Deciphering new components of Ca2+-signaling in endolysosomes

近年来，内溶酶体成为细胞内重要的钙信号中枢，内溶酶体内的钙释放被证明具有重要的生理和病理生理意义。胞浆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。溶酶体钙含量或释放的紊乱与疾病病理密切相关，影响或导致神经退行性和溶酶体储存性疾病、癌症和免疫学、代谢、肺部或传染病。尽管已经积累了关于溶酶体钙释放及其分子介体的知识，但与溶酶体钙稳态相关的几个关键问题仍然没有得到解答，本研究旨在解决其中的两个问题：1.钙释放后如何被带回溶酶体？2.溶酶体如何应对渗透压的变化和管腔的收缩和扩张，以及在溶酶体膜上哪些分子成分是感知机械刺激和拉伸介导的刺激的？在哺乳动物细胞的质膜上，PMCAs(质膜钙ATPase)起着将胞浆中的钙离子泵回细胞外间隙的作用，而在肌内质网膜中，PMCAs(肌内质网钙ATPase)则负责将胞浆中的钙离子泵回SER。类似的机制可能在溶酶体中起作用，来自植物的证据支持这一观点，其中钙-ATPase(ACAs)特别是ACA4和ACA11定位于液泡中。然而，哺乳动物溶酶体膜中潜在的等同蛋白的分子同一性仍然是个谜。我们的目标是通过使用膜片钳玻璃移液管急性分离溶酶体，结合质谱学/蛋白质组学分析，执行一种无偏见的方法来识别钙离子流入溶酶体的候选者。同样，溶酶体中的机械敏感离子通道在很大程度上仍然是个谜。在哺乳动物中，机械感觉过程，如触觉和血管发育，是由机械激活的非选择性、钙离子可渗透的阳离子通道的压电体家族介导的，在内耳，毛细胞中的机械转导复合体包括TMC1作为致孔离子通道。OSCA/TMEM63/OCAR/CSCL钙离子通道蛋白家族是一个在细胞膜上过度表达时具有机械敏感性的蛋白质家族，如我们所示，该家族主要在内溶酶体细胞器和内源性颗粒中表达，因此是内溶酶体机械感觉的一个很好的候选者。总之，通过结合电生理学、钙成像、分子生物学、基因敲除模型和急性分离溶酶体的创新方法，结合蛋白质组学分析，我们的目标是识别内溶酶体中新的、长假设的钙信号和机械感觉成分。

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.