Constantin B, Meerschaert K, Vandekerckhove J, Gettemans J
Flanders Interuniversity Institute for Biotechnology (V.I.B.) and Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganckstraat 35, B-9000 Gent, Belgium.
J Cell Sci. 1998 Jun;111 ( Pt 12):1695-706. doi: 10.1242/jcs.111.12.1695.
Fragmin from Physarum polycephalum is a gelsolin-like actin-binding protein and interferes with the growth of actin filaments in vitro by severing actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragmin dimer is phosphorylated in vivo and in vitro on the actin subunit by the actin-fragmin kinase. We have studied the properties of these capping proteins and their regulation by actin phosphorylation and Ca2+ ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection or by expression in conjunction with immunostaining and fluorescence microscopy. Microinjection of the actin-fragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect could be blocked by phosphorylation of the actin subunit by the actin-fragmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin. This suggests that in Physarum microplasmodia, actin polymerization can be controlled in a Ca2+-dependent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytoskeleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containing buffer. The cells were able to revert to their normal phenotype which indicates that endogenous regulatory mechanisms counteracted fragmin activity, probably by uncapping fragmin from the barbed ends of filaments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2+ concentration and this provides a basis for a more general mechanism in higher organisms to regulate microfilament organization.
多头绒泡菌的凝溶胶蛋白样肌动蛋白结合蛋白Fragmin通过切断肌动蛋白丝并以Ca2+依赖的方式形成肌动蛋白-Fragmin二聚体来封闭其带刺末端，从而在体外干扰肌动蛋白丝的生长。肌动蛋白-Fragmin二聚体在体内和体外被肌动蛋白-Fragmin激酶在肌动蛋白亚基上磷酸化。我们通过显微注射或与免疫染色和荧光显微镜结合表达，研究了这些封闭蛋白的特性以及它们在活的PtK2、CV1和NIH3T3培养细胞中受肌动蛋白磷酸化和Ca2+离子的调节。显微注射肌动蛋白-Fragmin二聚体使肌动蛋白细胞骨架解体并改变细胞形态。在低Ca2+条件下，肌动蛋白-Fragmin激酶对肌动蛋白亚基的磷酸化可阻断这种体内效应，高Ca2+浓度可能通过激活Fragmin中的第二个肌动蛋白结合位点来恢复封闭活性。这表明在多头绒泡菌微原质团中，肌动蛋白聚合可通过肌动蛋白的磷酸化以Ca2+依赖的方式受到控制。显微注射或过表达的重组Fragmin不会影响静息细胞基于肌动蛋白的细胞骨架或细胞形态，除非通过显微注射含钙缓冲液增加胞质游离Ca2+浓度。细胞能够恢复到正常表型，这表明内源性调节机制可能通过从丝的带刺末端解开Fragmin来抵消其活性。Fragmin还拮抗溶血磷脂酸诱导的应力纤维形成。我们的研究结果表明，肌动蛋白和Fragmin之间的相互作用受胞质Ca2+浓度的严格调节，这为高等生物中更普遍的调节微丝组织的机制提供了基础。
