Programmed cell death: Cytochemical and X-ray microanalytical characterization of calcium compartments in neuromuscular junctions during the normal breakdown of the intersegmental muscles in the giant silkmothAntheraea polyphemus

Summary Calcium stores were cytochemically demonstrated using a combined oxalate—pyroantimonate method in the neuromuscular junctions of the degenerating intersegmental muscles in the giant silkmothAntheraea polyphemus. The elemental composition of punctate precipitates of the reaction product was determined by electron probe X-ray microanalysis of unstained thin sections by energy-dispersive spectrometry and wavelength-dispersive spectrometry. The wavelength-dispersive spectra collected over terminal axons demonstrate a significant calcium signal and a trace of antimony.During the rapid lytic phase of spontaneous muscle degeneration, the calcium punctate deposits were detected in presynaptic terminals in the following sites: the synaptic vesicles and the mitochondria. Calcium precipitates were also found in the dense bodies and the mitochondria encountered in the glial convolutions. No calcium deposit was seen in the synaptic clefts and intercellular spaces of the subsynaptic reticulum of type I and type II. A comparison of calcium to antimony ratios between the terminal axons and the sarcoplasmic lysosomes revealed highly significant differences (P<0.001). Such a variability of the calcium to antimony ratio may be related to different conditions of precipitation or antimony diffusion in the different cell compartments. It was concluded that such synaptic terminals do not appear damaged in spite of the muscle degeneration and presumably continue to perform vital functions while the muscles are no longer contractile 20 h after adult ecdysis.     

Autophagy of mitochondria in rat liver assessed by immunogold procedures

Glutamate dehydrogenase and carbamoyl phosphate synthase-I were localized in rat liver by immunogold procedures, using monoclonal and polyclonal antibodies. As expected, there was extensive labeling in mitochondria. Label was also found in lysosomal autophagic vacuoles. When autophagy was stimulated by in vivo administration of the anti-microtubular agent vinblastine we found that: (a) carbamoyl phosphate synthase-I and glutamate dehydrogenase could be found in mitochondria within autophagic vacuoles; (b) the carbamoyl phosphate synthase-I and glutamate dehydrogenase content of the mitochondria sequestered into autophagic vacuoles is the same as that of the nearby "free" mitochondria; and (c) in the whole liver, autophagic vacuoles contain c. 1.5 times more glutamate dehydrogenase than carbamoyl phosphate synthase-I, in contrast to mitochondria which have c. three times more carbamoyl phosphate synthase-I than glutamate dehydrogenase. The latter finding could explain, at least partially, the difference in half-lives of these enzymes.     

Evaluation of the pyroantimonate method for detecting intracellular calcium localization in smooth muscle fibers by the X-ray microanalysis of cryosections

Summary The validity of the pyroantimonate method, which has been used for detecting intracellular Ca localization and translocation in smooth muscles, was examined by making cryosections of the relaxed anterior byssal retractor muscle (ABRM) of Mytilus edulis at various stages of procedures for preparing ordinary Epon-embedded sections and determining the elemental concentration ratios of the pyroantimonate precipitate, localized along the inner surface of the plasma membrane, with an energy dispersive X-ray microanalyzer. The concentration of Ca (relative to that of Sb) in the precipitate stayed constant after the procedures of fixation, dehydration and Epon-embedding, while the concentrations of K, Mg, Na and Os showed their respective characteristic changes after the above procedures, being lower than that of Ca in the Epon-embedded sections. The presence of Ca in the precipitate was also demonstrated with an electron energy-loss spectrometer. The localization of Ca underneath the plasma membrane was also observed in the cryosections of the ABRM fibers prepared after mild fixation with acrolein vapor without using pyroantimonate. These results indicate that the pyroantimonate precipitate serves as a valid measure of intracellular Ca localization.     

Autophagic response to strenuous exercise in mouse skeletal muscle fibers

Strenuous physical exercise induces necrosis of skeletal muscle fibers and increases lysosomal enzyme activities in surviving muscle fibers. This study examines the ultrastructural basis of the stimulation of the lysosomal system in mouse vastus medialis muscle during the appearance and repair of exercise-induced (9 h of running) injuries. Necrotic fibers appeared the day after exercise and an inflammatory response with the replacement of necrotic fibers by phagocytes was highest 2-3 days after exertion. Ultrastructural study of surviving muscle fibers revealed numerous autophagic vacuoles, residual bodies, and spheromembranous structures at the periphery of myofibers, especially in fibers adjacent to necrotic fibers. The autophagic response was most prominent between 2 and 7 days after exertion. Autophagic vacuoles with double or single limiting membranes contained mitochondria at various stages of degradation. Vacuolar and multilamellar structures were also observed in regenerating muscle fibers. The structure of injured skeletal muscle fibers returned to normal within 2 weeks. It is proposed that increased autophagic activity could be related to the breakdown of cellular constituents of surviving muscle fibers to provide structural elements for regenerating muscle fibers.     

Evaluation of the pyroantimonate method for detecting intracellular calcium localization in smooth muscle fibers by the X-ray microanalysis of cryosections

The validity of the pyroantimonate method, which has been used for detecting intracellular Ca localization and translocation in smooth muscles, was examined by making cryosections of the relaxed anterior byssal retractor muscle (ABRM) of Mytilus edulis at various stages of procedures for preparing ordinary Epon-embedded sections and determining the elemental concentration ratios of the pyroantimonate precipitate, localized along the inner surface of the plasma membrane, with an energy dispersive X-ray microanalyzer. The concentration of Ca (relative to that of Sb) in the precipitate stayed constant after the procedures of fixation, dehydration and Epon-embedding, while the concentrations of K, Mg, Na and Os showed their respective characteristic changes after the above procedures, being lower than that of Ca in the Epon-embedded sections. The presence of Ca in the precipitate was also demonstrated with an electron energy-loss spectrometer. The localization of Ca underneath the plasma membrane was also observed in the cryosections of the ABRM fibers prepared after mild fixation with acrolein vapor without using pyroantimonate. These results indicate that the pyroantimonate precipitate serves as a valid measure of intracellular Ca localization.     

Effects of parathyroid hormone on osteoclasts in vivo

Summary Young rats were treated with high doses of parathyroid hormone (PTH). Osteoclasts from these animals revealed characteristic alterations in comparison to control cells: a) The cytoplasm contained large vacuoles with phagocytosed cells, some of which resembled osteoblasts or osteocytes. The vacuoles were interpreted as lysosomes because the engulfed cells often appeared partly digested and the vacuoles contained acid phosphatase as demonstrated histochemically, b) lipid droplets were present in the cytoplasm and usually located close to the endoplasmic reticulum and/or in regions with many free ribosomes, c) the Golgi complex was more frequently separated from the nuclei than in control cells, d) small coated cytoplasmic bodies were numerous in the peripheral cytoplasm, e) the membranes of the endoplasmic reticulum were fused in some places, f) cytoplasmic regions with numerous free ribosomes were frequent, g) large ring-shaped granules occurred in some mitochondria. Energy dispersive X-ray analysis of these granules provided evidence that they contained calcium and probably phosphorus, h) in some osteoclasts the mitochondria were enlarged. — The findings are consistent with an increased activity of osteoclasts and in particular a stimulation of the lysosomal system in these cells.This research was supported by grants no. 512-819, 512-1545 and 512-1912 from the Danish Research Council. The observations were reported in part at the annual meeting of the Scandinavian Society for Electron Microscopy in Aarhus 1972 (Lucht, 1973). — The authors thank Mr. K. Ibe for cooperation in the energy dispersive X-ray analysis, which was carried out at the JEOL (Europe) S. A. Application Center, Paris.     

Role of LAMP-2 in lysosome biogenesis and autophagy

In LAMP-2-deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2-deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2-deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.     

Zinc Dyshomeostasis Is Linked with the Loss of Mucolipidosis IV-associated TRPML1 Ion Channel

Chelatable zinc is important in brain function, and its homeostasis is maintained to prevent cytotoxic overload. However, certain pathologic events result in intracellular zinc accumulation in lysosomes and mitochondria. Abnormal lysosomes and mitochondria are common features of the human lysosomal storage disorder known as mucolipidosis IV (MLIV). MLIV is caused by the loss of TRPML1 ion channel function. MLIV cells develop large hyperacidic lysosomes, membranous vacuoles, mitochondrial fragmentation, and autophagic dysfunction. Here, we observed that RNA interference of mucolipin-1 gene (TRPML1) in HEK-293 cells mimics the MLIV cell phenotype consisting of large lysosomes and membranous vacuoles that accumulate chelatable zinc. To show that abnormal chelatable zinc levels are indeed correlated with MLIV pathology, we quantified its concentration in cultured MLIV patient fibroblast and control cells with a spectrofluorometer using N-(6-methoxy-8-quinolyl)-p-toluene sulfonamide fluorochrome. We found a significant increase of chelatable zinc levels in MLIV cells but not in control cells. Furthermore, we quantified various metal isotopes in whole brain tissue of TRPML1^−/− null mice and wild-type littermates using inductively coupled plasma mass spectrometry and observed that the zinc-66 isotope is markedly elevated in the brain of TRPML1^−/− mice when compared with controls. In conclusion, we show for the first time that the loss of TRPML1 function results in intracellular chelatable zinc dyshomeostasis. We propose that chelatable zinc accumulation in large lysosomes and membranous vacuoles may contribute to the pathogenesis of the disease and progressive cell degeneration in MLIV patients.     

Rimmed Vacuoles in Becker Muscular Dystrophy Have Similar Features with Inclusion Myopathies

Rimmed vacuoles in myofibers are thought to be due to the accumulation of autophagic vacuoles, and can be characteristic in certain myopathies with protein inclusions in myofibers. In this study, we performed a detailed clinical, molecular, and pathological characterization of Becker muscular dystrophy patients who have rimmed vacuoles in muscles. Among 65 Becker muscular dystrophy patients, we identified 12 patients who have rimmed vacuoles and 11 patients who have deletions in exons 45–48 in DMD gene. All patients having rimmed vacuoles showed milder clinical features compared to those without rimmed vacuoles. Interestingly, the rimmed vacuoles in Becker muscular dystrophy muscles seem to represent autophagic vacuoles and are also associated with polyubiquitinated protein aggregates. These findings support the notion that rimmed vacuoles can appear in Becker muscular dystrophy, and may be related to the chronic changes in muscle pathology induced by certain mutations in the DMD gene.     

Leucine inhibition of autophagic vacuole formation in isolated rat hepatocytes

An electron microscopic, morphometric analysis of isolated rat hepatocytes revealed a 70% decrease in the early forms of autophagic vacuoles after administration of leucine. The lysosomal degradation of protein was reduced by only about 30% under the same conditions. These observations suggest that leucine is a major regulator of the bulk autophagy observable in the electron microscope, but that this type of autophagy contributes only about one-half of the total amount of protein degraded in lysosomes. Asparagine inhibited lysosomal protein degradation more strongly than did leucine, but had no significant effect on the amount of autophagic vacuoles. Leucine and asparagine would therefore seem to exert their effects on lysosomal protein degradation through different mechanisms.     

Ultrastructural Localization of Calcium in the Presumptive Ectodermal Cells in Gastrulae of the Newt, Cynops pyrrhogaster, as Revealed by Cytochemistry and X-Ray Microanalysis

The ultrastructural localization of calcium in the presumptive ectodermal cells of gastrulae of the newt, Cynops pyrrhogaster , was examined by cytochemical methods and X-ray microanalysis (XMA). The cells were fixed with solutions that contained potassium oxalate, potassium ferricyanide and potassium pyroantimonate to preserve the localization of intracellular calcium in situ and for the analysis of electron density due to calcium. Electron-dense deposits associated with the localization of calcium were observed under the electron microscope. Specificially, pigment granules, round vacuoles, endoplasmic reticulum and mitochondria as well as the extracellular matrix were observed to contain calcium. In addition, XMA clearly demonstrated the localization of calcium in all of these electron-dense organelles and yolk granules.     

Biogenesis of multilamellar bodies via autophagy

Transfection of Mv1Lu mink lung type II alveolar cells with beta1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the beta1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.     

Localization artefacts in ultracytochemical ion precipitation reactions

Summary The precipitation patterns of the following ultracytochemical methods in rat muscle cells were compared and examined critically: the potassium pyroantimonate method for calcium demonstration; the calcium phosphate technique for the Ca²⁺ — ATPase reaction; the formazan reaction for the demonstration of creatine kinase activity (all performed on heart muscle); and the lead phosphate technique for the Mg²⁺ — ATPase reaction in skeletal muscle. Using X-ray microanalysis, it was found that the antimonate precipitate contains only calcium as the precipitated ion in the vast majority of cases. Most probably it consists of pure calcium pyroantimonate. However, in myocytes showing the well-established precipitation pattern, the concentration of calcium was estimated to be about two orders of magnitude higher than the native concentration of total intracellular calcium. It is concluded that calcium ions diffuse freely from the extracellular space and from adjacent cells into cells containing antimonate and are precipitated mostly at sites where heterogeneous nucleation is facilitated by intracellular catalysts (biopolymers).As shown by the similar precipitation patterns for the four reactions compared, these catalysts are not specific to any of these reactions and are most probably neither calcium-binding sites nor sites of any one of the enzymes examined in the native cell.     

Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.     

An electron microscopic and biochemical study of the effects of propranolol on the glycogen autophagy in newborn rat hepatocytes

The effects of propranolol on the glycogen autophagy in newborn rat hepatocytes were studied by using biochemical determinations, electron microscopy and morphometric analysis. Propranolol lowered the liver cyclic AMP and cyclic AMP-dependent protein kinase activity. It also decreased the formyl-methionyl-leucyl-phenylalanine (FMLP)-inhibitable Ca2+-ATPase activity including lysosomal calcium uptake pump. The normal postnatal increase in the volume of autophagic vacuoles and the activity of acid glycogen-hydrolyzing alpha glucosidase were inhibited. Also, the degradation of glycogen inside the autophagic vacuoles was apparently inhibited. The activity of acid mannose 6-phosphatase was increased. These findings indicate that propranolol influences several steps in the sequence of events leading to the breakdown of glycogen in the autophagic vacuoles of newborn rat hepatocytes. This supports our previous studies suggesting that cyclic AMP regulates glycogen autophagy.     

THE SUBCELLULAR LOCALIZATION OF CALCIUM ION IN MAMMALIAN MYOCARDIUM

This study was designed to investigate the proposition that subcellular calcium is sequestered in specific sites in mammalian myocardium. 29 functioning dog papillary muscles were fixed through the intact vascular supply by means of osmium tetroxide containing a 2% concentration of potassium pyroantimonate (K₂H₂Sb₂O₇·₄H₂O). Tissue examined in the electron microscope showed a consistent and reproducible localization of the electron-opaque pyroantimonate salts of sodium and calcium to distinct sites in the tissue. Sodium pyroantimonate was found exclusively in the extracellular space and clustered at the sarcolemmal membrane. Calcium pyroantimonate, on the other hand, identified primarily by its susceptibility to removal by chelation with EGTA and EDTA, was consistently found densely concentrated in the lateral sacs of the sarcoplasmic reticulum and over the sarcomeric I bands. M zones were virtually free of precipitate. The implications of these findings with respect to various parameters of muscle function are discussed.     

大鼠睾丸间质细胞的自体吞噬活动

本文结合超微结构和细胞化学观察,研究大鼠睾丸间质细胞(Leydig细胞)中溶酶体的结??构与功能。观察结果表明,大鼠睾丸间质细胞中高尔基体非常发达,在高尔基体的成熟面存在着CMP酶阳性反应的GERL系统,说明这种细胞有不断产生溶酶体的能力。细胞化学结果也证实在睾丸间质细胞有较多的初级和次级溶酶体。睾丸间质细胞不仅有较多的溶酶体,而且还有相当数量的自噬小体,存在着活跃的自体吞噬活动。自噬小体的界膜来源于特化的光面内质网或高尔基体膜囊,包围的内容物主要是光面内质网和少量线粒体。当自噬小体与溶酶体融合后即成为自体吞噬泡,由于酶的消化作用,自体吞噬泡内的细胞器有一系列形态变化。根据CMP酶细胞化学反应,可以区分自噬小体和自体吞噬泡,后者是一种次级溶酶体,呈CMP酶阳性反应。睾丸间质细胞是分泌雄性激素的内分泌细胞,其光面内质网和线粒体在类固醇激素分泌中起重要作用,自体吞噬活动的结果是去除部分内质网和线粒体,可能在细胞水平上起着对雄性激素分泌的调节作用。     

Distribution of calcium ions in the muscle fibers of the rat diaphragm depending upon their state and the method of histochemical treatment

Distribution of calcium ions in the rat diaphragm muscle fibers has been studied electron histochemically using various fixation techniques and chemical treatment of the tissue. When potassium pyroantimonate in water solution is used after a short perfusate fixation with aldehydes, the reaction product granules are revealed in mitochondria, in the disk I, in the center of the disk A, more seldom the precipitate is found in the sarcoplasmic reticulum (SR) and in the T-system. The presence of calcium ions in the precipitate is proved by means of treatment the preparations with ethylenglycol- and ethylen-diamine-tetra-acetic acids. When contracture is resulted from potassium rhodanide administration, in mitochondria the reaction product granules decrease in their number, the precipitate disappears from the central part of the disk A, while the number of the granules increases in the SR terminal cisterns. The data obtained are compared with calcium ions distribution observed at the freezing-substitution method without an additional chemical fixation, as well as the histochemical fixations after Oschman method and at a usual fixation with OsO4. Certain similarity is revealed in distribution of the calcium pyroantimonate granules at aldehyde fixation and when the freezing-substitution method is used.     

Increased autophagy in chloroquine-treated tonic and phasic muscles: An alternative view

Ultrastructural and cytochemical techniques were used to investigate autophagy in the tonic anterior (ALD) and phasic posterior (PLD) latissimus dorsi muscles of the chicken following chloroquine administration. Autophagic vacuoles were seen in the ALD after 1 day of chloroquine administration while no change was seen in the PLD until 3 days. In both muscles, autophagic vacuoles and myeloid bodies were found at the level of the I band. Myeloid bodies usually were found in the longitudinal rows of mitochondria in the ALD muscle. Some, but not all, of the autophagic vacuoles and myeloid bodies were cytochemically acid phosphatase positive, while the portion of the sarcoplasmic reticulum of both muscles which is normally acid phosphatase positive was devoid of activity following chloroquine administration. These observations are discussed in regard to accepted mechanisms of autophagy and the possible inhibition of autophagy in skeletal muscle tissue by chloroquine.     

盘基网柄菌细胞分化和凋亡的形态特征

本文用透射电镜和DAPI荧光染色法研究了盘基网柄菌(Dictyosteliumdiscoideum)细胞分化和柄细胞的凋亡特征,结果显示:细胞丘中绝大部分细胞的线粒体内出现一小空泡,随着发育进程,空泡逐渐增大,线粒体的嵴随之变少,直至线粒体完全空泡化,最后形成单层膜的空泡。据此我们推测前孢子细胞特有的空泡来源于线粒体,并且这种细胞器水平上的内自噬现象与前孢子细胞分化密切相关。在前柄细胞分化阶段,前柄细胞中出现数个自噬泡,最初吞噬的线粒体嵴结构完整;随着前柄细胞进一步分化,部分线粒体内出现类似于前孢子细胞中的内自噬现象,并且自噬泡只吞噬这种线粒体。在凋亡后期,细胞核内核仁消失,染色体固缩形成高电子密度斑块,自噬泡采用与细胞核膜融合的方式来完成核的清除,最后柄细胞完全空泡化且包被一层纤维素壁。作者认为前柄细胞凋亡过程实质上是一种分化过程,所以有其鲜明特点:细胞出现自噬泡,标志着凋亡开始,用自噬而不是凋亡小体来清除胞内各种细胞器,直到分化最后阶段才清除细胞核和形成纤维素壁。这些特点不仅是前柄细胞凋亡的形态学指标,也和细胞发育和分化相关。