Daniel Colon-Ramos (Yale/HHMI) 2: Mechanisms of neuronal synapse assembly and function

[@thisnicklldo]

Very interesting. I'm still not sure I've fully understood the process, maybe somebody could confirm whether I'm getting it right: In a normal neuron vesicles cluster at the synapses. After synaptic release, the vesicle proteins are briefly within the cell membrane, but rapidly reform vesicles - that is, the same proteins that were incorporated into the membrane for neurotransmitter release quickly come out of the membrane to reform vesicles which can then be reloaded with neurotransmitter. This reformation of vesicles from the same proteins is the 'recycling' referred to in the video, and is the 'endocytosis' referred to in the video. This process requires energy, and so local metabolon sites have evolved to ensure anaerobic (specifically) glycolysis can take place close to the synapse if the cell is sufficiently hypoxic that there is insufficient ATP produced from aerobic processes (i.e. ATP coming from mitochondria) - so presumably most of the time vesicle recycling uses ATP floating in the cytosol produced in some nearby mitochondrion and only uses the anaerobic glycolytic process in case of dire need. In the mutants in the video, this process was disrupted - the metabolon sites are still there, but if the cell is made hypoxic, there is no ATP from the mitochondria and the metabolon sites can't produce the ATP because they are mutant, so after release of the neurotransmitter there is insufficient energy to allow reformation of the vesicles from the proteins now within the cell wall. The next bit I'm really not clear on: These vesicle proteins, now within the cell wall, and with insufficient ATP around to ensure they can recycle, fairly rapidly (on the order of 10 minutes) 'diffuse' along the cell wall to be evenly dispersed rather than localised. Thus the mutants show a diffuse picture of the distribution of vesicle proteins, once the mitochondrial ATP has been used up, as there is no local anaerobic ATP to take up the job of fuelling vesicle recycling. Is this right? This lateral diffusion of vesicle proteins seems very odd to me. Do task-specific proteins in the cell wall really laterally diffuse along the wall that fast? Once oxygen is available, apparently the localisation of proteins resumes. Does this mean that the proteins, diffused along the cell wall, now recycle back into the cell by formation of a vesicle from the cell wall (using general diffused cytosol ATP now available from mitochondria) , and then somehow travel within the cell back to their home near the synapse? How do they know where home is? How do they get there? I kind of understand that there are complex transport mechanisms moving neurotransmitters within the cell to be available for incorporation into vesicles, but are the vesicles themselves also subject to transport? Presumably the vesicle proteins have to be transported to the right place before vesicle formation in the first place, but I thought 'recycling' meant that a new vesicle reforms from the cell wall, rather than vesicle proteins being somehow moved from the cell wall to the cytosol and then incorporated into new vesicles. Maybe this is a key misunderstaning, sorry if this is so.

[@danielcolon-ramos4489]

Yes, you got it mostly right! Vesicles fuse with the plasma membrane (the term cell wall is used to refer to a specific structure in plant cells, so this is really a plasma membrane and not a cell wall) to release neurotransmitter. Once they fuse, they become part of the membrane. Vesicular proteins then difuse in the membrane unless they are recovered by endocytosis. Recovery by endocytosis requires ATP, and one of the main findings from our study was that these glycolytic proteins can dynamically relocalize to synpases to, we believe, locally supply the ATP. Like mobile/smart "generators". You are asking some good questions (how do they know where home is?) that are key in the field of endocytosis, some unanswerd or not fully understood. The process is referred to as recycling because all of this happens after vesicles first fuse with the plasma membrane. You are correct that "new" vesicles are first transported to the synapse. The molecular machines used for the transport, called kinesins, are very neat, and there are some great videos in iBio describing how they work. I hope that helps clarify and thanks for your questions!