Molecular chaperones in the regulation of the intermediate filament cytoskeleton

细胞骨架是一个动态的细丝网络，遍布动物细胞的细胞质。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细丝组成，微管和肌动蛋白细丝是单一类型蛋白质(分别为肌动蛋白和微管蛋白)的聚合物，中间细丝由一系列具有细胞类型特异性表达的相关蛋白(如波形蛋白、角蛋白、结蛋白和神经细丝)组成。例如，在哺乳动物中，甲型角蛋白在形成角、蹄、指甲和头发的表皮细胞中表达。了解中间丝是如何组织和调节的，对于我们理解细胞生物学的许多方面非常重要，例如癌症的发生、发展和转移扩散。此外，中间纤维新陈代谢和/或组织的变化与疾病有关。这包括神经退行性疾病，如肌萎缩侧索硬化症、夏科-玛丽-图斯病、巨大轴索神经病和帕金森氏病。最近，我们发现一种名为Sacsin的蛋白质功能丧失会导致波形蛋白中间丝细胞骨架的组织发生戏剧性变化，而其他研究人员则报告了神经丝的变化。糖蛋白的功能尚不清楚，但此前已证明它具有与蛋白质同源的区域，称为分子伴侣。这些蛋白调节其他蛋白质的折叠、降解和复杂的组装/拆解。这导致了这一提议的总体假设，即糖素是正常的中间丝动态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相互独立的研究目标来检验。我们将确定细胞骨架蛋白以及与Sacsin相互作用的细胞骨架蛋白的调节因子。对已知改变波形蛋白网络组织和动力学的机制上的损失的影响也将被测试。伴侣并不单独发挥作用，而是作为伴侣机器的组成部分，因此我们还将确定Sacsin是否与其他伴侣一起影响波形蛋白。最后，我们将研究不同类型的中间细丝的光谱，以确定哪些受到糖损失的影响。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学在内的技术，在我们之前建立的细胞模型中，确定Sacsin的丢失影响Vimentin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了sacsin的细胞，以及来自患有神经退行性疾病常染色体隐性遗传性共济失调(ARSACS)的细胞，在ARSACS中，sacsin发生了突变。这项工作的关键预测结果将是阐明一种调节中间丝组织的新机制。这可能与多种类型的中间细丝有关，因为最初的研究表明，波形蛋白和神经细丝都失去了糖蛋白的作用。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，患者手部灵活性降低，言语困难，行走问题日益严重--以至于他们通常需要轮椅--以及预期寿命缩短。此外，这项研究还可能对神经退行性疾病和其他以中间丝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.