The role of SNAREs in post-Golgi trafficking.

Lead Research Organisation: University of Cambridge
Department Name: Clinical Biochemist

Abstract

The inside of a cell is organized into a series of compartments (organelles) with each organelle having a defined function. Once a protein has been made or modified in one organelle, its function or activity is often required somewhere else. The cell therefore has to transport the protein to the site where it is needed, this is accomplished by packaging the proteins into a small lipid bound packages (vesicles) that brake off from one organelle and fuse with another. The aim of my research is to understand how these vesicles know with which organelles they should fuse. This research will increase our fundamental understanding of how cells function and may give insight into how defects in these processes cause diseases.

Technical Summary

One of the most fundamental unresolved questions in cell biology is how cells manage to preserve the organization and integrity of the endocytic and exocytic systems, while maintaining the transport of lipids and proteins between these specialized organelles. Proteins and lipids are transported between these organelles via small membrane bound transport vesicles, which bud from one compartment and fuse with another, thereby delivering their contents. The targeting and fusion of these vesicles with membranes is regulated in part by the specific interactions of a family of molecules known as SNAREs. In mammalian cells there are at least 38 SNAREs, each one being localized to a different compartment and involved in a subset of transport pathways. The aim of my research is to functionally define the SNAREs involved in Trans Golgi Network (TGN) to endosome and TGN to cell surface transport, by inhibiting SNARE function in combination with quantitative transport assays. To achieve this, I have generated a library of antibodies and siRNAs specific to 14 post-Golgi SNAREs and will use them in combination with flow cytometry based assays for measuring post-TGN trafficking. I also plan to identify the function of the vesicle SNARE VAMP4, by characterizing a VAMP4 knock out mouse that I am currently having generated. The proposed research will provide insight into how specificity is achieved in membrane transport and may also provide a framework for better understanding diseases caused by defects in membrane trafficking to lysosome and lysosome related organelles.

Publications

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Urano Y (2008) Transport of LDL-derived cholesterol from the NPC1 compartment to the ER involves the trans-Golgi network and the SNARE protein complex. in Proceedings of the National Academy of Sciences of the United States of America

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Dacks JB (2009) Evolution of specificity in the eukaryotic endomembrane system. in The international journal of biochemistry & cell biology

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Winslow AR (2010) a-Synuclein impairs macroautophagy: implications for Parkinson's disease. in The Journal of cell biology

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Seaman MN (2010) Ricin toxin hits a retrograde roadblock. in Cell

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Gokhale A (2015) The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Gordon DE (2021) Quantitative Flow Cytometry-Based Assays for Measuring Constitutive Secretion. in Methods in molecular biology (Clifton, N.J.)

 
Description BBSRC project grant
Amount £549,679 (GBP)
Funding ID BB/S009566/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2019 
End 04/2022
 
Description Research Grant
Amount £358,756 (GBP)
Funding ID BB/L022389/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2014 
End 04/2018
 
Description Research Grant
Amount £358,756 (GBP)
Funding ID BB/L022389/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2013 
End 02/2017
 
Title Pharmacologically regulated constitutive secretion cell line 
Description We have developed a cell line that expresses a pharmacologically regulated reporter construct that can be used to measure constitutive secretion. In conjunction with this system we have a developed a high throughput flowcytometry based assay for measuring this process. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact We have used this cell line to identify new genes involved in constitutive secretion. This cell lines has been used in over 10 publications and is being used in over 20 laboratories around the world. 
 
Title SNARE siRNA and antibody library 
Description We have generated an siRNA library to all of the SNAREs encoded in the human genome. In addition we have obtained and generated antibodies to the majority of these proteins. 
Type Of Material Antibody 
Year Produced 2008 
Provided To Others? Yes  
Impact We have used this siRNA library to define the role of SNAREs in constitutive secretion (manuscript in preparation). Collaborators are using this library to define the role of SNAREs in endosome to TGN transport and autophagosome lysosome fusion. 
 
Title V4 genetrap mouse 
Description We have created a V4 genetrap mouse in collaboration with Lexicon Pharmaceuticals. Disruption of V4 causes hypercholesterolemia indicating that SNAREs play a novel role in cholesterol homeostasis. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact The V4 genetrap mouse will be a powerful tool for investigating the role of SNAREs in cholesterol biology and will be a useful model of hypercholesterolemia. Currently, we are preparing a manuscript focused on the characterisation of these mice. 
 
Description Autophagy and secretion 
Organisation University of Cambridge
Department Cambridge Institute for Medical Research (CIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been investigating the role of the secretory pathway in autophagy.
Collaborator Contribution Providing tools and reagents.
Impact We have published a paper in 2010 (20855506).
Start Year 2008
 
Description Charcot-Marie-Tooth disease 
Organisation University of Cambridge
Department Cambridge Institute for Medical Research (CIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution We helped analyse the function of the protein SH3TC2.
Collaborator Contribution They performed the localisation experiments on SH3TC2.
Impact We have published a paper in 2010 (20028792).
Start Year 2009
 
Description Secretion screen 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution We analysed the function of several genes identified in a genome wide dsRNA screen.
Impact We have published a manuscript in 2010 (19942856).
Start Year 2008
 
Title A novel method for isolating transfected cells based on SBP-streptavidin interaction 
Description This patent relates to a new approach which allows cells to be magnetically purified using a cell surface tagged with streptavidin binding peptide and sstreptavidin beads. 
IP Reference WO2015189604 
Protection Patent granted
Year Protection Granted 2014
Licensed No
Impact Currently in discussions with companies to licence this technology.
 
Description Talks for school children 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach International
Primary Audience Schools
Results and Impact I have given several talks for school children in 2010/2011. I have communicated my research to approximately 80 children.

The children seemed interested in biomedical research.
Year(s) Of Engagement Activity 2010,2011