Multiscale interrogation of mechanically driven stromal cell behaviors
Embargo Date
2025-01-16
OA Version
Citation
Abstract
Cells throughout the body sense mechanical forces and respond to such forces to control tissue organization and maintain homeostasis in the face of perturbations like an injury. The transduction of these signals takes place across a vast temporal and spatial scale: cells must sense tissue level changes through the activation of sub-cellular signaling events and must coordinate tissue level responses through precise coordination of cellular level behaviors. In this work, we investigated how mechanical forces shape fibroblast behavior at two different scales. We first investigated how tissue level stretching can influence the depth-dependent alignment of fibroblasts in stromal microtissues over the course of many hours. Second, we generated genetically-encoded tools in an attempt to measure the sub-cellular transduction of mechanical forces through the activation of ERK signaling on the actin cytoskeleton on the scale of minutes.
Mechanical forces are responsible for shaping the organization and alignment of tissues throughout the body. Many tissues such as the corneal stroma and articular cartilage have complex multilayered alignment which is required for their function. Since natural regeneration programs are insufficient to restore proper multilayered tissue alignment after injury, methods to engineer multilayer alignment in tissue constructs are of great interest. In chapter 2 of this thesis, we report a new device to induce distinct multilayer alignment of fibroblasts in stromal microtissues based on depth-dependent cyclic stretching. We find that, after alignment, fibroblast migration is enhanced along the direction of alignment and that cyclic stretching increases tissue compaction. These findings highlight the complex role of mechanical forces in generating relevant tissue structures, as well as how these tissue-level structures dictate the behaviors of their constituent cells.
Despite being able to control cellular behaviors with mechanical forces, how these tissue level responses arise through the precise regulation of mechanotransducive signaling pathways is unclear. It has been proposed that one way cells encode information about their mechanical environment is through the activation of extracellular signaling-regulated kinase (ERK) signaling on the actin cytoskeleton, however tools to measure the actin-localized dynamics of ERK signaling are needed. Chapter 3 of this thesis describes the development of a genetically-encoded biosensor to detect ERK signaling on the actin cytoskeleton. We determined that no actin-specific signaling dynamic could be identified with actin-linked FRET sensors. Instead, we revealed inconsistencies in the ability to detect actin-localized ERK signaling with common techniques like immunofluorescence. Our work sheds light on the complexity of detecting localized signaling and provides insights for the future development of targeted biosensors. In summary, the work presented here provides insights on and new tools for studying the complex interactions between mechanical forces, intracellular signaling, and cellular behavior at multiple spatial and temporal scales.