Dissecting the Biomechanical Feedback Between Plasma Membrane Curvature and Endocytic Proteins in Mammalian Cells using Nanostructured Substrates

Abstract

Plasma membrane curvature is the hallmark of endocytosis. A mechanistic feedback loop between endocytic proteins and the plasma membrane drives clathrin-mediated endocytosis from pit to vesicle; however, the macro-scale relationship between the endocytic protein complex and membrane curvature is still not understood. We previously used a novel quartz “nanostructured” substrate to induce stable plasma membrane curvature, showing that endocytic proteins are preferentially recruited to curvatures < 200 nm. Here we use a polymer molded on to a glass substrate and live genome edited cells to define the relationship between curvature-sensing endocytic proteins and the curved plasma membrane. MDA-MB231 and human fibroblasts, endogenously expressing the fluorescent fusion proteins AP2, clathrin, and/or dynamin2 were used to monitor endocytosis by time-lapse fluorescence microscopy. Cells serially depleted for the late stage endocytic BAR domain proteins SNX9, Bin1, and EndophilinA2 were imaged to determine the molecular requirements for clathrin-mediated endocytosis in cells with a pre-curved plasma membrane. We also monitored how actin destabilization, and the depletion of N-WASP or the Arp2/3 complex affected the outcome of clathrin-mediated endocytosis in the context of induced membrane curvature. Finally, the size of the curvature-generating substrates was varied to determine the relevant range of membrane curvatures for clathrin-mediated endocytosis under depletion conditions.