Myosin va bound to phagosomes binds to F-actin and delays microtubule-dependent motility

We established a light microscopy-based assay that reconstitutes the bindin g of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosi n Va from chicken brain stimulated the phagosome-F-actin interaction. Myosi n Va association with phagosomes correlated with their ability to bind F-ac tin in an ATP-regulated manner and antibodies to myosin Va specifically blo cked the ATP-sensitive phagosome binding to F-actin. The uptake and retrogr ade transport of phagosomes from the periphery to the center of cells in bo ne marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in di lute-lethal macrophages the accumulation of phagosomes in the perinuclear r egion occurred twofold faster than in normal macrophages. Motion analysis r evealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin V a. mediates phagosome binding to F-actin, resulting in a delay in microtubu le-dependent retrograde phagosome movement toward the cell center. We propo se an "antagonistic/cooperative mechanism" to explain the saltatory phagoso me movement toward the cell center in normal macrophages.