Experimental and Modeling Approaches to Investigate Molecular-Scale Mechanosensitive Processes in Collective Cell Migration

Abstract

The coordinated movement of groups of cells, called collective cell migration (CCM), plays important roles in many developmental, physiological, and pathological processes. During CCM, cells remain mechanically coupled to their neighbors, which enables both long-range coordination and local rearrangements. This coupling requires the ability of cell adhesions to transmit and adapt to mechanical forces. However, the molecular mechanisms that underly these mechanosensitive processes remain poorly understood, hindering efforts to manipulate CCM for therapeutic or engineering purposes. To address this gap, this dissertation develops and applies a combination of experimental and modeling approaches to investigate molecular scale mechanosensitive processes. In the first part of this dissertation, we asked how mechanical forces and biochemical regulation interact to control mechanical coupling during CCM. We focused on the mechanical linker protein vinculin, which is known to mediate adhesion strengthening. Using a set of Förster resonance energy transfer (FRET)-based biosensors, we probed the mechanical function and biochemical regulation of vinculin, elucidating a switch that toggles both the activation and molecular loading of vinculin at cell adhesions. We found that the vinculin switch controlled both the speed and coordination of CCM, resulting in a covariation of these variables that suggested changes in adhesion-based friction. To bridge molecular and cellular measurements, we developed molecularly specific models of frictional forces at cell adhesions based on the force-sensitive bond dynamics of key proteins. In these models, increases in vinculin activation and loading produced increases in friction at adhesion structures, and this was due to the engagement of vinculin-actin catch bonding. Together, this work reveals how the biochemical regulation of a linker protein (vinculin) affects a cell-level mechanical property (adhesion-based friction) to control a multicellular behavior (CCM).In the second part of this dissertation, we focused on how cells sense mechanical forces at the molecular scale. This is thought to occur by force-induced changes in the structure/function of proteins. However, how forces affect protein function inside cells remains poorly understood due to a lack of tools to probe this inside cells. Motivated by in vitro work showing that the mechanical loading of fluorescent proteins (FPs) causes a reversible switching of their fluorescence, we investigated if this phenomenon could be detected inside cells to directly visualize force-sensitive protein function. Using a mathematical model of FP mechanical switching, we developed a framework to detect it inside FRET-based biosensors. Applying this framework, we observed FP mechanical switching in two sensors, a synthetic actin-crosslinker and the linker protein vinculin, and we found that mechanical switching was altered by manipulations to cellular forces on the sensor as well as force-dependent bond dynamics of the sensor. Together, this work develops a new framework for assessing the mechanical stability of FPs and enables visualizing the effect of forces on protein function inside cells.Overall, the work in this dissertation advances our basic understanding of mechanosensitive processes, addressing knowledge gaps in CCM and mechanobiology. The frameworks we have developed for integrating molecular- and cellular-level experiments with mathematical models will facilitate new mechanistic studies into mechanosensitive processes involving other proteins and biological contexts.