Biochemical and Biological Properties of Actin

由于细胞培养、遗传操作和实验设计的方便性等原因，盘状网囊阿米巴长期以来一直是研究肌动蛋白细胞骨架的生理作用的模型系统。近年来，我们感兴趣的是肌动蛋白交叉连接蛋白Cortex illins I、II和III的功能。我们已经证明，ctxI和ctxII的双重敲除通过减少cAMP的分泌而完全抑制细胞的流动和发育。最近，我们把重点放在ctxIII上。 与重组皮质菌素I和重组皮质菌素II形成同源二聚体和异源二聚体不同，我们发现在大肠杆菌中表达的皮质菌素III形成不稳定的单体，但不形成同源二聚体，当与皮质菌素I或皮质菌素II共表达时，异源二聚体非常稳定。重组皮质菌素同源和异源二聚体都不影响肌动蛋白的体外聚合，只有皮质菌素II同源二聚体与F-肌动蛋白具有高亲和力。野生型或皮质素III缺失型细胞中表达的皮质菌素III与皮质菌素I和皮质菌素II形成异源二聚体，并与DGAP1(DictyostelialGAP蛋白)结合，但仅以异二聚体形式结合，即在皮质菌素I和皮质菌素II双零细胞中表达的皮质菌素III不与DGAP1结合。表达的皮质菌素III异二聚体与F-肌动蛋白共定位于营养细胞的皮质、运动细胞的前沿和分裂细胞的分裂沟，但不存在于DGAP1缺失的细胞中；即皮质菌素III，也可能还有皮质菌素I和皮质菌素II，只有在与DGAP1络合时才能在体内与F-肌动蛋白结合。 正常情况下，皮质素III-零细胞产生化学效应，但在发育周期中，零细胞形成不稳定的流，形成比野生型细胞更小的土丘和更小的子实体。皮质菌素III可能是吞噬、吞噬、胞质分裂和细胞生长的负调节因子，因为在皮质菌素III缺失的细胞中，所有这些都被增强。

For a number of reasons including ease of cell culture, genetic manipulation and experimental design the social amoeba Dictyostelium discoideum has long been a model system for investigating the physiological roles of the actomyosin cytoskeleton. One of our interests in recent years has been the functions of the actin cross-linking proteins cortexillins (ctx) I, II and III. We had shown that the double knockout of ctxI and ctxII completely inhibits, cell streaming and development by decreasing the secretion of cAMP. More recently, we have focused on ctxIII. Unlike recombinant cortexillins I and II, which form both homodimers and heterodimers, we find that cortexillin III expressed in E. coli forms unstable monomers but not homodimers, and significantly stable heterodimers when coexpressed with either cortexillin I or II. None of the recombinant cortexillin homo- or hetero-dimers affects actin polymerization in vitro and only the cortexillin II homodimers bind to F-actin with high affinity. Cortexillin III expressed in Dictyostelium wild-type or cortexillin III-null cells forms heterodimers with both cortexillin I and II and binds to DGAP1 (a Dictyostelium GAP protein) but only as a heterodimer, i.e. expressed cortexillin III does not bind to DGAP1 in cortexillin I and II double-null cells. Heterodimers of expressed cortexillin III colocalize with F-actin in the cortex of vegetative cells, the leading edge of motile cells and the cleavage furrow of dividing cells but not in DGAP1-null cells; i.e. cortexillin III, and possibly also cortexillins I and II, binds to F-actin in vivo only when complexed to DGAP1. Cortexillin III-null cells chemotax normally but in the developmental cycle the null-cells form unstable streams and smaller mounds and smaller fruiting bodies than wild-type cells. Cortexillin III may be a negative regulator of pinocytosis, phagocytosis, cytokinesis and cell growth as all are enhanced in cortexillin III-null cells.