The Molecular Basis for Cellular Contractility Driven Mechanosensing

Abstract

Extensive literature is available on how focal adhesions sense and respond to environmental cues during fast migration in polarized cells. In contrast, little is known about how cells feel and translate environmental cues during slow mesenchymal migration through tissues (e.g. dendritic cells, macrophages). These cells form many more podosomes than focal adhesions and use them as mechanosensors to probe and remodel the extracellular matrix. Each podosome consists of a dense, protrusive actin core and an adhesive ring of integrins and cytoskeletal adaptor proteins such as vinculin and talin. Podosomes are highly dynamic, and their actin content continuously fluctuates mediating their protruding activity. Podosomes are organized in large clusters. Electron microscopy showed that podosomes are interconnected by cytoskeletal fibers, suggesting the existence of a mesoscale organization. By combining image correlation spectroscopy (ICS) techniques such as raster ICS (RICS) and timeresolved spatiotemporal ICS (trSTICS), we investigated the collective dynamic behavior of podosome clusters. We compared the dynamic behavior of the mechanosensitive protein vinculin and the mechano-insensitive protein talin. RICS showed similar diffusion (D=2.4 and 2.0mm 2 /s) and binding (t=0.024 and 0.026s) properties for vinculin and talin. In contrast trSTICS revealed significant differences in their flow patterns. While waves of correlated flow, with speeds ranging from 0.01-0.16 mm/min, are visible throughout the podosome cluster for vinculin, talin dynamics show no clear directionality. Moreover, podosome formation and dissolution are accompanied by characteristic flow patterns of vinculin, suggesting that localized recruitment of mechanosensitive proteins could coordinate podosome protrusive forces. Finally, using cross correlation STICS we show that vinculin, but not talin, dynamics correlate to flow patterns in the actin network. Taken together, our data demonstrate coordinated behavior of mechanosensitive adhesion proteins in podosome clusters providing evidence for mesoscale coordinated protrusive dynamics.