Probing the Spatiotemporal Relationship Between Membrane Fusion and Endocytosis

The cell membrane is a complex, liquid-crystal system. Its organization is driven by the interactions between its constituents—featuring charged and non-charged molecules—and the surrounding polar medium. These interactions govern the membrane's physical dynamics, such as tension and curvature, managed by endo- and exocytosis to maintain homeostasis. Multivesicular endosome (MVE) exocytosis releases cargo vesicles and adds significant surface area to the plasma membrane. To maintain balance, this excess membrane is retrieved by endocytosis, but the coupling's nature and degree remain unclear. Here, we characterize the spatiotemporal relationship between MVE fusion and subsequent endocytic events, focusing on the roles of Clathrin and the MVE marker, CD63. Using Total Internal Reflection Fluorescence microscopy, we monitored CD63 and Clathrin dynamics in live human lung cancer cells. We examined spatiotemporal colocalization of Clathrin and CD63 following MVE fusion, with Clathrin endocytosing near and within the MVE fusion site, retrieving lipids and proteins diffused from the MVE. We quantified and compared the rates and vesicle sizes of exocytic and endocytic events to determine the net membrane change. To probe underlying mechanisms, we applied osmotic shock and temperature trials, perturbations known to modulate membrane tension and disrupt Clathrin-mediated endocytosis, to explore the correlation of the observed coupling with membrane tension, a known stimulant of exocytosis.