Date of Award

Fall 10-1-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology

First Advisor

Rothman, James

Abstract

The overarching theme of my thesis work is centered around the understanding of how the Synaptotagmin (Syt) family of proteins, which are key players in neurotransmission, is regulated in the brain. Upon very briefly introducing other key molecular players, firstly, I summarize the discovery of the regulation of Syts by self-oligomerization. This work is focused on the characterization of the physiologically relevant Syt1 ring-like oligomers, which are formed in the presence of negatively charged lipids and dissociate upon calcium binding. Using the approach of hypothetical structure-guided mutagenesis, phenylalanine in position 349 is identified as critical for the oligomer formation. Importantly, an alanine mutation in this position abolishes the oligomer formation, while no other molecular properties are affected (which I established using a variety of biochemical and biophysical techniques). Taken together, we hypothesize that the oligomer can act as a “stopper” of spontaneous fusion between the opposing membranes; yet it also acts to synchronize the arrival of action potential to the evoked vesicular fusion and neurotransmitter release. In addition, the relevance of the Syt1 oligomers is shown in model cell cultures and the hypothesis is further refined using other structural and in vitro reconstitution approaches. A family of Synaptotagmins (Syts) undergo several types of post-translational processing, which regulates neuronal growth and plasticity. Protein kinase C (PKC), which is highly, yet differentially expressed in the brain, has been previously shown to potentiate synaptic vesicle release via phosphorylation of the Syt1 isoform. However, the precise mechanism and functional effect of phosphorylation on Syt1 function remains unclear. In this work, we identified a new site of phosphorylation (T328/T329), located within the polybasic patch of the C2B domain which is functionally important for binding to PIP2 and other negative lipid headgroups. To study the effect of phosphorylation at this site, we introduced a phospho-mimetic amino acid substitution (Syt1T328/329E). We used a single-vesicle fusion assay to monitor the docking, clamping, fusion, and release of a single vesicle. This fluorescence-based assay tracks individual v-SNARE containing vesicles (vSUVs) as they approach the physiologically relevant mimic of the target membrane. vSUVs containing 24 copies of wild-type Syt1 (Syt1WT) and 12 copies of VAMP2 remain stably docked and fuse after addition of 1 mM calcium ions. To the contrary, Syt1T328/329E containing vSUVs abolished clamping ability thereby leading to spontaneous fusion. The role of phosphorylation of Syt1 by PKC was further corroborated by a γ-32P radiography-based protocol for the in vitro phosphorylation of Syt1WT-containing vSUVs. Phosphorylated vesicles recapitulated the loss-of-clamping behavior exhibited by the Syt1T328/329E -containing vesicles. Expectedly, phosphorylated Syt1 exhibited a dramatic reduction in its ability to bind negatively charged membrane in the basal (EDTA/Mg2+) state and in the presence of physiologically relevant calcium concentrations. As PIP2 binding is crucial for Syt1 to oligomerize and clamp vesicle fusion, we posit that phosphorylation of Syt1 at residue 328/329 abrogates clamping function by disrupting the ability of Syt1 to oligomerize. Our findings suggest a novel mechanism of in situ regulation of Syt1 function by PKC.

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