Core - Structure and Assembly Mechanisms of Centrioles

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

Centrioles are large cylindrical cell organelles. They are strictly required for the formation of cilia and flagella, hair-like projections of cells that act as cellular antennae and also act as movement devices. Due to their essential role, it is not surprising that centrioles and their dysfunctions are linked with many human diseases like microcephaly, ciliopathies and cancer.

A better understanding of centrioles is therefore important but will require a detailed understanding of their exact molecular architecture. Many centriole components have been identified in the past. However, there is a very limited understanding about how these components act and come together to make functional centrioles. My lab aims to shed light on this question using a combination of cell biological, biochemical, X-ray crystallographic and electron microscopic methods.

A first step towards this goal was achieved through our characterisation of a key centriolar protein. We showed how this protein organises a critical structure found in the early stages of centriole formation and how this structure helps making functional centrioles. We currently expand our approach to other crucial centriolar components. As an outcome we hope that our work will make us better understand the function of centrioles in health and in disease.

Technical Summary

Centrioles are barrel shaped, 9-fold symmetric cell organelles that constitute the base of cilia and flagella and are essential for their formation. Centrioles also form the core of centrosomes that they help to structure. Due to their important cellular roles, centrioles and their dysfunctions are associated with numerous human diseases ranging from microcephaly and ciliopathies to cancer and infertility.
In the past, successful efforts have been made to identify the key proteins involved in the assembly and function of centrioles. However, what is so far largely lacking is a detailed understanding of how these components are structurally organized within the centriole, what their exact assembly mechanisms are and how their assembly leads to a faithful formation of functional centrioles.

My lab aims to shed light on these questions by combining biochemical and biophysical approaches with the structural characterisation of centriolar components using X-ray crystallography and electron microscopy. Specifically, we try to make key centriolar components or fragments of these recombinantly using different expression systems. The purified components are characterised biochemically and biophysically to obtain information on their ability to self-associate and associate with each other. The components or their complexes are also subjected to protein crystallographic methods to derive high-resolution structures or, if diffraction grade crystals cannot be obtained, are studied by electron microscopy. The combined information from these approaches is then used to derive detailed models of the self-association and mutual interactions of the centriolar components. Subsequently, the significance of these interactions for the formation of centrioles is tested using targeted mutations of these components in vivo and the effect of these mutations on the architecture and function of centrioles is assessed using light microscopy and electron microscopy. The nature of the resulting assembly defects and specifically the resulting presence or absence of other centriolar components will then reveal the hierarchy of interactions that make up centrioles.

The power of this approach is highlighted by our previous characterization of the conserved centriolar protein SAS-6. We could show that SAS-6 self-associates to organise a key assembly intermediate in centriole formation, elucidate its assembly mechanism and show how it contributes to the establishment of centrioles of the right symmetry in vivo. A detailed study of other key centriolar components can therefore be expected to yield information about how this assembly intermediate is extended and how the downstream assembly steps of centrioles are achieved on a molecular level.
 
Description Cambridge Cancer Center Scholarship
Amount £57,000 (GBP)
Organisation Cambridge Cancer Centre 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 09/2017
 
Title Atomic coordinates of a centriolar complex mutated in microcephaly 
Description We deposited the high resolution structure of a centriolar protein complex that is mutated in a human neurodevelopmental disease (microcephaly). Our structure explains how this complex is mechanistically disturbed in some human patients. Furthermore, we also deposited the high resolution structure of one of this proteins carrying a mutation the equivalent of which is found in human microcephaly patients. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2013 
Provided To Others? Yes  
Impact Other groups can use these coordinates to try to find ways to stabilise complex formation that we showed to be disturbed in some human microcephaly patients. 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4bxr
 
Description Collaboration with the CRUK Cambridge Institute, UK 
Organisation Cancer Research UK Cambridge Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We solved the high-resolution structures of a centriolar component that is mutated in several ciliopathies (developmental diseases in humans). Our structures of the corresponding mutants and further biophysical experiments established the likely disease mechanism.
Collaborator Contribution My collaboration partners extended our structural and biophysical work by characterising one of the mutants in tissue culture cells, which allowed us to further understand how the corresponding disease is caused.
Impact This collaboration is multi-disciplinary. We focus on the structural biology and biophysics, while our collaboration partners cover the cell biology of the underlying processes. We will publish a paper on our findings this year.
Start Year 2016
 
Description Collaboration with the Dunn School of Pathology, Oxford 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution I set-up this collaboration to test our structural models of a centriolar protein complex that is disturbed in microcephaly patients (a human neurodevelopmental disease). We had shown how this complex works on a structural level, but needed to confirm the relevance of our findings in an in vivo model.
Collaborator Contribution My collaborators tested point mutations that I suggested in the fruit fly Drosophila melanogaster. The corresponding mutants showed defects in the formation of centrioles as our structural models predicted. My collaborators also confirmed our structural models with the fruit fly proteins, based on my construct suggestions and previous work.
Impact Our work has been published in 2013 with me as the senior and corresponding author. This collaboration is multidisciplinary and spans from structural biology, biophysics and biochemistry (mainly our part) to cell biology and genetics (the part worked on by our collaborators).
Start Year 2011
 
Description Collaboration with the Ludwig Institute for Cancer Research 
Organisation Ludwig Institute for Cancer Research
Department San Diego Branch
Country United States 
Sector Charity/Non Profit 
PI Contribution I set-up this collaboration to test our structural models of a centriolar protein complex that is disturbed in microcephaly patients (a human neurodevelopmental disease). We had shown how this complex works on a structural level, but needed to confirm the relevance of our findings in an in vivo model. In a parallel collaboration we used the fruit fly, as the proteins of this complex are well conserved compared to humans. In order to check whether the complex also exists in organisms where these proteins are much less well conserved we also wanted to test it in the nematode worm C.elegans.
Collaborator Contribution My collaborators tested partial deletions of the relevant C.elegans proteins in vivo and also confirmed that the two proteins bind each other in vitro. This work confirms that the complex still exists in C.elegans and has a similar role, but that the overall binding mode may be different as suspected from the poor sequence conservation.
Impact Our work has been published in 2013 with me as the senior and corresponding author. This collaboration is multidisciplinary and spans from structural biology, biophysics and biochemistry (mainly our part) to biochemistry, cell biology and genetics (the part worked on by our collaborators).
Start Year 2011
 
Description Collaboration with the Lunenfeld-Tanenbaum Research Institute, Toronto, Canada 
Organisation Mount Sinai Hospital (Canada)
Department Samuel Lunenfeld Research Institute
Country Canada 
Sector Hospitals 
PI Contribution We have identified a putative novel protein complex involved in centriole duplication and solved its high-resolution structure. Furthermore, we characterised the binding of the corresponding components to each other in-vitro and an in-vivo.
Collaborator Contribution My collaboration partner tests our structural model in vivo using point-mutations suggested by us. The goal is to establish the in-vivo effects of disrupting the complex that we identified.
Impact This is a multi-disciplinary collaboration - we do mainly biochemical and structural work, my collaboration partner does cell-biological work. The work done by our collaboration partner has so far suggested that the novel protein complex identified by us is relevant for centriole duplication in-vivo and we are currently trying to pin down the exact effector mechanism.
Start Year 2015
 
Description Development of a science inspired board/card game by my post-doc in his free time 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact My post-doc Caezar al Jassar developed a science inspired board/card game (Lab Wars) in his free time and funded its production through a kick-starter campaign. This game was covered by several science related journals.
Year(s) Of Engagement Activity 2015,2016
URL https://www.lab-wars.com
 
Description MRC LMB open day activity support 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Open day at the LMB to inform the general public about the research done in our institute. Plenty of activities centered around the science done here. More than 2000 visitors came and the LMB received wonderful feedback about the level of education and insight that was provided by this event. My PhD student was involved as an internal volunteer to support scientific activities/demonstrations during the day.
Year(s) Of Engagement Activity 2017
 
Description Seminar for Non Scientific Staff 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact The LMB Seminars for Non-Scientific Staff are friendly science talks given by LMB Group Leaders uniquely for the non-scientific support staff, where the research in each laboratory is explained in accessible language (i.e. directed to a lay audience).
Year(s) Of Engagement Activity 2016
URL http://www2.mrc-lmb.cam.ac.uk/internal/index.php/key-information/public-engagement/lay-talks-for-sup...