Cutting edge novel technologies to investigate lysosomal channels and transporters important in recovery post-autophagy

研究溶酶体通道和转运蛋白的尖端新技术对自噬后的恢复很重要

• 批准号: 2750007
• 金额: --
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2750007
• 关键词: Cutting; edge; novel; technologies; investigate

Autophagy is a fundamental function of cells that governs the response to starvation, infection and protein mis-processing. It is central to cellular and organism health, directly impacts upon healthy ageing and is demonstrably impaired in cancers, metabolic conditions and neurodegenerative diseases. The process of clearing damaged mitochondria or toxic protein aggregates within the cell is called macroautophagy. Here the cell engulfs the target within a double membrane that originates from the endoplasmic reticulum and transports it to the lysosome where it fuses in a calcium (Ca2+) dependent manner; the cargo is destroyed by proteases contained within the acidic lysosomal lumen. This is where our current knowledge of autophagy ends. However, it is clear that the lysosome must somehow recover from this process, as the macroautophagy delivers a cargo of cytoplasm that would de-acidify and depolarise the lysosome. This would shut down the degradative and recycling capacity of the lysosome and impair one of the fundamental roles of autophagy, to allow the cell to survive during periods of starvation. Clearly, in a healthy cell, the lysosome does recover, the question we are asking with this project is how does it do this?It is remarkable that this process has never been studied, in part, we believe, owing to the difficulty in isolating and studying lysosomal ion channel and transporter function. The process of maintaining membrane potential depends upon ion transport, as occurs during neuronal action potentials. The lysosomal membrane functions in the same way, but is under-studied and has never been followed during an event such as autophagy and post-autophagy recovery. We have developed cutting edge techniques to magnetically purify lysosomes and utilise them across a host of automated electrophysiology/patch clamping instruments that, in the UK, exists only in Cardiff. With this project we will measure how lysosomal ion fluxes are altered during and post-autophagy in magnetically purified lysosomes from cells where chemical treatments are used to induce or arrest autophagy. We will determine how these ion fluxes alter lysosomal membrane potential, and how this impacts on lysosomal solute transporter function. Together, these data will provide the first evidence not only of how the lysosome recovers post-autophagy, but how this process governs the cellular response to starvation by restoring lysosomal transporter function to enable macromolecular recycling and protein synthesis. We will use our data to generate models to fill this gap in our understanding of autophagy, a truly critical cellular process.

自噬是细胞的一种基本功能，它控制着细胞对饥饿、感染和蛋白质错误加工的反应。它是细胞和有机体健康的核心，直接影响健康的老龄化，并在癌症，代谢疾病和神经退行性疾病中明显受损。清除细胞内受损线粒体或有毒蛋白质聚集体的过程称为宏自噬。在此，细胞将靶标吞噬在源自内质网的双膜内，并将其转运至溶酶体，在溶酶体中以钙（Ca 2+）依赖性方式融合;货物被酸性溶酶体腔中包含的蛋白酶破坏。这就是我们目前对自噬的知识的终点。然而，很明显，溶酶体必须以某种方式从这个过程中恢复，因为大自噬提供了细胞质的货物，这将使溶酶体去酸化和去极化。这将关闭溶酶体的降解和再循环能力，并削弱自噬的基本作用之一，使细胞在饥饿期间存活。很明显，在一个健康的细胞中，溶酶体确实会恢复，我们在这个项目中问的问题是它是如何做到这一点的？值得注意的是，这一过程从未被研究过，我们认为，部分原因是分离和研究溶酶体离子通道和转运蛋白功能的困难。维持膜电位的过程取决于离子转运，如在神经元动作电位期间发生的。溶酶体膜以相同的方式发挥功能，但研究不足，并且在自噬和自噬后恢复等事件中从未被跟踪。我们已经开发出尖端技术来磁性纯化溶酶体，并将其用于许多自动电生理学/膜片钳仪器，这些仪器在英国仅存在于卡迪夫。通过这个项目，我们将测量溶酶体离子通量如何在自噬期间和自噬后在磁性纯化的溶酶体中改变，其中化学处理用于诱导或阻止自噬。我们将确定这些离子通量如何改变溶酶体膜电位，以及这如何影响溶酶体溶质转运功能。总之，这些数据将提供第一个证据，不仅说明溶酶体如何在自噬后恢复，而且还说明这个过程如何通过恢复溶酶体转运蛋白功能来控制细胞对饥饿的反应，以实现大分子再循环和蛋白质合成。我们将使用我们的数据来生成模型，以填补我们对自噬的理解中的这一空白，自噬是一个真正关键的细胞过程。