A BIOENGINEERED APPROACH TO STUDY THE INTRACELLULAR MOLECULAR FORCES THAT REGULATE CELL STRUCTURE AND FUNCTION

Abstract

Mechanical forces are fundamental regulators of cell structure and function, yet how and where cells sense these forces at subcellular scales to affect cell behavior is poorly understood. In particular, adhesion protein complexes are the structures that physically link the internal cytoskeletal structures to either the extracellular matrix or neighboring cells and are known to undergo conformational changes and respond to applied force. This thesis leverages genetically encoded Förster Resonance Energy Transfer (FRET) tension sensors to spatially resolve cell-matrix and cell-cell adhesion forces with subcellular precision. We developed advanced imaging and analysis pipelines to quantify focal adhesion tension in three dimensions using human induced pluripotent stem cells (iPSCs) endogenously expressing a vinculin-based FRET tension sensor. Our approach revealed that different spatial patterns of adhesion force form unique cells states correlate with lineage commitment. To determine the spatial distribution of mechanical sensing in cardiomyocytes due to contraction, we measured focal adhesion tension using both live-cell imaging and a novel fixed-cell method as a function of subcellular location. Focal adhesion tension increased with sarcomere shortening, and depended on adhesion morphology and localization; more elongated adhesions toward the distal end of the cell had higher force in contracted cells. Furthermore, we investigated the role of mechanical contraction for the development and maintenance of myofibril structure in cardiomyocytes using a D65A mutation in cardiac troponin C that impairs calcium-activated contraction. Despite the elimination of contractility, mutant cells formed sarcomeres, yet the mutant cells had less organized myofibril structure and defects in proteomic maturation. These deficits were partially rescued by external mechanical cues from nanopatterned substrates, suggesting that substrate topography can overcome internal cues to modulate mechanosensitive pathways that regulate intracellular structure. Collectively, these studies establish a framework for quantifying subcellular adhesion forces in 3D and demonstrate how mechanical signatures are integrated by a cell to regulate development and structure. These insights contribute to our understanding of fundamental force sensing and mechanotransduction and may inform future studies of mechanically induced pathological cellular remodeling.