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

自噬是细胞的一项基本功能，它控制着对饥饿、感染和蛋白质错误加工的反应。它对细胞和机体健康至关重要，直接影响健康老龄化，并在癌症、代谢状况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聚集体的过程称为巨噬。在这里，细胞将靶标吞没在起源于内质网的双层膜内，并将其运送到溶酶体，在那里以依赖钙（Ca2+）的方式融合；货物被酸性溶酶体腔内的蛋白酶破坏。这就是我们目前对自噬的了解。然而，很明显，溶酶体必须以某种方式从这一过程中恢复，因为巨噬提供了大量的细胞质，这些细胞质会使溶酶体去酸和去极化。这将关闭溶酶体的降解和再循环能力，并损害自噬的基本作用之一，使细胞能够在饥饿期间存活。很明显，在健康细胞中，溶酶体会恢复，我们在这个项目中要问的问题是它是如何恢复的？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由于分离和研究溶酶体离子通道和转运体功能的困难。维持膜电位的过程依赖于离子运输，就像发生在神经元动作电位期间一样。溶酶体膜以同样的方式起作用，但研究不足，从未在自噬和自噬后恢复等事件中进行过跟踪研究。我们开发了最先进的技术来磁性纯化溶酶体，并在英国仅存在于卡迪夫的一系列自动电生理/贴片夹紧仪器中使用它们。在这个项目中，我们将测量在使用化学处理诱导或阻止自噬的细胞中磁性纯化的溶酶体在自噬期间和自噬后溶酶体离子通量的改变。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蛋白的功能。总之，这些数据将提供第一个证据，不仅是自噬后溶酶体如何恢复，而且这个过程如何通过恢复溶酶体转运体功能来控制细胞对饥饿的反应，从而实现大分子循环和蛋白质合成。我们将使用我们的数据来生成模型，以填补我们对自噬理解的空白，自噬是一个真正关键的细胞过程。

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.