RESUMO
Studies on fixed preparations have shown that vacuoles containing zymosan (Z) particles internalized by infected macrophages can selectively fuse with the large parasitophorous vacuoles (PVs) that shelter Leishmania amazonensis. To examine the kinetics of vacuolar fusion in individual cells, particles were followed by time-lapse cinemicrography from their uptake to their entry in a PV. Newly formed Z-containing vacuoles moved centripetally and, if they contacted a PV, the two vacuoles remained closely apposed for variable, often extended, periods of time before they eventually fused. Transmission electron microscopy confirmed that the cytoplasm separating the partner vacuoles could be reduced to a very thin layer. Initiation of fusion was indicated by reduced refractility of the boundary between Z vacuoles and target PVs. Within a few minutes the PV enlarged and encompassed the Z particles, which remained immobile throughout. The interval between phagocytosis and fusion, 50 +/- 7.4 min (N = 17; range, 4 to 108 min), suggests that most but not all Z vacuoles underwent significant maturation by the time of fusion. Some particles were transferred singly, others entered PVs in groups of 2 or more, and additional clustered transfers to the same vacuole were also observed. These observations provide a baseline for studies of the biochemical mechanisms and the pharmacological control of the fusion of Leishmania PVs, and for the comparison of the fusion behavior of the PVs with that of other phagocytically derived vacuoles.