Matrix Of Lysosomes Research Articles

Mutation of sec63 in zebrafish causes defects in myelinated axons and liver pathology.

SUMMARYMutations in SEC63 cause polycystic liver disease in humans. Sec63 is a member of the endoplasmic reticulum (ER) translocon machinery, although it is unclear how mutations in SEC63 lead to liver cyst formation in humans. Here, we report the identification and characterization of a zebrafish sec63 mutant, which was discovered in a screen for mutations that affect the development of myelinated axons. Accordingly, we show that disruption of sec63 in zebrafish leads to abnormalities in myelinating glia in both the central and peripheral nervous systems. In the vertebrate nervous system, segments of myelin are separated by the nodes of Ranvier, which are unmyelinated regions of axonal membrane containing a high density of voltage-gated sodium channels. We show that sec63 mutants have morphologically abnormal and reduced numbers of clusters of voltage-gated sodium channels in the spinal cord and along peripheral nerves. Additionally, we observed reduced myelination in both the central and peripheral nervous systems, as well as swollen ER in myelinating glia. Markers of ER stress are upregulated in sec63 mutants. Finally, we show that sec63 mutants develop liver pathology. As in glia, the primary defect, detectable at 5 dpf, is fragmentation and swelling of the ER, indicative of accumulation of proteins in the lumen. At 8 dpf, ER swelling is severe; other pathological features include disrupted bile canaliculi, altered cytoplasmic matrix and accumulation of large lysosomes. Together, our analyses of sec63 mutant zebrafish highlight the possible role of ER stress in polycystic liver disease and suggest that these mutants will serve as a model for understanding the pathophysiology of this disease and other abnormalities involving ER stress.

Morphological and functional analysis of PTA granules in human NK cells.

Human natural killer (NK) cells contain unique granules with parallel tubular arrays (PTA granules) of approximately 30 nm diameter that can be seen only by electron microscopy. In order to clarify the role of PTA granules in NK cell-mediated cytolysis we examined these structures with regard to frequency and expression of lytic proteins (perforin, granzymes). NK cells (CD3-, CD16+, CD56+) were obtained from heparinized blood of healthy donors and enriched by double-step negative selection using mAb coupled to magnetic beads. PTA granules were found in 31.3% of freshly separated NK cells. When NK cells were cultivated, even in the presence of various stimulating agents (rhIL-2, rhIL-4, rhIL-6, rhIL-12, GM-CSF, rhIFNalpha, anti-CD16 mAb, dexamethasone), PTA granules disappeared and transformed into conventional primary lysosomes. By immune electron microscopy using antibodies directed against perforin and granzyme B we observed distinct immuno-reactivity in the tubules and in the tubule-associated faintly electron-dense matrix of PTA granules. Immuno-labelling for perforin and granzyme B was also found in the fine granular matrix of primary lysosomes. Finally, we tested the distribution of chondroitin 4-sulfate which is suggested to inactivate lytic proteins. Immuno-reactivity for chondroitin 4-sulfate was detected only in the moderately electron-dense matrix but not in the tubules of PTA granules. These observations indicate that perforin and granzyme B are stored in an inactive form in PTA tubules due to highly ordered paracrystalline protein folding. As soon as the tubules decay the lytic proteins are kept in an environment of chondroitin 4-sulfate for inactivation.

Ca2+-ATPase in mucous and oxyntico-peptic cells of the fowl proventriculus.

Calcium adenosine triphosphatase (Ca(2+)-ATPase) was localized by means of histo-and ultracytochemistry in the secretory cells of the proventriculus of the domestic fowl. The mucous cells exhibited plasmalemmal-associated enzyme activity on the external aspect of the basolateral cell membrane. Intracellularly, the luminal aspect of Golgi-membranes and of secretory vesicle membranes reacted positively for Ca(2+)-ATPase activity, as did the apical cytosol and the matrix of lysosomes. Oxyntico-peptic cells were characterized by apical and apico-lateral plasmalemmal activity and by an organelle-associated distributional pattern similar to that in the mucous cells. In addition, Ca(2+)-ATPase was associated either with the matrix of mitochondria or with tubuli of the rough-surfaced endoplasmic reticulum. The results are discussed with respect to messenger and effector functions of calcium in the process of proventricular mucus secretion. In addition, Ca(2+)-ATPase distributional patterns in the oxyntico-peptic cell are related to the unique structure and function of these cells.

Diacylglycerol hydrolysis in rat liver lysosomes

The matrix of rat liver lysosomes exhibits high hydrolytic activity towards 1,2-diacylglycerol with an optimum at pH 4.0. The lipolytic reaction follows Michaelis-Menten kinetics (apparent V max 470 nmol hydrolysed min per mg protein; apparent K m71 μM 1,2-dioleoylglycerol). Formation of 1- and 2-monooleoylglycerols indicates an initial attack at both the primary and secondary ester bonds. The lysosomal matrix also catalyses (re)acylation reactions, i.e. the formation of 1,2-diacylglycerol from 2-monoacylglycerol and free fatty acid. However, (re)acylation proceeds at a far lower rate than deacylation of diacylglycerol. Lysosomal diacylglycerol hydrolysis is sensitive towards non-ionic detergents, cationic amphiphilic drugs and the lipase inhibitor RHC 80267.

Immunocytochemical localization of cathepsin H in rat kidney. Light and electron microscopic study.

Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For light microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.

Ultrastructural localization of carbonic anhydrase in lysosomes.

Ultrastructural localization of carbonic anhydrase was determined by applying Hansson's histochemical method to glutaraldehyde-fixed frozen sections of guinea pig peritoneal polymorphonuclear leukocytes (PMNs) and lysosomes isolated from rat liver tissue after the animal had been injected with Triton WR-1339. A positive histochemical reaction for carbonic anhydrase in PMNs was found in the matrix of lysosomes. After PMNs phagocytized polystyrene latex particles or emulsified paraffin oil droplets, a positive reactivity for carbonic anhydrase was found in the space between the lysosomal membrane and the particle. Liver lysosomes also revealed positive carbonic anhydrase histochemical reactivity. To confirm the histochemical reaction, indirect immunoferritin labeling was conducted with rabbit antibody to human red blood cell carbonic anhydrase C on glutaraldehyde-fixed, freeze-thawed human PMNs. Immunolabeling was observed in lysosomes. These results suggest that carbonic anhydrase is a constituent of lysosomes of PMNs and liver cells.

A coat of glycoconjugates on the inner surface of the lysosomal membrane in the rat kidney

After perfusion fixation of the rat kidney with glutaraldehyde, and postfixation of the renal cortex with osmium-low ferrocyanide (40 mM OsO4 +6 mM K4Fe(CN)6 in 0.135 M phosphate buffer, pH 8.0), secondary lysosomes of proximal tubule cells carry a coat of electron dense material on the inner surface of the lysosomal membrane. This coat separates matrix and membrane of lysosomes, and corresponds in location and width to the electron translucent halo of conventionally processed lysosomes in TEM. The material which forms the coat, is stained by phosphotungstic acid at pH 0.3, and by periodic acid - thiocarbohydrazide - silver proteinate more intensively than the cell surface coat of the same cell; it contains a high concentration of hydroxyl, vicinal-glycol and alpha-aminoalcohol groups.

Isolation and characterization of a rough microsomal fraction from rat kidney that is enriched in lysosomal enzymes.

1. A special population of rough microsomal material (microsomes) rich in lysosomal acid hydrolases was separated by isopycnic centrifugation as a discrete fraction (RM(2)) from the bulk of rough microsomal material in rat kidney because of its greater density. 2. The specific activities of five acid hydrolases in the RM(2) fraction were approximately one-half those of a purified lysosomal (L) fraction and 10- to 30-fold greater than those of an ordinary rough microsomal (RM(1)) fraction. 3. These special rough microsomes have a distinctive ultrastructure and electron-cytochemical properties. Their cisternal content resembles the matrix of lysosomes in that it is electron-dense, osmiophilic and plumbophilic and gives a positive reaction for acid phosphatase activity. 4. Polyacrylamide-gel electrophoresis of soluble proteins from the L fraction resolved nine anionic glycoproteins, most of which exhibit acid hydrolase activities (Goldstone & Koenig, 1970, 1973; Goldstone et al., 1971a). The most anionic glycoprotein is the acidic lipoglycoprotein of the lysosomal matrix (Goldstone et al., 1970). 5. Polyacrylamide-gel electrophoresis of soluble proteins from the RM(2) fraction resolved two cationic glycoproteins with acid hydrolase activities (Goldstone & Koenig, 1973) and an anionic glycoprotein with the same electrophoretic mobility as the lysosomal lipoglycoprotein, but without its lipid constituents or capacity to bind the basic fluorochrome Acridine Orange. These constituents are considered to be the precursors of the lysosomal glycoproteins.

Ultracytochemistry of Intracellular Membranes

The modified KUSHIDA and FUJITA method (1967) for in situ PTA staining was applied to the kidney, heart, spleen and brain of the normal adult albino rat. In general, intracellular membranes such as plasma membranes, sarcolemma, membranes constituting junctional areas (synapses, macula adherens, macula occludens etc.), mitochondrial membranes, limiting membranes of lysosomes, membranes of the endoplasmic reticulum including the nuclear envelope were in the negative image, while the mitochondrial matrix, myofilaments, substances in the Z line, the matrix of lysosomes, and nucleoproteins such as ribosomes, chromatin and perichromatin substance were in the positive image. Substances in the cisternae of the endoplasmic reticulum and the basement membrane were also stained moderately positive. Structural similarities observed among the erythrocytic subsurface PTA-positive zone, the subpostsynaptolemmal PTA-positive zone and the macula adherens (grouped as “subplasmalemmal PTA-positive zone”) were suggested in relation to their possible role in ionic movement.The electron lucent layer of membranes seems to correspond to the classical osmiophilic layer. The membrane showed, however, a triple-layered unit membrane-like structure following poststaining with uranyl acetate and/or lead citrate. The PTA-negative electron lucent leaflet of the membrane may be composed of substances with non-polar groups or with a very low concentration of polar groups. Some doubts on the ROBERTSON's unit membrane theory based on the DAVSON-DANIELLI model were expressed.It can be concluded in the present study that ultracytochemistry sine osmium tetroxide is promising for some specific purposes in electron microscopy.