Localization, dynamics, and function of survivin revealed by expression of functional survivinDsRed fusion proteins in the living cell

Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.     

Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence

A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.     

Conserved stem II of the box C/D motif is essential for nucleolar localization and is required,along with the 15.5K protein,for the hierarchical assembly of the box C/D snoRNP

The 5' stem-loop of the U4 snRNA and the box C/D motif of the box C/D snoRNAs can both be folded into a similar stem-internal loop-stem structure that binds the 15.5K protein. The homologous proteins NOP56 and NOP58 and 61K (hPrp31) associate with the box C/D snoRNPs and the U4/U6 snRNP, respectively. This raises the intriguing question of how the two homologous RNP complexes specifically assemble onto similar RNAs. Here we investigate the requirements for the specific binding of the individual snoRNP proteins to the U14 box C/D snoRNPs in vitro. This revealed that the binding of 15.5K to the box C/D motif is essential for the association of the remaining snoRNP-associated proteins, namely, NOP56, NOP58, fibrillarin, and the nucleoplasmic proteins TIP48 and TIP49. Stem II of the box C/D motif, in contrast to the U4 5' stem-loop, is highly conserved, and we show that this sequence is responsible for the binding of NOP56, NOP58, fibrillarin, TIP48, and TIP49, but not of 15.5K, to the snoRNA. Indeed, the sequence of stem II was essential for nucleolar localization of U14 snoRNA microinjected into HeLa cells. Thus, the conserved sequence of stem II determines the specific assembly of the box C/D snoRNP.     

The proteasome regulatory particle subunit Rpn6 is required for Drosophila development and interacts physically with signalosome subunit Alien/CSN2

The eukaryotic 26S proteasome plays a central role in ubiquitin-dependent intracellular protein metabolism. The multimeric holoenzyme is composed of two major subcomplexes, known as the 20S proteolytic core particle and the 19S regulatory particle (RP). The RP can be further dissected into two multisubunit complexes, the lid and the base complex. The lid complex shares striking similarities with another multiprotein complex, the COP9 signalosome. Several subunits of both complexes contain the characteristic PCI domain, a structural motif important for complex assembly. The COP9 signalosome was shown to act as a versatile regulator in numerous pathways. To help define the molecular interactions of the signalosome during Drosophila development, we performed a yeast two-hybrid screen to identify proteins that physically interact with subunit 2 of the complex, namely Alien/CSN2. Here, we report that Drosophila Rpn6, a non-ATPase subunit of the RP lid complex, interacts with Alien/CSN2 via its PCI domain. The temporal and spatial expression patterns of Rpn6 and alien/CSN2 overlap on a large scale during development providing additional evidence for their interaction in vivo. Analyses of an Rpn6 P element insertion mutant and newly generated Rpn6 alleles reveal that Rpn6 is essential for Drosophila development.     

Regulation of outside-in signaling in platelets by integrin-associated protein kinase C beta

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin alpha(IIb)beta(3) in platelets, but the responsible PKC isoforms and mechanisms are unknown. Alpha(IIb)beta(3) interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether alpha(IIb)beta(3) might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKC beta co-immunoprecipitated with alpha(IIb)beta(3) in response to the interaction of platelets with soluble or immobilized fibrinogen. PKC beta recruitment to alpha(IIb)beta(3) was accompanied by a 9-fold increase in PKC activity in alpha(IIb)beta(3) immunoprecipitates. RACK1, an intracellular adapter for activated PKC beta, also co-immunoprecipitated with alpha(IIb)beta(3), but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKC beta recruitment to alpha(IIb)beta(3) and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKC beta spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKC beta I to associate with alpha(IIb)beta(3) and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the beta(3) cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to beta(3). These studies demonstrate that the interaction of alpha(IIb)beta(3) with activated PKC beta is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through alpha(IIb)beta(3). Furthermore, the studies extend the concept of alpha(IIb)beta(3) as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.     

Acetic acid esters and permeable weak acids induce active proton extrusion and extension growth of coleoptile segments by lowering the cytoplasmic pH

In Avena coleoptile segments a decrease of cytoplasmic pH activates energy-dependent H⁺ extrusion into the apoplast, thereby triggering extension growth. This sequence of events cannot be inhibited by cycloheximide and is induced by the following conditions and compounds. (i) A short anaerobic treatment of coleoptile segments results in the formation of lactic acid and an intracellular decrease of pH. For a period of 20 min after transfer to normal air, the growth rate is up to six times higher than the rate before anaerobiosis. (ii) Similarly, incubation of segments with CN^– (0.1 mM) in the presence of oxygen causes and accumulation of lactic acid and a fall in cell-sap pH. After removing CN^– a growth burst occurs. (iii) Higher concentrations of permeable acids (10 mM in buffer pH 5.8) induce extension growth. This growth is O₂-dependent and therefore differs from the acid growth, which can be triggered under anaerobic conditions by acid buffers of pH5 via the direct increase of cell-wall plasticity. (iv) A short application of CO₂-saturated buffer (pH 5.8) causes CO₂-induced elongation growth; after a 3-min pulse the growth rate is enhanced for about 15 min. (v) Lipophilic esters of acetic acid or propionic acid, such as naphthylacetate, naphthylpropionate, phenylacetate, benzylacetate induce elongation growth. These compounds, when taken up into the cell, are hydrolized by esterases; the acids released lower the cytoplasmic pH (shown by the pH indicator, fluorescein). The highest esterase activity was found in a microsomal membrane fraction of coleoptiles. While the carboxyester-induced extension growth is completely inhibited under anoxia, the initial acidification of the bathing solution can still be observed. This decrease in external pH is obviously the result of ester hydrolysis, caused by damaged cells, and is not the result of pH changes within the cell-wall compartment. It is suggested that a fast uptake of carboxyesters and the shift in equilibrium caused by their internal hydrolysis leads to a continuous formation of acids which lowers the cytoplasmic pH and activates the ATP-dependent H⁺ extrusion. In most experiments fusicoccin (a diacetic acid ester) acts similarly to naphthylacetate and the other carboxyesters, although quantitative differences exist. Therefore, it is possible that fusicoccin is effective partly on the basis of its ester characteristic. The effects observed are discussed with regard to the very narrow pH optimum of plasma-membrane H⁺-ATPases exhibiting their highest levels of activity at pH 6.5 (Hager and Biber 1984, Z. Naturforsch. C 39, 927–937).Abbreviations CHM cycloheximide - DMO dimethadione (5.5-dimethyl-2,4-oxazolidinedione) - FC fusicoccin - IAA indole-3-acetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - NA (or )-naphthylacetate (acetic acid-1(or-2-)naphthylester) - NAA (or )-naphthaleneacetic acid - PA phenylacetate (acetic acid phenylester)     

Spatial,temporal and interindividual epigenetic variation of functionally important DNA methylation patterns

DNA methylation is an epigenetic modification that plays an important role in gene regulation. It can be influenced by stochastic events, environmental factors and developmental programs. However, little is known about the natural variation of gene-specific methylation patterns. In this study, we performed quantitative methylation analyses of six differentially methylated imprinted genes (H19, MEG3, LIT1, NESP55, PEG3 and SNRPN), one hypermethylated pluripotency gene (OCT4) and one hypomethylated tumor suppressor gene (APC) in chorionic villus, fetal and adult cortex, and adult blood samples. Both average methylation level and range of methylation variation depended on the gene locus, tissue type and/or developmental stage. We found considerable variability of functionally important methylation patterns among unrelated healthy individuals and a trend toward more similar methylation levels in monozygotic twins than in dizygotic twins. Imprinted genes showed relatively little methylation changes associated with aging in individuals who are >25 years. The relative differences in methylation among neighboring CpGs in the generally hypomethylated APC promoter may not only reflect stochastic fluctuations but also depend on the tissue type. Our results are consistent with the view that most methylation variation may arise after fertilization, leading to epigenetic mosaicism.     

Therapeutic antibody engineering by high efficiency cell screening

In recent years, several cell-based screening technologies for the isolation of antibodies with prescribed properties emerged. They rely on the multi-copy display of antibodies or antibody fragments on a cell surface in functional form followed by high through put screening and isolation of cell clones that carry an antibody variant with the desired affinity, specificity, and stability. Particularly yeast surface display in combination with high-throughput fluorescence-activated cell sorting has proven successful in the last fifteen years as a very powerful technology that has some advantages over classical generation of monoclonals using the hybridoma technology or bacteriophage-based antibody display and screening. Cell-based screening harbours the benefit of single-cell online and real-time analysis and characterisation of individual library candidates. Moreover, when using eukaryotic expression hosts, intrinsic quality control machineries for proper protein folding and stability exist that allow for co-selection of high-level expression and stability simultaneously to the binding functionality. Recently, promising technologies emerged that directly rely on antibody display on higher eukaryotic cell lines using lentiviral transfection or direct screening on B-cells. The combination of immunisation, B-cell screening and next generation sequencing may open new avenues for the isolation of therapeutic antibodies with prescribed physicochemical and functional characteristics.