A Lysosomal Calcium Channel Regulates Membrane Trafficking During Phagocytosis.

Abstract

Macrophages participate in two main biological functions; they actively participate in apoptotic body removal and are involved in pathogen elimination. Interestingly, lysosomal membrane trafficking events have been shown to be vital for both processes. Ingestion of large extracellular particles, such as apoptotic bodies, requires the fusion of lysosomal membranes with the plasma membrane (i.e. lysosomal exocytosis) in order to provide excess membrane for the formation of plasmalemmal pseudopod necessary for phagosome sealing and, consequently, particle ingestion. Conversely, pathogen elimination by macrophages involves the trafficking of the ingested pathogens to lysosomes for degradation. Here I identified Mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating both lysosomal trafficking events in macrophages. Both lysosomal exocytosis and particle ingestion are inhibited by synthetic TRPML1 blocker and are defective in macrophages isolated from TRPML1 knockout mice. Additionally, TRPML1 overexpression and TRPML1 agonists induce both lysosomal exocytosis and particle uptake. By using time-lapse confocal imaging I found that particle binding induces the production of lysosomal PI(3,5)P2 in order to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of particle uptake. This then promotes the fusion of TRPML1-containg lysosomal membranes with the plasma membrane at the site of particle uptake through lysosomal exocytosis. Thus, phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+- dependent exocytosis pathway in order to provide the membrane necessary for pseudopod extension. In correlation with this, accumulation of apoptotic cells was observed in various tissues obtained from TRPML1 knockout mice, suggesting defective clearance of apoptotic cells in vivo. Moreover, I observed that bacteria elimination is inhibited by synthetic TRPML1 blocker and is defective in macrophages isolated from TRPML1 knockout mice. Time-lapse confocal imaging showed that the fusion of bacteria-containing phagosomes with the lysosomes is defective in macrophages lacking TRPML1 activity. Collectively, I have showed that TRPML1 regulates various lysosomal membrane trafficking events within macrophages by releasing lysosomal calcium into the cytoplasm and providing the calcium necessary for mediating the fusion machinery involved in membrane trafficking.