Greenberg S, el Khoury J, di Virgilio F, Kaplan E M, Silverstein S C
Rover Laboratory of Cellular Physiology, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York 10032.
J Cell Biol. 1991 May;113(4):757-67. doi: 10.1083/jcb.113.4.757.
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.
巨噬细胞对IgG包被颗粒的吞噬作用被认为涉及基于肌动蛋白的细胞骨架，因为F-肌动蛋白在正在形成的吞噬体下方积聚，并且颗粒吞噬被细胞松弛素（一种抑制肌动蛋白丝组装的药物）所阻断。然而，尚不清楚Fc受体连接是否会影响IgG包被颗粒吞噬过程中F-肌动蛋白组装的速率或程度。为了研究这个问题，我们使用了一种定量光谱荧光法来检测炎症小鼠巨噬细胞对IgG包被红细胞进行同步吞噬波期间的F-肌动蛋白动力学。我们观察到在颗粒吞噬过程中巨噬细胞F-肌动蛋白含量呈双相上升，在吞噬开始后1分钟和5分钟达到最大值。F-肌动蛋白在30分钟时降至静息水平，此时颗粒吞噬已完成。巨噬细胞F-肌动蛋白的这些定量增加反映在F-肌动蛋白分布的局部变化中。先前的研究表明，巨噬细胞吞噬的IgG包被颗粒的数量不受细胞外钙缓冲或将细胞质游离钙浓度（[Ca2+]i）钳制到非常低水平的影响（迪·维尔吉利奥，F.，B.C.迈耶，S.格林伯格，和S.C.西尔弗斯坦。1988.《细胞生物学杂志》106: 657 - 666）。为了确定钳制巨噬细胞中的[Ca²⁺]i是否会影响颗粒吞噬的速率，或吞噬过程中F-肌动蛋白的组装或拆卸，我们在使用fura 2/AM和EGTA的乙酰氧基甲酯将[Ca²⁺]i钳制到约小于3 nM的巨噬细胞中检测了这些参数。我们发现，在富含Ca²⁺和缺乏Ca²⁺的巨噬细胞中，吞噬的初始速率以及组装和拆卸的F-肌动蛋白量相似。我们得出结论，小鼠巨噬细胞中Fc受体介导的吞噬作用伴随着F-肌动蛋白组装和拆卸的有序序列，该序列对[Ca²⁺]i不敏感。
