UC Irvine

PIEZO1 is a mechanically-activated ion channel critical for physiological function and development, and can be activated by externally- and internally-generated mechanical cues. This cation channel is widely expressed in the body and in a variety of organisms, and is mobile in the plasma membrane. Using TIRF microscopy and cell biology techniques, we demonstrate that PIEZO1 mobility is heterogeneous and complex. We classified “mobile” and “immobile” populations of PIEZO1 using trajectory spread, and manipulated membrane composition and channel activity to further explore the role of the membrane and activity in PIEZO1 mobility. PIEZO1 became more mobile when cholesterol was depleted from the membrane using methyl-β-cyclodextrin and when cells were treated with Yoda1, a PIEZO1 agonist. Cholesterol supplementation and incubation with GsMTx-4, a PIEZO1 antagonist, decreased PIEZO1 mobility. Further analysis of the “mobile” class by fitting the time-averaged mean-squared displacement versus lag time to a power-law model revealed that PIEZO1 puncta exhibit anomalous subdiffusion. We used micropatterned substrates to control cell size and shape (disc, crossbow, H, Y, and square-shaped) and found that PIEZO1 mobility is influenced by cellular geometry. Using square-shaped micropatterned substrates and FLIM, we found that membrane tension is highest at the edges and corners of square-shaped cells. We similarly found that PIEZO1 activity is highest at the edges and corners of square-shaped cells using TIRF microscopy. Taken together, our work sets the foundation for future studies to understand the mechanisms through which PIEZO1 interacts with the membrane and transduces mechanical forces.