Bengtsson T, Särndahl E, Stendahl O, Andersson T
Department of Medical Microbiology, University of Linköping, Sweden.
Proc Natl Acad Sci U S A. 1990 Apr;87(8):2921-5. doi: 10.1073/pnas.87.8.2921.
The motility of human neutrophils, which is of vital importance for the role of these cells in host defense, is based on rapid and dynamic changes of the filamentous actin F-actin) network. Consequently, to understand how neutrophils move and ingest particles, we need to know how polymerization and depolymerization of actin are regulated. Previous studies by several investigators have, based on indirect evidence obtained with pertussis toxin, suggested a role for GTP-binding protein(s) (G protein) in chemotaxis-induced, but not phagocytosis-induced, reorganization of the F-actin network. The aim of the present investigation was to study the effects of directly activated G proteins (i.e., without prior ligand-receptor complex formation) on the F-actin content in human neutrophils. AlF4- induced a pronounced and sustained increase in F-actin in intact neutrophils. This effect coincided with an increase in cytosolic free Ca2+, indicating that phospholipase C and the subsequent transduction mechanism were also activated. Inhibition of phospholipase C activity by extensive depression of the cytosolic free Ca2+ level (less than 20 nM) only marginally affected the AlF4(-)-induced rise in F-actin content. The major part of the AlF4(-)-induced rise in F-actin content was also resistant to pertussis toxin, suggesting that pertussis toxin-insensitive G proteins in neutrophils are also able to trigger actin polymerization. The specificity of AlF4- in activating G proteins was also tested in permeabilized cells. In this case the effect was more rapid and could be totally abolished by guanosine 5'-[beta-thio]diphosphate. In analogy, in permeabilized cells guanosine 5'-[gamma-thio]triphosphate mimicked the effect of AlF4- on actin polymerization, and the effect induced by this nonhydrolyzable GTP analogue could also be totally abolished by guanosine 5'-[beta-thio]diphosphate. In summary, the present data support our previous hypothesis that G proteins are intimately linked to actin polymerization in human neutrophils.
人类中性粒细胞的运动能力对这些细胞在宿主防御中的作用至关重要，其运动能力基于丝状肌动蛋白（F-肌动蛋白）网络的快速动态变化。因此，为了理解中性粒细胞如何移动和摄取颗粒，我们需要知道肌动蛋白的聚合和解聚是如何被调控的。几位研究者之前的研究基于用百日咳毒素获得的间接证据，表明GTP结合蛋白（G蛋白）在趋化作用诱导而非吞噬作用诱导的F-肌动蛋白网络重组中起作用。本研究的目的是研究直接激活的G蛋白（即无需事先形成配体-受体复合物）对人类中性粒细胞中F-肌动蛋白含量的影响。AlF4-可诱导完整中性粒细胞中F-肌动蛋白显著且持续增加。这种效应与胞质游离Ca2+的增加同时出现，表明磷脂酶C及随后的转导机制也被激活。通过大幅降低胞质游离Ca2+水平（低于20 nM）来抑制磷脂酶C活性，仅对AlF4-诱导的F-肌动蛋白含量升高产生轻微影响。AlF4-诱导的F-肌动蛋白含量升高的主要部分也对百日咳毒素具有抗性，这表明中性粒细胞中对百日咳毒素不敏感的G蛋白也能够触发肌动蛋白聚合。还在通透细胞中测试了AlF4-激活G蛋白的特异性。在这种情况下，效应更迅速，并且可被鸟苷5'-[β-硫代]二磷酸完全消除。类似地，在通透细胞中，鸟苷5'-[γ-硫代]三磷酸模拟了AlF4-对肌动蛋白聚合的效应，并且这种不可水解的GTP类似物诱导的效应也可被鸟苷5'-[β-硫代]二磷酸完全消除。总之，目前的数据支持我们之前的假设，即G蛋白与人类中性粒细胞中的肌动蛋白聚合密切相关。
