The role of spectraplakins as key integrators of axonal microtubule networks

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.

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.

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.