Abstract
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.
Highlights
Focal adhesions (FAs) are the main cellular structures linking the intracellular cytoskeleton to the extracellular matrix (ECM)
We reveal that the binding dynamics of vasodilator-stimulated phosphoprotein (VASP), zyxin, vinculin and paxillin differ with FA location and FA orientation relative to the closest edge of the ventral, or adherent, portion of the plasma membrane
Fluorescence Recovery After Photobleaching (FRAP)-experiments we examined the binding dynamics of fluorescently-labelled paxillin, vinculin, VASP and zyxin at FAs in two different cell-types from two different species; U2OS cells, a human bone cancer cell line, and MDCK dog kidney cells (Fig. 1)
Summary
Focal adhesions (FAs) are the main cellular structures linking the intracellular cytoskeleton to the extracellular matrix (ECM). We investigated FA location and FA orientation dependent dynamics of four FA proteins, the large scaffold proteins paxillin and vinculin, and two FA proteins that are closely linked to the actin associated with FAs, zyxin and vasodilator-stimulated phosphoprotein (VASP). Taking advantage of a photoconvertible fluorescent protein in combination with a Fluorescence Recovery After Photobleaching (FRAP) set-up on a confocal microscope, we developed a dedicated assay to reveal the spatial location of the stably bound fraction of a protein We applied this technique to paxillin and vinculin because FRAP experiments showed both these proteins have strikingly large stably bound fractions of nearly 50%. By using Monte Carlo based simulations we were able to provide a detailed quantification of the binding dynamics of these four proteins, as well as of the differences seen in FAs with different orientations or cellular locations[41]
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