Molecular mechanisms of Synaptotagmin 1 mediated synaptic vesicle fusion

Lead Research Organisation: University College London
Department Name: Cell and Developmental Biology


The goal of this project, which falls into the Bioscience and Biotechnology priority area, is to obtain new fundamental insights into the molecular mechanism of synaptic transmitter release. This project features the development of new experimental tools (with Scientifica) via integration of optogenetics, patch-clamp electrophysiology and fluorescence imaging.
The timing of synaptic vesicle fusion is precisely controlled by vesicular Ca2+ sensor synaptotagmin (Syt1, 2, and 9 in different synapses), yet the mechanism of its action remains enigmatic. We propose to test the hypothesis that formation of Ca2+-sensitive Syt1 oligomeric rings plays a major role in synchronisation of evoked vesicular release. We will capitalise on recent findings of our collaborator J. Rothman (Yale) who produced a Syt1 mutant (F349A) that exerts a dominant-negative effect on Syt1 oligomersation in biochemical assays. If Syt1 oligomeric rings are essential for synchronisation of transmitter release then overexpression of the F349A mutant in presynaptic terminals should desynchronise vesicle fusion.
Testing this hypothesis requires measurements of evoked vesicle release with a submillisecond precision. This is difficult to achieve in randomly connected neurons in culture and thus requires the use of organised brain tissue. We will use transgenic mice where EYFP tagged channelrhodopsin-2 (ChR2) is expressed in pyramidal neurons under the control of CaMKII promoter (the colony is established in our lab). We will inject lentiviral constructs carrying either WT or F349A Syt1 fused with mCherry into mouse neocortex in vivo. Several weeks later we will prepare acute brain slices and use yellow and red fluorescence to identify ChR2 expressing cells which also express recombinant Syt1 constructs. Identified individual neurons will be stimulated with 477 nm laser and optically evoked post-synaptic currents (EPSCs) will be recorded from a post-synaptic cell. In this way, we will compare the effects of F349A and WT Syt1 constructs on fast evoked transmitter release and will establish whether Syt1 oligomerisation is required for synchronisation of vesicle fusion.
These experiments pose several technical challenges that are squarely within the expertise of our industrial partner. First, we need to restrict photostimulation to a single presynaptic neuron avoiding spike generation in the neighbouring neurons and their fibres. To address this, the student will develop new software capabilities that will implement precise control of the spatio-temporal stimulation patters in the recently marketed Scientifica Laser Applied Stimulation and Uncaging (LASU) system.
Second, we need to ensure that EPSCs are generated by spiking of a single neuron. Imaging of spikes in neurons co-expressing a genetically encoded Ca2+ indicator (e.g. GCaMP6) potentially provides a powerful control for the specificity of photostimulation. Therefore, along with Syt1 constructs we will co-inject GCaMP6 viral constructs. The challenge here is the overlap between the excitation spectra of ChR2 and GCaMP6. To overcome this we will integrate an EM-CCD camera and an LED-based epifluorescence imaging system with the LASU. The use of EM-CCD will allow us to reduce the intensity of GCaMP6 excitation light below the ChR2 activation threshold.
The use of the above approach should allow parallel stimulation of Syt1 expressing neurons. As a contingency we will also use paired whole-cell patch-clamp recordings.


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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M009513/1 30/09/2015 29/09/2023
1946223 Studentship BB/M009513/1 30/09/2017 30/03/2022 Damandeep Rathore
BB/R50600X/1 30/09/2017 30/12/2021
1946223 Studentship BB/R50600X/1 30/09/2017 30/03/2022 Damandeep Rathore
Description Scientifica's LASU system is able to stimulate neurones with sufficient spatial resolution to characterise brain function.
A second avenue of work has been initiated investigating epileptic events in awake mice. A new platform to study seizures and spreading depolarisation has been created utilising imaging and electrographic techniques.
A powerful novel automated analysis pipeline has been created. This has allowed powerful insight into epileptic events.
Several contributions to work characterising and developing graphene-based neural interfaces.
Exploitation Route Development of novel technology and accompanying techniques that can be applied to specific research questions in the future.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Guiding technology development for Scientifica Ltd. Data supporting the commercialisation of graphene-based neural interfaces.
First Year Of Impact 2022
Sector Electronics,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Description BBSRC LIDo Doctoral Career Development Fellowship
Amount £50,152 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2022 
End 12/2022
Title Analysis pipeline for widefield imaging epilepsy datasets 
Description Automated pipeline to process and extract information from large widefield imaging datasets and characterise epilepsy-related events. 
Type Of Material Data analysis technique 
Year Produced 2022 
Provided To Others? No  
Impact Analysis of epilepsy data in high detail- developments still underway. 
Description Development of analysis pipeline for widefield imaging in epilepsy 
Organisation University of Warwick
Department Department of Computer Science
Country United Kingdom 
Sector Academic/University 
PI Contribution At UCL I have collected complex imaging data. Through this collaboration with University of Warwick, we have built a powerful, novel analysis pipeline to extract epilepsy-related event properties from this data.
Collaborator Contribution Provided expertise in Python-based analysis. Implemented complex analysis tasks and helped guide my development of computational skills to allow in-house analysis with greater depth.
Impact Cross-over between neuroscience and computational science.
Start Year 2019
Description Development of graphene-neural interfaces for imaging in epileptic animals 
Organisation Spanish National Research Council (CSIC)
Department Barcelona Institute of Microelectronics
Country Spain 
Sector Public 
PI Contribution Our collaborators fabricate graphene solution-gated field effect transistor arrays which I use to record epileptiform activity in awake rodents.
Collaborator Contribution They are microelectronics experts with experience in fabricating this technology.
Impact I have contributed to papers characterising this technology and worked on multiple projects surround the development and optimisation of these devices.
Start Year 2019