Myosin-driven mechanical stress triggers curvature-dependent membrane phase separation within cell-like model blebs*

The membrane phase behavior in the cell plasma membrane plays pivotal physical and biochemical roles in controlling membrane stability, rigidity, viral infection, and ion channel functions. The physical interaction between the plasma membrane and the underlying cytoskeleton has been hypothesized to be a crucial regulator of membrane phase behaviors; however, the impact of membrane deformation on membrane phase behaviors, coupled with cytoskeleton-generated mechanical stress, remains unclear. Here, by constructing a membrane-bound actomyosin layer within liposomes, we demonstrate that myosin contractility drives cortical actin flow and cell-like blebbing, leading to membrane phase separation within blebs. Through laser ablation of the actin cortex and physical modeling, we established the mechanical link between the actomyosin cortex and the membrane. Remarkably, laser ablation-induced blebbing revealed that cell-like blebs accumulate more cholesterol than the mother liposome. Furthermore, we observed that cholesterol accumulation increases as bleb size decreases, indicating a curvature-dependent redistribution of cholesterol within the membrane. These results provide valuable mechanical insights into the coordination among cytoskeleton-generated stress, membrane curvature, and membrane phase separation. This study sheds light on membrane phase behaviors regulated by cytoskeletal forces in mammalian cells.