Rivero F, Furukawa R, Noegel A A, Fechheimer M
Max-Planck-Institute for Biochemistry, Martinsried, Germany.
J Cell Biol. 1996 Nov;135(4):965-80. doi: 10.1083/jcb.135.4.965.
Cells lacking the Dictyostelium 34,000-D actin-bundling protein, a calcium-regulated actin cross-linking protein, were created to probe the function of this polypeptide in living cells. Gene replacement vectors were constructed by inserting either the UMP synthase or hygromycin resistance cassette into cloned 4-kb genomic DNA containing sequences encoding the 34-kD protein. After transformation and growth under appropriate selection, cells lacking the protein were analyzed by PCR analyses on genomic DNA, Northern blotting, and Western blotting. Cells lacking the 34-kD protein were obtained in strains derived from AX2 and AX3. Growth, pinocytosis, morphogenesis, and expression of developmentally regulated genes is normal in cells lacking the 34-kD protein. In chemotaxis studies, 34-kD- cells were able to locomote and orient normally, but showed an increased persistence of motility. The 34-kD- cells also lost bits of cytoplasm during locomotion. The 34-kD- cells exhibited either an excessive number of long and branched filopodia, or a decrease in filopodial length and an increase in the total number of filopodia per cell depending on the strain. Reexpression of the 34-kD protein in the AX2-derived strain led to a "rescue" of the defect in the persistence of motility and of the excess numbers of long and branched filopodia, demonstrating that these defects result from the absence of the 34-kD protein. We explain the results through a model of partial functional redundancy. Numerous other actin cross-linking proteins in Dictyostelium may be able to substitute for some functions of the 34-kD protein in the 34-kD cells. The observed phenotype is presumed to result from functions that cannot be adequately supplanted by a substitution of another actin cross-linking protein. We conclude that the 34-kD actin-bundling protein is not essential for growth, but plays an important role in dynamic control of cell shape and cytoplasmic structure.
为了探究这种多肽在活细胞中的功能，构建了缺乏盘基网柄菌34000-D肌动蛋白束蛋白（一种钙调节的肌动蛋白交联蛋白）的细胞。通过将尿苷一磷酸合酶或潮霉素抗性盒插入包含编码34-kD蛋白序列的4-kb克隆基因组DNA中，构建基因置换载体。在适当的选择条件下转化并生长后，通过对基因组DNA进行PCR分析、Northern印迹和Western印迹，对缺乏该蛋白的细胞进行分析。在源自AX2和AX3的菌株中获得了缺乏34-kD蛋白的细胞。缺乏34-kD蛋白的细胞在生长、胞饮作用、形态发生以及发育调控基因的表达方面均正常。在趋化性研究中，缺乏34-kD蛋白的细胞能够正常移动和定向，但运动的持续性增加。缺乏34-kD蛋白的细胞在移动过程中也会丢失一些细胞质。根据菌株的不同，缺乏34-kD蛋白的细胞要么表现出过多的长而分支的丝状伪足，要么丝状伪足长度减少且每个细胞的丝状伪足总数增加。在源自AX2的菌株中重新表达34-kD蛋白导致运动持续性缺陷以及过多的长而分支的丝状伪足的“挽救”，表明这些缺陷是由于缺乏34-kD蛋白所致。我们通过部分功能冗余模型来解释这些结果。盘基网柄菌中的许多其他肌动蛋白交联蛋白可能能够替代缺乏34-kD蛋白的细胞中34-kD蛋白的某些功能。推测观察到的表型是由其他肌动蛋白交联蛋白替代无法充分完成的功能导致的。我们得出结论，34-kD肌动蛋白束蛋白对生长不是必需的，但在细胞形状和细胞质结构的动态控制中起重要作用。
