Homeostasis and Functions of Ca2+ and Metal Stores in Lysosomes and Lysosome-related Organelles

Abstract

Lysosomes and lysosome-related organelles (LROs) are involved in many intracellular signaling pathways as well as in a variety types of membrane trafficking processes such as exocytosis and autophagy. These organelles serve as intracellular stores for Ca2+, Zn2+ and Fe2+. Impairment of lysosomal Ca2+ homeostasis and membrane trafficking has been implicated as a likely cause of lysosomal storage diseases (LSDs), and more broadly in neurodegeneration, and cancer. Lysosomal membrane proteins, particularly ion channels, are crucial for lysosomal Ca2+ signaling. This thesis is focused on the establishment and regulation of Ca2+ homeostasis in lysosomes and LROs, and their contributions to diseases, but also explores the roles of other lysosomal cations. The first two data chapters of this thesis focused on the major Ca2+ release channel of lysosomes, transient receptor potential mucolipin 1 (TRPML1 or ML1), which regulates various aspects of lysosomal function, including trafficking, fusion, and fission. In chapter II, we studied tubulovesicles, the organelles within parietal cells of the stomach that secrete acid in response to histamine stimulation. We identified TRPML1 as the tubulovesicular Ca2+ release channel, and showed that in response to histamine stimulation of parietal cells, TRPML1 is activated by cyclic AMP/protein kinase A (cAMP/PKA). This activation is required for the apical-directed trafficking of tubulovesicles, which results in the delivery of the hydrogen potassium ATPase (H+/K+-ATPase) to the apical membrane and an increase in acid secretion. The identification and functional analysis of TRPML1 on tubulovesicles provides a novel target for acid-related gastric diseases. In chapter III, we showed that in a melanoma cancer model, hyper-activity of TRPML1 leads to elevated cell death of metastatic melanoma cells. Release of Zn2+, instead of Ca2+, is required for elevating cell death, potentially through direct disruption of mitochondrial functions. Collectively, these findings may advance our understanding of the functional roles of lysosomes and LROs as stores for signaling ions such as Ca2+ and Zn2+, and how they engage in disease pathologies. The molecular mechanisms that establish and maintain the 5000-fold Ca2+ gradient across the lysosome membrane remains an enigma, but endoplasmic reticulum (ER) Ca2+ has been suggested to be critical for lysosomal Ca2+ store refilling. In chapter IV, by using a physiological assay which monitors lysosomal Ca2+ store refilling, and super resolution imaging to visualize lysosome behavior upon luminal Ca2+ release and depletion, we confirmed that ER Ca2+ is required for the acute refilling of lysosomes and identified several molecules required for this function. Lysosome Ca2+ stores are refilled using membrane contact sites (MCSs) with ER tubules, in which the ER Ca2+ releasing channel IP3R participate. VPS13D is recruited to ER-lysosome MCSs sites for MCS formation and stabilization. Such recruitment is supported by a cytosolic Ca2+ sensor, potentially CETN3, which was suggested to directly interact with VPS13D in previous literature. Taken together, the results of this thesis add important new information on the machinery regulating lysosomal [Ca2+], as well as the physiology of Ca2+ channeling MCSs. Disruption of lysosomal Ca2+ homeostasis is commonly seen in LSDs and neurodegenerative diseases. Therefore, ER to lysosome Ca2+ transfer may serve as a potential therapeutic target.