The Nanoscale Organization of Focal Adhesion Signaling Complexes can Reflect Changes in Cellular Contractility and Motility

Abstract

Focal adhesions are the conduits through which cells receive and interpret mechanical signals. It is not known if nanoscale protein organization is altered to accommodate changes in mechanical inputs from the cytoskeleton and extracellular matrix components. We hypothesized that the relative position of specific focal adhesion proteins could correspond to the engagement of a physical protein clutch for different adhesion functions. To this end we employed Scanning Angle Interference Microscopy to determine the 3D organization of proteins comprising focal adhesions with a precision of ∼5nm. We found paxillin, FAK, vinculin, talin, and zyxin to be stratified in distinct layers over a vertical range of 60 nm. We then compared nascent versus focal adhesions at the cell leading edge, and found that paxillin localized ∼7nm towards the cell membrane in developing adhesions. We inhibited intracellular contractility to see how adhesion architecture dynamically responds to changes in mechanical input, and observed that paxillin and zyxin, but not vinculin, undergoes a marked increase in height of >15nm. Conversely, vinculin without a force dependent auto-inhibition domain, T12; undergoes dramatic reorganization at the nanoscale after contractility inhibition. Overexpression of vinculinT12 resulted in increased intramolecular forces as seen in a vinculinT12 FRET tension sensor, targeting of vinculin to an architecture that corresponded with talin and actin engagement, but not changes in cellular traction. When we reduced cellular motility through overexpression of a constitutively active Rac1 mutant, adhesions at the lamella-lamellipodia border had different vinculin architecture than other adhesions in the cell. Our results suggest that elimination of vinculin force dependent auto-inhibition can dictate focal adhesion architecture and morphology, but not cellular traction; and that there are specific vinculin architectures that reflect distinct states of cellular contractility and motility.