The spatial organization of proteins within the plasma membrane is a fundamental regulator of biological processes, yet the biophysical principles governing their subcellular distribution remain incompletely understood. This study elucidates how membrane geometry and lipid composition interact to dictate the localization and function of Piezo1 and NINJ1. Piezo1 is a bona fide mechanosensitive ion channel ubiquitously expressed in mammalian cells. The distribution of Piezo1 within a cell is essential for various biological processes including cytokinesis, cell migration, and wound healing. NINJ1 is recently identified as an active modulator of plasma membrane fragility. Activation of NINJ1 is critical in multiple lytic cell death pathways including pyroptosis, necrosis and ferroptosis. First, we demonstrate that membrane curvature serves as a key regulator of Piezo1. In living cells, Piezo1 depletes from highly curved membrane protrusions such as filopodia and enriches to nanoscale membrane invaginations. Quantification of the curvature-dependent sorting of Piezo1 directly reveals the in-situ nano-geometry of the Piezo1-membrane complex. Piezo1 density on filopodia increases upon activation, independent of calcium, suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that diminishes with channel activation.Beyond geometry, we identify membrane phase separation as a dominant driver of Piezo1 sorting. In giant plasma membrane vesicles (GPMVs) and fixed cells, Piezo1 exhibits a strong preference for liquid-disordered (Ld) phase. Activation of Piezo1 doesn’t change the Piezo1 sorting neither chemically nor mechanically. The strong lipid preference orchestrates with membrane curvature effect and regulates the distribution of Piezo1. These findings suggest that the local lipid environment is a critical determinant of Piezo1-mediated mechanotransduction. Finally, we apply these biophysical principles to understand the process of membrane rupture. We propose a model where phase separation drives NINJ1 clustering and polymerization. In GPMVs, activated NINJ1 translocate from the Ld phase to the interface between Ld and Lo phases, acting as a protein linactant. This interface localization prepares NINJ1 for both “Pore” and “Cutter” rupture pathways highlighting a lipid composition dependent lysis mechanism. Loss of function mutant NINJ1-K45Q and inactive homolog NINJ2 shows reduced translocation which further supports the functional relevance of interface localization. Collectively, our research establishes that the interplay between membrane curvature, lipid phase separation and protein conformational changes regulates the distribution of mechanosensitive membrane proteins. These insights provide a new framework for understanding cellular mechanics and inflammatory responses, offering potential therapeutic targets to modulate membrane protein functions.
Shilong Yang (Thu,) studied this question.