Intracellular Electrophysiology: An electrochemical atlas of organelles

项目摘要 这项建议的长期目标是建立一个细胞器的电化学图谱， 在神经退行性疾病的背景下合理操纵细胞器间的接触。新模式 细胞内通讯出现在细胞器的水平，从而两个并列的膜 细胞器通过其细胞质表面上的蛋白质-蛋白质相互作用物理连接，称为 细胞器间的联系离子和小分子从一个细胞器主动转移到另一个 这些接触穿过两个密封膜。细胞器间的接触对细胞功能、组织 因为它们调节从脂质代谢到细胞凋亡的过程。 然而，我们仍然不知道是什么信号启动了接触形成，或者是什么开启了化学运输 我们也不能区分功能性和功能性障碍的接触。因此，虽然我们 我们知道蛋白质中的特定突变会破坏接触，导致各种神经退行性疾病， 仍然不知道如何恢复这些接触和治疗这些疾病。 我认为，细胞器的电化学状态，单独和接触，将告知哪些途径和 分子应该被靶向以纠正疾病状态中的异常接触。我的基本原理是，如果我们抽象 从分子细节来看，细胞器间的接触类似于神经元突触。即使在突触中， 线索穿过两个密封的、邻接的膜。正如跨神经元膜的离子转运机制一样 由Hodgkin和Huxley的电化学模型揭示， 将揭示跨细胞器间接触的离子流机制，以及疾病中哪些特定流受到影响。 我建议建立一个细胞器的电化学图谱，作为研究健康接触的通用参考， 疾病该图谱将是一个方程式的概要，包括主要细胞器的电化学模型， 单独和联系。通过使我们能够区分正常和异常的接触，我设想地图集将揭示 可以有针对性地恢复与受影响的离子流的接触的疾病之间的共同途径。 由于无法测定细胞器中的离子或电压， 它们的细胞膜模型。在过去的十年里，我的实验室开发了一个化学平台来量化离子， 细胞器中的电压通过将电生理学整合到这个平台上，我建议现在绘制出 在隔离和接触的细胞器膜的电化学特性，并作出电化学 细胞器图谱我们将应用该图谱来阐明Ca 2+是如何流过细胞之间的异常接触的。 在帕金森病中，内质网和溶酶体可以被整流以恢复溶酶体Ca 2+。 溶酶体Ca 2+失调是许多神经退行性疾病的共同因素， 电化学图谱是它的开创性能力，揭示了共同的途径，可以针对治疗， 疾病交叉的方式。

PROJECT SUMMARY The long-term objective of this proposal is to build an electrochemical atlas of organelles to guide the rational manipulation of inter-organelle contacts in the context of neurodegenerative diseases. A new mode of intracellular communication is emerging at the level of organelles, whereby the membranes of two juxtaposed organelles are physically connected, via protein-protein interactions on their cytoplasmic faces, referred to as inter-organelle contacts. Ions and small molecules are actively transferred from one organelle to the other across these contacts, traversing two sealed membranes. Inter-organelle contacts are vital to cell function, tissue homeostasis and physiology because they regulate processes ranging from lipid metabolism to apoptosis. However, we still do not know what signals initiate contact formation or what switches on chemical transport across contacts, nor can we discriminate between functional and dysfunctional contacts. Hence, although we know of specific mutations in proteins that disrupt contact, leading to diverse neurodegenerative diseases, we still do not know how to restore these contacts and treat those diseases. I posit that the electrochemical states of organelles, alone and in contact, will inform which pathways and molecules should be targeted to rectify aberrant contacts in disease states. My rationale is that, if we abstract out the molecular details, inter-organelle contacts resemble neuronal synapses. Even in synapses, ions flow on cue across two sealed, abutting membranes. Just as ion-transport mechanisms across neuronal membranes were revealed by Hodgkin and Huxley’s electrochemical model, an analogous model of organelle membranes will reveal ion flow mechanisms across inter-organelle contacts and which specific flows are impacted in disease. I propose to build an electrochemical atlas of organelles as a universal reference to study contacts in health and disease. This atlas will be a compendium of equations comprising electrochemical models of major organelles, alone and in contact. By enabling us to discriminate normal and aberrant contacts, I envisage the atlas will reveal common pathways across diseases that can be targeted to restore contacts with impacted ion flows. The inability to assay ions or voltage in organelles has prevented the development of electrochemical models of their membranes. Over the last decade, my lab developed a chemical platform to quantify ions and voltage in organelles. By integrating electrophysiology to this platform, I propose to now map out the electrochemical characteristics of organelle membranes in isolation and in contact, and make an electrochemical atlas of organelles. We will apply the atlas to elucidate how Ca2+ flow across aberrant contacts between the endoplasmic reticulum and the lysosome can be rectified to restore lysosomal Ca2+ in parkinsonism. Dysregulated lysosomal Ca2+ is a common factor across many neurodegenerative diseases and the value of the electrochemical atlas is its pioneering ability to reveal common pathways that can be targeted for treatment in a disease cross-cutting manner.