Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium.
The role of changes in cytosolic free calcium concentration ([Ca2+]i) in the assembly and disassembly of actin during adhesion and phagocytosis was evaluated. Rhodamine-phalloidin staining combined with quantitative fluorescence and confocal laser scanning microscopy was used to measure local F-actin changes in single adherent human neutrophils phagocytosing yeast particles on different surfaces and under different calcium conditions. Cells were suspended in a) calcium-containing medium (CCM) or b) calcium-free medium (CFM) or c) were first depleted of calcium (i.e., MAPT/AM-loaded in CFM) and then suspended in CFM (MAPT). In parallel, local [Ca2+]i changes were monitored using a fura-2 ratio imaging system. In CCM or CFM, attachment to the substrate and formation of pseudopods around a yeast particle generated, within a few seconds, rises in [Ca2+]i, both around the phagosome and in the cell body. During continued phagocytosis, [Ca2+]i was more elevated around the phagosome compared to the rest of the cell. No [Ca2+]i fluctuations were observed in MAPT cells. Adhesion and phagocytosis led to a several-fold increase in F-actin. The increase was transient in cells in CCM and CFM, but remained high in Ca-depleted neutrophils. A distinct ring of F-actin was formed around a phagosome with a yeast particle. Twenty min after ingestion the amount of this actin decreased more than 50% in CCM and CFM cells but increased by 40 to 100% in MAPT cells. The accumulation of F-actin in MAPT cells was reduced to resting levels by adding Ca2+ and ionomycin after ingestion. This treatment reestablished the periphagosomal [Ca2+]i rises, as observed in CCM cells. In conclusion, the present study shows that the actin polymerization, occurring in human neutrophils during adhesion and phagocytosis, is not influenced by changes in [Ca2+]i, whereas the subsequent depolymerization is. The accumulation of actin filaments around the phagosome in calcium-depleted cells could be involved in the inhibition of phagolysosome fusion seen in the absence of [Ca2+]i changes (Jaconi et al., J. Cell Biol. 110, 1555-1564 (1990)). This suggests that the actin network, controlled by [Ca2+]i, regulates the movement of granules during phagocytosis.
Bengtsson
T
T
Department of Medical Microbiology, Faculty of Health Sciences, University of Linköping, Sweden.
Jaconi
M E
ME
Gustafson
M
M
Magnusson
K E
KE
Theler
J M
JM
Lew
D P
DP
Stendahl
O
O
D000199:Actins / Q000097:blood*; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D006801:Humans; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism
D000199:Actins; D002118:Calcium