Mechanical forces, originating both intracellularly and extracellularly, must be transduced into biological signals through a process known as mechanotransduction. Numerous biological systems, ranging from proprioception to stem cell development and differentiation, depend on proper mechanotransduction, which is facilitated by mechanical force-sensing proteins on the cell surface, including components of focal adhesions and ion channels. Multiple mechanical force-sensing proteins have been identified in plants and bacteria, but for many years, it remained unknown whether similar mechanosensing ion channels existed in mammals until the discovery of the PIEZO channels in a mouse neuroblastoma cell line. This family of two novel ion channels, PIEZO1 and PIEZO2, is capable of converting mechanical forces into electrochemical signals. PIEZO1 has known involvement in human diseases, including cancer, chronic pain, and migraines. Function-altering mutations in PIEZO1 cause circulatory and lymphatic developmental defects. Previous research in PIEZO mechanotransduction has sought to understand how the channel's mechanosensitivity is achieved by examining the protein’s structure in combination with functional assays of PIEZO1 activity.
Traditionally, the study of PIEZO1 channel function has centered around patch clamp electrophysiology, though electrophysiology has several limitations. Whole-cell patch clamp dialyzes the cell, disrupting cellular structures and physiology, while cell-attached patch clamp imparts a large resting tension on the patch that may alter channel behavior. The regulation of PIEZO1 function is complex and context-dependent, and it must be assayed using tools that do not perturb the native cellular milieu. Microscopy is another technique that facilitates visualization of the channel in a native cell, without disturbing the mechanical environment of the channel. When visualizing channel localization with microscopy, several PIEZO1 antibodies are available for detecting the protein in fixed samples. However, the sensitivity and specificity of these antibodies are debated. The process of fixing and permeabilizing samples, which is necessary for antibody use, can alter the density of plasma membrane proteins (\cite{Cheng2019-rp}), lead to mislocalization, or mask epitopes of target proteins, potentially confounding the observed signals. Alternatively, many have observed PIEZO1 using cells with a PIEZO1 tagged with a fluorescent protein. However, fluorescent proteins have several limitations, including rapid photobleaching, providing only localization information, and most often such systems have used overexpression or non-human models. Finally, since PIEZO1 is Ca\textsuperscript{2+}-permeable, Ca\textsuperscript{2+} imaging modalities can be used to visualize and quantify the channel’s activity. However, these methods are generally limited to genetically encoded Ca\textsuperscript{2+} indicators or organic dyes that are not specific to PIEZO1. As a result, no single tool allows for the simultaneous visualization of PIEZO1 activity and localization.
Within my dissertation, I describe my efforts from the last five years to develop a novel tool, the PIEZO1-HaloTag hiPSC line, to fill this gap. This tool enables the investigation of the endogenous localization and activity of the channel in single cells and in vitro tissue organoids. In Chapter 2, I describe the development and validation of the PIEZO1-HaloTag hiPSC line. This novel molecular tool allowed us to quantify PIEZO1 motility behaviors with high signal-to-noise ratio, reduced photobleaching, and improved localization precision. Furthermore, we found the PIEZO1-HaloTag system to be well-suited for imaging endogenous channel activity dynamics in single cells and in vitro models of early neural development. In Chapter 3, I illustrate the use of the PIEZO1-HaloTag system to uncover the relationship between motility and activity of the channels concerning tension generation and propagation. We found that PIEZO1 is less motile when tension propagation and generation are inhibited through inhibition of actomyosin and actin cortical attachments to the membrane, respectively. Overall, we present the novel PIEZO1-HaloTag hiPSC line, enabling the study of endogenous PIEZO1 activity and mobility in response to a variety of perturbations.
