THE ARCHITECTURE OF ACTIN-FILAMENTS AND THE ULTRASTRUCTURAL LOCATION OF ACTIN-BINDING PROTEIN IN THE PERIPHERY OF LUNG MACROPHAGES
THE ARCHITECTURE OF ACTIN-FILAMENTS AND THE ULTRASTRUCTURAL LOCATION OF ACTIN-BINDING PROTEIN IN THE PERIPHERY OF LUNG MACROPHAGES
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DOI：
10.1083/jcb.103.3.1007
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发表时间：
1986-09-01
影响因子：
7.8
通讯作者：
SHEVLIN, P
中科院分区：
文献类型：
--
作者：
HARTWIG, JH;SHEVLIN, P
高度分支的细丝网络是巨噬细胞的洗涤剂提取的细胞骨架外围的主要结构，所述巨噬细胞的细胞骨架已经铺展在表面上并且冷冻或临界点干燥，然后用铂-碳旋转遮蔽。这种细丝阵列完全填充从细胞延伸的薄片和纤维，在细胞体的顶部和底部形成0.2-0.5 μ m厚的层。巨噬细胞骨架与抗肌动蛋白IgG和与胶体金结合的抗IgG的反应产生这些细丝的致密染色，并且与肌球蛋白亚片段I孵育均匀地装饰这些细丝，将它们鉴定为肌动蛋白。45%的总细胞肌动蛋白和. apprx. 70%的肌动蛋白结合蛋白保留在洗涤剂不溶性细胞残留物中。可溶性肌动蛋白不是丝状的，通过沉降分析，DNA酶I抑制试验，和电子显微镜，表明细胞骨架不被洗涤剂提取片段化。肌动蛋白网络的分支之间的间距为94 ± 100。47 nm和118 ±. 72 nm的细胞骨架制备的电子显微镜冷冻干燥和临界点干燥。游离丝端是罕见的，除了少数从网络体向上突出或向下延伸到基底的丝端。网络的细丝主要以直角相交，形成具有显著平行性的T形和X形重叠，或者由以120 - 130 °相交的细丝组成的Y形相交。的角度膜层中肌动蛋白丝浓度较高，平均值为12.5mg/ml。浓度是更均匀的冻干制剂比临界点干燥的标本，表明有较少的崩溃与冷冻技术。巨噬细胞皮质细胞质的正交肌动蛋白网络类似于肌动蛋白结合蛋白制成的肌动蛋白凝胶。反应的细胞骨架和肌动蛋白凝胶与肌动蛋白结合蛋白与抗肌动蛋白结合蛋白IgG和抗IgG包被的金珠导致沉积的金簇在丝交叉的点和丝的末端，可能已经与膜接触之前，其去除洗涤剂。在肌动蛋白凝胶与肌动蛋白结合蛋白，75%的肌动蛋白纤维的交叉标记，和交叉点之间的丝间距是一致的，如果每个添加的肌动蛋白结合蛋白分子交联两个丝，形成一个交叉点的凝胶上的理论依据预测。另一方面，只有38%的所有丝交叉标记的巨噬细胞皮质细胞骨架，和连接点之间的丝间距是远远小于预测的肌动蛋白结合蛋白的摩尔比肌动蛋白在细胞骨架。尽管肌动蛋白凝胶与肌动蛋白结合蛋白和巨噬细胞骨架之间的差异，这可能是由于更大的复杂性相比，纯化的蛋白质组装的细胞，网络是定性相似。
A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and fiburcates to form 0.2-0.5 .mu.m thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment I uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and .apprx. 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAse I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 .+-. 47 nm and 118 .+-. 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130.degree. angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of cell cytoskeletons and of an actin gel made with actin-binding protein with anti-actin-binding protein IgG and anti-IgG-coated gold beads resulted in the deposition of clusters of gold at points where filaments intersect and at the ends of filaments that may have been in contact with the membrane before its removal with detergent. In the actin gel made with actin-binding protein, 75% of actin-fiber intersections labeled, and the filament spacing between intersections is consistent with that predicted on theoretical grounds if each added actin-binding protein molecule cross-links two filaments to form an intersection in the gel. On the other hand, only 38% of all filament intersections in the macrophage cortical cytoskeletons labeled, and the filament spacing between junctions is much smaller than that predicted from the molar ratio of actin-binding protein to actin in the cytoskeletons. Despite the differences between actin gels made with actin-binding protein and macrophage cytoskeletons, which presumably arise from the greater complexity of the cell compared with the purified protein assembly, the networks were qualitatively similar.