Abstract:
Membrane proteins are sorted to different cellular compartments by a process termed as vesicular transport; wherein the membrane buds out and is severed to form a vesicle. This process allows cells to take up nutrients, ensures inheritance of organelles after cell division and manages synaptic transmission thus making it fundamental to life. Protein machines implicated in the severing reaction belong to a highly conserved family of GTPases. Dynamin represents the paradigmatic member of this family and functions to generate synaptic vesicles for fast neurotransmission. The mechanism by which dynamin manages to sever membranes remains debated. The severing reaction is typically carried out in a confined region of the membrane enclosed within a 10 nm wide, 2-rung scaffold comprised of ~26 molecules of dynamin. Thus, events leading to membrane severing have been difficult to probe. We have developed an assay system of supported membrane tubes (SMrT) that topologically mimics the necks of budded vesicles (~30 nm wide), the physiological membrane substrate for dynamin. Using real-time fluorescence microscopy with high temporal resolution, we find that dynamin forms a scaffold, which upon GTP hydrolysis, undergoes progressive constriction leading to severing of the underlying membrane. This assays allows us to interrogated precise structure-function relationships that render dynamin capable of severing membranes. Dynamin engages with the membrane through a unique pleckstrin-homology domain (PHD) - yet the basis of it's evolutionary conservation remains unclear. We engineered a dynamin mutant where PHD functions are replaced by generic membrane association. Remarkably, we find that this mutant manages a severing reaction that displays highly variable rates and with long-lived pre-scission intermediates. Our results thus reveal that the physiological requirement for a fast-acting membrane fission apparatus appears to have been fulfilled by adoption of the PHD. Ourwork has contributed both to technology development to monitor membrane-remodeling events and critical re-evaluation of dynamin function that substantially improves our understanding of the important process of membrane fission.
