Investigating Focal Adhesion Substructures by Localization Microscopy

Cells rely on focal adhesions (FAs) to carry out a variety of important tasks, including motion, environmental sensing, and adhesion to the extracellular matrix. Although attaining a fundamental characterization of FAs is a compelling goal, their extensive complexity and small size, which can be below the diffraction limit, have hindered a full understanding. In this study we have used single-molecule localization microscopy (SMLM) to investigate integrin β3 and paxillin in rat embryonic fibroblasts growing on two different extracellular matrix-representing substrates (i.e., fibronectin-coated substrates and specifically biofunctionalized nanopatterned substrates). To quantify the substructure of FAs, we developed a clustering method based on expectation maximization of a Gaussian mixture that accounts for localization uncertainty and background. Analysis of our SMLM data indicates that the structures within FAs, characterized as a Gaussian mixture, typically have areas between 0.01 and 1 μm2 , contain 10–100 localizations, and can exhibit substantial eccentricity. Our approach based on SMLM opens new avenues for studying structural and functional biology of molecular assemblies that display substantial varieties in size, shape, and density.

Cell shape-dependent early responses of fibroblasts to cyclic strain

Jürgen Brugger, Mattia Marelli

Randomly spread fibroblasts on fibronectin-coated elastomeric membranes respond to cyclic strain by a varying degree of focal adhesion assembly and actin reorganization. We speculated that the individual shape of the cells, which is linked to cytoskeletal structure and pre-stress, might tune these integrin-dependentmechanotransduction events. To this aim, fibronectin circles, squares and rectangles of identical surface area (2000 μm2) were microcontact printed onto elastomeric substrates. Fibroblasts plated on these patterns occupied the corresponding shapes. Cyclic 10% equibiaxial strain was applied to patterned cells for 30min, and changes in cytoskeleton and cell-matrix adhesionswere quantified after fluorescence staining. After strain,megakaryocytic leukemia-1 protein translocated to the nucleus in most cells, indicating efficient RhoA activation independently of cell shape. However, circular and square cells (with radial symmetry) showed a significantly greater increase in the number of actin stress fibers and vinculin-positive focal adhesions after cyclic strain than rectangular (bipolar) cells of identical size. Conversely, cyclic strain induced larger changes in pY397-FAK positive focal complexes and zyxin relocation from focal adhesions to stress fibers in bipolar compared to symmetric cells. Thus, radially symmetric cells responded to cyclic strain with a larger increase in assembly, whereas bipolar cells reacted with more pronounced reorganization of actin stress fibers and matrix contacts. We conclude that integrin-mediated responses to external mechanical strain are differentially modulated in cells that have the same spreading area but different geometries, and do not only depend on mere cell size.

Elsevier2013

Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

Hendrik Georges Deschout, Lely Feletti, Theo Lasser, Tomas Lukes, Aleksandra Radenovic, Azat Sharipov

Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM–SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

Spie-Int Soc Optical Engineering2017

Nano-Stenciled RGD-Gold Patterns That Inhibit Focal Contact Maturation Induce Lamellipodia Formation in Fibroblasts

Jürgen Brugger, Mattia Marelli, Kristopher Pataky

Cultured fibroblasts adhere to extracellular substrates by means of cell-matrix adhesions that are assembled in a hierarchical way, thereby gaining in protein complexity and size. Here we asked how restricting the size of cell-matrix adhesions affects cell morphology and behavior. Using a nanostencil technique, culture substrates were patterned with gold squares of a width and spacing between 250 nm and 2 mu m. The gold was functionalized with RGD peptide as ligand for cellular integrins, and mouse embryo fibroblasts were plated. Limiting the length of cell-matrix adhesions to 500 nm or less disturbed the maturation of vinculin-positive focal complexes into focal contacts and fibrillar adhesions, as indicated by poor recruitment of alpha 5-integrin. We found that on sub-micrometer patterns, fibroblasts spread extensively, but did not polarize. Instead, they formed excessive numbers of lamellipodia and a fine actin meshwork without stress fibers. Moreover, these cells showed aberrant fibronectin fibrillogenesis, and their speed of directed migration was reduced significantly compared to fibroblasts on 2 mu m square patterns. Interference with RhoA/ROCK signaling eliminated the pattern-dependent differences in cell morphology. Our results indicate that manipulating the maturation of cell-matrix adhesions by nanopatterned surfaces allows to influence morphology, actin dynamics, migration and ECM assembly of adhering fibroblasts.

Public Library of Science2011