Structural mechanisms of centriole assembly during cell duplication

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

From embryo to adulthood and every day of our lives we rely on the normal growth and division of millions of cells. For each cell division our genetic material, packed in chromosomes, must also properly divide. In humans and animals this is achieved by an elaborate structure, called the nuclear spindle, organized by copies of the centrosome at each end of the cell. After division, each new cell has only one centrosome; therefore it must duplicate in order for further cell divisions to occur.
Centrosome duplication is a process for which we have relatively little information. We know that any abnormalities in this process can cause diseases or even cancer; however we do not know how this process is regulated, what is the role of each individual centrosomal component or how they assemble together. If we could understand how centrosome duplication takes place and how abnormalities can occur, then we could possibly devise treatments or new drugs. Therefore, there is much interest in understanding this process at the most detailed level possible.
We propose to use a combination of approaches, including cell biology, microscopy and biophysics to answer specific questions about the centrosome duplication process. We are interested in how their elaborate 9-fold symmetric shape is defined, and whether this is achieved by the same method in all organisms or whether there are significant differences. We would like to study how the cells control the start of the duplication process to ensure that it only happens once per cell division. Finally, we aim to find the correct place in the assembly for the various centrosomal components, similar to pieces in a puzzle.
The centrosome is a molecular machine, composed of many large proteins. To be able to study these components in sufficient detail, we first aim to find smaller, functional subunits, which we can analyze with biophysical tools. We can then make predictions for the properties of whole components based on these individual fragments, and test these predictions using electron microscopy and cell biology. Some important aspects of this work require expertise not present in our core group. Therefore we collaborate with internationally recognized groups in the UK and abroad to access the broadest base of expertise possible.

Technical Summary

Centrioles are structures in eukaryotic cells with almost universal 9-fold symmetry. They are large, typically 400 nm by 150 nm in size, and contain symmetric sets of microtubules. Centrioles serve primarily two roles: near the cell membrane they organize flagella and cilia, and in animal cells a pair of centrioles forms the centrosome, the primary microtubule organizing centre. To maintain their numbers centrioles must duplicate once per cell cycle. Defects in this process are linked to medical conditions such as ciliopathies, male sterility or even cancer. As a result, there is widespread interest in understanding how centrioles duplicate, how this process is controlled and what determines their unique architecture.
A relatively small number of proteins, including SAS-6, SAS-5 and SAS-4 in C. elegans and their equivalents in higher organisms, was identified by genetic analysis as critical for the initial steps of the duplication process. The most conserved member of this group, SAS-6, was recently studied by biophysical and cell biology methods, and shown to form oligomers similar to early ultrastructures in centriole duplication. Here, we propose to study how SAS-6 is regulated, how it interacts with other centriolar components, and how these complexes recruit microtubules to the growing centriole.
We plan to use a dissection approach, whereby centriolar components will be initially studied at the level of individual domains. We will use biophysical methods (CD, fluorescence, AUC and ITC) to characterize these domains and their interactions, and crystallography and NMR to obtain high-resolution structures. Information from individual components will be used to create models of higher-order assemblies, which will be studied through EM. The functional significance of complexes observed will then be examined in C. elegans embryos or human cell lines through residue substitutions designed to interfere with the assembly process.

Planned Impact

We expect our activities to have societal and economic impact through three distinct pathways: direct relevance to medical conditions, education and training of people, and outreach into the community.
Centrioles are important for human cells as they organize flagella, cilia, and the mitotic spindle during cell division. Aberrations in centriole structure or numbers can affect human health in a number of ways. Abnormal cilia formation can disrupt cell migration during development, giving rise to genetic diseases collectively known as ciliopathies. This is an emerging class of multi-sympton conditions that can affect liver, kidneys, gut and the respiratory track. In adults, epithelial cells and sperm also use cilia and flagella for function, thus defects in centriole duplication can directly lead to male sterility or ectopic pregnancies. In addition, centrioles, in the guise of the centrosome, organize the mitotic spindle and are thus necessary for correct chromosome segregation across cell divisions.
Our current understanding of the origins of many of these conditions is limited, since our knowledge of centriole structure is incomplete. Our research aims to provide a mechanistic view of how centrioles are put together, what are the structural and functional roles of their components, and how they are regulated. Eventually, our research may yield diagnostics and tools to control centriole duplication and structure. We may achieve this kind of control in a not too-distant timeframe: already our work on the structural role of SAS-6 in centriole assembly suggests a possible avenue of inhibiting centriole duplication by limiting oligomerization of SAS-6.
In addition to long-term considerations, academic basic research provides benefits through education and training. The PDRAs involved in this project will be trained in new techniques that will enhance their skills. Our collaboration with cell biologists and electron microscopists, as well as the multi-disciplinary nature of the project ensures their exposure to new ideas. In addition, they will be mentored towards independence, by giving them the chance of designing as well as implementing research, by allowing self-management and by encouraging their exposure to large audiences through presentations and conferences. Whether they choose to remain in academia or move to industry, this course should allow them to assume leadership positions in the future.
The PI is also keen to recruit postgraduate and undergraduate students to participate in this project, and he and the PDRAs will provide appropriate personal training and mentoring. For students, this project can offer exposure to a wide range of experimental methods and a corresponding increase in skills. At the same time care will be taken that the students develop their ability to frame scientific questions and pursue answers with all tools available, as well as troubleshoot when problems arise. In collaboration with other departments, Oxford Biochemistry offers a number of Ph.D. studentships funded by BBSRC, EPSRC and the Wellcome Trust. Furthermore, the department requires undergraduate students to complete a 6-month research project at their final year. The PI is active in student recruitment from both of these pools.
Finally, we recognize that we have a responsibility to reach out to the community and explain our goals and prospects. We will engage in outreach activities, such as open days and scientific blogs, aimed at making our research accessible to the public, explaining why this research is important, how it can contribute to healthcare, and why they should support basic research.
 
Description Our research grant on studying the structural mechanisms of centriole assembly is now concluded. In the grant's first year we focused on C. elegans, a nematode worm that serves as model animal system in centriole studies. We addressed how C. elegans' centrioles differ from those of higher organisms; this information will prove critical when trying to compare experiments across C. elegans and other model systems. In year two we addressed how centriole elongate as a result of the protein CPAP, mutations of which lead to primary microcephaly. In the final year of the grant we returned to C. elegans centrioles, this time studying a protein seemingly unique to this class of organisms, SAS-5. We combined biophysical and biological information on SAS-5 to propose the first molecular mechanism of function.
Exploitation Route Our findings could inform academic research seeking to compare centriole across organisms. We have demonstrated significant variation in C. elegans, a model system, compared to vertebrates or algae. We have made a number of key predictions on centriolar organization, which could form the underlying hypotheses for further research.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Our work was primarily one of basic research, with major impact on the way we view centrioles from model organisms. However, at the same time the grant provided us with opportunities to engage with the wider public, and to train students and young scientists in cutting-edge methods. In particular, we note the training of two PhD students and three post-graduate researchers that took part in the work proposed.
First Year Of Impact 2013
Sector Education
Impact Types Societal,Economic

 
Description MRC grant of centriolar research (2016-19)
Amount £530,081 (GBP)
Funding ID MR/N009274/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 07/2016 
End 06/2019
 
Title Chemical shifts of the CPAP-interacting epitope of Danio rerio STIL 
Description NMR chemical shifts of the CPAP-interacting epitope of Danio rerio STIL 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact Research publication 
URL http://www.bmrb.wisc.edu/data_library/summary/index.php?bmrbId=19318
 
Title NMR chemical shifts of C. elegans SAS-5 N-terminus 
Description Sequence-specific NMR chemical shift assignments of the C. elegans SAS-5 N-terminal, microtubule-interacting domain 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Facilitated the characterisation of the SAS-5 / microtubule interaction 
URL http://www.bmrb.wisc.edu/data_library/summary/index.php?bmrbId=27056
 
Title Structure of C. elegans SAS-6 D123 variant 
Description Crystallographic structure of an engineered variant of C. elegans SAS-6 N-terminal domain 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact No non-academic impact 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4G79
 
Title Structure of a stabilised C. elegans SAS-6 N-terminal dimer 
Description Crystallographic structure of a stabilised dimer formed by the C. elegans SAS-6 N-terminal domain 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact No non-academic impact 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4GEU
 
Title Structure of a stabilised C. elegans SAS-6 N-terminal dimer, second form 
Description Crystallographic structure of a stabilised C. elegans SAS-6 dimer, second crystal form 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact No non-academic impact 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4GEX
 
Title Structure of the C. elegans SAS-5 Implico dimerization domain 
Description Crystallographic structure of the C. elegans SAS-5 Implico dimerization domain 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact No non-academic impacts 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4YNH
 
Title Structure of the C. elegans SAS-5 coiled coil domain 
Description Crystallographic structure of the C. elegans SAS-5 coiled coil domain 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact No non-academic impacts 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4YV4
 
Title Structure of the coiled-coil dimer of C. elegans SAS-6 
Description Crystallographic structure of a C. elegans SAS-6 dimer mediated by the coiled coil 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact No non-academic impact 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4GFA
 
Title Structure of the coiled-coil dimer of C. elegans SAS-6, second form 
Description Crystallographic structure of a C. elegans SAS-6 dimer mediated by the coiled coil, second form 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact No non-academic impact 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4GFC
 
Title Structure of the zebrafish CPAP G-box 
Description Crystallographic structure of the zebrafish CPAP G-box domain 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact No non-academic impact 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4LZF
 
Title Structure of the zebrafish CPAP G-box in complex with STIL 
Description Crystallographic structure of a complex between the zebrafish proteins CPAP and STIL 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact No non-academic impact 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4LD3
 
Description Oxford - EPFL collaboration 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Country Switzerland 
Sector Public 
PI Contribution We have developed a collaboration with a group at the EPFL, Switzerland, to evaluate structural insights in cellular systems.
Start Year 2010
 
Description Oxford - LMB collaboration 
Organisation University of Cambridge
Department Department of Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of recombinant protein samples from our laboratory to that of Prof. David Glover. Sharing of unpublished information on centriolar protein structure.
Collaborator Contribution Sharing of unpublished information on centriolar protein interactions. Provision of genetic constructs for our laboratory.
Impact No outputs to date. Multidisciplinary collaboration between structural biology and biophysics (our group) and cell biologists (Prof. Glover).
Start Year 2017
 
Description Oxford-PSI collaboration 
Organisation Paul Scherrer Institute
Country Switzerland 
Sector Public 
PI Contribution Collaboration with a group at the Paul Scherrer Institut, Switzerland, for electron microscopy
Start Year 2010
 
Description Oxford Open Days 
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 Public/other audiences
Results and Impact Open Days in Oxford Biochemistry, targeting prospective undergraduate students as well as their parents. The Open Days feature a mixture of face-to-face meetings, research demos, and organised talks.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017
 
Description Research talk to prospective DPhil students 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Research talks to prospective structural biology DPhil students to inform their decisions on which research topic(s) to devote their studies on.

Two students decided to do their PhDs on centriole biology.
Year(s) Of Engagement Activity 2012,2013,2014,2015
 
Description School outreach 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation of research demos to 30-60 primary school children. The children were engaged directly by asking them to participate (i.e. perform) some simple chemistry experiments.
Year(s) Of Engagement Activity 2017,2018
 
Description Talk at Biochemical Society meeting; Repetitive, Non-Globular Proteins: Nature to Nanotechnology (York 30/03/2015-01/04/2015) 
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 initiated many discussions and prompted the publication of our results

No realisable impact
Year(s) Of Engagement Activity 2015
URL https://www.biochemistry.org/Events/tabid/379/View/Programme/MeetingNo/SA168/Default.aspx
 
Description Talk at ETH Zurich 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk on centriolar research at the ETH Zurich, Switzerland
Year(s) Of Engagement Activity 2016
 
Description Talk at UCL - Birbeck 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Talk on centriolar research at UCL / Birbeck, London
Year(s) Of Engagement Activity 2016
 
Description Talk at University of Edinburgh 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Talk on centriolar research at the University of Edinburgh
Year(s) Of Engagement Activity 2016
 
Description Talk at the Australian National University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk on centriolar research at the Australian National University, Canberra
Year(s) Of Engagement Activity 2016
 
Description Talk at the SEE-DRUG 2015 conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited presentation at the 2015 SEE-DRUG conference, Patras, Greece, June 17-20 2015
Year(s) Of Engagement Activity 2015
URL http://www.seedrug.upatras.gr/index.php?option=com_content&view=article&id=104&Itemid=221
 
Description Talk at the Structural Biology 2017 conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited presentation at the Structural Biology 2017 conference in Zurich, Switzerland, Sept. 18-20 2017
Year(s) Of Engagement Activity 2017
URL https://structuralbiology.conferenceseries.com/2017/
 
Description Talk at the University of Wurzburg 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited seminar at the Institute of Physiology of the University of Wurzburg (Germany) on 28/01/2013. Title: "Structural basis of centriolar 9-fold symmetry"

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Talk at the University of Wurzburg 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited presentation at the University of Wurzburg on Dec. 1st 2015
Year(s) Of Engagement Activity 2015
 
Description Undergraduate research presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Recruited two undergraduate students to work on this project

Helped undergraduate students decide what research to pursue as part of their Biochemistry degree.
Year(s) Of Engagement Activity 2012,2013,2014