Temporary disturbance of actin stress fibers in swine kidney cells during pseudorabies virus infection

Rounding and loosening of cells is a consequence of infection with pseudorabies virus (PrV), both in vitro and in vivo. These changes in the normal structure of the cell may be the result of cytoskeletal changes. Immunofluorescence staining of actin filaments and microtubule bundles was performed to examine whether PrV induces a reorganization of these cytoskeletal components in infected swine kidney (SK) cells. Every 2h until 12h post-inoculation (p.i.), cells were washed in cytoskeleton stabilizing buffer (CSB), fixed with paraformaldehyde and washed again with CSB. Cells were permeabilized with a 1/1000 dilution of Triton X-100 and actin filaments were stained by incubating cells with phalloidin-Texas Red. Staining of microtubules was done by incubating the cells subsequently with mouse monoclonal anti-alpha-tubulin and goat anti-mouse IgG-FITC. During the course of infection, actin fibers of SK cells were rearranged in the following sequence: (1) disappearance of thick actin stress fibers between 4 and 6h p.i., (2) complete loss of stress fibers between 6 and 8h p.i., and (3) reappearance of thin stress fibers starting from 10h p.i. In contrast to herpes simplex virus 1 (HSV1) or equine herpesvirus 1 (EHV1), PrV infection did not induce changes in the cellular microtubule network. PrV infection induces a temporary disassembly of actin stress fibers.     

Actin polymerization enhances Pasteurella haemolytica leukotoxicity

Pasteurella haemolytica leukotoxin is cytotoxic to bovine leukocytes, causing increased cell membrane permeability, osmotic swelling, release of cytosolic proteins and cell lysis. These studies were designed to test if leukotoxin causes release of the cytoskeletal protein, actin, from bovine leukemia cells and if purified actin-influenced bacterial growth or leukotoxin production. Culture supernatants caused a 7-fold decrease in viability of bovine leukemia cells and increased cell permeability that was accompanied by release of beta-actin into the cell culture supernatant. Exposing P. haemolytica to purified actin solutions induced the conversion of monomeric G-actin to polymerized F-actin. This conversion was partially inhibited by bovine P. haemolytica immune, but not pre-immune, serum. Loss of streptomycin resistance following treatment of the organism with acridine orange ablated the polymerizing activity. Incubation of P. haemolytica in the presence of purified F-actin did not affect growth but resulted in culture supernatant that had 3.0-3.9-fold greater leukotoxicity compared to medium alone or medium containing G-actin, heat-denatured actin or albumin. The effect of actin on leukotoxicity was concentration-dependent and directly associated with increases in secreted leukotoxin. The interaction between P. haemolytica and actin is potentially detrimental to the host by inducing polymerization of actin into insoluble filaments and by enhancing leukotoxicity.     

Histochemischer Nachweis von Aktin in den terminalen Darmgäßen des Huhnes*

Using fluorescence microscopy, cytoskeletal actin filaments were found in the terminal blood vessels of chicken intestine. Direct detection with Tetramethylrhodaminyl-Phalloidin showed a high concentration of actin filaments in the tunica media and a low concentration in the endothelium. Actin filaments were also present in the endothelial cells of capillaries and to a higher degree also in the pericytes. The wall of the veins in the villi, which were concentrically surrounded by bundles from the tunica muscularis mucosae, had a greater number of actin filaments than the wall of the venules present in the villi. The discussion touches on the possibility that the microfilaments play a role in blood regulation, especially in the capillaries.     

Analysis of selective mobilization of L-selectin and Mac-1 reservoirs in bovine neutrophils and eosinophils

Following activation of granulocytes, L-selectin (CD62L) is generally shed from the cellular surface, whereas Mac-1 (CD11b/CD18) expression is well known to increase. However, a number of studies in bovines and humans show that the expression of L-selectin may increase as well. This urged us to examine the possible existence of both L-selectin and Mac-1 reservoirs in bovine neutrophil and eosinophil populations through the use of flow cytometry in combination with an optimized method for cell membrane permeabilization. Augmented L-selectin and Mac-1 expression was detected in both granulocyte populations upon saponin treatment. Confocal microscopic studies indicated that both molecules exhibit a different pattern of subcellular localization. Incubation with sialidase revealed the existence of hidden L-selectin epitopes at the cell surface, while no additional Mac-1 epitopes were exposed. Platelet-activating factor stimulation decreased surface and total expression of L-selectin to the same extent in both populations, but solely affected Mac-1 surface expression on eosinophils. Moreover, cytoskeletal actin filaments and microtubules were found to be involved in the regulation of Mac-1 surface expression on bovine neutrophils and eosinophils. In marked contrast, expression of L-selectin was minimally affected by cytoskeleton perturbing agents. The present study indicates that L-selectin and Mac-1 adhesion molecules reside in distinctly located reservoirs in bovine granulocytes and can be selectively mobilized upon in vitro stimulation.     

Comparison of bovine and human platelet deformability, using micropipette elastimetry

We evaluated the deformability of bovine platelets and contrasted the effects of pharmacologic and thermal perturbations on cytoskeletal structure of human and bovine platelets. Platelets were aspirated into micropipettes (0.7 to 0.8 micron in diameter) by stepwise increments in tension. The resulting lengths of the cell extensions were recorded. The cell extensions aspirated from bovine platelets were shorter than the extensions drawn from human platelets. Disassembly of the circumferential microtubule coil allowed human platelets to pass through the pipette, but the same treatments only slightly increased the deformability of bovine platelets. Alteration of the actin filament cytoskeleton caused increased mechanical fragility of human platelets. In contrast, even the combined use of microtubule and actin filament-disrupting agents only modestly increased the deformability of bovine platelets and did not cause premature fragmentation of the cells. Unusual cytoskeletal structure, absence of an open canalicular system, and disparity in granule size may all contribute to the variance in deformability between the platelets of the 2 species. Reduced cell deformability may impair bovine platelet surface interactions by diminishing the ease of cell spreading and formation of areas of contact between the platelet and other cell surfaces.     

Immunocytochemical Identification of Different Cell Types in Bovine Nasolabial Glands with Particular Emphasis on Cytoskeletal Protein Expression

Nasolabial glands are serous glands forming a thick subcutaneous layer in the bovine muzzle. In order to identify the different epithelial cell types, both immunofluorescent and immunoperoxidase techniques were employed on frozen and fixed sections using monoclonal antibodies to cytoskeletal proteins and S-100. Actin was also detected with phalloidin. The results show that four cell types can be identified on the ground of the different composition of the cytoskeletal filaments: acinar, basket, luminal duct and basal duct cells. Acinar, luminal duct cells and basal duct cells express different patterns of cytokeratins, as shown by the 12 anti-cytokeratin monoclonal antibodies used, and both basket cells and the basal cells of intercalated ducts are also reactive to phalloidin and anti-x-smooth muscle actin monoclonal antibody. The presence of actin supports the conclusion that basal duct cells are also contractile elements, i. e. myoepithelial cells. Vimentin, glial fibrillary acidic protein (GFAP), and S-100, molecules considered to be markers of myoepithelial cells by many AA., were not found. The intermediate filaments of the duct epithelium appear more complex and heterogeneous in comparison with those present in the acinar cells.     

Immunolocalization of cytoskeletal components in the tegument of the 3-week-old juvenile and adult Fasciola gigantica

Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments have been localised in the tegument of the 3-week-old juvenile and adult Fasciola gigantica by means of immunofluorescence and immunoperoxidase techniques, using mouse monoclonal anti-alpha-tubulin, anti-cytokeratin antibodies and biotinylated-phalloidin, respectively. The immunostaining with the above probes were also performed in adult Schistosoma mansoni for comparison. The presence of tubulin, indicative of microtubules, was demonstrated in the tegumental cell bodies, their cytoplasmic processes, and the basal layer of the tegumental syncytium of F. gigantica. While in S. mansoni, tubulin appeared as vertical lines stretching across the whole thickness of the syncytium. Cytokeratin, representing one type of intermediate filaments, was detected in the tegumental cell bodies, their cytoplasmic processes, tegumental syncytium and spines of F. gigantica. In contrast, cytokeratin was evident only in the syncytium of S. mansoni, but not in the spines. Phalloidin, which could bind to actin, a subunit of microfilament, was detected in the tegumental cell bodies, their processes, and the microtrabecular network which form the scaffold of the tegumental syncytium of F. gigantica. In S. mansoni, actin was localized in similar tissues except the syncytium was not stained while spines exhibited intense staining. In F. gigantica, the presence of microtubules and actin filaments in the tegumental cells, their processes and in the syncytium could mediate the movement of secretory granules from the cell bodies towards the basal as well as the apical layer of the tegument. Cytokeratin filaments may serve to reinforce the integrity of the tegumental syncytium as well as the spines.     

Studies on synchronous egress of coccidian parasites (<Emphasis Type="Italic">Neospora caninum,Toxoplasma gondii,Eimeria bovis)</Emphasis> from bovine endothelial host cells mediated by calcium ionophore A23187

Neospora caninum, Toxoplasma gondii and Eimeria bovis are coccidian parasites of veterinary importance. Tachyzoites of N. caninum and T. gondii and sporozoites of E. bovis are able to invade and replicate in endothelial cells in vivo and in vitro. As it holds true for all eukaryotic cells, the survival of parasitized host cells and the parasites themselves should be dependent on ion balances, especially on extra- and intracellular calcium concentrations. Addition of the calcium ionophore A23187 reliably did release merozoites from mature N. caninum and T. gondii meronts grown in cultured primary bovine umbilical vein endothelial cells (BUVEC). Extent and time course of merozoite release depended on both, maturity of the meronts and concentration of the calcium ionophore. Attempts, however, to achieve synchronous release of merozoites from E. bovis first generation meronts by ionophore treatment failed, suggesting a different biological behaviour of this parasite. According to microscopical observations, the quite variable time of E. bovis macromeront maturation and a hampered merozoite exit owing to dense parasite-induced cytoskeleton elements surrounding the meront may be a reason for the lack of inducible synchronous release. Electronic supplementary materials The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Evidence for cytoskeletal changes secondary to plasma membrane functional alterations in the in vitro cell response to Clostridium perfringens epsilon-toxin

To investigate the mode of action of Clostridium perfringens epsilon-toxin, MDCK cells were treated with purified toxin and incubated at 37 degrees C for up to 24h. Exposure to epsilon-toxin caused a time-dependent decrease in cell-cell and cell-substrate interactions. After 30min of treatment retraction of the cell body and the emission of filopodia were detectable in a number of cells. Longer exposure resulted in cell rounding and cell blebbing which reached a maximum after 5h of toxin treatment. A parallel modification in the cytoskeleton was also detected. Actin marginalization and the entanglement of microtubules and intermediate filaments were observed by fluorescence microscopy after 30min of toxin exposure. Functional alterations of the plasma membrane of MDCK cells were assessed by flow cytometry. After 10 or 30min of intoxication an increase in cell volume was detected, indicating an alteration in plasma membrane permeability. These findings provide evidence for cytoskeletal changes and plasma membrane functional alterations in the in vitro cell response to C. perfringens epsilon-toxin.     

Myosins 1 and 6, myosin light chain kinase,actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes

Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.     

Actinfilaments in the Ruminal Epithelium of Cattle

Phalloidin and immunogold labeling were used to localize actinfilaments in the ruminal pilar epithelium of adult catde on the light and electron microscopic level. Incubation with rhodaminyl-phalloidin permitted an overview of general actin distribution pattern. Distinct subcellular localization of actin was revealed with the biotin-streptavidin bridge technique in a postembedding procedure. Superior fixation of membranes and filaments was achieved with tannic acid-glutaraldehyde. Surface-etching prior to immunoincubation ensured restoration of actin immunoreactivity. Patterns of distribution in non-keratinized and keratinized epithelial cells as well as in pericytes and endothelial cells point to cell-specific cytoskeletal and motile functions of actinfilaments.     

Escherichia coli isolated from bovine mastitis invade mammary cells by a modified endocytic pathway

Escherichia coli is a major pathogen in the aetiology of bovine mastitis. Although classically considered to be an environmental pathogen causing mainly transient infection, the incidence of persistent E. coli mastitis infections may be increasing, suggesting an adaptation of this pathogen to the bovine udder environment. Mastitis E. coli strains have been demonstrated to enter bovine mammary cells in vitro but little is known about the invasion mechanism or the intracellular fate of the bacteria. In order to further understand the pathogenesis of persistent E. coli bovine mastitis we investigated the intracellular trafficking of mastitis E. coli isolates in primary bovine mammary cells using confocal microscopy and fluorescent markers of endocytic compartments. Consistent with other studies, mastitis E. coli were found to invade primary bovine mammary cells in vitro. This process did not involve in the rearrangement of the actin cytoskeleton. Intracellular bacteria were observed within membrane-bound compartments that labelled with the early endosomal marker phosphatidylinositol 3-phosphate (PtdIns(3)P) and also within late endosome-like compartments labelled with the small GTPase Rab7, indicating an endocytic mechanism of bacterial internalization. Bacteria were not observed within acidified lysosomal compartments or autophagic vacuoles, suggesting that the internalized bacteria are not targeted for lysosomal degradation via either the classical endocytic pathway or the autophagic response. Our findings are consistent with an endosomal survival niche for the internalized bacteria, allowing them to evade host immune responses and establish an infection reservoir that could later re-emerge as a recurrent clinical mastitis episode.     

Adherence to various host cell lines of Mycoplasma bovis strains differing in pathogenic and cultural features

Mycoplasma bovis is known to be responsible for pneumonia and arthritis in calves, as well as mastitis in dairy cows. Despite clear evidence of its pathogenic potential, little is known about mechanisms of cytadherence and the molecular factors involved. The purpose of this work was to compare adherence rates of M. bovis field strains to different host cell lines and study the effects of cloning and sub-culturing M. bovis strains on their adherence properties. Eighteen metabolically labeled M. bovis strains isolated from different pathological backgrounds were examined in adherence trials using four different host cell lines, i.e. embryonic bovine lung (EBL), embryonic bovine trachea (EBTr), Madin Darby bovine kidney (MDBK) and rabbit kidney (RK) cells. Although large interstrain variations in adherence rates (3.4-19.1%) were measured they could not be correlated to the pathological background (pneumonia, arthritis or mastitis). Adherence rates to the fibroblast cell line (EBTr) were significantly lower than those to the three epithelial cell lines (EBL, MDBK and RK). The only non-pathogenic strain (221/89) exhibited lower adherence rates than three isolates from clinical mastitis. Interestingly, adherence rates were significantly reduced after in vitro passaging. In contrast, no effect of single cloning of strains on adherence was observed. There was no general correlation between expression of variable surface proteins (Vsps) as monitored by immunoblotting and adherence rates, although alterations in Vsp expression profiles were seen as a consequence of passaging. As there is probably a large number of adhesins, variable and non-variable, on the surface of M. bovis cells the issue is very complex, and the most active components have yet to be identified.     

MSTN基因对牛肌动蛋白细胞骨架调节通路的影响

为探究肌肉生长抑制素（myostatin,MSTN）对牛骨骼肌生长发育的作用机制,本研究前期利用定量蛋白质组学与磷酸化蛋白质组学分析野生型蒙古牛（MG.WT）和MSTN^+/-蒙古牛（MG.MSTN^+/-）腿臀肌肌肉组织中蛋白质水平和磷酸化修饰水平的差异变化,使用已建立的牛骨骼肌卫星细胞体外诱导成肌分化模型,检测设计合成的MSTN siRNA （si-MSTN）干扰效果;采用实时荧光定量PCR和Western blotting方法检测转染si-MSTN的增殖期（GM）和分化第3天（DM3）牛骨骼肌卫星细胞中肌动蛋白细胞骨架调节通路相关基因的mRNA和蛋白水平的表达变化,研究敲低MSTN表达对肌动蛋白细胞骨架调节通路的影响。结果显示,在MSTN^+/-蒙古牛肌肉组织中共鉴定到16个肌动蛋白细胞骨架调节通路相关基因表达丰度上调;转染si-MSTN细胞中的MSTN表达水平极显著降低（P<0.01）;在转染si-MSTN的GM期牛骨骼肌卫星细胞中,肌动蛋白细胞骨架调节通路相关基因ENAH、ACTN4和Cdc42的mRNA水平均显著升高（P<0.05）,PFN1、RhoA和ACTN4的蛋白水平均显著或极显著升高（P<0.05;P<0.01）;在转染si-MSTN的DM3牛骨骼肌卫星细胞中,ENAH、CFL1、SCIN和Cdc42基因mRNA水平均显著升高（P<0.05）,RhoA基因mRNA水平极显著升高（P<0.01）,PFN1和ACTN4的蛋白水平均显著升高（P<0.05）。结果表明,干扰MSTN可以促进肌动蛋白细胞骨架调节通路相关基因的表达,探明了MSTN可能通过介导肌动蛋白细胞骨架调节通路影响牛骨骼肌卫星细胞增殖和成肌分化的分子机制,为进一步研究MSTN对牛成肌分化的调控机制提供参考。     

Development and characterization of monoclonal antibodies to first-generation merozoites of Eimeria bovis.

Merozoites of Eimeria bovis were harvested from bovine monocyte cell cultures and used to immunize BALB/C mice. Spleens from immunized mice were removed and the cells fused with mouse myeloma cells. Supernates from resulting hybridoma cell lines were examined for antibodies to first-generation E. bovis merozoites using an indirect immunofluorescent antibody (IFA) assay. Three positive cell lines were identified and cloned by limiting dilution. All three cell lines produced immunoglobulins of the IgG1 isotype that recognized antigens in the anterior half to two-thirds of the merozoites. Specificity of the monoclonal antibodies was examined with the IFA assay against sporozoites of E. bovis, sporozoites and merozoites of Eimeria papillata from mice and Eimeria tenella from chickens, sporozoites of Isospora suis from pigs, and tachyzoites of Toxoplasma gondii and Neospora caninum from cell cultures. Monoclonal antibodies from the three clones reacted with the anterior end of E. bovis sporozoites, but did not react with the other parasites examined. None of the monoclonal antibodies reacted with merozoite antigens in immunoblots.