High-resolution three-dimensional views of membrane-associated clathrin and cytoskeleton in critical-point-dried macrophages
High-resolution three-dimensional views of membrane-associated clathrin and cytoskeleton in critical-point-dried macrophages
复制标题
临界点干燥巨噬细胞中膜相关网格蛋白和细胞骨架的高分辨率三维视图
作者：
J. Aggeler;R. Takemura;Z. Werb
我们获得了高分辨率的拓扑信息网格蛋白和细胞骨架丝的细胞质膜表面的巨噬细胞扩散到玻璃盖玻片上的临界点干燥破碎的开放细胞和旋转铂副本的制备。不规则补丁的质膜的粘附腹面暴露在这些细胞中，这些暴露的膜大面积覆盖网格蛋白包被的补丁，坑，和囊泡。不同数量的细胞骨架附着在这些伸展细胞的质膜上，或者作为不同的星形焦点，或者作为从细胞骨架网辐射出来的单个细丝和束。在新贴壁细胞中，观察到发育良好的Golgi-GERL复合体，其特征在于与粗面内质网相关的平滑的盘状池。有许多被膜小泡从高尔基池出芽，这些主要是大的类型（150 nm）通常与质膜。在临界点干燥的样品中，细胞骨架和细胞膜在适当固定后，与速冻样品的细胞骨架和细胞膜的细节相当。临界点干燥细胞的旋转复制提供了一种快速、易于控制且通常易于进行的方法，用于获得足够大的暴露膜样品，以允许在各种实验条件下定量膜相关网格蛋白和细胞骨架。
We obtained high-resolution topographical information about the distribution of clathrin and cytoskeletal filaments on cytoplasmic membrane surfaces of macrophages spreading onto glass coverslips by both critical-point drying of broken-open cells and preparation of rotary platinum replicas. Irregular patches of the adherent ventral surface of the plasma membrane were exposed in these cells, and large areas of these exposed membranes were covered with clathrin-coated patches, pits, and vesicles. Various amounts of cytoskeleton were attached to the plasma membranes of these spreading cells, either as distinct starlike foci, or as individual filaments and bundles radiating out from the cytoskeletal meshwork. In newly adherent cells a well developed Golgi-GERL complex, characterized by smooth, dish-like cisternae associated with rough endoplasmic reticulum, was observed. There were many coated vesicles budding off from the Golgi cisternae, and these were predominantly of the large type (150 nm) usually associated with the plasma membrane. In critical-point-dried samples, both cytoskeleton and membranes were preserved in detail comparable to that of quick-frozen samples, after appropriate fixation. Rotary replication of critical-point-dried cells provides a rapid, easily controlled, and generally easy to perform method for obtaining samples of exposed membrane large enough to permit quantification of membrane- associated clathrin and cytoskeleton under various experimental conditions.
影响因子：
56.9
作者：
ROTHMAN, JE
通讯作者：
ROTHMAN, JE