Molecular chaperones in the regulation of the intermediate filament cytoskeleton

细胞骨架是遍布动物细胞细胞质的动态纤维网络。它的作用是调节细胞形状和内部组织，同时提供使细胞分裂和移动的机械支持。它由微管和肌动蛋白丝组成，肌动蛋白丝是单一类型蛋白质（分别是肌动蛋白和微管蛋白）的聚合物，以及由具有细胞类型特异性表达的相关蛋白质家族（如vimentin，角蛋白，desmin和神经丝）组成的中间丝。例如，在哺乳动物中，α -角蛋白在形成角、蹄、指甲和毛发的上皮细胞中表达。了解中间丝是如何组织和调节的，对我们理解细胞生物学的许多方面都很重要，比如癌症的发生、发展和转移扩散。此外，代谢和/或中间纤维组织的改变与疾病有关。这包括神经退行性疾病，如肌萎缩性侧索硬化症、沙克-玛丽-图斯病、巨大轴索神经病和帕金森病。最近，我们发现一种叫做sacsin的蛋白质的功能丧失导致了波形蛋白中间丝细胞骨架的组织发生了巨大的变化，而其他研究人员也报道了神经丝的变化。sacsin的功能是未知的，但它以前被证明具有同源区域的蛋白质称为分子伴侣。它们调节其他蛋白质的折叠、降解和复杂的组装/拆卸。这导致了该提案的总体假设，即sacsin是正常中间纤维动力学和功能所需的分子伴侣系统的一部分。这一假设将通过四个相互关联但又独立的研究目标进行检验。我们将鉴定细胞骨架蛋白和与囊蛋白相互作用的细胞骨架蛋白的调节因子。sacsin的损失对机制的影响，已知的改变静脉蛋白网络组织和动力学也将被测试。伴侣蛋白不是单独起作用的，而是作为伴侣蛋白机器的组成部分，因此我们还将确定sacsin是否与其他伴侣蛋白一起作用以影响蛋白。最后，我们将研究不同类型的中间细丝的光谱，以确定哪些受到囊蛋白损失的影响。拟议的研究是多学科的，将利用我们在分子细胞生物学和蛋白质组学方面的专业知识。它将利用包括活细胞成像和质谱在内的技术，来定义我们之前生成的细胞模型中，囊蛋白损失影响静脉蛋白网络组织的机制。这些细胞包括通过一种称为基因组编辑的技术敲除沙棘蛋白的细胞，以及来自患有沙棘常染色体隐性共济失调（ARSACS）神经退行性疾病的个体的细胞，其中沙棘蛋白发生突变。这项工作的关键预测结果将是阐明一种调节中间细丝组织的新机制。这可能与多种类型的中间丝有关，因为初步研究表明，囊蛋白的损失对静脉蛋白和神经丝都有影响。这项工作还将有助于确定ARSACS的分子发病机制。ARSACS是一种儿童期发病的疾病，患者的手灵活性降低、语言困难、行走困难增加（通常需要轮椅）以及预期寿命缩短。此外，该研究还可能为神经退行性疾病和其他以中间纤维异常为特征的疾病提供见解。

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.