EXOSYTS

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

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 Obiettivo del progetto (Objective)

'Exocytosis is at the base of fundamental physiological processes such as neurotransmission and hormone release and elucidating its molecular mechanisms is a major step towards deciphering the immense complexity of brain function. It is well established that synaptotagmins (syts) are essential components of the release machinery that couple the entry of Ca2 to the fusion of vesicles and subsequent release of their contents. However, many aspects of syt function, and of the interplay between different syt isoforms, in triggering the release process are as yet unknown. In the present project I propose to investigate the roles of different syt isoforms on exocytosis in chromaffin cells and hippocampal neurons. Since both syt-1 and syt-7 cooperatively participate in exocytosis I will first study the modulation of fusion pore properties by these isoforms. This will be done by studying fusion pore formation through single-spike amperometry in chromaffin cells obtained from syt-1, syt-7 and syt-1/syt-7 knockout mice. I will also virally re-express wild type syts and chimeras between both proteins to identify the domains involved in slow and fast secretion. Additionally, I expect to clarify whether vesicles containing syt-1 and syt-7 are recruited during separate phases of release (fast vs. slow) using constructs that express these isoforms tagged with a pH-sensitive YFP. Finally, I will seek to identify a possible function of syt-7 in hippocampal neurons by first studying its subcellular distribution in vesicles vs plasma membrane. Additionally, I will study whether syt-7 plays a differential role in release between GABAergic and glutamatergic neurons. My proposed multidisciplinary approach offers great potential to tackle these questions and is expected to yield original results that may help in deciphering the precise role of different syt isoforms in exocytosis. This will contribute, on the longer term, to our understanding of neuronal communication.'

Introduzione (Teaser)

Exocytosis is the process through which a cell directs the contents of secretory vesicles into the extracellular space. Exocytosis is at the base of fundamental physiological processes such as neurotransmission and hormone release.

Descrizione progetto (Article)

The elucidation of the molecular mechanisms of exocytosis is a major step towards deciphering the immense complexity of brain function. The EU-funded 'Dissecting synaptotagmin isoform function: from vesicle docking to fusion pore formation' (EXOSYTS) project goal is to understand the regulation of the fast and slow phases of calcium ion-triggered exocytosis.

The nervous system performs immensely complex functions through precisely defined and timed communication between neurons that are dependent on exocytosis. These events are mediated by dedicated proteins that are localised on the neuronal synaptic vesicles themselves and the plasma membrane.

Some isoforms of the protein synaptotagmin act as calcium ion sensors. They are involved in early synaptic vesicle docking to the presynaptic membrane and late steps of synaptic vesicle fusion. Researchers focused on syt1 and syt7 isoforms and experiments revealed no change in any of the properties of single spikes in the absence of syt7.

At the next stage, research shifted towards protein Doc2b that is also involved in exocytosis. The protein also binds calcium but exists in a soluble state. Experiments in chromaffin cells using Doc2b knock-out mice revealed that the calcium dependence of vesicle priming and release remained unchanged. The conclusion was that Doc2b acts to inhibit vesicle priming during prolonged calcium exposure and protects vesicles from premature fusion.

Finally researchers identified a novel role for protein interacting with C Kinase 1 (Pick1) in the biogenesis of secretory vesicles in mouse chromaffin cells. In its absence, chromaffin cells display reduced exocytosis in response to calcium release or membrane depolarisation. In the absence of Pick1, the vesicles had reduced size and number but remained fully functional. The results of the project were disseminated through several high impact publications. The project in general made an important contribution towards a better understanding of the complexity of exocytosis.