Ultrastructural studies of hepatocyte cytoskeleton in experimental cholestasis by quick-freezing and deep-etching method

The ultrastructural association between the cytoskeleton and other organelles was studied by the quick-freezing and deep-etching method in rats treated with alpha-naphthylisothiocyanate (ANIT), or phalloidin, and in rats with obstructive jaundice. Cytoplasmic filaments were classified by measuring their diameters, and actin filaments were identified by specific decoration with myosin subfragment 1 (S1). S1-positive actin filaments and S1-negative intermediate filaments (12-14 nm in diameter) were observed to form a three-dimensional network around bile canaliculi, and were more numerous than in controls, not only in phalloidin-treated rats and rats with obstructive jaundice, but also in ANIT-administered rats. In all cholestatic rats, vesicular structures were also more numerous than in controls in the pericanalicular regions, and were closely associated with the microfilaments and the intermediate filaments. Filaments of a new type were localized between the lamellae of rough-surfaced endoplasmic reticulum and mitochondria, and between the lamellae of Golgi sacs and vesicles. Other thin filaments were also observed within the network of actin filaments. These filaments were 4-6 nm in diameter on replica membranes and were never decorated with S1. They were also directly connected with the canalicular membranes. Cytoskeletal components associated with membrane-bound organelles, including these new filaments, were suggested to be involved in the localization and migration of organelles.     

Ultrastructural studies of hepatocyte cytoskeletons of phalloidin-treated rats by quick-freezing and deep-etching method

We observed hepatocyte cytoskeletons in phalloidin-treated rats by the quick-freezing and deep-etching method in three dimensions and compared them with the ultrastructural findings on conventional ultrathin sections. The numbers of microvilli in dilated bile canaliculi were decreased in the rats treated with phalloidin for 1 wk. In hepatocytes the cytoplasm around bile canaliculi could be divided into three layers, increased microfilament layer, cell organelle layer of secretory system and increased smooth surface endoplasmic reticulum layer. In the rats treated with phalloidin for 4 wk, microfilaments were extended into the cytoplasm near the nucleus in addition to the increased number of large lysosomes and microtubules. In both groups, three-dimensional structures of microfilaments could be visualized around bile canaliculi and along cell borders by the quick-freezing and deep-etching method. The branching microfilaments with the diameters of 7 to 10 nm were directly attached to other filaments, cell organelles or cytoplasmic sides of cell membranes. Moreover, bundled intermediate filaments were increased around peribiliary microfilaments associated with long-term cholestasis. It is suggested that excessive accumulation of peribiliary microfilaments disturb the secretion of bile components into bile canaliculi. The cytoskeletal reorganizations of intermediate filaments seem to alter the arrangements of various cell organelles.     

Immunoelectron microscopic observation of alpha-fetoprotein synthesis in human non-malignant liver tissues using immunoperoxidase methods

Alpha-fetoprotein (AFP) synthesis in non-malignant liver tissue of 34 patients with chronic hepatitis or liver cirrhosis, some of whom also had hepatocellular carcinoma (HCC), was studied by light and ultrastructural immunohistochemistry using peroxidase-labeled anti-human AFP. Simultaneously, the serum level of AFP was measured in these patients by radioimmunoassay. AFP-positive cells were identified in non-malignant liver tissue of 7 patients with elevation of serum AFP. AFP was demonstrated in several hepatocytes which were clustered in hepatic lobules, and also in some bile ductular cells which were distributed in the periphery of portal tracts. In an immunoelectron microscopic study of AFP-positive hepatocytes, dense reaction products of anti-AFP were localized in the membranes and cisternae of rough endoplasmic reticulum (r-ER), perinuclear space (PNS) and Golgi apparatus. The prominent feature of AFP-positive hepatocytes was abundant r-ER encompassing many mitochondria. As to AFP-positive bile ductular cells, they had scanty cytoplasm and few intracytoplasmic organelles and were surrounded by basement membrane. AFP was focally localized in the r-ER of such bile ductular cells. These observations suggest that AFP can be produced by malignant and non-malignant liver cells and that in non-malignant liver tissues, AFP can be produced by two distinct cell types; bile ductular cells and hepatocytes themselves.     

Polarized thyroid cells in monolayers cultured on collagen gel: their cytoskeleton organization, iodine uptake, and resting membrane potentials

Porcine thyroid cells were cultured on collagen gel-coated cover glasses. They were reorganized into polarized monolayer cells; the basal cell membranes were in contact with the collagen gel, and the apical ones faced the culture medium. We studied cytoskeleton organization, resting membrane potentials, and iodine uptake of these cells. The quick-freezing and deep-etching replica method provided three-dimensional images of the cytoskeleton organization. Networks of microfilaments were observed under the apical cell membrane. In the deep cytoplasm and near the basal cell membranes, intermediate filaments predominated and were interlinked with the microfilaments. When the cells were cultured in the presence of TSH, TSH induced the formation of microvilli at the apical cell membranes and the accumulation of microfilaments under these membranes; in the deep cytoplasm, the intermediate filaments were more closely interlinked with the microfilaments. The microfilaments were immunostained with antiactin antibody. Thus, collagen is a factor in determining the cell polarity, and TSH further augments polarization through reorganizing the cytoskeletons. Electrophysiological study revealed that the resting membrane potential of cells cultured in the absence of TSH was -46 mV, and that of cells cultured in the presence of TSH was -58 mV. TSH hyperpolarized resting membrane potentials. These cells took up iodine. TSH in the medium augmented this uptake. TSH augments thyroid cell polarization through reorganizing the cytoskeletons and hyperpolarizing the resting membrane potentials and enhances iodine uptake by the cells.     

Anti-liver-kidney microsome antibody is a marker for the rat hepatocyte endoplasmic reticulum

Human sera, containing anti-liver-kidney microsome antibody as demonstrated by indirect immunofluorescence, were obtained from a subgroup of young patients with autoimmune chronic hepatitis. The anti-liver-kidney microsome antibody-positive sera were used to study the localization of the liver-kidney microsome antigen in hepatocytes. Immunoblot analysis of microsomal subfractions, lysosomal membranes, plasma membranes, mitochondria and purified ribosomes obtained from rat liver demonstrated that this antibody recognizes a protein of 50 kD present only in endoplasmic reticulum membranes. Immunogold labeling of ultrathin frozen sections and immunoperoxidase staining of 11 to 15 micron cryostat sections were used to detect the liver-kidney microsome antigen in rat liver tissue. The anti-liver-kidney microsome antibody binds to antigenic domains on the cytoplasmic face of smooth and rough endoplasmic reticulum membranes of hepatocytes. No labeling was observed of the Golgi apparatus, peroxisomes, mitochondria, lysosomes, nuclei or plasma membranes. Not only was the antigen recognized by the anti-liver-kidney microsome antibody specific for endoplasmic reticulum membranes, but it was also specific for the endoplasmic reticulum of hepatocytes only, since no labeling was observed in any organelle of Kupffer or endothelial cells. Therefore, the anti-liver-kidney microsome antibody can be considered as a marker for endoplasmic reticulum in rat hepatocytes.     

A morphometric study of the endoplasmic reticulum in human hepatocytes : Correlation between morphological and biochemical data in subjects under treatment with certain drugs

The distribution of the endoplasmic reticulum in human hepatocytes is defined in quantitative terms using the techniques of morphometry. The subjects of the study are liver biopsies from normal, untreated subjects and patients being treated with various drugs. In contrast to rat hepatocytes, the amount of smooth endoplasmic reticulum (SER) in man exceeds that of the rough endoplasmic reticulum (RER) and accounts for 76.3% of the total endoplasmic reticulum. This is to be taken into consideration in pharmacological or toxicological studies. In addition, two components of the SER have been identified: more prominent is the type 1 or vesicular which has a regular honeycomb pattern, made up of cisternae with patent lumina and a mean width of 1500 A; the type 2, or non-vesicular, occurs in discrete foci of densely packed smooth membranes with a spacing of about 140 A. In subjects under short-term treatment with Benzodiazepin (diazepam) the RER remained unchanged but the SER membranes were significantly increased with a remarkable, two-to threefold increase of the SER type 2 in three out of four patients. A rise in incorporation of (14)C-acetate into digitonin-precipitable sterols as measured in liver biopsy material was also noted in these three patients. The suggestion is made that the SER 2 represents the newly formed membranes whereas the SER 1 would represent ;adult' membranes. No changes were observed in two patients under short-term treatment with phenobarbital or Dilantin.     

Morphological findings in the liver of children with cystic fibrosis: a light and electron microscopical study

Liver tissue from five children with cystic fibrosis, obtained through percutaneous liver biopsies, have been investigated via light and electron microscopy. None of the patients had clinical evidence of liver disorder, and their blood chemistry was mainly normal. Light microscopy showed slight fibrosis in three cases, more advanced fibrosis in one case and focal cirrhotic changes in one case. All patients had fatty infiltration in the hepatocytes and glycogen in the nuclei of these cells. Electron microscopy showed an increase in the number of Ito cells around the portal tracts and also fibrosis in all patients. In the majority of hepatocytes, no evident necrosis was seen. Hypertrophy of the smooth endoplasmic reticulum and the Golgi apparatus were noted. Large lysosomes containing lipofuscin and lipids were also present. No direct evidence of cholestasis could be seen in the hepatocytes. The bile canaliculi were not dilated and did not contain bile plugs. No bile pigment was seen in the cells, and direct evidence of cholestasis was thus not found in the hepatocytes. Other organelles, such as the rough endoplasmic reticulum, peroxisomes and mitochondria, had a normal appearance. Bile ducts, even when seen in fibrotic portal tracts, were not dilated. The ultrastructural findings cannot explain the basis for the liver cell damage. Cholestasis does not seem to be a presumable etiological factor as judged from the findings in the present study.     

Ultrastructural localization of cations in the rat pars distalis under various experimental conditions.

Modifications of the pyroantimonate technique were used to localize intracellular sites of bound cations in the pars distalis of normal and hypothyroid rats and in rats with increased levels of plasma calcium. In the normal animal, cations were localized within most intracellular organelles and sometimes on the membranes involved in exocytosis. The amount of bound cations within pituitary intracellular organelles was considerably augmented in rats injected intravenously with calcium chloride. However, in the thyroidectomy cells of hypothyroid rats, the amount of cation precipitate appeared to be selectively increased in the dilated rough endoplasmic reticulum (RER). The calcium chelator, EGTA, and X-ray microprobe analysis revealed the presence of calcium in cation deposits.     

Ultraviolet radiation (UV) induces reorganization of actin cytoskeleton in CHOAA8 cells

Effect of UV radiation on actin cytoskeleton was studied in CHOAA8 cells by fluorescence and electron microscopy. UV irradiated cells showed impaired adherence, disruption of the actin filaments and stronger F-actin labeling in the center of the cell. Attached cells, especially enlarged ones showed rather weak labeling of stress fibers and bundles of F-actin in the cytoplasm, but some cells with intensive labeling of these structures were also observed. Detached cells were rounded, showed strong F-actin labeling and often had buds. At the ultrastructural level UV-irradiated cells showed segmented nuclei, bodies resembling micronuclei, dilatation of endoplasmic reticulum, swollen and disturb mitochondria. Immunogold labeling of actin at the ultrastructural level was observed in non-radiated and UV irradiated cells. Actin labeling was seen in nuclei and cytoplasm. In nuclei gold particles were localized in the area of condense chromatin. Labeling for actin was not found after control incubation. Our observations show that UV radiation promotes changes in the distribution of actin in CHOAA8 cells. The results also suggest that not only reorganization of actin but changes in organelles are involved in the process of apoptosis initiated by UV radiation.     

Effect of vanadate on intracellular distribution and function of 10-nm filaments

Earlier reports from this laboratory suggested that 10-nm filaments and microtubules act together in the movement and positioning of nuclei and centrioles. Sodium vanadate has been found to alter the distribution of 10-nm filaments and separate them from microtubules in virus-induced syncytia and uninfected cells. Accompanying this change in cytoskeletal elements in an alteration in the distribution of nuclei, centrioles, and other organelles. Nuclei in vanadate-treated syncytia were found in a circle or horseshoe arrangement, and 10-nm filaments were aggregated within the circle, whereas microtubules, were found in a network throughout the cytoplasm. Vanadate also caused a perinuclear aggregation of 10-nm filaments in single uninfected cells, whereas microtubules were throughout the cytoplasm, as in syncytia. Centrioles, mitochondria, rough endoplasmic reticulum, and lysosomes were scattered in the perinuclear area, with mitochondria and rough endoplasmic reticulum frequently closely associated, whereas the peripheral region of vanadate-treated cells contained ribosomes, microfilament bundles, and microtubules, but not 10-nm filaments. Vanadate limited virus-induced fusion of cells to polykaryocytes with 5--20 nuclei, in contrast to the massive syncytia found in untreated cells. These results indicate that vanadate separates 10-nm filaments and microtubules topologically and functionally, and support previous evidence that 10-nm filaments and microtubules act together in the movement and positioning of nuclei and other organelles.     

Effects of chlorpromazine and low calcium on the cytoskeleton and the secretory function of hepatocytes in vitro

It has been established that the cytoskeleton plays an important role in the secretory function of hepatocytes. We studied the effect of chlorpromazine (CPZ) and low calcium (LC) on the cytoskeleton of primary cultured hepatocytes using double-labelling immunofluorescence and secretion of fluorescein diacetate (FD) into the bile canaliculi (BC). The hepatocytes were obtained from 14-day-old male rats. They were cultured for 24 h in serum-free William's E medium with insulin and dexamethasone added to induce differentiation including bile canaliculus formation. After incubation with CPZ (200 microM) for 1 h, the BC became dilated and distorted and formed diverticula. Actin filaments around the BC became more prominent and the stress fibers decreased. CPZ did not affect the microtubules or cytokeratin filaments. Exposure to LC (20 microM) for 24 h caused a slight dilation of the BC. Actin spread out over the cell periphery and appeared non-filamentous. Actin filaments around the BC appeared unchanged and the stress fibers disappeared. Microtubules and cytokeratin filaments were unaffected. Secretion of FD into the BC occurred normally after treatment with CPZ or LC. These results support the idea that the integrity of actin is not necessary for secretory function and that microtubules and intermediate filaments play a role in this process. The dilatation and diverticular formation in the BC induced by CPZ treatment suggests that a cytochalasin-like loss of contraction of the BC may explain the CPZ-induced decrease in bile flow observed in vivo.     

Lovastatin, an inhibitor of cholesterol synthesis, induces hydroxymethylglutaryl-coenzyme A reductase directly on membranes of expanded smooth endoplasmic reticulum in rat hepatocytes.

Lovastatin is a potent competitive inhibitor of the rate-limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (NADPH) [HMG-CoA reductase; (S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. We determined the subcellular distribution of HMG-CoA reductase at high resolution by means of immunoelectron microscopy on ultrathin frozen liver sections of rats treated with lovastatin and cholestyramine. High concentrations of reductase were located on the outer (cytoplasmic) surfaces of smooth endoplasmic reticulum (SER) membranes induced in hepatocytes by acute drug administration. The enzyme was specifically localized over the whorled SER membranes and was absent from nonwhorled SER, rough endoplasmic reticulum, and peroxisomes. Intense HMG-CoA reductase labeling was only observed in hepatocytes containing high levels of HMG-CoA reductase activity; no staining was detected in untreated livers. These observations show that HMG-CoA reductase is induced as an integral component of the SER membranes that form in rat hepatocytes subsequent to lovastatin treatment and suggest that the formation of SER whorls in rat hepatocytes is due to mechanism-based effects of lovastatin.     

线粒体内质网结构偶联的蛋白组成和功能

线粒体内质网结构偶联(MAM)是线粒体与内质网二者之间形成的一个动态膜偶联结构,并且MAM可以参与这两个细胞器之间信息交流。研究证实MAM参与调控钙信号、脂质平衡、线粒体动态变化、线粒体自噬和内质网应激反应等。MAM与心血管疾病、神经退行性疾病和代谢性疾病等密切相关。本文综述了MAM的蛋白组成、功能以及与疾病的关系。     

Immunoelectron microscopic observation of intrahepatic HBeAg in patients with chronic hepatitis B

Immune light and electron microscopic studies using monoclonal antibodies have been applied to localize HBeAg in liver biopsy specimens of 19 patients with chronic hepatitis B. Under the light microscope, HBeAg was demonstrated in nuclei, cytoplasm and on the cell surface of hepatocytes. The number of HBeAg-positive hepatocytes correlated well with the serum levels of HBeAg (enzyme immunoassay) and DNA-polymerase. Of 11 patients in whom high numbers of HBeAg-positive hepatocytes were found at the light microscopic level, HBeAg was also studied in hepatocytes at the electron microscopic level. The HBeAg in nuclei was either found as aggregates or dispersed diffusely. In the aggregates of HBeAg, the 27-nm core particles were frequently found. In addition, the antigen was found in the cytosol of some hepatocytes as amorphous mass and in some hepatocytes in the cisternae of perinuclear space, endoplasmic reticulum and Golgi saccules. Occasionally the antigen was found on the membranes of the cell organelles and on the plasma membranes that faced the intercellular and the Disse spaces. These findings suggest that cytoplasmic HBeAg in hepatocytes may be ultrastructurally classified into two different patterns by its distribution, mainly in endoplasmic reticulum or in cytosol. The ratio varied between hepatocytes with these two types of patterns. The titer of serum HBeAg tended to be higher when the corresponding liver biopsy specimens contained more hepatocytes with HBeAg in endoplasmic reticulum than those with HBeAg in the cytosol.     

High resolution ultrastructural mapping of total calcium: electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve-muscle preparation.

We report on a procedure for tissue preparation that combines thoroughly controlled physical and chemical treatments: quick-freezing and freeze-drying followed by fixation with OsO4 vapors and embedding by direct resin infiltration. Specimens of frog cutaneous pectoris muscle thus prepared were analyzed for total calcium using electron spectroscopic imaging/electron energy loss spectroscopy (ESI/EELS) approach. The preservation of the ultrastructure was excellent, with positive K/Na ratios revealed in the fibers by x-ray microanalysis. Clear, high-resolution EELS/ESI calcium signals were recorded from the lumen of terminal cisternae of the sarcoplasmic reticulum but not from longitudinal cisternae, as expected from previous studies carried out with different techniques. In many mitochondria, calcium was below detection whereas in others it was appreciable although at variable level. Within the motor nerve terminals, synaptic vesicles as well as some cisternae of the smooth endoplasmic reticulum yielded positive signals at variance with mitochondria, that were most often below detection. Taken as a whole, the present study reveals the potential of our experimental approach to map with high spatial resolution the total calcium within individual intracellular organelles identified by their established ultrastructure, but only where the element is present at high levels.     

Fine structural changes in the liver of young and old rats as influenced by microsomal enzyme inducers

When given orally to young and old rats, pregnenolone-16 alpha-carbonitrile, spironolactone, or phenobarbital, known microsomal enzyme inducers, caused an increase in smooth endoplasmic reticulum. Dexamethasone, while a potent microsomal enzyme inducer, did not cause smooth endoplasmic reticulum increase. In both untreated and treated old rats, there was dilatation and vesiculation of rough endoplasmic reticulum with occasional granular material present in vesicles. Cytoplasmic lipid droplets of various sizes were frequent. Some mitochondria exhibited polymorphism and a variation in matrical density. Lysosomes and autophagic vacuoles as well as lipid droplets of various sizes were frequent in all groups. These results show that microsomal enzyme inducers influence the subcellular structure of hepatocytes in old rats.     

Alcoholic hyalin-containing hepatocytes — a characteristic morphologic appearance

ABSTRACT— We studied the morphologic appearance of alcoholic hyalin (AH)-containing hepatocytes in liver biopsies from 14 patients with alcoholic liver disease. Most hepatocytes had a characteristic appearance. The cells were swollen and hydropic with an intact cell membrane. The mitochondria had variable-sized cristae which were both shortened and elongated. The smooth endoplasmic reticulum was markedly decreased. The rough endoplasmic reticulum was bizarre, with detachment of the ribosomes that surrounded the AH. The hepatocytes that contained AH bodies had lost almost all the glucose-6-phosphate activity but had variable amounts of succinic dehydrogenase and diphosphopyridine nucleotide diaphorase activities. The neutrophils admixed with mononuclear cells attached themselves to the hepatocytes and then invaginated into the hepatocytic cytoplasm with focal lysis of the cell membrane mediated via the release of neutrophilic lysosomes. The distortion of protein-synthesizing organelles and decrease in glucose-6-phosphatase activity suggest that the AH-containing hepatocyte is metabolically decompensated. The final cell death may be related to the neutrophilic attack, rather than the metabolic derangement.     

Hepatocellular transplantation --morphological study on hepatocytes transplanted into rat spleen--.

Hepatocellular transplantation into the spleen was investigated as a new attempt in utilizing isolated hepatocytes to compensate for impaired liver function. Present study was undertaken to evaluate morphological and histochemical alterations up to 6 weeks following transplantation in hepatocytes transplanted into the splenic parenchyma. Light microscopic studies revealed viable hepatocellular islets in the splenic parenchyma up to 6 weeks, although minimal cytoplasmic changes were observed. Electron microscopic studies demonstrated moderate changes in organelles, which developed gradually as the time after transplantation proceeded. Distortion and fragmentation of the membranes around the cytoplasm and organelles were not recognized. Moreover, newly formed bile canaliculi and tight junctions which indicate reconstruction of hepatic plates were observed between adjacent cell membranes, and enzyme activities were detected by cytochemical determination of glucose-6-phosphatase in the hepatocytes even 6 weeks after transplantation. The transplanted hepatocytes preserved their characteristic enzyme and fine structures as hepatocytes up to 6 weeks. Our present study based on the persistence of cellular viability suggests that inoculated hepatocytes do maintain their hepatocellular functions after transplantation.     

Alcoholic hyalin-containing hepatocytes--a characteristic morphologic appearance

We studied the morphologic appearance of alcoholic hyalin (AH)-containing hepatocytes in liver biopsies from 14 patients with alcoholic liver disease. Most hepatocytes had a characteristic appearance. The cells were swollen and hydropic with an intact cell membrane. The mitochondria had variable-sized cristae which were both shortened and elongated. The smooth endoplasmic reticulum was markedly decreased. The rough endoplasmic reticulum was bizarre, with detachment of the ribosomes that surrounded the AH. The hepatocytes that contained AH bodies had lost almost all the glucose-6-phosphate activity but had variable amounts of succinic dehydrogenase and diphosphopyridine nucleotide diaphorase activities. The neutrophils admixed with mononuclear cells attached themselves to the hepatocytes and then invaginated into the hepatocytic cytoplasm with focal lysis of the cell membrane mediated via the release of neutrophilic lysosomes. The distortion of protein-synthesizing organelles and decrease in glucose-6-phosphatase activity suggest that the AH-containing hepatocyte is metabolically decompensated. The final cell death may be related to the neutrophilic attack, rather than the metabolic derangement.     

Electron Microscope Studies on Normal Human Myeloid Elements

The development of representative myeloid elements is traced by correlatedlight and electron microscopy. Cytoplasmic changes during maturation ofgranulocytes from the myeloblast include loss of basophilia, development ofthe endoplasmic reticulum complex, decrease in number of mitochondria, andgranule formation. The endoplasmic reticulum vesicles increase in size andnumber during the promyelocyte and myelocyte stages, accompanied by theappearance of non-specific and specific granules, and decrease again duringthe cytosomal maturation of the metamyelocyte. A reduction in number ofmitochondria is noted through the metamyelocyte stage. The apparent continuity of the limiting membranes of both the granules and mitochondria withthose of the cisternae of endoplasmic reticulum suggests a direct connectionamong cytosomal organelles. The role of the endoplasmic reticulum in granulogenesis is discussed. Maturation of the nucleus involves a loss of nucleolar differentiation by a loosening of the compact fibrillar aggregates, and progressivechromatin condensation.

Submitted on April 23, 1963 Accepted on September 6, 1963