Understanding the conformational changes of proteins in their native cellular context is crucial for clarifying their physiological function. Förster resonance energy transfer (FRET) is a widely used fluorescence-based method for detecting nanometer-scale distance changes, providing a non-invasive means to monitor structural dynamics at high spatial resolution. However, large and flexible membrane proteins remain challenging to study in living cells. Among them, Piezo1 is a particularly large mechanosensitive ion channel with important roles in processes such as cardiovascular development and mechanosensation, yet its conformational dynamics in cells remain poorly understood. Here, we describe progress toward establishing a FRET-based approach designed to monitor these structural changes. To label Piezo1 in cells, we used a site-directed fluorescent tagging strategy at selected residues, enabling measurement of FRET signals that reflect changes in molecular proximity. Initial experiments with labeled Piezo1 constructs revealed measurable FRET signals, indicating sensitivity to conformational changes. When cells were subjected to mechanical stimulation, fluorescence changes were observed that are consistent with, and likely reflect, structural changes of Piezo1. These preliminary findings provide a basis for ongoing experiments aimed at further validating the observations and refining this platform. This developing methodology could be applied broadly to investigate the dynamics of other mechanosensitive membrane proteins and study force-dependent signaling pathways in live cells. By providing direct, real-time readouts of structural changes, the platform has the potential to advance quantitative mechanobiology studies and contribute to understanding the molecular basis of mechanotransduction in diverse physiological contexts.
Mukai et al. (Sun,) studied this question.