Enterohemorrhagic Escherichia coli induces apoptosis which augments bacterial binding and phosphatidylethanolamine exposure on the plasma membrane outer leaflet

Enterohemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes watery diarrhea and hemorrhagic colitis and can lead to serious and even fatal complications such as hemolytic uremic syndrome. We investigated the ability of EHEC to kill host cells using three human epithelial cell lines. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA and morphological changes detected by electron microscopy changes revealed evidence of apoptotic cell death. The rates and extents of cell death were similar for both verotoxin-producing and nonproducing strains of EHEC as well as for a related gastrointestinal pathogen, enteropathogenic E. coli (EPEC). The induction of apoptosis by bacterial attachment was independent of verotoxin production and greater than that produced by a similar treatment with verotoxin alone. Expression of phosphatidylethanolamine, previously reported to bind EHEC and EPEC, was also increased on apoptotic cells but with little correlation to phosphatidylserine expression. Phosphatidylethanolamine levels but not phosphatidylserine levels on dying cells correlated with EHEC binding. Cells treated with phosphatidylethanolamine-containing liposomes also showed increased EHEC binding. These results suggest that bacterial induction of apoptosis offers an advantage for bacterial attachment by augmenting outer leaflet levels of the phosphatidylethanolamine receptor.     

Calcineurin is implicated in the regulation of the septation initiation network in fission yeast

BACKGROUND: In fission yeast, calcineurin has been implicated in cytokinesis because calcineurin-deleted cells form multiple septa and cell separation is impeded. However, this mechanism remains unclear. RESULTS: We screened for mutations that confer synthetic lethality with calcineurin deletion and isolated a mutant, its 10-1/cdc7-i10, a novel allele of the cdc7+ gene involved in the septation initiation network (SIN). The mutation created a termination codon, resulting in the truncation of Cdc7 by 162 amino acids, which is not localized in the spindle pole body. Following treatment with the immune suppressive drug FK506, cdc7-i10 and the original cdc7-24 mutant cells showed highly elongated multinuclear morphology with few visible septa, closely resembling the phenotype at the restrictive temperature. Other SIN mutants, cdc11, spg1, sid2 and mob1 showed similar phenotypes following FK506 treatment. Consistent with this, expression of the constitutively active calcineurin suppressed the growth defects and septum initiation deficiency of these SIN mutants at the restrictive temperature. Moreover, electron microscopy revealed that calcineurin-deleted cells had very thick multiple septa which were partially and ectopically formed. CONCLUSION: These results suggest that calcineurin is involved in the regulation of the SIN pathway, and is required for the proper formation and maturation of the septum in fission yeast.     

Caveolae membrane domains, specialized transmembrane exchange zones implicated in cell signalling

Caveolae are small pockets or invaginations localized at the plasma membrane. They are enriched in glycosphingolipids, cholesterol, sphingomyelin and lipid-enchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4 C. Caveolins are the principal protein components of caveolae and play an important structural role in the formation of caveolae membranes. Numerous molecules involved in cell signalling have been identified in caveolae, suggesting that these structures may serve to compartimentalize, modulate and integrate signalling events at the cell surface. Depletion of membrane cholesterol disrupts the formation and function of caveolae, suggesting that these membrane microdomains are involved in a range of biological processes. Moreover, exposure of endothelial cells to high levels of cholesterol upregulates the caveolin abundance in caveolae, and decreases nitric oxide synthesis, suggesting that this may be an early event in atherogenesis. Alteration in the expression of caveolin genes has also been implicated in human diseases such as cancers, diabetes, Alzheimer's disease and muscular distrophy.     

Plasma membrane appearance of phosphatidylethanolamine in stimulated macrophages

Mouse peritoneal macrophages were labeled with [1-3H]ethanolamine, and the presence of radioactive [3H]phosphatidylethanolamine (PE) at the plasma membrane was monitored by reacting the cells with trinitrobenzene sulfonic acid (TNBS) under nonpenetrating conditions. Macrophages stimulated with either the calcium ionophore A23187 or zymosan demonstrated a larger proportion of radiolabeled PE in the plasma membrane than control, nonstimulated cells. In experiments in which macrophages were labeled with ethanolamine for increasing times, appearance of membrane 3[H]PE was stimulated as early as after 2 hr of labeling. Macrophages labeled for 24 hr, then stimulated and returned to fresh medium still reflected a higher amount of membrane 3[H]PE at 2 hr after the stimulation, suggesting stimulation results in long-term alterations in plasma membrane lipids. Protease-peptone-elicited macrophages, which are not stimulated by zymosan or ionophore, did not exhibit an increase in membrane 3[H]PE upon stimulation. The size of the TNBS-accessible radiolabeled PE pool increased proportionately with a second stimulation; however, a subsequent labeling of the cells with TNBS after brief warming increased the TNBS-accessible pool in control cells only. As shown in previous studies, macrophage stimulation resulted in an increased incorporation of lipid precursors into phospholipid. The mass of plasma membrane Tnp-PE relative to mass of PE was not increased in ionophore-treated macrophages in contrast to a small (approximately 22%) increase in zymosan-treated cells. These results are suggestive of alterations in lipid synthesis in stimulated macrophages and possible long-term changes in the structure and function of the plasma membrane of macrophages following stimulation.     

Granulophysin is located in the membrane of azurophilic granules in human neutrophils and mobilizes to the plasma membrane following cell stimulation.

Granulophysin, a protein described in platelet dense granule membranes, has been shown to be similar or identical to CD63, a lysosomal membrane protein. We have previously shown granulophysin to be present in neutrophils using immunofluorescence. We now localize granulophysin to the neutrophil azurophilic granules by fine structural immunocytochemistry. Granulophysin expression on the surface membrane of the neutrophil is increased following stimulation of the cells, demonstrated by flow cytometry and fine structural immunocytochemistry. A similar pattern is shown for an anti-CD63 antibody. Incubation of activated neutrophils with D545, a monoclonal antibody to granulophysin, blocks subsequent binding of anti-CD63 antibodies to the cell surface, and anti-CD63 antibodies prevent subsequent binding of D545 as assessed by flow cytometry and immunoblotting. Our results support the homology of CD63 and granulophysin previously demonstrated in platelets and confirm CD63 as an activation marker in neutrophils and the first azurophilic granule membrane marker of neutrophils.     

Unipolar membrane association of Dishevelled mediates Frizzled planar cell polarity signaling

Drosophila epithelia acquire a planar cell polarity (PCP) orthogonal to their apical-basal axes. Frizzled (Fz) is the receptor for the PCP signal, and Dishevelled (Dsh) transduces the signal. Here, I demonstrate that unipolar relocalization of Dsh to the membrane is required to mediate PCP, but not Wingless (Wg) signaling. Dsh membrane localization reflects the activation of Fz/PCP signaling, revealing that the initially symmetric signal evolves to one that displays unipolar asymmetry, specifying the cells' ultimate polarity. This transition from symmetric to asymmetric Dsh localization requires Dsh function, and reflects an amplification process that generates a steep intracellular activity gradient necessary to determine PCP.     

Insulin receptor is implicated in triple-negative breast cancer by decreasing cell mobility

Triple-negative breast cancer (TNBC) has a poor prognosis and typically earlier onset of metastasis in comparison with other breast cancer subtypes. It has been reported that insulin receptor (INSR) is downregulated in TNBC, however, its clinical significance and functions in TNBC remain to be elucidated. In this study, we found that INSR expression was significantly downregulated in TNBC, and overexpression of INSR suppressed cell migration and invasion in TNBC. In addition, the survival rate of breast cancer patients with low INSR expression was lower than that of patients with high INSR expression. INSR expression was significantly correlated with lymph node metastasis, clinical tumor stages, ER status, PR status, and the proliferation index Ki-67 expression. In summary, our study suggests that INSR may serve as a biomarker for breast cancer prognosis and it may be a potential target for TNBC treatment.     

Cell surface biotinylation in the determination of epithelial membrane polarity

Summary The plasma membranes of polarized epithelial cells are divided into two distinct domains separated by tight junctions and characterized by distinct polypeptide compositions. Study of the cellular mechanisms involved in generating this anisotropy has recently been facilitated through the introduction of a surface labeling technique employing NHS-biotin (29). Incorporation of the biotin marker allows proteins present at one or the other cell surface domain to be identified and isolated. This methodology is applicable to investigations of both the steady state and the dynamic aspects of epithelial polarity. We discuss protocols through which this technique can be applied to various experimental situations. Methodologic details and special considerations are discussed. The utility of surface biotinylation for studies of epithelial polarity depends on this technique's capacity to report quantitatively on the surface distributions of plasmalemmal proteins. We find that under certain circumstances, surface biotinylation is extremely inefficient. Furthermore, in some cases the efficiency of surface biotinylation is dramatically different at the two plasmalemmal domains of epithelial cells grown on permeable filter supports. Thus, surface biotinylation does not always provide an accurate picture of plasma membrane protein distributions. These situations are discussed and methods to assess the efficiency and accuracy of surface biotinylation are proposed.     

Rod-shaped particles in the plasma membrane of the mitochondria-rich cell of amphibian epidermis

A freeze-fracture study has revealed rod-shaped intramembra-nous particles on the plasma membrane P-face (cytoplasmic leaflet) of the mi-tochondria-rich cell (or flask cell) of Xenopus laevis and Rana ridibunda epidermis. Such particles have previously been found in all other mitochondria rich cells examined by this technique, namely, the MR-cell of toad bladder epi-thelium, the dark cèll of rat kidney collecting tubule, and the flask cell of Xenopus kidney collecting tubule. These particles are assumed, therefore, to be closely connected with the function of this cell type.     

Angiopoietin-2 is implicated in the regulation of tumor angiogenesis

We addressed the effect of angiopoietin expression on tumor growth and metastasis. Overexpression of angiopoietin-2 (Ang-2) in Lewis lung carcinoma and TA3 mammary carcinoma cells inhibited their ability to form metastatic tumors and prolonged the survival of mice injected with the corresponding transfectants. In contrast, angiopoietin-1 (Ang-1) overexpression had no detectable effect on the ability of either tumor type to disseminate. Tumors derived from Ang-2-overexpressing cells displayed aberrant angiogenic vessels that took the form of vascular cords or aggregated vascular endothelial cells with few associated smooth muscle cells. These vascular cords or aggregates were accompanied by endothelial and tumor cell apoptosis, suggesting that an imbalance in Ang-2 expression with respect to Ang-1 and vascular endothelial growth factor may disrupt angiogenesis and tumor survival in vivo. Our observations suggest that Ang-2 may play an important role in regulating tumor angiogenesis.     

Dysferlin is a plasma membrane protein and is expressed early in human development

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.     

Fusion protein vesicle-associated membrane protein 2 is implicated in IFN-gamma-induced piecemeal degranulation in human eosinophils from atopic individuals

BACKGROUND: Exocytosis is an integral event during IFN-gamma-induced piecemeal degranulation in eosinophils. In many tissues soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including vesicle-associated membrane protein (VAMP), act as specific intracellular receptors to allow granule fusion with the membrane during degranulation. However, the mechanisms underlying eosinophil piecemeal degranulation induced by IFN-gamma are not well understood. OBJECTIVE: We sought to assess whether eosinophils express the vesicular SNARE protein VAMP-2 and to determine the involvement of VAMP-2 in IFN-gamma-induced piecemeal degranulation. METHODS: Human peripheral blood eosinophils (> or =97%) from atopic subjects were subjected to RT-PCR and sequence analysis with specific primers for VAMP-2 mRNA. Western blotting and flow cytometric analysis were carried out to confirm the identity of VAMP-2 and its susceptibility to cleavage by tetanus toxin. Confocal laser scanning microscopy imaging was conducted on double-labeled cytospin preparations of eosinophils at 0, 5, 10, 30, and 60 minutes and 16 hours of IFN-gamma (500 U/mL) stimulation. RESULTS: Eosinophils expressed VAMP-2 mRNA (n = 4 donors), which exhibited 100% homology with human VAMP-2 cDNA on sequencing. Eosinophils were also found to express tetanus toxin-sensitive VAMP-2 protein. RANTES and VAMP-2 immunofluorescence were observed to colocalize to similar intracellular structures by means of confocal imaging. IFN-gamma induced a rapid translocation of VAMP-2(+) organelles toward the cell membrane in correlation with RANTES. CONCLUSIONS: These findings suggest that exocytosis in human eosinophils is regulated by SNAREs, with a specific role indicated for VAMP-2 in piecemeal degranulation.     

Factors implicated in the generation and persistence of long-lived plasma cell-mediated autoimmunity

Autoimmune diseases are frequently associated with the production of autoantibodies by cells that escaped the protective mechanisms that control self-tolerance. Some of these cells develop into long-lived plasma cells which are predominantly located in the bone marrow. The generation of this particular type of cell requires specific migration, differentiation, and survival signals. The identification of some of the factors involved in these pathways has permitted the development of specific therapeutic approaches and may even provide investigators with further new therapeutic targets, particularly in autoimmune diseases associated with persistent autoantibody production. We reviewed the existing evidence for the mechanisms implicated in the perpetuation of long-lived plasma cells and the most recent therapeutic proposals in this context.     

Geranylgeranyltransferase Cwg2‐Rho4/Rho5 module is implicated in the Pmk1 MAP kinase‐mediated cell wall integrity pathway in fission yeast

Pmk1, a fission yeast homologue of mammalian ERK MAPK, regulates cell wall integrity, cytokinesis, RNA granule formation and ion homeostasis. Our screen for vic (viable in the presence of immunosuppressant and chloride ion) mutants identified regulators of the Pmk1 MAPK signaling, including Cpp1 and Rho2, based on the genetic interaction between calcineurin and Pmk1 MAPK. Here, we identified the vic2‐1 mutants carrying a mis‐sense mutation in the cwg2⁺ gene encoding a beta subunit of geranylgeranyltransferase I (GGTase I), which participates in the post‐translational C‐terminal modification of several small GTPases, allowing their targeting to the membrane. Analysis of the vic2‐1/cwg2‐v2 mutant strain showed that the localization of Rho1, Rho4, Rho5 and Cdc42, both at the plasma and vacuolar membranes, was impaired in the vic2‐1/cwg2‐v2 mutant cells. In addition, Rho4 and Rho5 deletion cells exhibited the vic phenotype and cell wall integrity defects, shared phenotypes among the components of the Pmk1 MAPK pathway. Consistently, the phosphorylation of Pmk1 MAPK on heat shock was decreased in the cwg2‐v2 mutants, and rho4‐ and rho5‐null cells. Moreover, Rho4 and Rho5 associate with Pck1/Pck2. Possible roles of Cwg2, Rho4 and Rho5 in the Pmk1 signaling will be discussed.     

Modified method for evaluation of plasma membrane integrity in eukaryotic cell

We propose a method for evaluation of the number of viable cells by the content of bromcresol purple dye absorbed by dead cells from the incubation medium. Myramistin was used as a pore-forming agent. The number of live cells in yeast suspension inversely correlated with the percentage of dye absorbed by cells. The method is simple and requires no special equipment. The effect of myramistin on Candida albicans and Malassezia sympodialis cells and on epitheliocytes was evaluated. Two-hour treatment with myramistin in a concentration commonly used in clinical practice (100 mug/ml) decreased the number of viable cells by 2 and 1 order of magnitude, respectively. Epitheliocytes under the same conditions absorbed approximately the same mount of the dye as Candida cells.