Hallmarks of Reversible Separation of Living, Unperturbed Cell Membranes into Two Liquid Phases

Controversy has long surrounded the question of whether spontaneous lateral demixing of membranes into coexisting liquid phases can organize proteins and lipids on micron scales within unperturbed, living cells. A clear answer hinges on observation of hallmarks of a reversible phase transition. Here, by directly imaging micron-scale membrane domains of yeast vacuoles both in vivo and cell free, we demonstrate that the domains arise through a phase separation mechanism. The domains are large, have smooth boundaries, and can merge quickly, consistent with fluid phases. Moreover, the domains disappear above a distinct miscibility transition temperature (T-mix) and reappear below T-mix, over multiple heating and cooling cycles. Hence, large-scale membrane organization in living cells under physiologically relevant conditions can be controlled by tuning a single thermodynamic parameter.