Fig. 3.

Measuring Ca²⁺-dependent lysosomal exocytosis. A: Lysosomal exocytosis is a Ca²⁺-dependent process. Early endosomes are generated from nascent endocytic vesicles via endocytosis. Autophagosomes are generated via autophagy. Upon a series of sorting and maturation processes, autophagosomes and endosomes fuse with lysosomes to become endolysosomes and autolysosomes. Lysosomes are high Ca²⁺ (∼0.5 mM) compartments. Lysosomes fuse with the plasma membrane in response to an increase in extralysosomal [Ca²⁺]_i (>1 μM). B: Methods for the detection of lysosomal exocytosis. Method 1 (lysosomal enzyme release) is used to measure the release of lysosomal contents, such as hydrolytic enzymes, into the cell culture medium. Method 2 (Lamp1 surface staining) is to monitor the insertion of lysosomal membrane proteins into the plasma membrane by using a monoclonal antibody against a luminal epitope of lysosomal membrane proteins, such as Lamp1, in nonpermeabilized cells. Confocal images in the right panel show that ML-SA1 treatment resulted in the localization of Lamp1 (red) on the plasma membrane in nonpermeabilized WT, but not TRPML1 KO macrophages. The total amount of Lamp1 (green) proteins is detected using the same antibody in permeabilized cells [modified from (28) with permission]. Method 3 (electrophysiology-based exocytosis assay) is used to detect the plasma membrane insertion of lysosomal ion channels as a result of lysosomal exocytosis.