Spatial organization of cytoskeletal actin filaments and their regulatory proteins revealed by advanced methods in electron microscopy.

目前的研究旨在揭示核膜外围细胞骨架肌动蛋白细丝的空间组织。到目前为止，仅用荧光显微镜研究了活细胞中的细胞骨架肌动蛋白细丝。因此，观察仅限于细胞腹侧荧光较强的应激纤维。为了检测细胞骨架的真实空间结构，应用高压电子显微镜(1000KV)、高分辨率扫描电子显微镜和免疫冷冻蚀刻技术对无顶全细胞进行了观察。核膜上有许多细胞骨架，包括中间丝、肌动蛋白细丝和微管。特别是，中间丝，波形蛋白，从核孔像玫瑰花环一样延伸，形成复杂的网状结构，覆盖整个表面。许多肌动蛋白细丝也附着在核膜上。由于S1纹饰在细胞核表面清楚地显示出尖端和带刺的末端，因此在核膜上似乎有肌动蛋白细丝的终止和起源。然而，不幸的是，我们无法在核膜上追踪到整个肌动蛋白细丝，因为所有的肌动蛋白细丝都与细胞核表面的波形蛋白细丝联系在一起或覆盖着。在许多电子显微镜下仔细观察，许多肌动蛋白细丝似乎从核孔中延伸出来。事实上，Nesprin 1，肌动蛋白结合蛋白，在核孔的外围被检测到。从核膜延伸出来的肌动蛋白细丝逐渐成束，形成应力纤维。这些都是关于核相关肌动蛋白细丝的全新概念。

Current study aims to reveal the spatial organization of cytoskeletal actin filaments in the periphery of nuclear envelope. Cytoskeletal actin filaments in living cell have been investigated so far exclusively withfluorescent light microscopy. Therefore, observation was restricted on the stress fiber with relatively strong fluorescence and at ventral side of the cell. In order to detect real spatial structure of cytoskeleton, high voltage TEM (1000 KV), high resolution SEM and immuno-freeze etching technique were applied to unroofed whole cells. Nuclear envelope was supplied with many cytoskeleton including intermediate filaments, actin filaments and microtubules. In particular, intermediate filaments, vimentin, extending from nuclear pore like a rosette formed complicated meshwork and covered the total surface. Many actin filaments were attached to nuclear envelope as well. Since S1 decoration showed both pointed and barbed ends clearly in the surface of nucleus, there seemed to be the terminations and origins of actin filaments on the nuclear envelope. Unfortunately, however, we were not able to trace entire actin filaments on the nuclear envelope, because all actin filaments were associated or covered with vimentin filaments on the surface of nucleus. Under careful observation of many electron micrographs, many actin filaments appeared to extend from nuclear pores. Indeed, Nesprin 1, actin binding protein, was detected inthe periphery of nuclear pores. Actin filaments extending from nuclear envelope were bundled gradually to form stress fibers. These are completely new concept on nuclear related actin filaments.