Membrane order regulates clathrin-coated pit dynamics but not initiation

Clathrin-mediated endocytosis involves the remodeling of membranes via the initiation, maturation, and scission of clathrin-coated pits (CCPs). How CCP initiation and dynamics are regulated has been addressed primarily from the perspective of the core proteins that mediate endocytosis. Whether and how the physical properties of the membrane regulate CCP initiation and dynamics are heavily underexplored. Here, we used a solvatochromic probe to readout membrane order in the plasma membrane in live cells undergoing endocytosis, using total internal reflection fluorescence microscopy. Cholesterol depletion decreased membrane order, and reduced CCP initiation and increased lifetimes of CCPs. In unperturbed cells, however, membrane order was correlated to CCP lifetimes, but not their initiation. When membrane order was decreased or increased independently without extracting lipids, CCP lifetimes were affected, but CCP initiation was not. Together, by reading out membrane order in living cells undergoing endocytosis and manipulating membrane order in both directions, we show that membrane order primarily regulates CCP dynamics, and that cholesterol extraction has additional effects on CCP initiation independent of its effect on order.