Ca²⁺-sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress

Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that low concentrations of GPN rupture a small fraction of lysosomes, but surprisingly trigger Ca²⁺ release from nearly all. Chelating cytoplasmic Ca²⁺ makes lysosomes more sensitive to GPN-induced rupture, suggesting a role for Ca²⁺ in lysosomal membrane resilience. GPN-elicited Ca²⁺ release causes the Ca²⁺-sensor Apoptosis Linked Gene-2 (ALG-2), along with Endosomal Sorting Complex Required for Transport (ESCRT) proteins it interacts with, to redistribute onto lysosomes. Functionally, ALG-2, but not its ESCRT binding-disabled ΔGF¹²² splice variant, increases lysosomal resilience to osmotic stress. Importantly, elevating juxta-lysosomal Ca²⁺ without membrane damage by activating TRPML1 also recruits ALG-2 and ESCRTs, protecting lysosomes from subsequent osmotic rupture. These findings reveal that Ca²⁺, through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress.