Coordination and regulation of ER-to-Golgi transport by the microtubule cytoskeleton and protein phosphorylation.

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

My research concerns the way in which human cells are organized. The processes that I investigate are fundamental to all human cells and tissues. My work addresses the question of how components are moved between different compartments within the cell and how they are secreted (i.e. exported from of the cell). Many components including hormones and signalling molecules in the brain (cargo) have to be specifically packaged and delivered to the outside of the cell. For an individual part of the cell to function, all of its components parts must be assembled together and delivered to it. The work proposed seeks to address the way in which the formation and transport of these carriers is integrated and how these processes are regulated to ensure their correct function. Much is known about the basic machinery mediating each of these processes individually, but not how they integrate to form an entire functioning cell. My lab uses state-of-the-art imaging techniques to look at these events inside living cells. These experiments will help us understand the basic function of all cells. This is important because defects in these processes cause a wide range of diseases including motor neuron disease and disorders of fat metabolism.

Technical Summary

Research in my lab is focused on the organization and function of the early secretory pathway in mammalian cells. The aim of this work is to analyse the coordination of membrane traffic by the microtubule cytoskeleton and the way in which this is modulated by protein phosphorylation. These processes are fundamental to all human cells. While much is known about the molecular machinery involved, key questions remain unanswered. (i) What are the mechanisms that couple membranes of the early secretory pathway to microtubules; when and how is this link established? (ii) How does protein phosphorylation control the formation of productive transport carriers? (iii) What is the role of the early secretory pathway in establishing the downstream functional architecture of the cell? The work proposed will focus on the interrelationship between three protein machines: the COPII complex which generates the first set of membrane-bound carriers of secretory cargo, the dynactin complex, which couples these membranes to microtubules, and PCTAIRE protein kinases which regulate these and other processes through integrating upstream regulators and downstream effectors. Many distinct functional forms of these machines exist (arising from diversity of isoforms of individual components, inclusion or exclusion of specific accessory subunits and post-translational modification). A key part of this study will seek to define the specificity of individual states of these machines in specific cellular functions. I will address these questions using a combination of live cell imaging (including single molecule detection), biochemical analyses and electron microscopy. Key collaborations with world-leading researchers in the areas of electron microscopy (David Woolley and Ana Pombo) and single molecule imaging (Justin Molloy) enable me to incorporate these cutting edge techniques in to our work. I will also develop this programme towards analysis of neuronal system s in which they play a central role (collaboration with Jeremy Henley). My lab has identified the mechanism underlying functional coupling of COPII-coated membranes to microtubules, as well as a potential mechanism for its regulation by protein kinases. The work described here is directed towards an understanding of these events at a molecular level in complex mammalian systems, and how these related to important aspects of cell polarity, including neurite outgrowth. These experiments are directed towards a full understanding of basic biological processes that impinge on all aspects of cellular function. Failure of component parts of these systems can lead to many diseases including motor neuron disease, disorders of lipid metabolism and blood coagulation defects.

Publications

10 25 50
 
Description BBSRC Initiative Research Grant
Amount £352,316 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2007 
End 06/2010
 
Description BBSRC Research Grant
Amount £353,818 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2007 
End 06/2010
 
Description MRC Research Grant
Amount £478,125 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2009 
End 07/2014
 
Description Royal Society Incoming International Fellowship (India)
Amount £80,422 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2006 
End 01/2008
 
Title Antibodies 
Description Antibodies to COPII proteins 
Type Of Material Antibody 
Year Produced 2006 
Provided To Others? Yes  
Impact Further publications from other groups 
 
Title GFP-fusions 
Description Various intracellular biomarkers for discrete intracellular compartments. 
Type Of Material Biological samples 
Year Produced 2006 
Provided To Others? Yes  
Impact Many labs are now using and publishing with our reagents which we disseminate freely for academic use. 
 
Title LG186 
Description Small molecule inhibitor of GBF1 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Publications in Traffic and Molecular Biosystems. 
 
Title Lentivirus expressing cDNA 
Description Lentivirus constructs to stably express fluorescent markers of COPII activity in mammalian cells. 
Type Of Material Biological samples 
Year Produced 2010 
Provided To Others? Yes  
Impact Publication Hughes and Stephens J Cell Sci 2010. 
 
Title Tracking software 
Description Software to apply to the tracking of objects moving within cells at high resolution. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Two publications arising. 
 
Title LG186 
Description A small molecule inhibitor of GBF1 that acts on human and canine cells. 
IP Reference  
Protection Protection not required
Year Protection Granted 2010
Licensed Yes
Impact Publications.
 
Description Bristol Biochemistry "Schools Week" 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact This event runs annually hosted by my depatment and includes around 400 local school children providing them with lectures and hand on experience of lab science. Over 3 years 2005-2007, this included a talk from me that incorporated aspects of our MRC funded research.

Continued applications from the schools involved to our degree programme.
Year(s) Of Engagement Activity 2006,2007,2008,2009
 
Description Bristol University Open Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Annual, ongoing events tht include presenations and discussions relating to our work in the lab.

No data from this; impossible to derive
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016
 
Description Local school visit "careers in science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Gave talk in local school on careers in science including data from my own research and information about biomedical sciences research careers and the role of the MRC.

None followed up
Year(s) Of Engagement Activity 2006,2014,2015
 
Description Public Lecture, Bath 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public lecture for ~30 people at the Bath Royal Literary and Scientific Institution which included some work from this current project.

Developed further links with an artist currently training at Central St Martin's who attended this talk. I was able to link her up with more local (London-based) groups undertaking similar work.
Year(s) Of Engagement Activity 2011