Deciphering new components of Ca2+-signaling in endolysosomes

• Applicant: Professor Dr. Christian Michael Grimm
• Subject Area: Pharmacology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 536658766

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.

• DFG Programme: Research Grants