Molecular chaperones in the regulation of the intermediate filament cytoskeleton

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The cytoskeleton is a dynamic network of filaments that pervades the cytoplasm of animal cells. It acts to regulate cellular shape and internal organisation, while providing the mechanical support that enables cells to divide and move. It consists of microtubules and actin filaments, which are polymers of single types of proteins (actin and tubulin, respectively), as well as intermediate filaments, which are composed of a family of related proteins (e.g. vimentin, keratin, desmin and neurofilament) that have cell type specific expression. For example, in mammals, alpha-keratins are expressed in epitheial cells that make horn, hooves, nails and hair.Knowledge of how intermediate filaments are organised and regulated is important for our understanding of many aspects of cell biology, such as the initiation, progression and metastatic spread of cancers. Moreover, alterations in the metabolism and/or organisation of intermediate filaments is linked to disease. This includes neurodegenerative conditions such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, giant axonal neuropathy and Parkinson's disease.Recently we have identified loss of function of a protein called sacsin leads to dramatic alterations in the organisation of the vimentin intermediate filament cytoskeleton, while other researchers have reported alterations in neurofilaments. The function of sacsin is unknown but it has previously been shown to have regions of homology to proteins known as molecular chaperones. These modulate the folding, degradation and complex assembly/diassembly of other proteins.This has led to the overarching hypothesis of this proposal, that sacsin is part of a molecular chaperone system required for normal intermediate filament dynamics and function. This hypothesis will be tested through four interlinked, yet independent, research objectives. We will identity cytoskeletal proteins and regulators of cytoskeletal proteins that interact with sacsin. The effects of loss of sacsin on mechanisms that are known to alter vimentin network organisation and dynamics will also be tested. Chaperones do not function in isolation, but rather as components of chaperone machines, therefore we will also determine if sacsin functions with other chaperones to influence vimentin. Finally, we will investigate a spectrum of different types of intermediate filaments to determine which ones are effected by loss of sacsin.The proposed research is multi-disciplinary and will exploit our expertise in molecular cell biology and proteomics. It will utilise techniques including live cell imaging and mass spectrometry, to define the mechanism through which loss of sacsin impacts vimentin network organisation in cell models we have previously generated. These include cells where sacsin has been knocked out by a technique called genome editing and cells derived from individuals that have the neurodegenerative disease autosomal recessive ataxia of Charlevoix Saguenay (ARSACS), where sacsin is mutated.The key predicted outcome of this work will be elucidation of a novel mechanism regulating intermediate filament organisation. This is likely to be relevant to multiple types of intermediate filament as initial studies show loss of sacsin effects both vimentin and neurofilament. The work will also help to define the molecular pathogenesis of ARSACS, a childhood onset disease where patients suffer reduced manual dexterity, speech difficulties, increasing problems with walking - such that they normally require a wheelchair - and decreased life expectancy. Moreover, the research may also give insights into neurodegenerative and other diseases where intermediate filament abnormalities are a feature.