Immunoelectron microscopy reveals significant granule fusion upon stimulation of electropermeabilized human neutrophils

Although electropermeabilization has become an important tool for studying the signal requirements of exocytosis, relatively little is known about the morphological changes accompanying this response in electropermeabilized cells. In this study, we determined that electropermeabilization of human neutrophils by itself caused only minor changes in the morphology as determined by transmission electron microscopy. The structure of the plasma membrane did not show detectable changes, whereas the cytoplasm was more electron lucent as compared to intact cells. Activation of intact neutrophils with formyl-methionyl-leucyl-phenylalanine (FMLP), in the presence of cytochalasin-B, caused the development of invaginations of the plasma membrane. In contrast, activation of electropermeabilized cells with 1 microM Ca2+ and/or 50 microM GTP-gamma-S caused the development of vacuoles that did not seem to be in contact (or had previously been in contact) with the extracellular environment. However, fusion of azurophilic and specific granules with these vacuoles clearly had taken place. The response characteristics of this fusion induced by Ca2+ and GTP-gamma-S were quite similar to those of the direct fusion of granules with the plasma membrane. We conclude that in electropermeabilized human neutrophils, two processes involving granule fusion can be distinguished. First, a direct fusion of granules with the plasma membrane. Secondly, the fusion of granules leading to the formation of vacuoles, not in contact with the extracellular space.