽

Total Internal Reflection ◽

Focal Adhesions ◽

Living Cells ◽

Internal Reflection

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Reply to the ‘Comment on “Tensional homeostasis at different length scales” by J. Humphrey and C. Cyron, Soft Matter, 2022, 18, DOI: 10.1039/D1SM01151K’

Soft Matter ◽

10.1039/d1sm01495a ◽

2022 ◽

Author(s):

Dimitrije Stamenović ◽

Michael L. Smith

Keyword(s):

Soft Matter ◽

Focal Adhesions ◽

Living Cells ◽

Length Scales ◽

Tensional Homeostasis

In this Reply to the Comment, we discuss data from the literature which show that the idea that tensional homeostasis in focal adhesions (FAs) of living cells exists over “a central range of FAs”, which is promulgated in the Comment, is not tenable.

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Green fluorescent protein (GFP) tagged to the cytoplasmic tail of αIIb or β3 allows the expression of a fully functional integrin αIIbβ3: effect of β3GFP on αIIbβ3 ligand binding

Biochemical Journal ◽

10.1042/bj3570529 ◽

2001 ◽

Vol 357 (2) ◽

pp. 529-536 ◽

Cited By ~ 15

Author(s):

Sébastien PLANÇON ◽

Marie-Christine MOREL-KOPP ◽

Elisabeth SCHAFFNER-RECKINGER ◽

Ping CHEN ◽

Nelly KIEFFER

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Chinese Hamster ◽

Focal Adhesions ◽

Cytoplasmic Tail ◽

Receptor Activation ◽

Living Cells ◽

Cell Surface Expression ◽

Β3 Integrin ◽

Green Fluorescent

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a β3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either αIIb or β3 allowed normal expression, heterodimerization, processing and surface exposure of αIIbGFPβ3 and αIIbβ3GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced αIIbβ3 capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged αIIbβ3. GFP-tagged αIIbβ3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125FAK tyrosine phosphorylation similar to wild-type αIIbβ3 (where FAK corresponds to focal adhesion kinase). However, GFP tagged to β3, but not to αIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of αIIbβ3 receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged αIIbβ3 during the early stages of cell attachment and spreading, starting with αIIbβ3 clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of αIIbβ3 in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either αIIb or β3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the β3 integrin cytoplasmic tail, rather than the αIIb subunit, plays a major role in αIIbβ3 affinity modulation. With the successful direct visualization of functional αIIbβ3 receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.

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How we discovered fluorescent speckle microscopy

Molecular Biology of the Cell ◽

10.1091/mbc.e11-07-0646 ◽

2011 ◽

Vol 22 (21) ◽

pp. 3940-3942 ◽

Cited By ~ 2

Author(s):

E. D. Salmon ◽

Clare M. Waterman

Keyword(s):

Epithelial Cells ◽

Fluorescence Intensity ◽

Focal Adhesions ◽

Time Lapse ◽

Living Cells ◽

Variable Intensity ◽

Macromolecular Assemblies ◽

Dynamic Assembly ◽

Fluorescence Images

Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ∼10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual “speckled” appearance—there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.

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Viscoelasticity of basal plasma membranes and cortices derived from MDCK II cells

10.1101/2021.05.20.444971 ◽

2021 ◽

Author(s):

Andreas Janshoff

Keyword(s):

Mechanical Properties ◽

Epithelial Cells ◽

Plasma Membranes ◽

Basolateral Membrane ◽

Scaling Law ◽

Focal Adhesions ◽

Living Cells ◽

The Body ◽

Basolateral Membranes ◽

Apical Side

In mature epithelial cells, however, cells adhere to one another through tight junctions, adherens junctions and desmosomes thereby displaying a pronounced apical-basal polarity. In vivo, the apical membrane has a larger surface area and faces the outer surface of the body or the lumen of internal cavities, whereas the basolateral membrane is oriented on the side away from the lumen and forms focal adhesions with the extracellular matrix. The mechanical properties of cells are largely determined by the architecture and dynamics of their viscoelastic cortex, which consists of a contractile, cross-linked actin mesh attached to the plasma membrane via linker proteins. Measuring the mechanical properties of adherent, polarized epithelial cells is usually limited to the upper, i.e., apical side of the cells due to their accessibility on culture dishes. Moreover, contributions from the cell interior comprising various filament types, organelles, and the crowded cytoplasm usually impede examination of the cortex alone. Here, we investigate the viscoelastic properties of basolateral membranes derived from polarized MDCK II epithelia in response to external deformation and compare them to living cells probed at the apical side. Therefore, we grew MDCK II cells on porous surfaces to confluency and removed the upper cell body by sandwich cleavage. The free-standing, defoliated cortices were subject to force indentation and relaxation experiments permitting a precise assessment of cortical viscoelasticity. A new theoretical framework to describe the force cycles is developed and applied to obtain the time-dependent area compressibility modulus of cell cortices from adherent cells. Compared to the viscoelastic response of living cells the basolateral membranes are substantially less fluid and stiffer but obey to the same universal scaling law if excess area is taken into account.

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Spatial distribution and functional significance of activated vinculin in living cells

The Journal of Cell Biology ◽

10.1083/jcb.200410100 ◽

2005 ◽

Vol 169 (3) ◽

pp. 459-470 ◽

Cited By ~ 118

Author(s):

Hui Chen ◽

Daniel M. Cohen ◽

Dilshad M. Choudhury ◽

Noriyuki Kioka ◽

Susan W. Craig

Keyword(s):

Conformational Change ◽

Direct Evidence ◽

Resonance Energy Transfer ◽

Focal Adhesions ◽

Resonance Energy ◽

Time Lapse ◽

Living Cells ◽

Actin Binding ◽

Binding Conformation ◽

Time Lapse Imaging

Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin β by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics.

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