Membrane mechanisms during exo- and endocytosis in excitable cells

Sammanfattning: Excitable cells, like endocrine cells and neurons release hormones and transmitters through exocytosis to mediate many important functions, such as stress responses, immune responses, control of blood glucose, and synaptic transmission that mediates communication between neurons in the brain. This process relies on delicate endocytic mechanisms, to retrieve vesicular membranes and proteins previously fused at the plasma membrane and thus maintains the ability of exocytosis for excitable cells. Upon exocytosis, vesicle fusion with the plasma membrane generates an Ω-shape membrane profile with a narrow fusion pore (< ~5 nm), through which the vesicular content is released. For decades, it has been thought that the narrow pore of the Ω-profile either dilates till the Ω-profile flattens to facilitate content release (full-collapse) or closes to limit content release (kiss-and-run). Using a combination of confocal, super-resolution STED and electron microscopy, we found that this classical view needs to be modified significantly. We observed that the fusion pore may expand, constrict and/or close at different rates to regulate content release and that its size may vary between 0 and 490 nm. The cytoskeletal F-actin facilitates fusion pore opening. The merge between fused vesicles and the plasma membrane is not via the full-collapse fusion mode, but via Ω-profile shrinking while maintaining the Ω-shape (Ω-shrink fusion mode). F-actin provides sufficient membrane tension to facilitate Ω-shrink fusion in neuroendocrine chromaffin cells, and to facilitate vesicle merging at lamprey giant synapses. Thus, facilitation of vesicle merging by F-actin may be applicable to many secretory cells and synapses. Unlike the classical view that full-collapse and kiss-and-run facilitate and limit release, respectively, facilitation is mediated by shrink-fusion with a large non-dilating pore, whereas during inhibition, by enlarge-fusion that enlarges the Ω-profile, a small pore is maintained. Shrink and enlarge-fusion may account for diverse hormone and transmitter release kinetics observed in secretory cells. Clathrin-mediated endocytosis (CME), the most common form of endocytosis, is considered to retrieve vesicles from the flat plasma membrane. With conventional and platinum replica electron microscopy (EM), we found that clathrin-coated pits prefer to be localized at the bulk invagination of the plasma membrane instead of the flat plasma membrane. Using super-resolution STED microscopy, we observed for the first-time direct budding of vesicles from bulk membrane invagination. These results suggest that bulk membrane invaginations are a major platform for mediating clathrin-dependent endocytosis, calling for modification of the current view that clathrin-mediated endocytosis mostly originated from the flat plasma membrane. In summary, the studies described here improve our understanding of the regulation of hormone and transmitter release as well as endocytosis.