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Ca(2+)-independent F-actin assembly and disassembly during Fc receptor-mediated phagocytosis in mouse macrophages
S Greenberg1, J el Khoury, F di Virgilio
1Rover Laboratory of Cellular Physiology, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York 10032.
Abstract:
Phagocytosis of IgG-coated particles by macrophages is presumed to involve the actin-based cytoskeleton since F-actin accumulates beneath forming phagosomes, and particle engulfment is blocked by cytochalasins, drugs that inhibit actin filament assembly. However, it is unknown whether Fc receptor ligation affects the rate or extent of F-actin assembly during phagocytosis of IgG-coated particles. To examine this question we have used a quantitative spectrofluorometric method to examine F-actin dynamics during a synchronous wave of phagocytosis of IgG-coated red blood cells by inflammatory mouse macrophages. We observed a biphasic rise in macrophage F-actin content during particle engulfment, with maxima at 1 and 5 min after the initiation of phagocytosis. F-actin declined to resting levels by 30 min, by which time particle engulfment was completed. These quantitative increases in macrophage F-actin were reflected in localized changes in F-actin distribution. Previous work showed that the number of IgG-coated particles engulfed by macrophages is unaffected by buffering extracellular calcium or by clamping cytosolic free calcium concentration ([Ca2+]i) to very low levels (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106: 657-666). To determine whether clamping [Ca2+]i in macrophages affects the rate of particle engulfment, or the assembly or disassembly of F-actin during phagocytosis, we examined these parameters in macrophages whose [Ca2+]i had been clamped to approximately less than 3 nM with fura 2/AM and acetoxymethyl ester of EGTA. We found that the initial rate of phagocytosis, and the quantities of F-actin assembled and disassembled were similar in Ca(2+)-replete and Ca(2+)-depleted macrophages. We conclude that Fc receptor-mediated phagocytosis in mouse macrophages is accompanied by an ordered sequence of assembly and disassembly of F-actin that is insensitive to [Ca2+]i.
Insights
Phagocytosis of IgG-coated particles by macrophages involves actin cytoskeleton dynamics. This study shows that F-actin assembly and disassembly during particle engulfment are independent of intracellular calcium levels.
Area of Science:
- Cell Biology
- Immunology
- Cytoskeletal Dynamics
Background:
- Phagocytosis of IgG-coated particles by macrophages is crucial for immune responses.
- The actin cytoskeleton is known to be involved in phagocytosis, but the precise dynamics and calcium dependency were unclear.
Purpose of the Study:
- To investigate the dynamics of F-actin assembly and disassembly during Fc receptor-mediated phagocytosis.
- To determine the role of intracellular calcium concentration ([Ca2+]i) in these actin dynamics and phagocytic rates.
Main Methods:
- Quantitative spectrofluorometry was used to measure F-actin content during phagocytosis of IgG-coated red blood cells by mouse macrophages.
- Intracellular calcium levels were clamped to very low concentrations using fura 2/AM and EGTA to assess calcium's role.
Main Results:
- A biphasic increase in F-actin content was observed during particle engulfment, peaking at 1 and 5 minutes.
- Phagocytosis rate and F-actin assembly/disassembly kinetics were unaffected by clamped low intracellular calcium levels compared to calcium-replete conditions.
Conclusions:
- Fc receptor-mediated phagocytosis involves a regulated sequence of F-actin assembly and disassembly.
- These actin dynamics during phagocytosis are independent of intracellular calcium concentration in mouse macrophages.