Cell surface crowding is a tunable biophysical barrier to cell-cell fusion.

Cell-cell fusion is fundamental to developmental processes such as muscle formation, as well as to viral infections that cause pathological syncytia. An essential step in fusion is close membrane apposition, but cell membranes are crowded with proteins, glycoproteins, and glycolipids, all of which must be cleared before a fusion pore can be nucleated. Here, we find that cell surface crowding drastically reduces fusogenicity in multiple systems, independent of the method for driving fusion. We estimate that cell surface crowding presents an energetic barrier to membrane apposition on the scale of , greater than that of bare membrane fusion. We show that increasing cell surface crowding reduces fusion efficiency of PEG-mediated and fusogen-mediated cell-cell fusion, as well as synthetic membranes under force. Interestingly, we find that differentiating myoblasts naturally decrease cell surface crowding prior to fusion. Cell surface crowding presents an underappreciated biophysical barrier that may be tuned developmentally and could be targeted externally to control tissue-specific cell-cell fusion.