The role of spectraplakins as key integrators of axonal microtubule networks
Lead Research Organisation:
University of Manchester
Department Name: Life Sciences
Abstract
The ability of neurons to extend long processes (axons) towards other neurons, muscles or glands, is a key process underlying the formation of the neuronal networks that make up our brain and coordinate our behaviour. For example, failure of axonal growth is often fatal or causes paralysis upon spinal injury. To find cures we have to acquire a thorough understanding of the mechanisms underlying axonal growth. Growing axons follow reproducible paths signposted by chemical cues that direct the growing axon. Axonal elongation is essentially driven by filamentous skeletal elements of cells, called actin and microtubules. Actin and microtubules have to cooperate closely, and their activity must be adaptable to instructions through the guiding chemical cues. Unravelling how these different factors (actin, microtubules and signals) integrate and cross-coordinate each other during axonal growth is the key task that needs to be addressed. To this end, we focus our work on putative integrator molecules, called spectraplakins. They can physically link microtubules to actin and signalling components and, in their absence, axonal growth is severely inhibited. However, how the links of spectraplakins to actin, microtubules, and signalling components help them to perform their growth promoting function remains to be resolved. To facilitate this task, we study the spectraplakin Short stop (Shot) of fruitflies, which represents a most helpful model. Thus, Shot can be studied with powerful and rapid strategies available in fruitflies; the data obtained are relevant and can be translated into biomedical research, since the characteristics and functions of Shot are virtually identical to those of mammalian or human spectraplakins. As the starting point of our project, we have identified three regions/domains of the Shot molecule that can link Shot to microtubules, actin and, most likely, signalling factors. We have shown that all of them are absolutely required for Shot function in axonal growth. Here we will address the detail of their function, using advanced microscopy, structure-function studies, and state-of-the art technology (mass spectrometry) to identify yet unknown binding factors. As an essential further strategy we will make systematic use of combinatorial genetics. We will combine mutations of Shot with mutations in other genes known to contribute to axonal growth. The combined mutant defects will give essential insights into the functional relationships of Shot to other factors, thus mapping Shot function into the systemic context of axonal growth. Our results will provide essential new insights into the function of spectraplakins in health and disease and the regulatory networks underlying axonal growth.
Technical Summary
The growth of axons is a key process in the development and regeneration of neuronal networks. We will study fundamental mechanisms of axonal growth by focussing on an essential molecule in this process - the highly conserved Spectraplakins, a family of large cytoskeletal linker molecules. We recently reported that functional deficiency for the mouse spectraplakin ACF7 and its close Drosophila homologue Shot cause homologous neuronal phenotypes, including disorganised axonal microtubule networks and impairment of axon extension. Here we will decipher the molecular mechanisms underlying neuronal spectraplakin function, capitalising on Shot which can be studied with the power of Drosophila genetics and easily analysed in vivo and our recently established primary neuron culture system. We will focus on three domains of Shot (Gas2-Ctail, calponin homology and plakin domain), which we have shown to be essential for axonal growth, and believe to mediate Shot function through their essential links to the cytoskeleton and other structural or signalling factors. We will use i) refined live microscopy to pinpoint which aspects of MT regulation (polymerisation, bundling) are regulated by Gas-Ctail; ii) a series of approaches modifying the interaction of the calponin homology domain with F-actin to determine how this link influences Shot function, and iii) mass spectrometry to search for interactors of the plakin domain. Most importantly, we will complement these approaches with iv) systematic application of combinatorial genetics, i.e. analyse the combined effects of loss- and/or gain-of-function of Shot with that of other MT or actin regulators in the same cells - with a clear view to a systemic understanding of axonal growth and the role of Shot therein. Given the high degree of structural conservation between Drosophila and mammalian spetraplakins, our results will have wide relevance for the role of these proteins in health and disease.
Planned Impact
Given the importance of axonal growth in neuronal pathologies and regeneration processes, and the enormous range of clinically relevant, yet little understood functions of spectraplakins, our research has great potential to provide insights that ultimately lead to medical advances. The Drosophila primary neuron system developed by us is highly amenable to genetics. Notably, it shows features and phenotypes highly homologous to mammalian systems (Sánchez-Soriano et al., 2010, Dev. Neurobiol. 70, 58ff.), and our data provide clear proof that our findings can be translated into mammalian biology (Sánchez-Soriano et al., 2009, J Cell Sci 122, 2534ff.). Therefore, our studies are consistent with the 3Rs, by reducing the use of vertebrates in biomedical research. Furthermore, we see potential for industrial links in areas of neuronal de/regeneration, where our primary culture models would be ideal systems for cell based assays or small molecule screens, providing efficient means for drug target identification and characterisation. The feasibility of systematic screens on primary Drosophila cultures has been demonstrated (Sepp et al., 2008, PLoS Genet 4, e1000111ff.). To establish closer industrial links, we will use this project as an opportunity to seek industrial contacts with the goal of arranging CASE studentships to pursue these ideas. As bioscience becomes increasingly quantitative and predictive, new approaches and technologies are needed. Our mass spectrometry approach clearly acknowledges this fact. Even more, the consequent use of systematic combinatorial genetics in this project as a novel strategy to address systemic complexity of axonal growth and spectraplakin function, can develop into a promising platform for systems biology approaches. For instance, the ability to systematically analyse the role of a panel of microtubule regulators, singly or in combination, in a single cellular system, provides an ideal source of data with which to develop and then test a mathematical model of actin dynamics. Therefore, this project provides an opportunity to pioneer this approach, and we have had discussions with mathematicians with a view to future collaboration. For the dissemination of the Faculty's research to the national and international media, the Faculty has a dedicated media advisor supporting staff. The angle on nervous system development as a scientific topic is usually of great interest to the public. The research carried out by our labs generates striking images and movies, which lay audiences find fascinating. In particular, our time lapse movies can help to bring complicated biological processes to life. Our research is thus ideal for promoting cutting edge science to the public and we are active contributors to such activities, A.P. has given presentations to pupils, presented in the 'New Scientist' seminar series for lay audiences, and participated in the 'Wellcome to the Matrix' and 'Matrix has talent ' events organised by the Wellcome Trust Centre of Cell-Matrix Research at the University's successful museum (The Manchester Museum), which provides a direct and permanent link between our research activities and the local public. Furthermore, A.P. designed a 'Layman's guide to synapses' for his web page (http://www.prokop.co.uk) which receives around 500 visits per month. Finally, A.P provides A-level students the opportunity to join their laboratories for several days to experience real scientific work at the bench. All these forms of engagement will be continued and expanded during the project period, and a second layman's guide 'Neuronal growth - a layman's guide' will be developed and placed on the web.
Organisations
Publications
Alves-Silva J
(2012)
Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins).
in The Journal of neuroscience : the official journal of the Society for Neuroscience
Beaven R
(2015)
Drosophila CLIP-190 and mammalian CLIP-170 display reduced microtubule plus end association in the nervous system.
in Molecular biology of the cell
Broadie K
(2011)
Extracellular matrix and its receptors in Drosophila neural development.
in Developmental neurobiology
Fostier M
(2015)
A novel electronic assessment strategy to support applied Drosophila genetics training in university courses.
in G3 (Bethesda, Md.)
Hahn I
(2016)
Functional and Genetic Analysis of Spectraplakins in Drosophila.
in Methods in enzymology
Hahn I
(2021)
Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons.
in PLoS genetics
Description | The most significant achievements were numerous outputs of almost 10 publications where the grant is acknowledged, and the involvement in 13 outreach/school events. All objectives were successfully addressed: Work on objective 1 could demonstrate a guidance function of microtubules through binding to the microtubule plus end (a concept that still holds in our models of research), a role in MT stabilisation and co-laboration of with Tau in promoting synapse formation. Work on objective 2 clearly demonstrated the importance of cortical actin interaction of Shot in microtubule guidance, indicated by structure-function analyses (deletion or replacement of actin-binding domain). Work on objective 3 was less successful and also attempts to carry out enhancer-suppressor screens failed. The role of the plakin-like domain in fly remains elusive unfortunately. Work on this grant was crucial to most of the projects that followed and remains a pillar in our models of axon biology, e.g. our newer work on Efa6 recently published in bioRxiv. |
Exploitation Route | Conceptual understanding of MT regulation in axons as an essential prerequisite for unravelling mechanisms underlying axonopathies |
Sectors | Education Pharmaceuticals and Medical Biotechnology |
URL | https://www.research.manchester.ac.uk/portal/en/researchers/andreas-prokop(3e9ef9ff-37e0-4bdb-8c87-c185229b21eb)/publications.html |
Description | Disseminating our initiative at a European conference: Patel, S., Prokop, A. (2021b) Making the impossible possible through objective-driven, long-term initiatives. 18th European Drosophila Neurobiology Conference (Org.: S. Casas Tintó, F. A. Martin), online from Madrid (04-06 May) -- www.neurofly2020.com |
First Year Of Impact | 2021 |
Sector | Education |
Impact Types | Societal |
Description | BBSRC DTP studentship |
Amount | £50,000 (GBP) |
Funding ID | not known - Daniel Howard |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2012 |
End | 09/2014 |
Description | BBSRC DTP studentship |
Amount | £100,000 (GBP) |
Funding ID | not known - Cristina Melero |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2014 |
End | 09/2018 |
Description | Functional Biosystems Imaging award for supra-resolution microscopy |
Amount | £1,000 (GBP) |
Funding ID | 14230031 |
Organisation | Rutherford Appleton Laboratory |
Department | Central Laser Facility |
Sector | Academic/University |
Country | United Kingdom |
Start | 11/2014 |
End | 02/2015 |
Description | STFC Proof-of-principle award |
Amount | £1,000 (GBP) |
Funding ID | 13230046 |
Organisation | Rutherford Appleton Laboratory |
Department | Central Laser Facility |
Sector | Academic/University |
Country | United Kingdom |
Start | 05/2014 |
End | 08/2014 |
Description | Bollington Scibar, Bollington, 14 Oct 2019 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Discussing the importance of Drosophila research for the biomedical sciences at a local bar outreach event |
Year(s) Of Engagement Activity | 2019 |
URL | https://bollingtonscibar.wordpress.com/speakers/ |
Description | Calderón Segura, M. E, González Marín, B, Muraro, N, Galagovsky, D, Olguin, P, Prokop, A, Patel, S. (2020). droso4LatAm - Constuyendo una comunidad para la divulgación de Drosophila melanogaster |
Form Of Engagement Activity | Engagement focused website, blog or social media channel |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Schools |
Results and Impact | A webpage was designed to reach Spanish speaking audiences to raise awareness and disseminate resources to teach biology in schools based on Drosophila experiments |
Year(s) Of Engagement Activity | 2020 |
URL | https://droso4latam.wordpress.com |
Description | Educational YouTube video (part 1) |
Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Small fly, big impact: Why the fly > 6K views |
Year(s) Of Engagement Activity | 2014 |
URL | https://www.youtube.com/watch?v=qDbJnFLl3kU |
Description | Interview on Radio 4's Material World about the use of fruit flies in neuroscience |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Many people reported back to have heard the interview Success in the wider context of our work |
Year(s) Of Engagement Activity | 2010 |
URL | http://www.bbc.co.uk/programmes/b00tjrp4#synopsis |
Description | Lay articles about our Alves-Silva et al. 2012 paper in BBSRC blog, BBSRC Business Magazine and Wellcome Trust Blog |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Helped to spread the concepts of our science Questions by email |
Year(s) Of Engagement Activity | 2012 |
URL | http://blog.wellcome.ac.uk/2012/11/20/feature-the-portrait-of-a-fly-part-1/ |
Description | Organiser and active contributor to a science fair exhibition on the importance of Drosophila in science (9 fairs since 2011) |
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 | Great responses from the public Far more positive views |
Year(s) Of Engagement Activity | 2011,2012,2013 |
URL | http://www.flyfacility.ls.manchester.ac.uk/forthepublic/outreachactivities |
Description | Organiser and active contributor to extracurricular school days (7 school visits) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Increasingly improved resources get seen by pupils to help them with their curriculum More schools engage, ask for help or feedback. The concept gets wider recognition. |
Year(s) Of Engagement Activity | 2012,2013,2014 |
URL | http://www.flyfacility.ls.manchester.ac.uk/forthepublic/teachersandschools |
Description | Patel, S and Prokop, A. (2021) "Re-animating school biology lessons through teaching with living flies", submitted REF2021 impact case |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | The outreach activities since 2011 with respect to developing school resources based on our research activities were turned into an impact case for REF2021; successfully submitted. |
Year(s) Of Engagement Activity | 2021 |
Description | Presenting our concepts on STEM teacher conferences |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Teachers got very engaged with our teaching concepts to convey curriculum-relevant biology specifications using Drosophila Active collaboration with schools |
Year(s) Of Engagement Activity | 2014 |
URL | http://www.flyfacility.ls.manchester.ac.uk/forthepublic/teachersandschools |
Description | Supervising 4 placement students actively working on the droso4schools project |
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 | PIPS postgraduate students or undergraduate placement scheem students work as teaching assistants in our partner schools and generate teaching resources for biology classes, using the fruit fly Drosophila melanogaster as a teaching tool: • Sophie Demaine, University of Manchester - August 2016-June 2017 (UG placement) • Joshua Heafield, University of Mnachester - August 2016-June 2017 (UG placement) • Jennifer Harbottle, University of Aberdeen - January-March 2015 (PG PIPS) • Patrick Stangward, University of Manchester - January-March 2015 (PG PIPS) |
Year(s) Of Engagement Activity | 2015,2016,2017 |
URL | http://www.flyfacility.ls.manchester.ac.uk/forthepublic/teachersandschools/#Placements |