Molecular chaperones in the regulation of the intermediate filament cytoskeleton

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.

This proposal aims to understand how molecular chaperones regulate organisation of intermediate filament (IF). It originates from our research on ARSACS, a neurodegenerative disease caused by mutations in the protein sacsin, which contains domains linking it to chaperones and protein quality control systems.

Recently, it was reported that neurons lacking sacsin have an abnormal neurofilament network. To investigate if loss of sacsin impacts the organisation of IFs more generally we analysed vimentin localisation in ARSACS patient fibroblasts and sacsin knockout SH-SY5Y cells. This showed that loss of sacsin resulted in abnormal accumulations of vimentin that surrounded the microtubule organising centre. In addition, FRAP analysis showed that vimentin dynamics was altered in sacsin null cells. Importantly, preliminary analyses in sacsin null cells, to identify insoluble proteins by mass spectrometry-based proteomics, predominantly found IF and linked proteins.

Based on these data we hypothesis that sacsin is part of a molecular chaperone system regulating IF protein dynamics and function. To test this hypothesis, we will identify whether sacsin interacts directly with vimentin and/or modulates IF function through another protein-protein interaction. We will also investigate if alteration in vimentin post-translational modifications and trafficking contribute to the IF phenotype in sacsin null cells. Complementary to these experiments we will investigate if sacsin functions with Hsp70 or a wider network of proteostasis proteins. Finally, we will determine if sacsin is required for the normal organisation of other types of IFs and test if mechanisms are the same as for vimentin.

Understanding regulation of IFs is important as they participate in many key physiological functions (e.g. organelle distribution, signal transduction, cell polarity and gene regulation) and are dysregulated in human diseases.

This biomedical science project will have the generation of new knowledge and scientific advancement as its most significant immediate impacts. The research will deliver a comprehensive analysis of a novel chaperone system that regulates the organisation and dynamics of intermediate filaments (IFs). It is also likely to provide insights into the molecular pathogenesis of an inherited ataxia.

Conventional academic publication in high quality open access journals and presentation at conferences will be the main methods of ensuring impact within the research community. Our previous research on the cellular function of sacsin has been disseminated by these means and a key objective of this work will be to publish results in years 2 and 3. Some of the research findings will also be disseminated to non-scientific stakeholders including ARSACS patients and the clinicians treating them.

The potential long-term scientific opportunities are most closely allied with biomedical impacts. Dysregulated IF networks occur in multiple rare diseases where IF proteins are mutated and are also a feature of some more common diseases. Identifying a chaperone system that regulates IF dynamics and links to key regulators of proteostasis (e.g. Hsp70 and the UPS) may ultimately suggest therapeutic approaches for these conditions. Importantly, manipulation of molecular chaperone systems and cellular protein homeostasis networks is recognised as a strategy for intervention in human diseases and in particular neurodegenerations.

More specifically, this research will efine molecular pathogenic mechanisms for ARSACS, which will represent a first step towards the development of therapeutic strategies for this disease. The cytoskeletal network could be manipulated pharmacologically. For example, if we confirm altered post-translational modifications of IFs in sacsin null cells these could be targeted (e.g. if IF phosphorylation is altered in sacsin null cells, kinases and phosphates could represent drug targets for ARSACS).

This work may also be relevant to the development of anti-cancer drugs. Loss of sacsin causes dramatic alterations of the vimentin IF network, and vimentin IFs have been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-to-mesenchymal transition (EMT), and the metastatic spread of cancer. Thus, targeting sacsin may inhibit these processes.

Therefore, beyond academic beneficiaries, this research may ultimately lead to societal and economic impacts related to the development of novel disease treatment. Any commercial potential would be maximised by utilising the QMUL Innovation and Enterprise Unit and MRC commercialisation and development opportunities. Regular meetings with representatives from the QMUL Innovation and Enterprise Unit will be scheduled to identify such opportunities.

This project will also have an impact in the area of training and skills development for the postdoctoral research assistant and technician who will work on the project. Importantly, it will utilise a number of cutting edge technologies including advanced mass spectrometry approaches and super resolution microscopy.