Tardif M, Huang S, Redmond T, Safer D, Pring M, Zigmond S H
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018, USA.
J Biol Chem. 1995 Nov 24;270(47):28075-83. doi: 10.1074/jbc.270.47.28075.
To address the mechanisms through which agonists stimulate actin polymerization, we examined the roles of monomer sequestering proteins and free barbed ends on actin polymerization induced by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in neutrophils permeabilized with streptolysin O. Addition of profilin (without GTP gamma S) caused a net decrease in F-actin. Thus, merely making profilin available in the cell was not sufficient to induce actin polymerization. On the other hand, addition of profilin hardly affected the polymerization induced by GTP gamma S, while thymosin beta 4 or DNase I decreased this polymerization. These data suggested that GTP gamma S induced polymerization by increasing the availability of barbed ends. In the presence of cytochalasin B, profilin did inhibit polymerization induced by GTP gamma S, demonstrating that GTP gamma S did not inhibit profilin's monomer sequestering ability. The F-actin induced by GTP gamma S was not limited by a time-dependent loss of G-actin or G-proteins from permeabilized cells since, following stimulation with suboptimal concentrations of GTP gamma S, addition of more GTP gamma S induced further polymerization. Barbed ends remained free after F-actin reached plateau since (a) cytochalasin B caused depolymerization of induced F-actin and (b) profilin did not depolymerize induced F-actin unless the cells were first treated with cytochalasin to cap barbed ends. The data indicate that GTP gamma S maintains an increased level of F-actin by keeping at least a few barbed ends available for polymerization.
为了探究激动剂刺激肌动蛋白聚合的机制，我们研究了单体隔离蛋白和游离的肌动蛋白丝正极末端在由5'-3-O-（硫代）三磷酸鸟苷（GTPγS）诱导的、经链球菌溶血素O通透处理的中性粒细胞肌动蛋白聚合过程中的作用。添加肌动蛋白结合蛋白（无GTPγS）导致F-肌动蛋白净减少。因此，仅仅使细胞内有肌动蛋白结合蛋白并不足以诱导肌动蛋白聚合。另一方面，添加肌动蛋白结合蛋白几乎不影响GTPγS诱导的聚合，而胸腺素β4或脱氧核糖核酸酶I则降低了这种聚合。这些数据表明，GTPγS通过增加肌动蛋白丝正极末端的可用性来诱导聚合。在细胞松弛素B存在的情况下，肌动蛋白结合蛋白确实抑制了GTPγS诱导的聚合，这表明GTPγS并不抑制肌动蛋白结合蛋白的单体隔离能力。GTPγS诱导的F-肌动蛋白不受通透细胞中G-肌动蛋白或G蛋白随时间依赖性损失的限制，因为在用次优浓度的GTPγS刺激后，添加更多的GTPγS会诱导进一步聚合。当F-肌动蛋白达到平台期后，肌动蛋白丝正极末端仍保持游离状态，原因如下：（a）细胞松弛素B导致诱导的F-肌动蛋白解聚；（b）除非细胞先用细胞松弛素处理以封闭肌动蛋白丝正极末端，否则肌动蛋白结合蛋白不会使诱导的F-肌动蛋白解聚。数据表明，GTPγS通过保持至少一些可供聚合的肌动蛋白丝正极末端，维持F-肌动蛋白的高水平。
