Shoyer, T. C. | Search bioRxiv

bioRxiv2024

Detection of Fluorescent Protein Mechanical Switching in Cellulo

ABSTRACT/SUMMARYThe ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, the mechanisms by which forces affect protein function inside cells remain unclear. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated if force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Forster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo in a synthetic actin-crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cellular force generation as well as force-sensitive bond dynamics of the biosensor. Together, this work describes a new framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells. MOTIVATIONThe ability of cells to sense mechanical forces is critical in developmental, physiological, and pathological processes. Cells sense mechanical cues via force-induced alterations in protein structure and function, but elucidation of the molecular mechanisms is hindered by the lack of approaches to directly probe the effect of forces on protein structure and function inside cells. Motivated by in vitro observations of reversible fluorescent protein mechanical switching, we developed an approach for detecting fluorescent protein mechanical switching in cellulo. This enables the visualization of force-sensitive protein function inside living cells.

cell biology↗

bioRxiv2023

Coupling During Collective Cell Migration is Controlled by a Vinculin Mechanochemical Switch

Collective cell migration (CCM) plays important roles in development, physiological, and pathological processes. A key feature of CCM is the dynamic mechanical coupling between cells, which enables both long-range coordination and local rearrangements. This coupling requires the ability of cell adhesions to adapt to forces. Recent efforts have identified key proteins and implicated cellular-scale mechanical properties, but how key proteins give rise to these larger-scale mechanical processes is unclear. Using force-sensitive biosensors, cell migration assays, and molecular clutch models, we sought a molecular understanding of adhesion strengthening that could bridge this gap. We found that the mechanical linker protein vinculin bears substantial loads at AJs, FAs, and in the cytoplasm during epithelial sheet migration, and we identified a switch-like residue on vinculin that regulates its conformation and loading at the AJs during CCM. In vinculin KO-rescue, this switch jointly controlled the speed and coupling length-scale of CCM, which suggested changes in adhesion-based friction. To test this, we developed molecularly detailed friction clutch models of the FA and AJ. They show that open, loaded vinculin increases friction in adhesive structures, with larger affects observed in AJs. Thus, this work elucidates how load-bearing linker proteins can be regulated to alter mechanical properties of cells and enable rapid tuning of mechanical coupling in CCM.

cell biology↗