Ca ²⁺ -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 cathepsin C-metabolized osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that widely used concentrations of GPN rupture only a small fraction of lysosomes, but surprisingly trigger Ca ²⁺ release from nearly all. Chelating cytoplasmic Ca ²⁺ using BAPTA makes lysosomes more likely to rupture under GPN-induced stress, suggesting that Ca ²⁺ plays a role in protecting or rapidly repairing lysosomal membranes. Mechanistically, we establish that GPN causes the Ca ²⁺ -sensitive protein Apoptosis Linked Gene-2 (ALG-2) and interacting ESCRT proteins to redistribute onto lysosomes, improving their resistance to membrane stress created by GPN as well as the lysosomotropic drug chlorpromazine. Furthermore, we show that activating the cation channel TRPML1, with or without blocking the endoplasmic reticulum Ca ²⁺ pump, creates local Ca ²⁺ signals that protect lysosomes from rupture by recruiting ALG-2 and ESCRTs without any membrane damage. These findings reveal that Ca ²⁺ , through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress.

As the degradative hub of the cell, lysosomes are full of toxic content that can spill into the cytoplasm. There has been much recent interest in how cells sense and repair lysosomal membrane damage using ESCRTs and cholesterol to rapidly fix "nanoscale damage". Here, we extend understanding of how ESCRTs contribute by uncovering a preventative role of the ESCRT machinery. We show that ESCRTs, when recruited by the Ca ²⁺ -sensor ALG-2, play a critical role in stabilizing the lysosomal membrane against osmotically-induced rupture. This finding suggests that cells have mechanisms not just for repairing but also for actively protecting lysosomes from stress-induced membrane damage.