Differential expression and characterization of a member of the mucin-associated surface protein family secreted by Trypanosoma cruzi

We describe the characterization, purification, expression, and location of a 52-kDa protein secreted during interaction between the metacyclic form of Trypanosoma cruzi and its target host cell. The protein, which we have named MASP52, belongs to the family of mucin-associated surface proteins (MASPs). The highest levels of expression of both the protein and mRNA occur during the metacyclic and bloodstream trypomastigote stages, the forms that infect the vertebrate host cells. The protein is located in the plasma membrane and in the flagellar pockets of the epimastigote, metacyclic, and trypomastigote forms and is secreted into the medium at the point of contact between the parasite and the cell membrane, as well as into the host-cell cytosol during the amastigote stage. IgG antibodies specific against a synthetic peptide corresponding to the catalytic zone of MASP52 significantly reduce the parasite's capacity to infect the host cells. Furthermore, when the protein is adsorbed onto inert particles of bentonite and incubated with a nonphagocytic cell culture, the particles are able to induce endocytosis in the cells, which seems to demonstrate that MASP52 plays a role in a process whereby the trypomastigote forms of the parasite invade the host cell.     

Alcoholic hyalin, microfilaments and microtubules in alcoholic hepatitis.

The alcoholic hyalin which is composed of light and dark occasionally swollen and conglomerating filaments, is found to be surrounded by proliferated RER, hypertrophied Golgi apparatus and mitochondria containing enlarged matrical granules. Besides, microfilaments and some microtubules are seen in relation to the hyaline bodies. In biopsies where hyaline bodies are scarce, hepatocytes without alcoholic hyalin present similar changes and the Golgi apparatus is seen to contain very low density lipoprotein-like particles. These ultrastructural changes are suggested to be related to an early stage in the development of alcoholic hyalin. The abundance of microfilaments indicates an increased motility of the hepatocytes, possibly as a part of the regenerative processes. It can hardly be precluded that microfilaments disintegrate and accumulate in the alcoholic hyaline mass.     

Subpopulations of microtubules with differential sensitivity to nocodazole: role in the structural organization of the Golgi complex and the lysosomal system

The role of cytoplasmic microtubules (MTs) in the structural organization of the Golgi complex and the lysosomal system was studied in L929 mouse fibroblasts using a combination of cytochemical and electron microscopic methods. Immunocytochemical staining demonstrated a radiating pattern of MTs, originating in the juxtanuclear region containing the centrioles. The stacks of Golgi cisternae with associated vesicles and tubules (the trans Golgi network), as well as most of the endosomes and lysosomes, were also located in and immediately around this region. Wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) bound to the plasma membrane at 4 degrees C. After warming to 37 degrees C, the conjugate was rapidly internalized by endocytosis and via small vesicles transferred to endosomes and lysosomes in the juxtanuclear region. Slightly later it was also found in the trans Golgi network. A weaker but otherwise similar staining was obtained if the conjugate was applied to fixed and permeabilized cells. Treatment with 3.0 microM nocodazole induced depolymerization of MTs and rounding up of the cells. At the same time, the Golgi complex was disorganized with dispersion of its stacks of cisternae throughout the cytoplasm and atrophy of the trans Golgi network. The lysosomes gathered around the dispersed cisternal stacks and the endosomes became fewer in number. Moreover, the uptake of WGA-HRP was markedly inhibited. Following withdrawal of the drug, MTs reformed and the structural organization of the cell was again normalized. If the recovery was allowed to take place in the presence of low concentrations of nocodazole (0.03 or 0.15 microM), MTs of short and medium length were formed, the latter showing a curly path. In parallel, a partial or nearly complete normalization of cell morphology occurred. Internalization and transport of WGA-HRP labeled membrane to the lysosomes and the trans Golgi network was also resumed. In contrast, the cells were not able to pass through mitosis in a normal manner and multimicronucleated cells accumulated. Taken together, the findings indicate that L929 cells contain a subset of nocodazole-resistant MTs which play an important role in the structural organization of the cell, especially with regard to the Golgi complex and the lysosomal system. They support recent notions that there exists subpopulations of MTs with different physicochemical properties and functions.     

Immunocytochemical localization of neuraminidase in Trypanosoma cruzi.

A polyclonal antibody obtained against neuraminidase purified from Trypanosoma cruzi was used for the localization of the protein in whole cells by immunofluorescence microscopy and in thin sections of parasites (epimastigote, amastigote, and trypomastigote forms) embedded at a low temperature in Lowicryl K4M resin. The intensity of labeling, as evaluated by the number of gold particles associated with the parasite, varied according to the protozoan developmental stage. In the noninfective epimastigote forms, labeling of the cell surface was very weak. However, an intense labeling of some cytoplasmic vacuoles was observed. Labeling of the surfaces of most of the trypomastigote forms was weak, while gold particles were seen in association with the flagellar pockets of these forms, which suggests that the enzyme is secreted through this region. Intense labeling of the surfaces of many, but not all, transition forms between trypomastigote and amastigote forms was observed. Amastigote forms found in the supernatant of infected cell cultures had their surfaces intensely labeled, while few particles were seen on the surfaces of intracellular amastigotes. The results obtained are discussed in relation to the role played by T. cruzi neuraminidase in the process of parasite-host cell interaction.     

Ulrastructure of cytoskeletal element of endothelial and stromal cells of rat marrow

This study reports the distribution of microfilaments, microtubules, and intermediate filaments in endothelial cells, reticular cells, and macrophages of bone marrow of rats following fixation with glutaraldehyde, tannic acid, and saponin. In endothelial cells bundles of microfilaments are seen along the basal surface, where these cells adhere to underlying extracellular materials. The reticular cells, especially those that are closely associated with the endothelium of sinusoids, contain many intermediate filaments and microtubules as well as microfilaments. The reticular cell processes that partially cover the endothelium and extend among the blood cells have numerous microtubules and intermediate filaments arranged longitudinally within them; these cytoskeletal elements appear to provide mechanical support for the processes. Macrophages also have many microtubules and intermediate filaments but these organelles do not extend into the thin processes of these cells as is the case with reticular cells. Bundles of microfilaments are observed in the cytoplasm of adventitial and endothelial cells at sites where migrating blood cells are attached to these cells producing local regions of stress.     

Low cytoplasmic pH causes fragmentation and dispersal of the Golgi apparatus in human hepatoma cells

The centrosomal localization of the Golgi apparatus in interphase cells is thought to be maintained by retrograde microtubule-based motility. It is well established that, when intracellular pH is lowered, lysosomes and endosomes, also showing pericentrosomal localization, translocate towards the plus ends of microtubules within 15 min. In this study, we found that prolonged incubation in low pH medium (pH 6.6) with 20 mM Na acetate induced the fragmentation and dispersal of the Golgi apparatus in the human hepatoma cell line PLC/PRF/5. The fraction of Golgi-dispersed cells increased in a time-dependent manner, and reached over 60% after the 16-h incubation. The cytoplasmic pH was dropped to approximately 7.10. Replacement with normal pH medium restored the structure and localization of the apparatus within 30 min. In the low pH condition, the microtubular network and endoplasmic reticulum appeared normal, and cytoplasmic dynein was still bound to the fragmented Golgi membranes. These findings suggest that low cytoplasmic pH suppresses the retrograde movement of the Golgi apparatus as well as that of lysosomes and endosomes.     

Ultrastructure of cytoskeletal elements of endothelial and stromal cells of rat marrow

This study reports the distribution of microfilaments, microtubules, and intermediate filaments in endothelial cells, reticular cells, and macrophages of bone marrow of rats following fixation with glutaraldehyde, tannic acid, and saponin. In endothelial cells bundles of microfilaments are seen along the basal surface, where these cells adhere to underlying extracellular materials. The reticular cells, especially those that are closely associated with the endothelium of sinusoids, contain many intermediate filaments and microtubules as well as microfilaments. The reticular cell processes that partially cover the endothelium and extend among the blood cells have numerous microtubules and intermediate filaments arranged longitudinally within them; these cytoskeletal elements appear to provide mechanical support for the processes. Macrophages also have many microtubules and intermediate filaments but these organelles do not extend into the thin processes of these cells as is the case with reticular cells. Bundles of microfilaments are observed in the cytoplasm of adventitial and endothelial cells at sites where migrating blood cells are attached to these cells producing local regions of stress.     

Selective alterations of the host cell architecture upon infection with parvovirus minute virus of mice

During a productive infection, the prototype strain of parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations to the fibroblast host cell A9, resulting in cell lysis and progeny virus release. In order to understand the mechanisms underlying these changes, we characterized the fate of various cytoskeletal filaments and investigated the nuclear/cytoplasmic compartmentalization of infected cells. While most pronounced effects could be seen on micro- and intermediate filaments, manifest in dramatic rearrangements and degradation of filamentous (F-)actin and vimentin structures, only little impact could be seen on microtubules or the nuclear envelope during the entire monitored time of infection. To further analyze the disruption of the cytoskeletal structures, we investigated the viral impact on selective regulatory pathways. Thereby, we found a correlation between microtubule stability and MVM-induced phosphorylation of α/β tubulin. In contrast, disassembly of actin filaments late in infection could be traced back to the disregulation of two F-actin associated proteins gelsolin and Wiscott-Aldrich Syndrome Protein (WASP). Thereby, an increase in the amount of gelsolin, an F-actin severing protein was observed during infection, accounting for the disruption of stress fibers upon infection. Concomitantly, the actin polymerization activity also diminished due to a loss of WASP, the activator protein of the actin polymerization machinery the Arp2/3 complex. No effects could be seen in amount and distribution of other F-actin regulatory factors such as cortactin, cofilin, and profilin. In summary, the selective attack of MVM towards distinct host cell cytoskeletal structures argues for a regulatory feature during infection, rather than a collapse of the host cell as a mere side effect of virus production.     

Ultrastructure of the choroid plexus epithelium of pigeons treated with drugs. II. Effect of cytochalasin D and colchicine

A remarkable projection of bleblike protrusions, the expulsion of organelles into the protrusions formed on the apical surface, and the separation into the ventricular lumen of these protrusions was the general cellular response of choroidal epithelial cells to intravenous injection of cytochalasin D (CD). The compact microfilament mass and agglomeration of microtubules at the base of the cluster of protrusions reflect the results of cell contraction and displacement of microfilaments induced by CD. In earlier stages after intravenous injections of colchicine, an obvious increase in the number of varioussized vesicles, vacuoles, and lysosomes in the Golgi region was detected. In the later stages, these organelles were seen to accumulate in the basal portion of the epithelial cells. These changes were accompanied by an increase in vacuoles and the disorganization and displacement of the Golgi complex, and they coincided with a decrease in the number of microtubules in apical and basal cytoplasm. These findings suggest that the action of colchicine results in destruction of the three-dimensional architecture between cytoskeletal network and cell organelles. The present results suggest that the cytoskeletal network plays a role in the spatial coordination of the three-dimensional architecture of cell organelles. The study also indicates that the structural differences in the ventricles of the choroid plexus in drug-treated pigeons are manifestations of regional functional specialization in different parts of the ventricular system.     

Production of human influenza virus in a stabile line of guinea pig tongue cells expressing endogenous oncovirus: an electron microscopic study

Guinea pig tongue (GPT) cells represent a highly sensitive host system for influenza A/WSN (H1N1) infection as evidenced by numerous ultrastructural changes, considerable production of NS1 protein and widespread budding of viral particles at the cytoplasmic membrane. Vesicles of smooth endoplasmic reticulum and of the Golgi complex were transported to the apical area of cell membrane, where the budding of virions took place. Numerous microtubules were directed vertically to these portions of plasma membrane. In contrast, maturation of the endogenous oncovirus particles occurred at the lateral cytoplasmic membrane. Beneath the area of oncovirus maturation and release, a network was seen of microfilaments oriented towards the plasma membrane. The cytoplasm of GPT cells contained numerous nonstructural protein inclusions, which evidently accumulated at the periphery of nucleoli and were seen to reach the cytoplasm crossing the pores of nuclear membrane.     

Expression of cytoskeleton components in various Epstein-Barr virus infected cell lines

It has been shown that viruses can induce alterations in the content and distribution of cytoskeleton structures, particularly actin microfilaments and microtubules. An immunomorphologic study of the cytoskeleton components of various EBV-infected cell lines with expression of different functions of EBV genome has been performed using antibodies to each of its three major components. Intermediate filaments and microtubules were similarly represented in all examined lymphoblastoid cell lines. The distribution of actin microfilaments, on the contrary, differed significantly from cell line to cell line. It is concluded that the morphologic expression of actin might depend on the expression of EBV genome. Furthermore, some of these cell lines might represent a useful substrate for the identification of anticytoskeleton antibodies, mainly anti-actin antibodies, in human sera.     

Characterization of avascular corneal wound healing fibroblasts. New insights into the myofibroblast.

The characteristics and derivation of corneal wound healing fibroblasts (myofibroblasts) were evaluated by studying the temporal changes in the cellular actin distribution of corneal fibrocytes following full thickness 3-mm diameter central corneal wounds in the rabbit. Under certain conditions these wounds heal without neovascularization, allowing for the detailed analysis of invading fibroblasts with minimal contamination by other cell types. The authors employed transmission electron microscopy to localize microfilaments, fluorescent microscopy using NBD-phallacidin, a mushroom toxin which binds specifically to f-actin and oligomeres of g-actin, to localize actin filaments, and isoelectric focusing gels to characterize actin isotypes. During the early stages of wound healing (1-7 days) there is a gradual change in the corneal fibrocytes adjacent to the wound margin characterized by the development of extensive rough endoplasmic reticulum, microtubules, a prominent Golgi apparatus, and a cortical microfilament network. This is in contrast to the normal fibrocyte, which, for the most part, lacks these structures. The development of microfilaments correlated with increased NBD-phallacidin fluorescence of fibrocytes adjacent to the wound as compared with fibrocytes farther removed from the site of injury. Fibroblasts appearing within the wound from 7 days to 2 months after injury had ultrastructural characteristics similar to those of myofibroblasts, including parallel arrays of microfilaments, stress fibers and cell-cell, cell-matrix attachments. Furthermore, these cells stained intensely with NBD-phallacidin, supporting the ultrastructural findings. At 1 month after injury, cells contained within the wound possessed predominantly nonmuscle isoactins (gamma) as seen by silver staining of isoelectric focusing gels, but little or no (smooth muscle) isoactins could be detected. Moreover, no significant differences could be detected between electrophoretic profiles obtained from wounded versus normal corneas. These morphologic and biochemical data suggest that the corneal fibrocyte may develop into a fibroblastlike cell similar to the myofibroblast, and is characterized by a marked increase in filamentous actin.     

创伤弧菌感染树突状细胞的定位及对细胞骨架影响的实验研究

目的 探讨创伤弧菌(Vibrio vulnificus,Vv)感染小鼠树突状细胞株的侵袭过程、定位及其对细胞器的损伤与影响.方法建立Vv1.1758株侵入DC2.4细胞模型,电子显微镜观察不同时间段细菌定位、细胞形态及细胞器的变化过程;荧光显微镜观察细胞骨架—微丝、微管重排情况.结果 Vv1.1758株以双位点胞饮方式...     

Penetration and damage of endothelial cells by Candida albicans.

The mechanisms of phagocytosis of Candida albicans by human vascular endothelial cells and subsequent endothelial cell injury were examined in vitro. Both live and killed C. albicans cells were phagocytized by endothelial cells. This organism specifically induced endothelial cell phagocytosis because neither Candida tropicalis nor Torulopsis glabrata was ingested. Endothelial cell microfilaments polymerized around C. albicans as the organisms were phagocytized. Cytochalasin D inhibited this polymerization of microfilaments around C. albicans and blocked phagocytosis. The blocking of actin depolymerization with phalloidin had no effect on microfilament condensation around the organism, indicating that the microfilaments surrounding C. albicans are formed from a pool of G-actin. Intact microtubules were also necessary for the phagocytosis of C. albicans, since the depolymerizing of endothelial cell microtubules with nocodazole prevented the condensation of actin filaments around the organisms and inhibited phagocytosis. In contrast, microtubule depolymerization was not required for microfilament function because the blocking of microtubule depolymerization with taxol had no effect on microfilament condensation around C. albicans. The phagocytosis of C. albicans was pivotal in the induction of endothelial cell damage, since the blocking of candidal internalization significantly reduced endothelial cell injury. Endothelial cells were not damaged by phagocytosis of dead organisms, indicating that injury was caused by a factor associated with viable organisms. Therefore, C. albicans is uniquely able to induce endothelial cell phagocytosis by comparison with non-albicans species of Candida. Furthermore, at least two components of the endothelial cytoskeleton, microfilaments and microtubules, are necessary for the phagocytosis of C. albicans.     

Plectin deficiency on cytoskeletal disorganization and transformation of human liver cells in vitro

Plectin is a versatile cytoplasmic cross-linking protein that connects intermediate filaments to microfilaments, microtubules, and membrane adhesion sites. The cross-linking functions of plectin help organize the cytoskeleton into a stable meshwork important for maintaining uniformity in cell size and shape. As cells of hepatocellular carcinoma are morphologically different from normal human hepatocytes, we hypothesized that altered plectin expression and cytoskeletal organization underlies this pleomorphic transformation. To test this hypothesis, we analyzed expression levels and organization of all cytoskeletal elements, including intermediate filaments, microfilaments, and microtubules, after plectin knockdown in human Chang liver cells. We found that expression of cytokeratin 18, but not actin or tubulin, was downregulated by suppression of plectin protein. Furthermore, cytokeratin networks were partially collapsed and actin-rich stress fibers were increased. The organization of microtubule networks, by contrast, was unaltered. These findings support our hypothesis that, via effects on cytoskeletal organization, plectin deficiency might play an important role in the transformation of human liver cells.     

Cryptosporidium parvum induces host cell actin accumulation at the host-parasite interface

Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown that Cryptosporidium resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning microscopy to characterize this structure. These studies indicate that host filamentous actin is assembled into a plaque-like structure at the host-parasite interface during parasite invasion and persists during parasite development. The actin-binding protein alpha-actinin is also present in this plaque early in parasite development but is lost as the parasite matures. Other actin-associated proteins, including vinculin, talin, and ezrin, are not present. We have found no evidence of tyrosine phosphorylation within this structure. Molecules known to link actin filaments to membrane were also examined, including alpha-catenin, beta-catenin, plakoglobin, and zyxin, but none was identified at the host-parasite junction. Thus, Cryptosporidium induces rearrangement of the host cell cytoskeleton and incorporates host cell actin and alpha-actinin into a host-parasite junctional complex.     

Attachment of Trypanosoma cruzi to mammalian cells requires parasite energy, and invasion can be independent of the target cell cytoskeleton.

We have previously shown that the binding of Trypanosoma cruzi trypomastigotes to glutaraldehyde-fixed mammalian cells has the characteristics of a receptor-mediated process and that it mimics the attachment step of the invasion of live cells by this parasite. In this study we examined the metabolic requirements for the attachment of trypomastigotes to glutaraldehyde-fixed fibroblasts. The attachment of trypomastigotes to fixed cells is prevented when the energy conservation mechanisms are inhibited with the drugs 2-deoxyglucose, sodium azide, antimycin, crystal violet, oligomycin, N,N'-dicyclohexylcarbodiimide, and carbonyl cyanide 3-chlorophenylhydrazone. However, under the same experimental conditions, the movement of parasites is not significantly affected. Several of these drugs totally inhibit the penetration of the parasite into live target cells. We conclude that the attachment of trypomastigotes to mammalian cells is an active process that requires trypomastigote energy. In addition, we present evidence that penetration into nonphagocytic cells can also be an active process. Trypomastigotes can be seen in scanning electron micrographs traversing extended lamellipodia and entering paraformaldehyde-fixed epithelial cells. Cytochalasin D, a drug that disrupts microfilaments and prevents the formation of plasma membrane extensions mediated by actin, had little or no effect on trypomastigote invasion, while it inhibited Salmonella entry into epithelial cells.     

The expression of cytokeratin in hepatic stellate cells of the cod

In the current study, we examined the cytoskeletal architecture of cod hepatic stellate cells. We found that the cod hepatic stellate cells contain abundant cytoplasmic filaments. Deep-etch electron microscopy showed that the major component of the cytoplasmic filaments was intermediate filaments, although microtubules and microfilaments were also found in the cytoplasmic filament bundles. Immunoelectron microscopy revealed the presence of beta-tubulin, alpha-smooth muscle actin, smooth muscle type myosin, desmin and cytokeratin but not vimentin or glial fibrillar acidic protein. These results demonstrate that the cytoplasmic filaments of cod hepatic stellate cells are composed of desmin and cytokeratin intermediate filaments, acto-myosin complexes and microtubules, suggesting that the cod hepatic stellate cells have both contractile and structural functions. The expression of cytokeratin in cod hepatic stellate cells indicates that they serve for mechanical support in the extremely soft liver tissues of cods with their abundant lipids.     

The fine structural relationship betweenTheileria schizonts and infected bovine lymphoblasts from cultures

The fine structure ofTheileria-lymphoblast relationship was studied using cultured bovine lymphoblastoid cells infected withTheileria parva, T. lawrencei, orT. annulata. The major findings of this study were: (a) the presence of a very active Golgi complex with the associated annulate lamellae; (b) the presence of cytoplasmic microtubules which joined the parasites and host cell centriole during lymphoblast mitosis; and (c) the absence of morphological evidence to suggest that the host cell developed a reaction to the parasitic presence. The significance of these findings is discussed.Key to abbreviations used in Figs. 1–8 Al annulate lamellae - C centriole - Ch chromosome - G Golgi complex - Hc host cell cytoplasm - Mt microtubules - N nucleus - P parasite - S satellite bodies - V vacuole     

Electron microscopic studies on embryonic chick spinal ganglion cells: in vitro effects of antimicrotubular agents on the Golgi complex

Summary Spinal ganglia from 11 day chick embryos were incubated in media containing colchicine or vinblastine and subsequently examined by transmission electron microscopy. Both drugs caused a partial disappearance of cytoplasmic microtubules and a concomitant growth in the number of microfilaments in the neuroblasts. In ganglia treated with vinblastine for 4 h these effects were recorded morphometrically as a decrease of about 80% in the volume density of microtubules and a more than tenfold increase in the volume density of microfilaments. Furthermore, the cells displayed marked structural changes in the Golgi complex. The dictyosomes were mostly distinctly separated from each other and individual dictyosomes showed a decreased number of cisternae and an increased number of closely associated large vacuoles. The results are discussed with regard to the role of microtubules in the organization and function of the Golgi complex in nerve cells.