Nanopatterning Reveals an ECM Area Threshold for Focal Adhesion Assembly and Force Transmission that is regulated by Integrin Activation and Cytoskeleton Tension

Abstract

SummaryIntegrin-based focal adhesions (FA) transmit anchorage and traction forces between the cell and the extracellular matrix (ECM). To gain further insight into the physical parameters of the ECM that control FA assembly and force transduction in non-migrating cells, we used fibronectin (FN) nanopatterning within a cell adhesion-resistant background to establish the threshold area of ECM ligand required for stable FA assembly and force transduction. Integrin-FN clustering and adhesive force were strongly modulated by the geometry of the nanoscale adhesive area. Individual nanoisland area, not the number of nanoislands or total adhesive area, controlled integrin-FN clustering and adhesion strength. Importantly, below an area threshold (0.11 mm 2 ), very few integrin-FN clusters and negligible adhesive forces were generated. We then asked whether this adhesive area threshold could be modulated by intracellular pathways known to influence either adhesive force, cytoskeletal tension, or the structural link between the two. Expression of talin-or vinculinhead domains that increase integrin activation or clustering overcame this nanolimit for stable integrin-FN clustering and increased adhesive force. Inhibition of myosin contractility in cells expressing a vinculin mutant that enhances cytoskeleton-integrin coupling also restored integrin-FN clustering below the nanolimit. We conclude that the minimum area of integrin-FN clusters required for stable assembly of nanoscale FA and adhesive force transduction is not a constant; rather it has a dynamic threshold that results from an equilibrium between pathways controlling adhesive force, cytoskeletal tension, and the structural linkage that transmits these forces, allowing the balance to be tipped by factors that regulate these mechanical parameters.

Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1

“…Our observation that Y-27632 treatment accelerates cell movement with an accompanying loss of directionality is also in line with reports showing that RhoA/ROCK inhibition affects both the speed and directionality of cell movement by causing degradation of focal contacts and stress fibers [66][67][68]. The number and prominence of focal contacts have all been associated with an increase in the strength of cell-substratum adhesion [56,57,65,[69][70][71][72]. It thus came as a surprise that our MEFs that are devoid of focal contacts and stress fibers adhere more strongly to the substratum (Fig.…”

“…5 and 6, in which dark close contact areas cover nearly the entire undersurface. Close contacts associate with the cortical meshwork of actin microfilaments [57,83,83,84], so it is plausible that locally controlled contractility modulates cell surface geometry on a nanoscale, thus further contributing to cell adhesiveness [69,70,72,79,85]. It has also been observed that coated pits filled with ruthenium red-positive material seem to concentrate in areas of close contacts [86,87] possibly providing a wealth of charged molecules dangling from the cell surface.…”

Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification

“…Alteration in cell morphology as a consequence of cytoskeletal tension has an indirect effect on mechano-transduction pathways, as demonstrated by expression changes in stem cell responses [14,29]. In the current studies cytoskeletal rearrangement were observed, including altered expression patterns of vinculin, a key structural component of focal adhesions [30]. On the LASER topography, vinculin displayed arrangement around the cell cytoplasm periphery.…”

Vascular smooth muscle cells direct extracellular dysregulation in aortic stiffening of hypertensive rats

“…In addition, we found that activated integrin β1 was significantly increased in SHR TA VSMCs vs. WKY VSMCs. Given the evidence that integrins are the principal mediators of mechanical signaling in cell–ECM interactions (Chen, Tan & Tien, 2004; Ciobanasu, Faivre & Le Clainche, 2013; Coyer et al., 2012; Schwartz & DeSimone, 2008), the increased expression and activity of integrin β1 in SHR VSMCs provide a potential mechanism for higher cell–ECM interactions in the 3D reconstituted model and may represent a potential target for the amelioration of aortic stiffening.…”