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Dissecting synaptotagmin isoform function: from vesicle docking to fusion pore formation

Final Report Summary - EXOSYTS (Dissecting synaptotagmin isoform function: from vesicle docking to fusion pore formation.)

The EXOSYTS project aims at obtaining a better understanding of the structural requirements for the regulation of the fast and slow phases of calcium-triggered exocytosis by syt1 and syt7 in euroendocrine cells and neurons. For this we initially set out to clarify the implication of syt1 and syt7 isoforms in exocytosis and fusion pore dynamics, their possible differential implication in the release of different vesicle pools and also their functions in neurons. There were deviations to the objectives and work plan, as discussed in the Periodic Report, but these had no negative effect on the scientific and professional achievements.

The nervous system performs an immense complexity and variety of functions that depend on the precisely defined and timed communication between neurons. Such timing relies on the speed of a specialized process – exocytosis – by which neurotransmitters are released into the synaptic cleft to activate specific receptors on the target neurons. Such events are mediated by dedicated proteins, localized on both the vesicles themselves and on the
plasma membrane, the core of which is formed by a conserved set of proteins: the SNARE complex. In addition to SNAREs, exocytosis also requires Munc-18 and synaptotagmins (syts).
Both syt1 and syt7 knockouts have been generated and the general properties of chromaffin cell secretion reported. However, the properties of single fusion events have not been systematically investigated. This is an important issue since several recent reports postulated changed fusion pore duration or amplitude following syt mutations, but were conducted after expression of syt mutants in wild type PC12-cells, leaving it uncertain whether they dominate over the endogenous protein or change fusion pore dynamics indirectly. In other studies some of the mutants expressed were unable to reverse the knockout phenotype. At least two hypotheses can explain such results: 1) syt could have modified the properties of the fusion pore directly, by participating in it, or 2) indirectly, by titrating out other factors that are involved in the fusion pore. The only way to discriminate between these possibilities is to undertake investigations in knockout cells where the endogenous syts are no longer expressed. Towards this goal we have performed measurements of single amperometric spikes from syt1 and syt7 knockout mice. These experiments revealed no change in any of the properties of single spikes in the absence of syt7. Similar findings were reported in a publication by Segovia et al, 2010 (PNAS) using the same methodological approach and this particular task has therefore been down prioritized.

Role of Doc2B in vesicle fusion and exocytosis from chromaffin cells.
In recent years, another protein closely related to synaptotagmins, termed Doc2b, has received a lot of attention in high ranking journals. Similarly to synaptotagmins, it possesses two tandem C2 domains and the ability to bind calcium but, unlike synaptotagmins, it exists as a soluble protein. Despite the similarity to syts in in vitro lipid mixing assays, it remains unclear whether Doc2 proteins also actively trigger SNARE-dependent vesicle fusion, or whether they modulate secretion by acting on upstream steps. We have investigated the role of Doc2b in chromaffin cells using Doc2b knock-out mice and high temporal resolution measurements of exocytosis. We found that the calcium dependence of vesicle priming and release triggering remained unchanged, ruling out an obligatory role for Doc2b in those processes. However, in the absence of Doc2b, release was shifted from the readily-releasable pool to the subsequent sustained component. Conversely, upon overexpression of Doc2b, the sustained component was largely inhibited whereas the readily-releasable pool was augmented. Electron microscopy revealed an increase in the total number of vesicles upon Doc2b overexpression, ruling out vesicle depletion as the cause for the reduced sustained component. Further experiments showed that, in the absence of Doc2b, the refilling of the readily-releasable vesicle pools is faster, but incomplete. Faster refilling leads to an increase in the sustained component as newly primed vesicles fuse while the [Ca2+]i following stimulation is still high. We conclude that Doc2b acts to inhibit vesicle priming during prolonged calcium elevations, thus protecting unprimed vesicles from fusing prematurely, and redirecting them to refill the readily-releasable pool after relaxation of the calcium signal.
Further work, using selected mutations in Doc2b that hinder binding to several known cellular partners, is ongoing in order to decipher the molecular mechanisms by which it performs its modulatory role on priming.

Role of the BAR domain protein Pick1 in vesicle biogenesis in chromaffin cells.
Bar domain proteins have been implicated in the generation and stabilization of membrane curvature, a process that plays a key role in cellular physiology. Pick1 (protein interacting with C Kinase 1) is a BAR (Bin/Amphiphysin/Rvs) domain protein that has been intensively studied in the context of membrane receptor trafficking, where it’s PDZ binding domain - unique among BAR proteins - plays a key role. Here we identified a novel role for Pick1 in the biogenesis of Dense Core Vesicles (DCVs) in mouse chromaffin cells. In its absence, chromaffin cells display reduced exocytosis in response to calcium uncaging or membrane depolarization, without a change in the kinetics of release or its Ca2+ sensitivity. Single vesicle fusion events had a reduced frequency and lower amount of transmitter released per DCV. This was paralleled by a reduction in mean single vesicle capacitance, suggesting the presence of smaller DCVs. Electron microscopy confirmed that Pick1 KO chromaffin cells have significantly smaller and fewer DCVs and this was accompanied by a reduction in the levels of chromogranin A. At the cellular level, Pick1 was found to partly colocalize with the immature vesicle marker syntaxin 6, implicating a role in early steps of vesicle maturation. Therefore, in the absence of Pick1, DCVs have reduced size and number but remain fully functional.

Expected final results and their potential impact

Once the analysis of single spike data is concluded we expect to have clarified how syt1 and syt 7 participate in the modulation of fusion pore properties and exocytosis and publish an original research article. We have now published the first results on the function of Doc2b in modulating exocytosis (Pinheiro et al., J. Neuroscience; in press). Furthermore, we are working towards publishing the results describing the role of Pick1 in vesicle biogenesis, since this study is now complete and in manuscript form. This study reports the findings that a BAR domain protein can influence vesicle biogenesis, the absence of which leads to defects in vesicle number and size but, importantly, to no changes in fusogeneicity, and this work should have a large impact in the field.
The collaborations established during this IEF have also allowed me to participate in elucidating the interactions between synaptotagmin and SNAP25 that are involved in vesicle docking, priming and fusion triggering (Mohrmann et al., J. Neuroscience, 2013) and in helping defining a new mathematical model for secretion in chromaffin cells (PloS Computational Biology, resubmitted).