Small G proteins and the organisation of the Golgi apparatus

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

Our bodies are made up of billions of tiny cells that stick together to make our organs. To ensure that each cell maintains its individual identity, it is wrapped in an outer sheet or 'membrane' that stops its contents mixing with the rest of the body. However, the cells in our bodies also need to communicate. As a result cells have two special pathways to get things across the membrane that seals them. One pathway is an assembly line for making substances like insulin or antibodies and placing them in special containers for release outside the cell. The other pathway acts in reverse to bring into the cell nutrients such as iron and fats that come from our diet via the blood. We are studying the 'Golgi apparatus' which is the central sorting station for these pathways, with the aim of understanding how it works, and how its component parts are able to associate with the Golgi rather than other parts of the cell.
In some genetic diseases these uptake and release pathways do not function correctly. In addition, many bacteria and viruses get inside our cells by tricking the processes that are normally used to take up nutrients. Studying these pathways will help us to understand what occurs in our cells during diseases, and hence could suggest new treatments. Finally, some new types of therapeutics need get inside cells to reach their targets and understanding the normal processes of uptake may reveal better methods to direct these drugs to their targets.

Technical Summary

All eukaryotic cells have a set of internal compartments that act together to synthesise lipids and membrane proteins, to release secreted proteins, to take up nutrients, and to degrade membrane-bound and internalised molecules. Proteins move between these compartments by means of transport vesicles that bud from, and then fuse with, specific organelles. The accurate organisation of membrane traffic requires that these vesicles find and fuse with only the correct destination organelle. Vesicles are initially 'tethered' to the correct membrane, and then fusion is mediated by a set of fusogenic proteins called SNAREs. However, the function and even identity of many of the components involved in tethering remain unclear, as do the mechanisms by which they are accurately recruited to the correct vesicles and organelles.
We address these questions in the Golgi apparatus, an organelle that has a central role in the sorting and modification of proteins in the secretory pathway. The Golgi apparatus contains a number of long coiled-coil proteins called ‘golgins’ that have been suggested to act in vesicle tethering, and our recent work has show that this is correct for at least some. We are applying biochemical and genetic methods to identify interacting partners for these golgins, as well as using fluorescent microscopy in living cells to follow their dynamics. Most of these golgins are peripheral membrane proteins that are recruited to the Golgi directly from the cytoplasm. As with such proteins on other organelles, this recruitment is usually mediated via recognition of the active form of specific GTP-binding proteins. These ‘G proteins’ thus contribute to organelle identity, and the spatial accuracy of their activation underlies the organisation of membrane traffic. Therefore we are examining how the relevant G proteins are activated only on Golgi membranes, and what proteins they are recognising. One long term aim is to understand the fundamental principles by which the cell establishes a network of distinct but interconnected organelles.
For much of our work we use either mammalian tissue culture cells or the fruit fly Drosophila melanogaster to investigate the function of the proteins we identify in the different tissues of a multicellular organism.

Publications

10 25 50

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Boyle LH (2006) Selective export of HLA-F by its cytoplasmic tail. in Journal of immunology (Baltimore, Md. : 1950)

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Bretscher MS (2014) Asymmetry of single cells and where that leads. in Annual review of biochemistry

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Burkhardt P (2011) Primordial neurosecretory apparatus identified in the choanoflagellate Monosiga brevicollis. in Proceedings of the National Academy of Sciences of the United States of America

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Gillingham AK (2018) At the ends of their tethers! How coiled-coil proteins capture vesicles at the Golgi. in Biochemical Society transactions

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Gillingham AK (2014) Toward a comprehensive map of the effectors of rab GTPases. in Developmental cell

 
Description DFG Postdoctoral Fellowship
Amount £54,000 (GBP)
Organisation German Research Foundation 
Sector Public
Country Germany
Start 01/2010 
End 04/2012
 
Description DFG Postdoctoral Fellowship
Amount £43,845 (GBP)
Organisation German Research Foundation 
Sector Public
Country Germany
Start 11/2011 
End 10/2013
 
Description EMBO Long Term Fellowship
Amount £62,031 (GBP)
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 02/2014 
End 01/2016
 
Description EMBO Long Term Fellowship
Amount £54,943 (GBP)
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 01/2012 
End 01/2014
 
Description FCT, Phd Studentship
Amount £47,000 (GBP)
Organisation Government of the Portugese Republic 
Department Foundation of Science and Technology (FCT)
Sector Public
Country Portugal
Start 01/2007 
End 12/2010
 
Description MRC Centenary Early Career Award
Amount £38,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 11/2012 
End 04/2013
 
Description Wellcome Trust Strategic Award
Amount £419,587 (GBP)
Funding ID 095927/B/11/A 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2014 
End 10/2016
 
Title Antibodies to Drosophila organelles 
Description A panel antibodies that recognise the major organelles of the Drosophila secretory and endocytic pathways. 
Type Of Material Antibody 
Year Produced 2016 
Provided To Others? Yes  
Impact The antibodies were provided to the Developmental Studies Hybridoma Bank who have distributed them to a number of researchers. This has lead to the paper that describes them being cited in publications. 
URL http://bio.biologists.org/content/5/7/987
 
Title Arl8 antibodies 
Description Antibodies to Drosophila Arl8 
Type Of Material Antibody 
Year Produced 2007 
Provided To Others? Yes  
Impact It has appeared in several publications from other laboratories 
 
Description Analysis of Plant TMDs 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution I helped in the analysis of transmembrane domains (TMDs) of proteins from different organelles from plants, and helped to interpret the findings.
Collaborator Contribution The partners did most of the work in that they isolated plant organelles and identified membrane proteins in specific organelles.
Impact It resulted in a publication (PubMed ID 22923678). The work is multidisciplinary as it combined mass spectrometry, cell biology and computational biology.
Start Year 2011
 
Description Analysis of faecal outputs in Drosophila 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided data and some improvements to the method of analysis
Collaborator Contribution They devised the software tool for analysing Drosophila faecal output and wrote the paper.
Impact It resulted in two post-docs from my group (Joao Rocha and Satish Jayaram) appearing on a paper from our collaborator (PubMed ID 2490767). I declined the opportunity to add my name to the paper as it was entirely their work.
Start Year 2013
 
Description Cargo receptors for ER exit 
Organisation Weizmann Institute of Science
Department Department of Molecular Genetics
Country Israel 
Sector Academic/University 
PI Contribution We analysed the role of transmembrane domain length in exit from the ER and Golgi, and showed that the protein Erv14 appears to accelerate the exit of proteins with long transmembrane domains.
Collaborator Contribution Our collaborators screened known yeast membrane proteins in mutants of putative ER exit receptors and identifed the set of proteins that depend on Erv14 for ER exit.
Impact A publication in a high impact internationally peer reviewed journal (Herzig et al (2012); PubMed ID 22629230). A second paper was published after the collabration ended (PubMed ID 29527758).
Start Year 2011
 
Description Computational analysis of TMDs 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team performed most of the work on this collaboration, but we recieved expert technical advice on computer programming and TMD analysis from our collaborator in the University of Cambridge
Collaborator Contribution Provided vital technical input to a project which resulted in two publications
Impact It has produced a publications (PubMed ID 20603021) and an accompanying website (www.tmdsoline.com). It is a multidisciplinary combination of cell biology and computational biology.
Start Year 2007
 
Description Evolution of Rab G proteins 
Organisation University of Lausanne
Department Department of Cell Biology and Morphology
Country Switzerland 
Sector Academic/University 
PI Contribution We performed an analysis of Rab protein sequence from eukaryotes and used this to build evolutionary trees to reveal how the family emerged and diversified to generate the large set of 66 Rabs present in humans.
Collaborator Contribution Our partners helped with building a website and database management, and provided comments on the manuscript.
Impact Two publications international peer reviewed journals (Burkhardt et al 2011 (PubMed ID 21876177) and Kloepper et al 2012 (PubMed ID 22873208).
Start Year 2010
 
Description Genome-wide RNAi screen 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Public 
PI Contribution We analysed the data from an RNAi screen performed by our collaborators, and examined the role in secretion of the novel hits.
Collaborator Contribution They performed a genome-wide RNAi screen for proteins involved in secretion.
Impact The collaboration resulted in a publication - PubMed ID 19942856
Start Year 2006
 
Description Golgi coiled-coil proteins 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution We did most of the research, but they provided electron microscopy support
Collaborator Contribution They provided expert electron microscopy support for the project
Impact It was included in a publication PubMed Id 19001129
Start Year 2007
 
Description Golgi outposts in neuronal dendrites 
Organisation Penn State University
Country United States 
Sector Academic/University 
PI Contribution I provided plasmids and advice on how to perturb the Golgi in neurons
Collaborator Contribution The partners examined Golgi outputs in Drosophila neurons and determined the effect of perturbing them. They wrote the resulting publication.
Impact The publication resulted in a publication (PubMed ID 24807906)
Start Year 2010
 
Description HLA traffic 
Organisation University of Cambridge
Department Department of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution We helped with cell biological aspects of a project to study the trafficking of the immunological system regulator HLA-F
Collaborator Contribution Provided substantial research effort on project
Impact A publication Boyle et al (2006) PMID 16709803.
 
Description Investigation of conserved proteins of unknown function 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have analysed the function in Drosophila of selected highly conserved proteins of unknown function.
Collaborator Contribution Our partners have provided input to experimental design technical advice on working with Drosophila.
Impact No outcomes yet but we expect to publish a paper this year.
Start Year 2014
 
Description Map of Effectors of Rab GTPases 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution We did most of the work on a project to map comprehensively the effectors of Rab GTPases by protein purification, mass-spectrometric identification and experimental validation
Collaborator Contribution Our partners provided one half of the mass-spectrometric data
Impact It resulted in a publication in a high impact international peer-reviewed journal (Gillingham at al, 2014). The work is multidisciplinary as it combined mass-spectrometry, cell biology and protein biochemistry. Amongst the Rab interacting proteins identified were a number that are linked to human disease.
Start Year 2006
 
Description Role of the GTPase Arl4 
Organisation University of Liverpool
Department School of Life Sciences Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution We did most of the research, but they performed a yeast two-hybrid screen for us
Collaborator Contribution They performed a yeast two-hybrid screen for us
Impact It resulted in a publication PubMed ID 17398095
 
Description Strategic Award on Nanoscopy 
Organisation University of Cambridge
Department Gurdon Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We developed reagents to use super-resolution microscopy to study the Golgi apparatus in live cells.
Collaborator Contribution Yale and Oxford developed super-resolution microscopes and the Gurdon Institute developed systems for following cargo in polarized cells. Then we started to work together to understand how cargo moves through the Golgi in polarized cells and then gets sorted to different domains of the cell surface.
Impact We successfully applied for a Wellcome Trust Strategic Award to fund this research. This funding contributed to two publications, PMID 27256406 and 29273580. Following the end of the funding period, we have continued to collaborate with the St Johnston lab at the Gurdon Institute in Cambridge.
Start Year 2012
 
Description Strategic Award on Nanoscopy 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We developed reagents to use super-resolution microscopy to study the Golgi apparatus in live cells.
Collaborator Contribution Yale and Oxford developed super-resolution microscopes and the Gurdon Institute developed systems for following cargo in polarized cells. Then we started to work together to understand how cargo moves through the Golgi in polarized cells and then gets sorted to different domains of the cell surface.
Impact We successfully applied for a Wellcome Trust Strategic Award to fund this research. This funding contributed to two publications, PMID 27256406 and 29273580. Following the end of the funding period, we have continued to collaborate with the St Johnston lab at the Gurdon Institute in Cambridge.
Start Year 2012
 
Description Strategic Award on Nanoscopy 
Organisation Yale University
Department School of Medicine
Country United States 
Sector Academic/University 
PI Contribution We developed reagents to use super-resolution microscopy to study the Golgi apparatus in live cells.
Collaborator Contribution Yale and Oxford developed super-resolution microscopes and the Gurdon Institute developed systems for following cargo in polarized cells. Then we started to work together to understand how cargo moves through the Golgi in polarized cells and then gets sorted to different domains of the cell surface.
Impact We successfully applied for a Wellcome Trust Strategic Award to fund this research. This funding contributed to two publications, PMID 27256406 and 29273580. Following the end of the funding period, we have continued to collaborate with the St Johnston lab at the Gurdon Institute in Cambridge.
Start Year 2012
 
Description Super-resolution microscopy 
Organisation University of Cambridge
Department Gurdon Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided ideas and reagents for studies of membrane traffic through the Golgi in Drosophila cells by using super-resolution imaging.
Collaborator Contribution Daniel St Johnston at the Gurdon has provided expertise and development work on membrane traffic in Drosophila. James Rothman and Joerg Bewersdorf have provided expertise and equipment development for super-resolution imaging.
Impact The collaboration has received a Wellcome Trust Strategic Award for 2.5 years, and if this is renewed for a further 2.5 years my lab will receive some funding.
Start Year 2011
 
Description Super-resolution microscopy 
Organisation Yale University
Department Department of Cell Biology
Country United States 
Sector Academic/University 
PI Contribution Provided ideas and reagents for studies of membrane traffic through the Golgi in Drosophila cells by using super-resolution imaging.
Collaborator Contribution Daniel St Johnston at the Gurdon has provided expertise and development work on membrane traffic in Drosophila. James Rothman and Joerg Bewersdorf have provided expertise and equipment development for super-resolution imaging.
Impact The collaboration has received a Wellcome Trust Strategic Award for 2.5 years, and if this is renewed for a further 2.5 years my lab will receive some funding.
Start Year 2011
 
Description Yeast Grh1 and the Golgi apparatus 
Organisation Dartmouth College
Department Department of Biochemistry & Cell Biology
Country United States 
Sector Academic/University 
PI Contribution We performed most of the work, but they did some in vitro transport assays for us
Collaborator Contribution Technical support in form of ER to Golgi transport assays
Impact It produced a publication PubMed Id 17261844
Start Year 2006
 
Description Artists in Residence lunch time conversation 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Artists in residence lunch time conversation including the artists, a guest writer, a choreographer and several PhD students and technicians.
Year(s) Of Engagement Activity 2016
 
Description Durham University Seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talks stimulated questions and discussion

None to date
Year(s) Of Engagement Activity 2014
 
Description EMBO Workshop on Protein and lipid function in secretion and endocytosis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Talk sparked questions and much discussion

Discussed possible collaborations
Year(s) Of Engagement Activity 2014
URL http://events.embo.org/14-secretion-endocytosis/
 
Description Gordon Research Conference 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact 180 scientists attended the talk which led to suggestions for future work and discussion of possible collaborations.

Nothing particularly notable
Year(s) Of Engagement Activity 2013
URL http://www.grc.org/programs.aspx?year=2013&program=molmemb
 
Description Graham Warren Symposium 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact 100 academic scientists attended my scientific presentation and asked questions at the end of the talk and in discussions afterwards

no notable impacts
Year(s) Of Engagement Activity 2013
 
Description LMB Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Members of my laboratory participated in LMB Open Day by giving lab tours and presenting aspects of the research being done by the Division of Cell Biology. Many questions from members of the public about the research and about life as a scientist.
Year(s) Of Engagement Activity 2017
 
Description Lecture at EMBO Workshop on "Emerging concepts in cell organization" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An international scientific workshop on organelle cell biology attended by c 120 research scientists, post-docs and PhD students. A lot of discussion, exchange of ideas and discussion of possible future collaborations.
Year(s) Of Engagement Activity 2017
 
Description Membranes and Modules Meeting, Berlin, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk stimulated questions and discussion afterwards

None to date
Year(s) Of Engagement Activity 2014
 
Description Microscopes for Schools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact Isabel Torres in my lab organised visits to primary schools to demonstrate the use of affordable microscopes for learning biology. Members of my lab, and that of Simon Bullock also assisted. Simon and Isabel set up a website with information for schools (http://www2.mrc-lmb.cam.ac.uk/microscopes4schools/), and organised a stand at Cambridge University's Science Week 2011.

Positive feedback from school pupils about the visits. Enthusiasm amongst LMB post-docs and students to continue organising the visits after Isabel left my lab.
Year(s) Of Engagement Activity 2010,2011
 
Description Seminar Zentrum für Molekulare Neurobiologie (ZMNH), Hamburg 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Postgraduate students asked many questions about research topic and careers in science.

One postgraduate student applied to work in my research group
Year(s) Of Engagement Activity 2013
 
Description Seminars for non-scientific MRC staff 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact A series of bimonthly internal scientific seminars for non-scientific staff working at the MRC-LMB. These are presented by group leaders and are aimed at making their work understandable to the non-scientific support staff.
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Seminars for non-scientific MRC staff 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A series of bimonthly internal scientific seminars for non-scientific staff working at the MRC-LMB. These are presented by group leaders and are aimed at making their work understandable to the non-scientific support staff.
Year(s) Of Engagement Activity 2017,2018,2019
 
Description Warick University Seminar 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact 80 undergraduates, PhDs and more senior scientists attended my seminar, and asked questions afterwards and during informal discussions. I provided advice to a group collaborating with GSK on traffic of membrane proteins.

My talk was mention on Twitter by an audience member.
Year(s) Of Engagement Activity 2013