Cationized Ferritin Labeling Research Articles

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.

Toxicity, biodistribution, and ex vivo MRI detection of intravenously injected cationized ferritin

The goal of the work was to establish the toxicity and biodistribution of the superparamagnetic protein cationized ferritin (CF) after intravenous injection. Intravenously injected CF has been used to target the extracellular matrix with high specificity in the kidney glomerulus, allowing measurements of individual glomeruli using T2*-weighted MRI. For the routine use of CF as an extracellular matrix-specific tracer, it is important to determine whether CF is toxic. In this work, we investigated the renal and hepatic toxicity, leukocyte count, and clearance of intravenously injected CF. Furthermore, we studied CF labeling in several organs using MRI and immunohistochemistry. Serum measurements of biomarkers suggest that intravenous injection of CF is neither nephrotoxic nor hepatotoxic and does not increase leukocyte counts in healthy rats at a dose of 5.75 mg/100 g. In addition to known glomerular labeling, confocal and MRI suggest that intravenously injected CF labels the extracellular matrix of the hepatic sinusoid, extracellular glycocalyx of alveolar endothelial cells, and macrophages in the spleen. Liver T2* values suggest that CF is cleared by 7 days after injection. These results suggest that CF may serve as a useful contrast agent for detection of a number of structures and functions with minimal toxicity.

Antiadhesive Character of Mucin O-glycans at the Apical Surface of Corneal Epithelial Cells

Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal-limbal epithelial (HCLE) cells. Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-alpha-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell-cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber. Benzyl-alpha-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-alpha-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface. These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface-associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye).

Endocytosis in rat cultured astrocytes is inhibited by unconjugated bilirubin.

Excessive hyperbilirubinemia can cause irreversible neurological damage in the neonatal period. However, the complete understanding of the pathogenesis of unconjugated bilirubin (UCB) encephalopathy remains a matter of debate. This study investigates whether UCB inhibits the endocytosis of cationized ferritin (CF) by cultured rat astrocytes. The relationship between endocytosis and MTT reduction, as well as changes on tubulin and glial fibrillary acidic protein (GFAP) assembly, were also evaluated. Inhibition of endocytosis was complete in the presence of 171 microM UCB, while a marked decrease of CF labeling was noticed for 86 microM UCB. In addition, MTT reduction was inhibited by 60 to 76% as UCB concentrations changed from 17 to 171 microM, while alterations on both GFAP and microtubule morphology were only achieved by cell exposure to 171 microM UCB. These findings indicate that inhibition of CF endocytosis in rat cortical astrocytes by UCB is a concentration-dependent process that appears to be primarily related to a direct effect on the cell membrane and not to any alteration of cytoskeletal microtubules and intermediate filaments.

Albumin microbubble persistence during myocardial contrast echocardiography is associated with microvascular endothelial glycocalyx damage.

We hypothesized that the persistence of albumin microbubbles within the myocardium during crystalloid cardioplegia (CP) infusion and ischemia-reperfusion (I-R) occurs because of endothelial injury. The myocardial transit rate of albumin microbubbles was measured in 18 dogs perfused with different CP solutions and in 12 dogs undergoing I-R. Electron microscopy with cationized ferritin labeling of the glycocalyx was performed in 9 additional dogs after CP perfusion and in 3 additional dogs undergoing I-R. Microbubble transit was markedly prolonged during crystalloid CP perfusion. The addition of whole blood to the CP solution accelerated the transit rate in a dose-dependent fashion (P<0.05), which was greater with venous than with arterial blood (P<0.05). The addition of plasma or red blood cells to CP solutions was less effective in improving transit rate than addition of whole blood (P<0.05). Microbubble transit rate was independent of the temperature, K+ content, pH, PO2, osmolality, viscosity, and flow rate of the perfusate. Similarly, a proportion of microbubbles persisted in the myocardium after I-R, which was related to the duration of ischemia (P<0.01) but not of reflow. Crystalloid CP perfusion and I-R resulted in extensive loss of the endothelial glycocalyx without other ultrastructural changes. This effect was partially reversed in the case of crystalloid CP when it was followed by blood CP. Sonicated albumin microbubbles persist within the myocardium in situations in which the endothelial glycocalyx is damaged. The measurement of the myocardial transit rate of albumin microbubbles may provide an in vivo assessment of endothelial glycocalyx damage.

Variations in surface charge distribution of leukemic and non-leukemic transformed cells

The surface negative charge distribution of fresh leukemic cells from patients with acute leukemia was examined with cationized ferritin (CF), an electron dense label of anionic sites and compared with leukemic cells from phase-cycle fractionated populations of a human leukemic cell line K-562. Normal-untreated and phytohemagglutinin (PHA)-transformed lymphocytes were also tested. A CF-induced redistribution of anionic sites into clusters and patches within a wide range of variations in density was observed electron microscopically both in fresh acute leukemic cells and in unseparated cells of the K-562 line. Cells of the G 0/G 1 fraction from a phase-cycle separated K-562 population exhibit a higher density of CF label per unit length of membrane as compared with the G 2 + M cell fraction. PHA-benign transformed normal lymphocytes exhibit an even and continuous CF labeling, similar to that of normal-untreated lymphocytes. An attempt was made to correlate the CF-induced charge redistribution with the rate of agglutination with the cationic Poly- l-lysine. Our observations indicate that the CF ligand does induce an anionic site redistribution, varying in density, both in fresh leukemic cells and in cells from a K-562 line, and does not in normal untreated and PHA-benign transformed lymphocytes. The particular pattern of anionic site redistribution observed in leukemic cells of this study is apparently phase-cycle dependent.

Distribution and modulation of surface charges of cells from human leukemia-lymphoma lines at various stages of differentiation.

Untreated and retinoic acid (RA) treated human leukemia-lymphoma cell lines reflecting hematopoietic cells at various stages of differentiation, were examined electron microscopically for their surface negative charge distribution using cationized ferritin (CF), an electron dense label of anionic sites. The results indicate that there is a correlation between the CF labeling density/distribution and the stage of lymphoid cell differentiation. Viable unfixed null cell lines show a low CF labeling density with few and small CF patches. A gradual increase in CF labeling density and increase in size and number of CF patches correlates with the stage of differentiation on cell lines of both T or B origin. Treatment of viable unfixed cells with 10(-5) MRA for 10 days seems to prevent the CF-induced formation of CF patches, resulting in a continuous and even distribution of the CF label, similar to that observed on the surface of cells fixed before CF labeling. Some correlation between the distribution of surface anionic sites and the malignant potential of the human leukemic lines could be detected.

Characterization of endothelial cell transport in the developing mouse blood-brain barrier.

The cerebromicrovasculature of immature mice ranging in age from 1-24 days after birth was studied by electron microscopy. A micromanipulator apparatus enabled us to facilitate direction of a needle cannula and injection into leg veins or hearts with horseradish peroxidase (HRP) or ferritin (anionic and cationic) tracers and subsequent perfusion fixatives. Numerous HRP-filled vesiculocanalicular transport structures appearing in endothelial cells (ECs) of brain microblood vessels (MBVs) were observed in time periods ranging from 1-14 days. In addition to HRP transport across the ECs by tubulovesicular profiles, some of these structures appeared to become connected to multivesicular bodies. Between 14 and 24 days after birth, limited HRP was transported across the ECs to the basement membrane in only a few short segments of subpial arterioles. The decoration pattern with cationized ferritin (CF) on the luminal surface of the ECs depends upon whether the surface was exposed to the ligand before or after fixation. Quenching of aldehyde groups in fixed brain tissue has critical importance for the decoration pattern of CF on the luminal plasmalemmal surface. The absence of CF labeling on the delimiting membranes of plasmalemmal vesicles and tubular structures suggests that these structures represent differentiated microdomains engaged in macromolecular transport in the ECs of the developing mouse brain.

Distribution of cell surface charges on mesothelium and lymphatic endothelium

The distribution of anionic sites on the luminal surfaces of the peritoneal mesothelium and lymphatic endothelium was investigated by injecting cationized ferritin (CF) intraperitoneally. After washing with phosphate-buffered saline, the diaphragm was fixed and processed for electron microscopy. CF label occurred in discontinuous patches along the mesothelial surface. Microvilli were heavily marked and often closely applied to the mesothelial surface. The intercellular cleft was also heavily labeled. The luminal aspect of the lymphatic endothelium was more extensively labeled, with the marker occurring in long discontinuous dense bands. The clefts of lymphatic endothelial intercellular junctions were extensively labeled especially along regions where cells were loosely apposed. The existence of a high density of anionic sites on membranes at the intercellular junctions of both mesothelial and lymphatic endothelial cells represent a salient feature which is very different from binding in blood capillary endothelium. The presence of a high density of anionic sites along the intercellular clefts of adjacent cells may play a role in the rapid movement of small solutes and molecules from interstitial spaces into the lymphatic lumen.

Prevention by retinoic acid of anionic site redistribution on the surface of cultured human sarcoma cells.

The distribution of cell surface negatively-charged macromolecules was determined electron microscopically on untreated and on retinoic acid (RA)-treated cultured human osteosarcoma Hs791 and chondrosarcoma Hs705 cells using cationized ferritin (CF), an electron-dense marker of anionic sites. Labeling on the surface of prefixed cells was continuous and uniform whether they were grown in the absence or presence of RA. In contrast, CF distribution on unfixed cells was markedly affected by RA; CF labeling of untreated cells occurred in patches and clusters whereas the label on RA-treated cells was continuous, as on prefixed cells. CF labeling of unfixed cells decreased considerably after incubation of the cells either with hyaluronidase or neuraminidase. There was also a reduction in patching and clustering. Changes induced by RA in the apparent membrane microviscosity, in neuraminidase-releasable sialic acid, or in transglutaminase activity could not be related to the effect of RA on CF-induced anionic site redistribution since these characteristics were modulated differently in the two cell lines. In contrast, RA increased the sialylation of specific cell surface membrane glycoproteins on both cell types. These results suggest that RA prevents redistribution of cell surface sialoglycoconjugates and glycosaminoglycans by CF. This effect may be the result of increased sialylation of specific surface components and may be related causally to the suppression of the transformed phenotype in the sarcoma cells.

Preferential distribution of anionic sites on the basement membrane and the abluminal aspect of the endothelium in fenestrated capillaries.

Cationized ferritin (CF) was injected interstitially to study the distribution of anionic sites on the basement membrane and abluminal aspect of the endothelium in the fenestrated capillaries of the mouse pancreas and intestinal mucosa. Extensive, but uneven removal of the basement membrane was obtained by collagenase perfusion of the vasculature before CF labeling. In the absence of collagenase treatment, CF label was essentially restricted to the lamina rara externa of the basement membrane and occurred in clusters distributed in a relatively ordered planar lattice. After collagenase digestion, labeling of the lamina rara interna and of the abluminal aspect of the endothelium became possible. In the lamina rara interna, the CF label occurred in clusters with a distribution comparable to that found in the lamina rara externa. On the abluminal aspect of the endothelium, the plasmalemma proper was extensively, though variably, labeled. Coated pits were heavily labeled, whereas the membranes and stomatal diaphragms of plasmalemmal vesicles and transendothelial channels remained free of CF decoration. In contradistinction with the heavy labeling of their luminal aspects, the abluminal surface of the fenestral diaphragms were free of any CF decoration. Pronase treatment removed all anionic sites detectable by CF binding. The findings establish the existence of differentiated microdomains on the abluminal aspect of the endothelial plasmalemma and suggest that the capillary wall selects permeant macromolecules according to charge, in addition to size.

Pinocytosis in mouse L-fibroblasts: ultrastructural evidence for a direct membrane shuttle between the plasma membrane and the lysosomal compartment.

Mouse L-fibroblasts internalized large amounts of cationized ferritin (CF) by pinocytosis. Initially (60-90 s after addition of CF to cell monolayers at 37 degrees C), CF was found in vesicles measuring 100-400 nm (sectioned diameter) and as small clusters adhering to the inner aspect of the limiting membrane of a few large (greater than 600 nm) vacuoles. After 5-30 min, CF labeling of large vacuoles was pronounced and continuous. Moreover, 70-80% of all labeled structures were tiny (less than 100 nm) vesicles. However, the absolute frequency of tiny vesicles increased more than twofold from 5 min to 30 min. When the cells were incubated with CF for 30 min, then washed and further incubated for 3 h without CF, almost all CF was present in dense bodies (100-500 nm). When L-cells were first incubated with horseradish peroxidase (HRP), then washed and incubated with CF, double-labeled vacuoles were observed. Tiny vesicles also contained HRP-CF, and small HRP-CF patches were localized on the cell surface. Distinct labeling of stacked Golgi cisterns was not observed in any experiment. These observations suggest that the numerous tiny vesicles are not endocytic but rather pinch off from the large vacuoles and move towards the cell surface to fuse with the plasma membrane. Thus, ultrastructural evidence is provided in favor of a direct membrane shuttle between the plasma membrane and the lysosomal compartment.

Regional differences in surface anionic sites in the rat thyroid follicle cells shown by cationized ferritin labeling.

Regional differences in surface anionic sites in the rat thyroid follicle cells shown by cationized ferritin labeling.

Response of the resident macrophage to concanavalin A: Alterations of surface morphology and anionic site distribution

We have characterized certain resident guinea-pig peritoneal macrophage surface alterations following treatment with concanavalin A (ConA) and succinyl-ConA (S-ConA). Studies employing scanning electron microscopy (SEM) have demonstrated that ConA, a tetrameric lectin, decreases dramatically the number of macrophage surface folds and ruffles although S-ConA, a dimeric derivative, is apparently not active. However, incubation of S-ConA-treated cells with rabbit anti-ConA (anti-ConA) restored this effect. The decrease in surface folds could not be observed in the presence of α-methyl- d-mannoside (αMM), a hapten sugar of ConA. We have performed several receptor-labeling transmission electron microscopy (TEM) studies employing ferritin-conjugated ConA (FT-ConA) and cationized ferritin (CF). These experiments indicate that the ConA receptor-FT-ConA complexes form (1) clusters on the plasmalemma and (2) adsorptive pinocytic vesicles lined with the ferritin label. At the same time scale, we have observed a redistribution of macrophage surface anionic sites following treatment with ConA or S-ConA plus anti-ConA but not S-ConA alone. The redistribution of anionic sites is abolished in the presence of αMM. The specificity of the CF label was checked by pre-incubating cells with poly- l-lysine (PLL) or neuraminidase and by employing normal ferritin. These studies provide evidence which support the concept of a directed movement of surface charges during adsorptive pinocytosis. We discuss the possible relevance of this concept with regard to regional alterations of pH at the plasmalemma and the contribution of these anions to the trans-pinosomal pH gradient through the Donnan potential.

Cell surface properties of ascites sublines of the 13762 rat mammary adenocarcinoma: Relationship of the major sialoglycoprotein to xenotransplantability

The relationship between cell surface sialoglycoprotein and xenotransplantation has been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma. Two of the five sublines (MAT-C and MAT-C1) can be transplanted into mice. These two sublines also have the greatest amounts of total, trypsin-releasable and neuraminidase-releasable sialic acid. Chemical labeling using periodate treatment followed by [ 3H]borohydride reduction indicates that most of the protein-bound sialic acid is associated with a single major sialoglycoprotein (or family of glycoproteins) with a low mobility on polyacrylamide gels in dodecyl sulfate (SDS). This glycoprotein, denoted ASGP-1, is also labeled by lactoperoxidase and 125I, indicating its presence at the cell surface. Metabolic labeling with [ 3H]glucosamine shows that ASGP-1 is the major glycosylated protein in both xenotransplantable (MAT-C1) and non-xenotransplantable (MAT-B1) sublines, representing >70% of the protein-bound label in each. The labeling studies indicate that the non-xenotransplantable subline does not have a substantially greater amount of ASGP-1 on its cell surface. Likewise cationized ferritin labeling and transmission electron microscopy (TEM) do not show substantially greater amounts of negatively charged groups distributed along the cell surfaces of MAT-C1 than of MAT-B1 cells. The results indicate that the transplantation differences between these sublines cannot be explained solely by the presence of a major sialoglycoprotein at the cell surface.

Redistribution of cell surface anionic sites on hepatoma cells after treatment with Concanavalin A

Chang rat hepatoma ascites cells were treated with Con A and subsequently labeled with cationized ferritin (CF). When ascites cells were treated with Con A, fixed with glutaraldehyde, then labeled with CF, the labeling on the surface of the microvilli and plasma membrane was clustered and irregular. The areas of the cell surface between CF clusters were either lightly labeled or completely free of label. Cells fixed before Con A and CF treatment or cells incubated with Con A and α-methyl- d-mannoside were labeled with a continuous, even layer of ferritin particles. Treatment of ascites cells with cetyl pyridinium chloride before CF labeling resulted in an almost complete absence of ferritin particles from the cell surface.

Internalization of cationized ferritin receptors by rat hepatoma ascites cells

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.