Endocytosis and membrane trafficking

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

The cell is enclosed in a fatty membrane, and contains a number of internal regions themselves enclosed by membranes. Because the membranes separate these different compartments, the cell needs a means of transport of molecules between them: from outside the cell to internal compartments (a process known as endocytosis); from inside to outside (secretion); and between internal compartments. A small region of the flat membrane is bent to form a little ball (vesicle) of membrane, containing the cargo, by coating the outside of the vesicle with a number of proteins which recognise the cargo, the membrane and each other. The cargo molecules transported are either embedded in the membrane, or bound to protein molecules which are themselves embedded in the membrane. Endocytosis is required for the cell to take up nutrients such as iron, and to switch off signals from growth factors which are telling the cell to divide (preventing the uncontrolled cell growth which causes cancer), and for reactivating nerves for repeated firing. It is also a route hijacked by some viruses to enter the cell. We are trying to understand the complex molecular machinery which drives the formation of vesicles, both to understand how it works normally, and what happens when it goes wrong.

Technical Summary

We aim to understand the mechanism by which the cell forms the small protein-coated membrane vesicles which serve to move around the cell cargo molecules in the membrane or attached to membranes. A number of proteins are involved in concentrating membrane receptors into the budding vesicle, shaping the membrane into a spherical bud, and detaching the bud to make a vesicle. Our principal approach to trying to understand the mechanism of membrane vesicle formation is as in the past to express recombinant proteins or domains, to crystallise them, wherever possible as complexes with their target ligands (lipids, peptides or other proteins), solve their crystal structures and then to use the structural insights to design mutants for binding and functional experiments. The process of vesicle formation must select the correct contents of cargo, and also the correct protein components required for the fusion of the vesicle with its target membrane (e.g. SNARE proteins), so molecular machinery must recognise endocytic signals in cargo proteins and also different signals on the SNAREs, and we have made some progress in understanding how these different signals are recognised by different adaptor proteins, to avoid competition between the different requirements. We have also investigated how some of the proteins bend membranes into the tight curve required for vesicle formation, by insertion into the membrane and by providing a curved scaffold. The processes of vesicle trafficking involve a complex network of many proteins, linked to other regulatory processes in the cell, with a good deal of redundancy: understanding the individual interactions provides both insight and valuable tools for disentangling the various pathways.

Publications


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Evans P (2008) An introduction to molecular replacement. in Acta crystallographica. Section D, Biological crystallography
Evans P (2006) Scaling and assessment of data quality. in Acta crystallographica. Section D, Biological crystallography
Evans PR (2007) An introduction to stereochemical restraints. in Acta crystallographica. Section D, Biological crystallography
Evans PR (2011) An introduction to data reduction: space-group determination, scaling and intensity statistics. in Acta crystallographica. Section D, Biological crystallography
Evans PR (2013) How good are my data and what is the resolution? in Acta crystallographica. Section D, Biological crystallography
Evans PR (2013) How good are my data and what is the resolution? in Acta crystallographica. Section D, Biological crystallography
Fridmann-Sirkis Y (2006) Structural analysis of the interaction between the SNARE Tlg1 and Vps51. in Traffic (Copenhagen, Denmark)
 
Description FEBS, Long Term Fellowship
Amount £26,022 (GBP)
Organisation Federation of European Biochemical Societies (FEBS) 
Sector Charity/Non Profit
Country European Union (EU)
Start 07/2009 
End 07/2012
 
Description FEBS, Long Term Fellowship
Amount £26,022 (GBP)
Organisation Federation of European Biochemical Societies (FEBS) 
Sector Charity/Non Profit
Country European Union (EU)
Start 01/2006 
End 01/2008
 
Description LMB Cambridge Scholarship
Amount £20,000 (GBP)
Organisation Medical Research Council (MRC) 
Department MRC Laboratory of Molecular Biology (LMB)
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2006 
End 09/2010
 
Description MRC Studentship
Amount £14,500 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2008 
End 09/2011
 
Description Endocytosis systems 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution research
Collaborator Contribution collaborative research
Impact published papers
 
Description Vesicle trafficking 
Organisation University of Cambridge
Department Cambridge Institute for Medical Research (CIMR)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution collaborative research
Collaborator Contribution collaborative research
Impact published papers
 
Title Programs for processing of crystallographic diffraction data 
Description Programs for processing of crystallographic diffraction data (data reduction) (1) determination of space group (program POINTLESS) (2) scaling and merging of data (AIMLESS). (3) an earlier scaling program SCALA, replaced by AIMLESS. Scala was developed continuously over a period of over 20 years, from 1998 to when it was replaced by AIMLESS around 2011, and was widely used (via the CCP4 project) throughout that time. POINTLESS (from 2006) and AIMLESS (from 2011) are more recent, and both are much used worldwide. Recent (2015-2016) work on this has been the construction of a data reduction pipeline in the new CCP4 interface ccp4i2 
Type Of Technology Software 
Impact These programs form an essential part of the CCP4 suite for X-ray crystallographic macromolecular structure determination, which is used worldwide by most structural biology groups and at most synchrotron light sources. CCP4 software is released free of charge to any academic group. I have been involved with the CCP4 project since its inception in 1979, chairing its "working group 2" from 1996 to 2013, and being a member of its executive committee since 1996. 
URL http://www.ccp4.ac.uk