Hallmarks of Reversible Phase Separation in Living, Unperturbed Cell Membranes

Abstract

Controversy has long surrounded the question of whether micron-scale lateral phase separation can organize proteins and lipids within the membranes of 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 in vivo, we demonstrate that the domains arise through a phase separation mechanism. The domains disappear above a distinct miscibility transition temperature, Tmix, and reappear below Tmix over multiple heating and cooling cycles. The domains are large, have smooth boundaries, and can merge, consistent with fluid phases. Hence, large-scale membrane organization in living cells under physiologically relevant conditions can be controlled by tuning a single thermodynamic parameter.