Video-rate dynamics of exocytotic events associated with phagocytosis in neutrophils

Exocytotic responses associated with phagocytosis were investigated in a single neutrophil with a special reference to their dynamic properties and their spatiotemporal relationships with ionic and chemical responses during phagocytosis. The real-time sequence of phagocytosis-exocytosis was directly visualized by video-enhanced contrast differential interference contrast (VEC-DIC) microscopy. The actual release of contents from such a granule was proven by examining a cell loaded with quinacrine with a dual imaging system that allowed us to observe DIC and fluorescence images simultaneously at a high magnification. During the process of phagosome formation in a neutrophil engulfing an opsonized zymosan, the exocytotic response was observed first in a granule located near the cell surface initially attached to the zymosan, and then in other granules sequentially along pseudopodia surrounding the zymosan. When the phagocytosis was induced in a medium containing luminol, a chemiluminescence due to active oxidants was detected exclusively in the region of phagosome, suggesting that exocytosis took place on the phagosomal membrane and not on the plasma membrane. Changes in cytosolic free calcium concentration ([Ca2+]i) were further measured using fura-2 under the dual imaging system. [Ca2+]i transients were more closely related to the extension of pseudopodia for engulfing zymosan and not directly to the exocytosis. These findings lead to a conclusion that exocytosis associated with phagocytosis is initiated by attachment of the cell membrane to the invading organism and mediated by local activation of the phagosomal membrane.