PHAGOSOME-LYSOSOME FUSION IS A CALCIUM-INDEPENDENT EVENT IN MACROPHAGES

Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms, Functionally, it represents a form of polarized regulated secretion, which is classi cally dependent on increases in intracellular ionized calcium ([Ca2+]( i)). Indeed, increases in [Ca2+](i) are essential for phagosome-granul e (lysosome) fusion in neutrophils and for lysosomal fusion events tha t mediate host cell invasion by Trypanosoma cruzi trypomastigotes, Sin ce several intracellular pathogens survive in macrophage phagosomes th at do not fuse with lysosomes, we examined the regulation of phagosome -lysosome fusion in macrophages. Macrophages (MO) were treated with 12 .5 mu M bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N',-tetraacetic ac id tetraacetoxymethyl ester (MAPT/AM), a cell-permeant calcium chelato r which reduced resting cytoplasmic [Ca2+]; from 80 nM to less than or equal to 20 nM and completely blocked increases in [Ca2+](i) in respo nse to multiple stimuli, even in the presence of extracellular calcium , Subsequently, MO phagocytosed serum-opsonized zymosan, staphylococci , or Mycobacterium bovis, Microbes were enumerated by 4',6-diamidino-2 -phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LA MP-1) as a membrane marker and rhodamine dextran as a content marker f or lysosomes, Confirmation of phagosomelysosome fusion by electron mic roscopy validated the fluorescence microscopy findings, We found that phagosome-lysosome fusion in MO occurs normally at very low [Ca2+](i) (less than or equal to 20 nM), Kinetic analysis showed that in MO none of the steps leading from particle binding to eventual phagosome-lyso some fusion are regulated by [Ca2+](i) in a rate-limiting way. Further more, confocal microscopy revealed no difference in the intensity of L AMP-1 immunofluorescence in phagolysosome membranes in calcium-buffere d vs, control macrophages, We conclude that neither membrane recogniti on nor fusion events in the phagosomal pathway in macrophages are depe ndent on or regulated by calcium.