Role of calcium in the shape control of human granulocytes

The possible role of cytoplasmic calcium in the shape control of human blood granulocytes was explored with two complementary sets of experiments. First, cells were stimulated with N-formyl methionyl leucyl phenylalanine (FMLP) with or without depletion of free intracellular calcium (by incubation with BAPTA/AM in calcium-deprived medium). Control cells displayed a rapid and transient rise of free intracellular calcium (peaked after a few seconds, returned to the basal level after 2-3 minutes), extensive morphological polarization (more than 90% polarized cells after 15 minutes), and reorganization of actin filaments (as assessed by image analysis on cells labeled with a fluorescent phallacidin derivative). Calcium-depleted cells displayed no calcium rise, but both morphological polarization and actin reorganization were indistinguishable from those of controls. In a second set of experiments, individual granulocytes were labeled with fluorescent calcium probes and aspirated in a micropipette on the stage of a confocal laser microscope. About half of the cells displayed a transient calcium rise, which was concomitant with the formation of a protrusion in most cases. However, extensive protrusions were shown by the cells that did not exhibit calcium flashes. Furthermore, if cells were treated with ionomycin, in calcium-containing or calcium-deprived medium, then aspirated, they showed protrusions comparable to those of controls. Finally, when cells were treated with ionomycin during micropipette aspiration, a calcium rise was followed by a moderate increase of the rate of protrusion growth. It is concluded that free calcium alone cannot play a major role in the control of cell shape.