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首页> 美国卫生研究院文献>The Journal of Biophysical and Biochemical Cytology >Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks
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Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks

机译：肌动蛋白在培养细胞前沿的组织：四氧化和脱水对肌动蛋白网状结构超微结构的影响

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The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X- 100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.
机译：在用Triton X-100-戊二醛混合物短暂提取，进一步固定在戊二醛中，然后进行负染色以进行电子显微镜检查的细胞中，容易显示出培养的成纤维细胞前缘（层状脂质）的有序结构。通过该程序，前缘区域显示了高度组织的肌动蛋白丝的三维网络以及可变数量的辐射肌动蛋白丝束或微穗。戊二醛固定后使用鬼笔环肽导致细丝顺序的改善。通过常规电子显微镜通常采用的附加步骤来处理细胞骨架，导致肌动蛋白丝网络的顺序显着恶化或完全破坏。相反，应力纤维束的肌动蛋白丝基本不受影响。因此，四氧化的后固定（室温下为1％，在室温下放置7分钟）将网络转变为扭结纤维的网状结构，类似于在体外将肌肉F-肌动蛋白暴露于OsO4所产生的网状结构（P. Maupin-Szamier和TD Pollard 1978.J.Cell Biol.77：837--852）。尽管有限地暴露于OsO4（在0摄氏度下于20分钟内为0.2+）避免了这种破坏，但在丙酮或乙醇中进行脱水，无论是否进行后渗透，都会导致网丝的进一步不可避免的混乱和聚集。然后，前缘的网状区域与迄今为止在培养细胞的关键点干燥制剂中描述的网络表现出惊人的相似性。我得出的结论是，Wolosewick和Porter（1979. J. Cell Biol。82：11​​4--139）在后一种制剂中描述的许多“微小梁网格”构成了肌动蛋白网状结构和肌动蛋白丝阵列，以及它们的相关组件。通过使用的准备程序进行扭曲和汇总。

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