Tetsuo Katsumoto

The role of the vimentin intermediate filaments in rat 3Y1 cells elucidated by immunoelectron microscopy and computer-graphic reconstruction.

Summary— The three‐dimensional arrangement of vimentin intermediate filaments (IF) was studied in 3Y1, rat fibroblastic cell line, to elucidate its biological role in the cell. While actin filaments were observed exclusively in the superficial part of the cell, vimentin IF were found to be abundantly present in the inside of the cell where microtubules were occasionally discovered. By whole‐mount immunoelectron microscopy and computer‐graphic reconstruction of serial thin sections, it was observed in more detail that vimentin IF are located very close to the nucleus, endoplasmic reticulum, and mitochondria. Vimentin IF were observed to be attached to these organelles laterally or terminally. Thus, we can reasonably assume that vimentin IF are major cytoskeletal structures deep inside the cell and that they play an important role in supporting the location of the organelles. This is the first report which has visualized the three‐dimensional relationship between vimentin IF and the organelles of the cell.

Cell-cycle dependent biosynthesis and localization of p53 protein in untransformed human cells

Summary— Localization of p53 in human cultured lymphocytes and in cultured skin fibroblasts was studied by immuno‐fluorescent microscopy and post‐embedded immunoelectron microscopy using Lowicryl K4M. In quiescent lymphocytes, p53 was found in small amounts in both the cytoplasm and the nucleus. p53 in the nucleus was found associated with the non‐chromatin structure. At 24 h or 72 h of PHA stimulation, p53 increased markedly just beneath the plasma membrane and in the nucleus, which stained diffusely with anti‐p53. In resting fibroblasts, small amounts of p53 were present in both the cytoplasm and the nucleus. After 16 h of stimulation of confluent‐resting fibroblasts by trypsinization and replating, a phase just prior to the initiation of DNA synthesis, p53 slightly increased in both the cytoplasm and the nucleus. Afterwards, p53 was present predominantly in the cytoplasm, closely associated with the cytoskeletal actin filaments. In mitotic cells, p53 was distributed throughout the cytoplasm. When fibroblasts were extracted with saponin, p53 was still associated with the actin filaments, as well as mitochondrial membranes and granular structures of the nuclear matrix. Our data suggest that the initial increase of p53 in cells that enter the cell cycle through G1 first bind to the actin cytoskeleton, and that some of the p53 then move into the nucleus to initiate gene activation and DNA synthesis for cell proliferation. This implies that there is some functionally significant interaction between p53 and actin in the cells.

Accumulation of cholesterol and GM2 ganglioside in cells cultured in the presence of progesterone: an implication for the basic defect in Niemann-Pick disease type C.

Cultured fibroblasts from patients with Niemann-Pick disease type C (NP-C) are characterized by lysosomal accumulation of unesterified cholesterol and a defect in intracellular trafficking of cholesterol. We have found the accumulation of GM2 ganglioside in NP-C fibroblasts [Yano T, Taniguchi M, Akaboshi S, Vanier MT, Tai T, Sakuraba H, et al. Proc Japan Acad 1996;72B:214-219]. In this communication we show that several inhibitors known to inhibit intracellular cholesterol transport, progesterone, imipramine and KN-62, elicit accumulation of not only unesterified cholesterol but also GM2 ganglioside. This finding suggests that intracellular transport of cholesterol may be coupled with that of GM2 ganglioside. The accumulation of free cholesterol and GM2 ganglioside may be a clue for understanding the basic defect of NP-C. Recently NPC1 gene is found by the positional cloning. The mechanism of accumulating of GM2 ganglioside should be further investigated by studying of the functions of NPC1 gene.

Immunocytochemical localization of actin in the nucleolus of rat oocytes

Summary— In order to determine the localization of actin, growing and fully grown rat oocytes were immunocytochemically examined using a post‐embedding ultrastructural protein‐A gold technique. In quiescent oocytes, the nucleoplasm showed slightly lower levels of actin signal when compared to the surrounding cytoplasm. The highest levels of labeling were found on nucleoli showing a reticular type morphology. In oocytes at the diakinesis stage in which nucleolar compaction had occurred, the levels of labeling increased by 5–6 times those found in quiescent oocytes. Except for conspicuous accumulation of actin under the plasma membrane, compact nucleoli had significantly higher levels of labeling when compared with those found on the general cytoplasm, while the nucleoplasm with homogeneously dispersed chromatin showed significantly lower levels of associated actin signal than the general cytoplasm. In oocytes at metaphase I, the cytoplasmic region had comparable or lower levels of labeling than the cytoplasm of oocytes at diakinesis. The meiotic spindle embedded in material with medium electron density showed a similar level of labeling as the surrounding cytoplasm. On the other hand, significantly higher levels of associated actin were observed on the chromosomes of metaphase I. The actin signals were dispersed over the chromosomes and not concentrated on a specific region. These results suggest that nuclear actin may be involved in the process of chromosome construction and also the formation of the compacted structure of the nucleolus.

Maturation of human immunodeficiency virus, strain LAV, in vitro

The budding process of human immunodeficiency virus (HIV), strain LAV, starts with the formation of a crescent electron-dense layer directly underneath the cell membrane of infected CCRF-CEM cells. After completion of the formation of the circle of inner dense layer, immature virions with an electron-lucent center are released from the cells. Serial thin sections and stereo observation of thick sections showed that most of the immature virions adjacent to the cell surface had already come off the cell and some still had very thin connections to the cell. However, on rare occasions, virions at an intermediate stage between immature stage and mature virions with bar-shaped electron-dense cores were observed. Virions with dense cores were never observed to be connected to the cell surface. These observations support the idea that the last step of the maturation of HIV occurs outside the cell and that the electron-dense core seems to develop by rearrangement and dispersion of the substance of the inner dense layer of immature virions.

The orientation of primary cilia during the wound response in 3Y1 cells

Summary— The behavior of the primary cilia of 3Y1 cells in the interphase was investigated by indirect immunofluorescence microscopy and transmission electron microscopy, using an antibody for tubulin. At 4.5 h after scraping a part of a confluent cell sheet, the primary cilia of cells facing the wound were located predominantly forward of the nucleus on the wounded side, and were oriented in the direction of the leading lamellae. Cytoplasmic microtubules (MTs), emanating from around the base of the cilia, were well developed in the leading lamellae on the wounded side. On the other hand, in the cells of an unperturbed area away from the wounded edge, the primary cilia remained randomly distributed near the nucleus. The position and a certain well‐defined orientation of a pair of centrioles seem to play an important role for the development of cytoplasmic MTs, and consequently the orientation of the centrioles is controlled by the primary cilia.

Morphological phenotype of temperature-sensitive mutants of simian virus 40 in productive infection

Abstract Temperature-sensitive mutants of simian virus 40 from the complementation groups I (viral-DNA positive and V-antigen positive), II (viral-DNA positive and V-antigen negative), and III (viral-DNA negative and V-antigen negative) were examined by electron microscopy for their ability to synthesize physical virus particles at the nonpermissive temperature (40°) in productive infection. Examinations of thin sections of infected cells revealed that all the group I mutants studied were not temperature-sensitive for the synthesis of physical virus particles in terms of the proportion of cells synthesizing virions, approximate number and mode of arrangement of virions in the virus-producing cells, and morphology of virions present in the cells. Representative mutants of the complementation groups II and III were temperature-sensitive for the synthesis of virions or virion-related structures. The virus particles of the group I mutants produced at 40°, in contrast to those produced at the permissive temperature (33°), were readily destroyed after lysing the cells by repeated freezing and thawing or by sonication, but the lysate still retained V antigen, the amount of which was similar to that in lysates from the mutant-infected cells at 33° and from wild-type-infected cells at 33 and 40°. Temperature-shift experiments suggested that: (1) The event that is temperature sensitive in a group I mutant infection occurs about the time of the appearance of new infectious virions, suggesting that the mutation affects the final stage of virus maturation; (2) the affected function of the group II mutant occurs at about 10 hr prior to the time of the appearance of new infectious virions at 33°, although the mutant employed may be regarded as a late mutant because of the heat lability of the virions produced at 33° and because viral DNA synthesis is normal at 40°; and (3) the group III mutant temperature presented suggest that the three genetically distinct groups of mutants represent three functionally distinct processes in the replicative cycle of simian virus 40.

HETEROTRANSPLANTATION OF ARGYROPHIL SMALL CELL CARCINOMA OF THE UTERINE CERVIX INTEGRATING HPV16 DNA INTO NUDE MICE

Argyrophil small cell carcinoma of the uterine cervix (ASCC) containing human papillomavirus type 16 (HPV16) DNA has been successfully transplanted in nude mice for the first time, and we have designated the resultant cell line as YIK‐1. Histology of YIK‐1 was similar to that of the original tumor. The original and YIK‐1 tumor cells contained argyrophil granules and neurosecretory granules in the cytoplasm, and were immunohistochemically stained positive for neuron‐specific enolase, serotonin and chromogranin. Both tumors contained HPV16 DNA in a multiple‐copy integrated form. Thus, YIK‐1 maintains the characteristics of the original ASCC, and may therefore be useful as an animal system for experimental studies of ASCC.

Cellular senescence of angiofibroma stroma cells from patients with tuberous sclerosis

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by epilepsy, mental retardation and hamartomatous lesions in multiple organs. It has been shown that the genes responsible for TSC, TSC1 and TSC2, act as tumor suppressors, but the mechanism of hamartomatous growth in several tissues is not completely understood. The TSC hamartomas are essentially benign and they rarely progress to malignant tumors. In this report, we cultured the angiofibroma stroma cells of three adult TSC patients and compared these cells with normal skin fibroblasts for their proliferative capacity, cell morphology and mitotic cycle using a stain for microtubules and the expression of the senescent associated beta-galactosidase (SA beta-Gal). Cultured angiofibroma stroma cells from TSC patients displayed several characteristics observed in human senescent fibroblasts; a low proliferative capacity, an increase in cell size, increased binucleated cells in association with abnormal cytokinesis and increased SA beta-Gal positives. Growth of facial angiofibromas in TSC may be caused by a gain in enhanced sensitivity toward some of the potential mitogens and forced multiplication without loss of the cellular senescent program; this may be the reason why TSC hamartomas rarely progress to malignancy and why the growths are limited to a finite size.

Establishment and characterization of a new cell line TC-YIK originating from argyrophil small cell carcinoma of the uterine cervix integrating HPV16 DNA.

A new cell line, designated TC‐YIK, was established from YIK‐1 tumor cells, derived from argyrophil small cell carcinoma (ASCC) of the uterine cervix, and serially heterotransplanted into nude mice, integrating human papillomavirus type 16 (HPV16) DNA. The population doubling time of TC‐YIK was approximately 21.6 hours at the 119th subculture. Subcutaneous injection of 1 × 108 TC‐YIK cells into nude mice yielded a solid tumor. The cytologic appearance of TC‐YIK was similar to that of YIK‐1. The TC‐YIK cells contained argyrophil granules and neurosecretory granules in the cytoplasm and showed positive immunohistochemical staining for neuron‐specific enolase, serotonin, and chromogranin. Thus, TC‐YIK retained the histochemical characteristics of ASCC. The TC‐YIK cells contained HPV16 DNA in a multiple‐copy integrated form and actively transcribed the integrated HPV16 genome. Amplification of the c‐myc oncogene was observed in the TC‐YIK cells. These data suggest that TC‐YIK is a useful in vitro experimental model of ASCC and that HPV16 and c‐myc may play some role in the genesis of this malignant tumor and/or maintenance of the transformed TC‐YIK phenotype.