Single-particle tracking reveals heterogeneous PIEZO1 diffusion

The mechanically activated ion channel PIEZO1 is critical to numerous physiological processes, and is activated by diverse mechanical cues. The channel is gated by membrane tension and has been found to be mobile in the plasma membrane. We employed single-particle tracking (SPT) of endogenous, tdTomato-tagged PIEZO1 using total internal reflection fluorescence microscopy in live cells. Application of SPT unveiled a surprising heterogeneity of diffusing PIEZO1 subpopulations, which we labeled "mobile" and "immobile." We sorted these trajectories into the two aforementioned categories using trajectory spread. To evaluate the effects of the plasma membrane composition on PIEZO1 diffusion, we manipulated membrane composition by depleting or supplementing cholesterol, or by adding margaric acid to stiffen the membrane. To examine effects of channel activation on PIEZO1 mobility, we treated cells with Yoda1, a PIEZO1 agonist, and GsMTx-4, a channel inhibitor. We collected thousands of trajectories for each condition, and found that cholesterol removal and Yoda1 incubation increased the channel's propensity for mobility. Conversely, we found that GsMTx-4 incubation and cholesterol supplementation resulted in a lower chance of mobile trajectories, whereas margaric acid incubation did not have a significant effect on PIEZO1 mobility. The mobile trajectories were analyzed further by fitting the time-averaged mean-squared displacement as a function of lag time to a power law model, revealing that mobile PIEZO1 puncta exhibit anomalous subdiffusion. These studies illuminate the fundamental properties governing PIEZO1 diffusion in the plasma membrane and set the stage to determine how cellular processes and interactions may influence channel activity and mobility.