Dictyostelium discoideum cells lacking the 34,000-dalton actin-binding protein can grow, locomote, and develop, but exhibit defects in regulation of cell structure and movement: a case of partial redundancy.
Dictyostelium discoideum cells lacking the 34,000-dalton actin-binding protein can grow, locomote, and develop, but exhibit defects in regulation of cell structure and movement: a case of partial redundancy.
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DOI：
10.1083/jcb.135.4.965
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发表时间：
1996-11
期刊：
影响因子：
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通讯作者：
Fechheimer M
中科院分区：
文献类型：
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作者：
Rivero F;Furukawa R;Noegel AA;Fechheimer M
细胞缺乏Dictyosteoprotein 34，000-D肌动蛋白捆绑蛋白，钙调节肌动蛋白交联蛋白，被创建来探测这种多肽在活细胞中的功能。通过将UMP合酶或潮霉素抗性盒插入克隆的含有编码34-kD蛋白的序列的4-kb基因组DNA中来构建基因置换载体。在转化和在适当选择下生长后，通过基因组DNA的PCR分析、北方印迹和Western印迹分析缺乏蛋白质的细胞。在来源于AX 2和AX3的菌株中获得缺乏34-kD蛋白的细胞。生长、胞饮、形态发生和发育调控基因的表达在缺乏34-kD蛋白的细胞中是正常的。在趋化性研究中，34-kD-细胞能够正常移动和定向，但表现出运动的持久性增加。34 kD-细胞在运动过程中也丢失了少量的细胞质。34-kD-细胞表现出要么过多的长和分支的丝状伪足，或丝状伪足长度的减少和丝状伪足的总数增加每个细胞取决于应变。AX 2衍生菌株中34-kD蛋白的再表达导致了运动持续性缺陷和长且分支的丝状伪足数量过多的“拯救”，表明这些缺陷是由于缺乏34-kD蛋白造成的。我们解释的结果，通过模型的部分功能冗余。网骨藻中的许多其他肌动蛋白交联蛋白可能能够替代34-kD细胞中34-kD蛋白的某些功能。推测观察到的表型是由不能被另一种肌动蛋白交联蛋白取代的功能引起的。我们的结论是，34-kD肌动蛋白捆绑蛋白是不是必不可少的增长，但在动态控制细胞的形状和细胞质结构中起着重要的作用。
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.