Piezo1 as a force-through-membrane sensor in red blood cells

Abstract

Piezo1 is the stretch activated Ca2+ channel in red blood cells that mediates homeostatic volume control. Here we study the organization of Piezo1 in red blood cells using a combination of super resolution microscopy techniques and electron microscopy. Piezo1 adopts a non-uniform distribution on the red blood cell surface, with a bias towards the biconcave 'dimple'. Trajectories of diffusing Piezo1 molecules, which exhibit confined Brownian diffusion on short timescales and hopping on long timescales, also reflect a bias towards the dimple. This bias can be explained by 'curvature coupling' between the intrinsic curvature of the Piezo dome and the curvature of the red blood cell membrane. Piezo1 does not form clusters with itself, nor does it co-localize with F-actin, Spectrin or the Gardos channel. Thus, Piezo1 exhibits the properties of a force-through-membrane sensor of curvature and lateral tension in the red blood cell.

Single-particle tracking and machine-learning classification reveals heterogeneous Piezo1 diffusion

“…The available evidence from fluorescence correlation spectroscopy, fluorescent recovery after photobleaching, and SPT indicates that Brownian motion is not the prevalent diffusive behavior of proteins in the membrane environment (reviewed in (28, 48, 49)). Although the mechanistic details of Piezo1 diffusion in the plasma membrane have yet to be elucidated, the underlying assumption made so far in the literature is that Piezo1 diffusion can be described as Brownian motion (26, 50). With the tagged particle’s trajectory denoted as , diffusive behavior can be characterized by the so-called time averaged mean squared displacement (TAMSD) (49) where T is the trajectory’s total length in time and Δ is the lag time.…”

“…According to our classification scheme, a class of trajectories nominally identified as “mobile” exhibit high mobility in the plasma membrane compared to the other two classes, “trapped” or “intermediate.” A recent pre-print by Vaisey et al reporting SPT on endogenous Piezo1 in red blood cells also observed heterogeneity in Piezo1 trajectories (50). Our detailed analysis of this class suggests that Piezo1 mobility is sensitive to changes in membrane composition and that activity may modulate the channel’s diffusion.…”

Membrane curvature governs the distribution of Piezo1 in live cells

“…Additionally, Piezo1 has been found to enrich at subcellular structures that are important for mechanotransduction, such as focal adhesion sites 27,29 and T-tubules of cardiomyocytes 30,31 . Lastly, Piezo1 has been reported to preferentially localize to the intercellular bridge during cytokinesis 32 , and to the biconcave 'dimples' in red blood cells 19,21 . While a general mechanistic explanation has yet to be established, these intriguing subcellular patterns of Piezo1 raise the question of whether this ion channel can be sorted by fundamental physical factors on the cell surface.…”

“…The large size and curved architecture of Piezo1 trimers make them directly sensitive to tension in the plasma membrane 7,[12][13][14] . Additionally, several studies indicate a cytoskeletal role in the activation of Piezo channels, while the presence of direct Piezo1-cytoskeleton interaction is still under debate [15][16][17][18][19][20][21][22] . Notably, the cortical cytoskeleton can drastically affect the extent of membrane tension propagation, thereby indirectly controlling Piezo1 activation via the lipid bilayer [23][24][25][26] .…”

“…While the structure and activation of individual Piezo1 channels have been extensively studied, the dynamics and distribution of the channel within a cell are only starting to be explored 19,21,25,27 . In addition, it is unclear whether the Piezo1 structures determined in vitro recapitulate its nanoscale conformations in living cells 7, 8,10,11 .…”