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Exploring the functional dynamics between PIEZO1 and cell-generated forces

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The conversion of mechanical stimuli into biochemical signals plays an integral role in regulating physiology and homeostasis. Cells utilize several specialized proteins to translate these mechanical forces into intracellular signaling. Of these, the mechanically-activated ion channel PIEZO1 has emerged as a key mechanosensor regulating a range of biological processes occurring in a variety of cell types. Despite the observed importance of PIEZO1, the mechanisms by which PIEZO1 is activated under native cellular contexts and scales to control physiologically important phenomena are largely unknown. Within this dissertation, I describe my efforts to fill this gap within the scientific literature by contributing towards the development of novel microscopy-based techniques and accompanying bioimage analyses which can be implemented to study PIEZO1 activity under endogenous cellular conditions. In Chapter 2, we find that mechanical forces initiated by cell generated, or Myosin II-based, traction forces are able to activate nearby PIEZO1 channels. Given that cell generated forces play a central role during cell migration we then asked whether PIEZO1 may regulate the migration of keratinocytes, the predominant cell type of the epidermis, during wound healing. Within Chapter 3, we show through molecular, cellular and organismal studies that PIEZO1 plays a regulatory role during keratinocyte migration and wound healing. Epidermal-specific Piezo1 knockout mice exhibited faster wound closure while gain-of-function mice displayed slower wound closure compared to littermate controls. By imaging the spatiotemporal localization dynamics of endogenous PIEZO1 channels we find that channel enrichment at regions along the wound edge induces a localized cellular retraction that slows the wound closure process. Efficient wound closure is driven by the formation of specialized cells called leader cells which transmit mechanical and biochemical cues to ensuing follower cells, ensuring their uniform polarization and coordinated direction of migration, or directionality. Despite the observed importance mechanical cues play in regulating both leader cell formation and directionality, the underlying biophysical mechanisms remain elusive. Given that PIEZO1 plays a regulatory role during wound healing, within Chapter 4 we set to elucidate PIEZO1's contributions to collective migration by taking an integrative experimental and mathematical modeling approach. Through numerical simulations and subsequent experimental validation, we found that directionality plays a key role in regulating epithelial sheet migration and is inhibited by PIEZO1 activity. We propose that PIEZO1-mediated retraction suppresses leader cell formation which inhibits the coordination of directionality between cells during collective migration.

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