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

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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.