Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

译文

肌动蛋白结合蛋白已知在体外调节肌动蛋白组装成超分子结构,但缺乏在活的非肌肉细胞中活性的证据。盘基网柄菌的Amebae是非肌肉细胞,其中已经描述了几种肌动蛋白结合蛋白有缺陷的突变体。在这里,我们描述了一个缺乏120-kD凝胶化因子的突变体,该因子是盘状D细胞最丰富的F-肌动蛋白交联蛋白之一。在突变细胞提取物中未检测到归因于120-kD蛋白的F-肌动蛋白交联活性,并且识别多肽上不同表位的抗体显示缺乏整个蛋白。在使用的条件下,消除凝胶化因子不会显着改变细胞向cAMP源的生长,形状,运动性或趋化方向。突变体的聚集体发育成子实体,由正常分化的孢子和茎细胞组成。在细胞骨架制剂中,可以识别出细胞皮质典型的肌动蛋白丝的密集网络,以及沿丝足动物轴延伸的束。发现的显着变化是,当用环状AMP刺激细胞时,肌动蛋白在突变体的细胞骨架中的积累增加。我们的结果表明,控制细胞形状和运动不需要通过体外测定鉴定为肌动蛋白结合蛋白的所有蛋白质的微调相互作用。

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