THE DICTYOSTELIUM-DISCOIDEUM 30,000-DALTON PROTEIN IS AN ACTIN FILAMENT BUNDLING PROTEIN THAT IS SELECTIVELY PRESENT IN FILOPODIA
THE DICTYOSTELIUM-DISCOIDEUM 30,000-DALTON PROTEIN IS AN ACTIN FILAMENT BUNDLING PROTEIN THAT IS SELECTIVELY PRESENT IN FILOPODIA
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DOI：
10.1083/jcb.104.6.1539
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发表时间：
1987-06-01
影响因子：
7.8
通讯作者：
FECHHEIMER, M
中科院分区：
文献类型：
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作者：
FECHHEIMER, M
研究了细胞黏菌盘状盘牙霉（Dictyostelium disideum） 30,000-D肌动蛋白结合蛋白与肌动蛋白的相互作用和细胞内定位，以分析该蛋白对细胞结构和运动的潜在贡献。通过电子显微镜、光散射、粘度和偏振显微镜观察到含有肌动蛋白和30,000-D蛋白的各向异性交联丝网络（束）的形成。共沉淀实验表明，最多一个30,000-D蛋白分子可以与10个肌动蛋白单体结合，其表观结合常数为1倍。107升/摩尔。通过粘度测定、光散射、偏振显微镜和直接结合实验观察到镁或钙对30000 - d蛋白与肌动蛋白相互作用的抑制作用。然而，减少相互作用所需的镁浓度是钙的100倍。30000 - d蛋白与肌动蛋白的结合常数为4.2倍。106升/mol，或者小于1倍。在Mg2+或Ca2+浓度增加的情况下，分别达到105升/mol。酶联免疫测定表明，30000 - d蛋白占盘状棘球蚴蛋白的0.04%。通过测量30000 - d蛋白的浓度及其对肌动蛋白的亲和力，预测了30000 - d蛋白与细胞质中肌动蛋白的广泛相互作用。采用单特异性亲和纯化多克隆抗体，免疫荧光显微镜分析30000 - d蛋白的分布。30,000-D蛋白在细胞质中呈弥漫性分布，不存在于突出的细胞器中，并且在细胞表面突出的细延伸中相当突出。这些含有30,000-D蛋白的提取物的数量、长度和分布与扫描电镜观察到的丝状伪足相似。为了分析细胞厚度和细胞器分布对免疫荧光定位的影响，将荧光素标记的牛血清白蛋白掺入活细胞的细胞质中，然后用超声加载技术进行固定和染色。结果表明，30000 - d蛋白被选择性地整合到丝状足中。这些结果提供了多种肌动蛋白交联蛋白之间的明确区别，并表明30,000-D蛋白有助于丝状足中肌动蛋白丝束的组织。
The interaction with actin and intracellular localization of the 30,000-D actin-binding protein from the cellular slime mold Dictyostelium discoideum have been investigated to analyze the potential contributions of this protein to cell structure and movement. The formation of anisotropic cross-linked filaments networks (bundles) containing actin and the 30,000-D protein has been observed by electron microscopy, light scattering, viscometry, and polarization microscopy. Cosedimentation experiments indicate that a maximum of one molecule of the 30,000-D protein can bind to 10 actin monomers in filaments with an apparent association constant of 1 .times. 107 liters/mol. Inhibition of the interaction of the 30,000-D protein with actin by either magnesium or calcium was observed by viscometry, light scattering, polarization microscopy, and direct binding assays. However, the concentration of magnesium required to diminish the interaction is > 100 times greater than that of calcium. The association constant of the 30,000-D protein for actin is 4.2 .times. 106 liters/mol, or < 1 .times. 105 liters/mol in the presence of increased concentrations of either Mg2+ or Ca2+, respectively. Enzyme-linked immunoassays indicate that the 30,000-D protein comprises 0.04% of the protein in D. discoideum. Extensive interaction of the 30,000-D protein with actin in cytoplasm is predicted from these measurements of the concentration of this protein and its affinity for actin. The distribution of the 30,000-D protein was analyzed by immunofluorescence microscopy using monospecific affinity-purified polyclonal antibody. The 30,000-D protein exhibits a diffuse distribution in cytoplasm, is excluded from prominent organelles, and is quite prominent in fine extensions protruding from the cell surface. The number, length, and distribution of these extractions containing the 30,000-D protein are similar to those of filopodia observed by scanning electron microscopy. To analyze the effects of cell thickness and the distribution of organelles on the immunofluorescence localization, fluorescein-labeled BSA was incorporated into the cytoplasm of living cells before fixation and staining using a sonication loading technique. The results indicate that the 30,000-D protein is selectively incorporated into filopodia. These results provide a clear distinction between the multiple actin-cross-linking proteins present in D. discoideum, and suggest that the 30,000-D protein contributes to organization of bundles of actin filaments in filopodia.