Deciphering new components of Ca2+-signaling in endolysosomes

破译内溶酶体中 Ca2 信号传导的新成分

• 批准号: 536658766
• 负责人: Professor Dr. Christian Michael Grimm
• 金额: --
• 依托单位: Walther-Straub-Institut für Pharmakologie und Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/536658766?language=en
• 关键词: Deciphering; new; components; Ca2+; signaling

In recent years endolysosomes emerged as important intracellular Ca2+ signaling hubs and Ca2+ release from endolysosomes proved to be of significant physiological and pathophysiological relevance. Endolysosomal calcium regulates cellular processes that are key for human health such as autophagy, membrane trafficking, exocytosis, nutrient adaptation, membrane repair, or cell migration. Disruption of lysosomal Ca2+ content or release is strongly associated with disease pathology, affecting or causing neurodegenerative and lysosomal storage diseases, cancer and immunological, metabolic, lung, or infectious diseases. Despite accumulating knowledge about lysosomal Ca2+ release and their molecular mediators, several key questions related to lysosomal Ca2+ homeostasis remain unanswered, two of which we aim to address in this proposal: 1. How is Ca2+ taken back up into lysosomes after release? and 2. How do lysosomes cope with osmolarity changes and shrinkage and expansion of the lysosome lumen, and which are the molecular components sensing mechanical and stretch-mediated stimuli in the lysosomal membrane? In the plasma membrane of mammalian cells, PMCAs (plasma membrane Ca2+ ATPases) fulfill the function of pumping back Ca2+ from the cytosol into the extracellular space and in the sarco-endoplasmic reticulum membrane SERCAs (sarco-endoplasmic reticulum Ca2+ ATPases) pump Ca2+ back from the cytosol into the SER. A similar mechanism may operate in lysosomes and evidence e.g., from plants, where Ca2+-ATPases (ACAs) in particular ACA4 and ACA11 are localized in the vacuole supports this idea. Yet the molecular identity of a potential equivalent protein in the lysosomal membrane of mammalian species remains enigmatic. We aim here to perform an unbiased approach to identify candidates for Ca2+ influx into lysosomes using acute isolation of lysosomes with patch-clamp glass pipettes in combination with mass-spectrometry/proteomics analyses. Likewise, mechanosensitive ion channels in lysosomes remain largely enigmatic. In mammals, mechanosensory processes such as touch sensation and vascular development are mediated by the PIEZO family of mechanically activated non-selective, Ca2+ permeable cation channels and in the inner ear, the mechanotransduction complex in hair cells includes TMC1 as the pore-forming ion channel. A protein family that confers mechanosensitivity in the plasma membrane when overexpressed, the family of OSCA/TMEM63/OCaR/CSCL Ca2+ permeable ion channel proteins is, as we show here predominantly expressed in endolysosomal organelles and in granules endogenously and is hence a good candidate for mechanosensation in endolysosomes. In sum, by using a combination of electrophysiology, calcium imaging, molecular biology, knockout models, and an innovative method for the acute isolation of lysosomes in combination with proteomics analyses, we aim to identify novel, long postulated components of Ca2+ signaling and mechanosensation in endolysosomes.

近年来，内溶酶体作为细胞内重要的钙信号枢纽而出现，并且内溶酶体的钙释放被证明具有重要的生理和病理生理学意义。内溶酶体钙调节对人类健康至关重要的细胞过程，如自噬、膜运输、胞吐、营养适应、膜修复或细胞迁移。溶酶体Ca 2+含量或释放的破坏与疾病病理学密切相关，影响或引起神经变性和溶酶体贮积病、癌症和免疫、代谢、肺或感染性疾病。尽管积累了关于溶酶体Ca 2+释放及其分子介质的知识，但与溶酶体Ca 2+稳态相关的几个关键问题仍未得到解答，我们的目标是在本提案中解决其中两个问题：1。Ca 2+释放后如何被吸收回溶酶体？和2.溶酶体如何科普渗透压的变化和溶酶体腔的收缩和扩张，以及哪些是溶酶体膜中感受机械和拉伸介导的刺激的分子组分？在哺乳动物细胞的质膜中，PMCA（质膜Ca 2 + ATP酶）履行将Ca 2+从胞质溶胶泵回细胞外空间的功能，并且在肌质内质网膜中，SERCA（肌质内质网Ca 2 + ATP酶）将Ca 2+从胞质溶胶泵回SER。来自植物，其中Ca 2 +-ATP酶（ACA），特别是ACA 4和ACA 11定位于液泡中支持了这一观点。然而，哺乳动物物种的溶酶体膜中潜在的等价蛋白的分子身份仍然是个谜。我们的目标是执行一个公正的方法来确定候选人的钙离子流入溶酶体使用急性分离的溶酶体与膜片钳玻璃移液器结合质谱/蛋白质组学分析。同样，溶酶体中的机械敏感离子通道在很大程度上仍然是谜。在哺乳动物中，机械感觉过程如触觉和血管发育由机械活化的非选择性Ca 2+可渗透阳离子通道的PIEZO家族介导，并且在内耳中，毛细胞中的机械转导复合物包括作为成孔离子通道的TMC 1。当过表达时，在质膜中赋予机械敏感性的蛋白质家族，OSCA/TMEM 63/OCaR/CSCL Ca 2+渗透性离子通道蛋白家族，如我们在此所示，主要在内溶酶体细胞器和颗粒中内源性表达，因此是内溶酶体中机械感觉的良好候选者。总之，通过使用电生理学，钙成像，分子生物学，敲除模型的组合，并结合蛋白质组学分析的溶酶体急性分离的创新方法，我们的目标是确定新的，长期假设的Ca 2+信号和机械感觉的组成部分内溶酶体。