Focal exocytosis of Syntaxin 3 and TRPML1 at pseudopodia of nascent phagosomes

Abstract

Macrophages clear invading pathogens by phagocytosis. Phagocytosis is a complex mechanism involving the local expansion of the membrane, cytoskeletal remodeling, and the delivery of phagosomal proteins to the nascent phagosomes. However, the organelle trafficking events underlying this are largely unclear. Here, we show in human blood monocyte-derived macrophages that TRPML1, a calcium channel involved in the phagocytic process, is delivered to phagosomes in Syntaxin 3-positive vesicles. Syntaxin 3 is a SNARE protein previously shown to mediate the secretion of IL-6 by macrophages. Total Internal Reflection Microscopy (TIRF) revealed that Syntaxin 3 positive compartments carry TRPML1 to pseudopodia for focal exocytosis at the nascent phagosomes during E. coli uptake. Using siRNA knockdown, we show that both Syntaxin 3 and TRPML1 are required for E. coli uptake. Moreover, using TRPML1 agonists we show that increased TRPML1 activity leads to increased E. coli uptake, whereas calcium chelation decreased intracellular E. coli load. Understanding the membrane trafficking pathways is critical for understanding how macrophages clear invading pathogens. Key findingsO_LISyntaxin 3 positive vesicles are delivered at the plasma membrane site of phagocytosis. C_LIO_LISyntaxin 3-positive vesicles carry TRPML1 to pseudopodia. C_LIO_LIBacterial phagocytosis correlates with Syntaxin 3 and TRPML1 expression levels. C_LIO_LIBacterial phagocytosis depends on calcium flux through TRPML1. C_LIO_LISyntaxin-3 vesicles carry the cytokine interleukin-6. C_LI