bluuebunny/crossref_metadata_2025 huggingface dataset:
Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition.
Authors: Fan Z , Lu Y , Wu X , DeBlasio A , Koff A , Mendelsohn J
Published: May 2004

Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.
Cell surface expression of receptor protein tyrosine phosphatase RPTP mu is regulated by cell-cell contact.
Authors: Gebbink M F , Zondag G C , Koningstein G M , Feiken E , Wubbolts R W , Moolenaar W H
Published: May 2004

RPTP mu is a transmembrane protein tyrosine phosphatase with an adhesion molecule-like ectodomain. It has recently been shown that RPTP mu mediates homophilic interactions when expressed in insect cells. In this study, we have examined how RPTP mu may function as a cell contact receptor in mink lung epithelial cells, which express RPTPmu endogenously, as well as in transfected 3T3 cells. We find that RPTP mu has a relatively short half-life (3-4 hours) and undergoes posttranslational cleavage into two noncovalently associated subunits, with both cleaved and uncleaved molecules being present on the cell surface (roughly at a 1:1 ratio); shedding of the ectodomain subunit is observed in exponentially growing cells. Immunofluorescence analysis reveals that surface expression of RPTPmu is restricted to regions of tight cell-cell contact. RPTPmu surface expression increases significantly with increasing cell density. This density-induced upregulation of RPTP mu is independent of its catalytic activity and is also observed when transcription is driven by a constitutive promoter, indicating that modulation of RPTPmu surface expression occurs posttranscriptionally. Based on our results, we propose the following model of RPTP mu function: In the absence of cell-cell contact, newly synthesized RPTP mu molecules are rapidly cleared from the cell surface. Cell-cell contact causes RPTPmu to be trapped at the surface through homophilic binding, resulting in accumulation of RPTP mu at intercellular contact regions. This contact-induced clustering of RPTPmu may then lead to tyrosine dephosphorylation of intracellular substrates at cell-cell contacts.
Sialomucin CD34 is the major L-selectin ligand in human tonsil high endothelial venules.
Authors: Puri K D , Finger E B , Gaudernack G , Springer T A
Published: May 2004

Peripheral node addressin (PNAd) is a complex mixture of glycoproteins with L-selectin ligand activity that functions in lymphocyte homing. We have investigated the contribution of the sialomucin CD34 relative to other components of PNAd in lymphocyte tethering and rolling in in vitro laminar flow assays. PNAd was isolated with MECA-79 mAb-Sepharose from tonsillar stroma, and the CD34 component (PNAd,CD34+) and CD34-negative component (PNAd,CD34-) separated on CD34 mAb-Sepharose. Lymphocytes on the PNAd,CD34- fraction tether less efficiently, roll faster and are less resistant to shear detachment than on PNAd. The PNAd,CD34+ fraction constitutes about half the total functional activity. These studies show that CD34 is a major functional component of PNAd. Ligand activity in both the PNAd,CD34+ and PNAd,CD34- fractions is expressed on mucin-like domains, as shown with O-sialoglycoprotease. The CD34 component of PNAd has about four times higher tethering efficiency than total tonsillar CD34. CD34 from spleen shows no lymphocyte tethering. Although less efficient than the PNAd,CD34+ fraction from tonsil, CD34 from the KG1a hematopoietic cell line is functionally active as an L-selectin ligand despite lack of reactivity with MECA-79 mAb, which binds to a sulfation-dependent epitope. All four forms of CD34 are active in binding to E-selectin. KG1a CD34 but not spleen CD34 are active as L-selectin ligands, yet both lack MECA-79 reactivity and possess E-selectin ligand activity. This suggests that L-selectin ligands and E-selectin ligands differ in more respects than presence of the MECA-79 epitope.
A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones.
Authors: Taniura H , Glass C , Gerace L
Published: May 2004

Interaction of chromatin with the nuclear envelope and lamina is thought to help determine higher order chromosome organization in the interphase nucleus. Previous studies have shown that nuclear lamins bind chromatin directly. Here we have localized a chromatin binding site to the carboxyl-terminal tail domains of both A- and B-type mammalian lamins, and have characterized the biochemical properties of this binding in detail. Recombinant glutathione-S-transferase fusion proteins containing the tail domains of mammalian lamins C, B1, and B2 were analyzed for their ability to associate with rat liver chromatin fragments immobilized on microtiter plate wells. We found that all three lamin tails specifically bind to chromatin with apparent KdS of 120-300 nM. By examining a series of deletion mutants, we have mapped the chromatin binding region of the lamin C tail to amino acids 396-430, a segment immediately adjacent to the rod domain. Furthermore, by analysis of chromatin subfractions, we found that core histones constitute the principal chromatin binding component for the lamin C tail. Through cooperativity, this lamin-histone interaction could be involved in specifying the high avidity attachment of chromatin to the nuclear envelope in vivo.
Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway.
Authors: Itin C , Schindler R , Hauri H P
Published: May 2004

ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. To identify the targeting signals that mediate this recycling, N-glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER-ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC-cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC-53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER-retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant.
Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin.
Authors: Damke H , Baba T , van der Bliek A M , Schmid S L
Published: May 2004

A stable HeLa cell line expressing a dynamin mutant, dynts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dynts carries a point mutation, G273D, corresponding to the Drosophila shibirets1 allele. The ts-defect in receptor-mediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 degrees C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wild-type levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin- and clathrin-independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dynts cells rapidly and completely compensate for the loss of clathrin-dependent endocytosis by inducing an alternate endocytic pathway.
Degradation of HMG-CoA reductase-induced membranes in the fission yeast, Schizosaccharomyces pombe.
Authors: Lum P Y , Wright R
Published: May 2004

Elevated levels of certain membrane proteins, including the sterol biosynthetic enzyme HMG-CoA reductase, induce proliferation of the endoplasmic reticulum. When the amounts of these proteins return to basal levels, the proliferated membranes are degraded, but the molecular details of this degradation remain unknown. We have examined the degradation of HMG-CoA reductase-induced membranes in the fission yeast, Schizosaccharomyces pombe. In this yeast, increased levels of the Saccharomyces cerevisiae HMG-CoA reductase isozyme encoded by HMG1 induced several types of membranes, including karmellae, which formed a cap of stacked membranes that partially surrounded the nucleus. When expression of HMG1 was repressed, the karmellae detached from the nucleus and formed concentric, multilayered membrane whorls that were then degraded. During the degradation process, CDCFDA-stained compartments distinct from preexisting vacuoles formed within the interior of the whorls. In addition to these compartments, particles that contained neutral lipids also formed within the whorl. As the thickness of the whorl decreased, the lipid particle became larger. When degradation was complete, only the lipid particle remained. Cycloheximide treatment did not prevent the formation of whorls. Thus, new protein synthesis was not needed for the initial stages of karmellae degradation. On the contrary, cycloheximide promoted the detachment of karmellae to form whorls, suggesting that a short lived protein may be involved in maintaining karmellae integrity. Taken together, these results demonstrate that karmellae membranes differentiated into self-degradative organelles. This process may be a common pathway by which ER membranes are turned over in cells.
Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1.
Authors: Motley A , Lumb M J , Oatey P B , Jennings P R , De Zoysa P A , Wanders R J , Tabak H F , Danpure C J
Published: May 2004

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-->Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg-mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.
A three-dimensional structural dissection of Drosophila polytene chromosomes.
Authors: Urata Y , Parmelee S J , Agard D A , Sedat J W
Published: May 2004

We have analyzed the three-dimensional structural details of Drosophila melanogaster polytene chromosome bands and interbands using three-dimensional light microscopy and a novel method of sample preparation that does not involve flattening or stretching the chromosomes. Bands have been visualized in unfixed chromosomes stained with the DNA specific dye 4,6-Diamidino-2-phenylindole (DAPI). Interbands have been visualized using fixed chromosomes that have been immunostained with an antibody to RNA polymerase II. Additionally, these structures have been analyzed using in situ hybridization with probes from specific genetic loci (Notch and white). Bands are seen to be composed of approximately 36 substructural features that measure 0.2-0.4 micron in diameter. We suggest that these substructural features are in fact longitudinal fibers made up of bundles of chromatids. Band shape can be a reproducible characteristic of a particular band and is dependent on the spatial relationship of these bundles, varying from bands with a uniform distribution of bundles to bands with a peripheral concentration of chromatin. Interbands are composed of bundles of chromatids of a similar size and number as those seen in the bands. The distribution of bundles is similar between a band and the neighboring interband, implying that there is a long range organization to the DNA that includes both the coding and the noncoding portions of genes. Finally, we note that the polytene chromosome has a circular shape when viewed in cross section, whether there are one or two homologs present.
A lysosomal targeting signal in the cytoplasmic tail of the beta chain directs HLA-DM to MHC class II compartments.
Authors: Marks M S , Roche P A , van Donselaar E , Woodruff L , Peters P J , Bonifacino J S
Published: May 2004

In human B cells, class II molecules of the major histocompatibility complex (MHC-II) accumulate in an endosomal/lysosomal compartment, the MIIC, in which they may encounter and bind peptides. An additional molecule required for MHC-II peptide binding, HLA-DM (DM), has also been localized to the MIIC. Neither the relationship of the MIIC to the endosomal system nor the mechanisms by which DM localizes to the MIIC are understood. To address these issues, DM localization was analyzed in cells that do or do not express MHC-II. DM alpha beta heterodimers were localized in transfected MHC-II-negative HeLa and NRK cells, in the absence of the MHC-II-associated invariant chain, to a prelysosomal/lysosomal compartment by immunofluorescence microscopy. To identify a potential targeting determinant, we analyzed the localization of a chimeric protein, T-T-Mb, in which the cytoplasmic tail of murine DM beta (Mb) was appended to the lumenal and transmembrane domains of a cell surface protein, Tac. Like intact DM, T-T-Mb was localized to a lysosomal compartment in HeLa and NRK cells, as judged by immunofluorescence and immunoelectron microscopy. T-T-Mb was rapidly degraded in this compartment by a process that was blocked by inhibitors of lysosomal proteolysis. The DM beta cytoplasmic tail also mediated internalization of anti-Tac antibody from the cell surface and delivery to lysosomes. Deletion from the DM beta cytoplasmic tail of the tyrosine-based motif, YTPL, resulted in cell surface expression of T-T-Mb and a loss of both degradation and internalization; alanine scanning mutagenesis showed that the Y and L residues were critical for these functions. Similarly, mutation of the same Y residue within full-length DM beta resulted in cell surface expression of DM alpha beta heterodimers. Lastly, T-T-Mb was localized by immunoelectron microscopy to the MIIC in a human B lymphoblastoid cell line. Our results suggest that a motif, YTPL, in the cytoplasmic tail of the beta chain of DM is sufficient for targeting either to lysosomes or to the MIIC.
The essential OST2 gene encodes the 16-kD subunit of the yeast oligosaccharyltransferase, a highly conserved protein expressed in diverse eukaryotic organisms.
Authors: Silberstein S , Collins P G , Kelleher D J , Gilmore R
Published: May 2004

Oligosaccharyltransferase catalyzes the transfer of a preassembled high mannose oligosaccharide from a dolichol-oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six non-identical subunits (alpha-zeta). The alpha, beta, gamma, and delta subunits of the oligosaccharyltransferase are encoded by the OST1, WBP1, OST3, and SWP1 genes, respectively. Here we describe the functional characterization of the OST2 gene that encodes the epsilon-subunit of the oligosaccharyltransferase. Genomic disruption of the OST2 locus was lethal in haploid yeast showing that expression of the Ost2 protein is essential for viability. Overexpression of the Ost2 protein suppresses the temperature-sensitive phenotype of the wbp1-2 allele and increases in vivo and in vitro oligosaccharyltransferase activity in a wbp1-2 strain. An analysis of a series of conditional ost2 mutants demonstrated that defects in the Ost2 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins. Microsomal membranes isolated from ost2 mutant yeast show marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Surprisingly, the Ost2 protein was found to be 40% identical to the DAD1 protein (defender against apoptotic cell death), a highly conserved protein initially identified in vertebrate organisms. The protein sequence of ost2 mutant alleles revealed mutations at highly conserved residues in the Ost2p/DAD1 protein sequence.
Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii.
Authors: Pazour G J , Sineshchekov O A , Witman G B
Published: May 2004

Chlamydomonas has two photobehavioral responses, phototaxis and photoshock. Rhodopsin is the photoreceptor for these responses and the signal transduction process involves transmembrane Ca2+ fluxes. This causes transient changes in flagellar beating, ultimately resulting in phototaxis or photoshock. To identify components that make up this signal transduction pathway, we generated nonphototactic strains by insertional mutagenesis. Seven new phototaxis genes were identified (ptx2-ptx8); alleles of six of these are tagged by the transforming DNA and therefore should be easily cloned. To order the mutants in the pathway, we characterized them electrophysiologically, behaviorally, and structurally, ptx5, ptx6, and ptx7 have normal light-induced photoreceptor currents (PRC) and flagellar currents (FC) but their pattern of swimming does not change in the normal manner when the intraflagellar Ca2+ concentration is decreased, suggesting that they have defects in the ability of their axonemes to respond to changes in Ca2+ concentration. ptx2 and ptx8 lack the FC but have normal PRCs, suggesting that they are defective in the flagellar Ca2+ channel or some factor that regulates it. ptx4 mutants have multiple eye-spots. ptx3 mutants are defective in a component essential for phototaxis but bypassed during photoshock; this component appears to be located downstream of the PRC but upstream of the axoneme.
Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation.
Authors: Meier T , Perez G M , Wallace B G
Published: May 2004

Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti-phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.
The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway.
Authors: Jedd G , Richardson C , Litt R , Segev N
Published: May 2004

Small GTPases of the rab family are involved in the regulation of vesicular transport. The restricted distribution of each of these proteins in mammalian cells has led to the suggestion that different rab proteins act at different steps of transport (Pryer, N. K., L. J. Wuestehube, and R. Sheckman. 1992. Annu Rev. Biochem. 61:471-516; Zerial, M., and H. Stenmark. 1993. Curr. Opin. Cell Biol. 5:613-620). However, in this report we show that the Ypt1-GTPase, a member of the rab family, is essential for more than one step of the yeast secretory pathway. We determined the secretory defect conferred by a novel ypt1 mutation by comparing the processing of several transported glycoproteins in wild-type and mutant cells. The ypt1-A136D mutant has a change in an amino acid that is conserved among rab GTPases. This mutation leads to a rapid and tight secretory block upon a shift to the restrictive temperature, and allows for the identification of the specific steps in the secretory pathway that directly require Ypt1 protein (Ypt1p). The ypt1-A136D mutant exhibits tight blocks in two secretory steps, ER to cis-Golgi and cis- to medial-Golgi, but later steps are unaffected. Thus, it is unlikely that Ypt1p functions as the sole determinant of fusion specificity. Our results are more consistent with a role for Ypt1/rab proteins in determining the directionality or fidelity of protein sorting.
Characterization of T cell mutants with defects in capacitative calcium entry: genetic evidence for the physiological roles of CRAC channels.
Authors: Fanger C M , Hoth M , Crabtree G R , Lewis R S
Published: May 2004

Prolonged Ca2+ influx is an essential signal for the activation of T lymphocytes by antigen. This influx is thought to occur through highly selective Ca2+ release-activated Ca2+ (CRAC) channels that are activated by the depletion of intracellular Ca2+ stores. We have isolated mutants of the Jurkat human T cell line NZdipA to explore the molecular mechanisms that underlie capacitative Ca2+ entry and to allow a genetic test of the functions of CRAC channels in T cells. Five mutant cell lines (CJ-1 through CJ-5) were selected based on their failure to express a lethal diphtheria toxin A chain gene and a lacZ reporter gene driven by NF-AT, a Ca(2+)- and protein kinase C-dependent transcription factor. The rate of Ca2+ influx evoked by thapsigargin was reduced to varying degrees in the mutant cells whereas the dependence of NF-AT/lacZ gene transcription on [Ca2+]i was unaltered, suggesting that the transcriptional defect in these cells is caused by a reduced level of capacitative Ca2+ entry. We examined several factors that determine the rate of Ca2+ entry, including CRAC channel activity, K(+)-channel activity, and Ca2+ clearance mechanisms. The only parameter found to be dramatically altered in most of the mutant lines was the amplitude of the Ca2+ current (ICRAC), which ranged from 1 to 41% of that seen in parental control cells. In each case, the severity of the ICRAC defect was closely correlated with deficits in Ca2+ influx rate and Ca(2-)-dependent gene transcription. Behavior of the mutant cells provides genetic evidence for several roles of ICRAC in T cells. First, mitogenic doses of ionomycin appear to elevate [Ca2+]i primarily by activating CRAC channels. Second, ICRAC promotes the refilling of empty Ca2+ stores. Finally, CRAC channels are solely responsible for the Ca2+ influx that underlies antigen-mediated T cell activation. These mutant cell lines may provide a useful system for isolating, expressing, and exploring the functions of genes involved in capacitative Ca2+ entry.
Exogenous glycosyl phosphatidylinositol-anchored CD59 associates with kinases in membrane clusters on U937 cells and becomes Ca(2+)-signaling competent.
Authors: van den Berg C W , Cinek T , Hallett M B , Horejsi V , Morgan B P
Published: May 2004

CD59, an 18-20-kD complement inhibitor anchored to the membrane via glycosyl phosphatidylinositol (GPI), can induce activation of T cells and neutrophils upon cross-linking with antibody. GPI-anchored molecules cocluster in high mol wt detergent-resistant complexes containing tyrosine kinases that are implicated in the signaling pathway. Exogenous, incorporated GPI-anchored molecules are initially unable to induce activation, presumably because they are not associated with kinases. Here we demonstrate that erythrocyte-derived CD59 incorporated in a CD59-negative cell line acquires signaling capacity in a time-dependent manner. Confocal microscopy revealed an initial diffuse distribution of CD59 that became clustered within 2 h to give a pattern similar to endogenous GPI-anchored molecules. Gel filtration of detergent-solubilized cells immediately after incorporation revealed that CD59 was mainly monomeric, but after 3 h incubation all was in high mol wt complexes and had become associated with protein kinases. Newly incorporated CD59 did not deliver a Ca2+ signal upon cross-linking, but at a time when it had become clustered and associated with kinase activity, cross-linking induced a large calcium transient, indicating that CD59 had incorporated in a specialized microenvironment that allowed it to function fully as a signal-transducing molecule.
GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes.
Authors: Melikyan G B , White J M , Cohen F S
Published: May 2004

Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI-HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). We extended the evidence for hemifusion by labeling the inner monolayer leaflets of RBCs with FM4-64 and observing that these inner leaflets did not become continuous with GPI-HA-expressing cells. The region of hemifusion-separated aqueous contents, the hemifusion diaphragm, appeared to be extended and was long-lived. But when RBCs hemifused to GPI-HA-expressing cells were osmotically swollen, some diaphragms were disrupted, and spread of both inner leaflet and aqueous dyes was observed. This was characteristic of full fusion: inner leaflet and aqueous probes spread to cells expressing wild-type HA (wt-HA). By simultaneous video fluorescence microscopy and time-resolved electrical admittance measurements, we rigorously demonstrated that GPI-HA-expressing cells hemifuse to planar bilayer membranes: lipid continuity was established without formation of fusion pores. The hemifusion area became large. In contrast, for cells expressing wt-HA, before lipid dye spread, fusion pores were always observed, establishing that full fusion occurred. We present an elastic coupling model in which the ectodomain of wt-HA induces hemifusion and the transmembrane domain, absent in the GPI-HA-expressing cells, mediates full fusion.
Kinetochore microtubule dynamics and the metaphase-anaphase transition.
Authors: Zhai Y , Kronebusch P J , Borisy G G
Published: May 2004

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.
Calcineurin functions in Ca(2+)-activated cell death in mammalian cells.
Authors: Shibasaki F , McKeon F
Published: May 2004

Calcineurin is a calcium-dependent protein phosphatase that functions in T cell activation. We present evidence that calcineurin functions more generally in calcium-triggered apoptosis in mammalian cells deprived of growth factors. Specifically, expression of epitope-tagged calcineurin A induces rapid cell death upon calcium signaling in the absence of growth factors. We show that this apoptosis does not require new protein synthesis and therefore calcineurin must operate through existing substrates. Co-expression of the Bcl-2 protooncogene efficiently blocks calcineurin-induced cell death. Significantly, we demonstrate that a calcium-independent calcineurin mutant induces apoptosis in the absence of calcium, and that this apoptotic response is a direct consequence of calcineurin's phosphatase activity. These data suggest that calcineurin plays an important role in mediating the upstream events in calcium-activated cell death.
Continual assembly of half-desmosomal structures in the absence of cell contacts and their frustrated endocytosis: a coordinated Sisyphus cycle.
Authors: Demlehner M P , Schäfer S , Grund C , Franke W W
Published: May 2004

It is widely assumed that the coordinate assembly of desmosomal cadherins and plaque proteins into desmosome-typical plaque-coated membrane domains, capable of anchoring intermediate-sized filaments (IF), requires cell-to-cell contacts and a critical extracellular Ca2+ concentration. To test this hypothesis we studied several cell lines grown for years in media with less than 0.1 mM Ca2+ to steady-state low Ca2+ medium (LCM) conditions, particularly the human keratinocyte line HaCaT devoid of any junctional cell contact (HaCaT-L cells). Using immunolocalization and vesicle fractionation techniques, we found that the transmembrane glycoprotein, desmoglein (Dsg), colocalized with the plaque proteins, desmoplakin and plakoglobin. The sites of coassembly of desmosomal molecules in HaCaT-L cells as well as in HaCaT cells directly brought into LCM were identified as asymmetric plaque-coated plasma membrane domains (half-desmosomes) or as special plaque-associated cytoplasmic vesicles, most of which had formed endocytotically. The surface exposure of Dsg in these half-desmosomes was demonstrated by the binding, in vivo, of antibodies specific for an extracellular Dsg segment which also could cross-bridge them into symmetric quasi-desmosomes. Otherwise, these half-desmosomes were shown in LCM to be taken up endocytotically. Half-desmosomal assemblies were also seen in uncoupled cells in normal Ca2+ medium. We conclude that, in the absence of intercellular contacts, assembly of desmosomal proteins at the cell surface takes place, resulting in transient half-desmosomes which then, in LCM and without a stable partner connection to the adjacent cell, can be endocytotically resumed. This frustrated cycle of synthesis and assembly maintains an ensemble of molecules characteristic of epithelial differentiation and the potential to form desmosomes, even when the final junctional structure cannot be formed. We propose that these half-desmosomal structures are general cell structures of epithelial and other desmosome-forming cells.
Manganese effectively supports yeast cell-cycle progression in place of calcium.
Authors: Loukin S , Kung C
Published: May 2004

Metal ion requirements for the proliferation of Saccharomyces cerevisiae were investigated. We used bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relatively acid tolerant chelator, to reduce the free metal ion concentrations in culture media. Chelatable metal ions were added back individually and in combination. In addition to a requirement for approximately 10 pM external free Zn2+ we found an interchangeable requirement for either 66 nM free Ca2+ or only 130 pM free Mn2+. Cells depleted of Mn2+ and Ca2+ arrested as viable cells with 2 N nuclei and tended to have very small minibuds. In the absence of added Mn2+, robust growth required approximately 60 microM total internal Ca2+. In the presence of added Mn2+, robust growth continued even when internal Ca2+ was < 3% this level. Chelator-free experiments showed that MnCl2 strongly and CaCl2 weakly restored high-temperature growth of cdc1ts strains which similarly arrest as viable cells with 2 N nuclear contents and small buds. Its much greater effectiveness compared with Ca2+ suggests that Mn2+ is likely to be a physiologic mediator of bud and nuclear development in yeast. This stands in marked contrast to a claim that Ca2+ is uniquely required for cell-cycle progression in yeast. We discuss the possibility that Mn2+ may function as an intracellular signal transducer and how this possibility relates to previous claims of Ca2+'s roles in yeast metabolism.
Differential targeting of protein kinase C and CaM kinase II signalings to vimentin.
Authors: Ogawara M , Inagaki N , Tsujimura K , Takai Y , Sekimata M , Ha M H , Imajoh-Ohmi S , Hirai S , Ohno S , Sugiura H
Published: May 2004

Hydrolysis of inositol phospholipids by receptor stimulation activates two separate signaling pathways, one leading to the activation of protein kinase C (C kinase) via formation of diacylglycerol. The other is the inositol trisphosphate (IP3)/Ca2+ pathway and a major downstream kinase which is activated is Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). To examine signaling pathways of C kinase and CaM kinase II to the cytoskeletal protein vimentin, we prepared monoclonal antibodies YT33 and MO82 which recognize the phosphorylation state of vimentin by C kinase and by CaM kinase II, respectively. Ectopic expression of constitutively active C kinase or CaM kinase II in primary cultured astrocytes by microinjection of the corresponding expression vectors induced phosphorylation of vimentin at each specific phosphorylation site, followed by reorganization of vimentin filament networks. In contrast, simultaneous activation of C kinase and CaM kinase II by inositol phospholipid hydrolysis with receptor stimulation led to an exclusive phosphorylation of vimentin at the CaM kinase II site, not at the site of C kinase. These results indicate that the intracellular targeting of C kinase and CaM kinase II signalings to vimentin is regulated separately, under physiological conditions.
Binding between the neural cell adhesion molecules axonin-1 and Nr-CAM/Bravo is involved in neuron-glia interaction.
Authors: Suter D M , Pollerberg G E , Buchstaller A , Giger R J , Dreyer W J , Sonderegger P
Published: May 2004

Neural cell adhesion molecules of the immunoglobulin superfamily mediate cellular interactions via homophilic binding to identical molecules and heterophilic binding to other family members or structurally unrelated cell-surface glycoproteins. Here we report on an interaction between axonin-1 and Nr-CAM/Bravo. In search for novel ligands of axonin-1, fluorescent polystyrene microspheres conjugated with axonin-1 were found to bind to peripheral glial cells from dorsal root ganglia. By antibody blockage experiments an axonin-1 receptor on the glial cells was identified as Nr-CAM. The specificity of the interaction was confirmed with binding studies using purified axonin-1 and Nr-CAM. In cultures of dissociated dorsal root ganglia antibodies against axonin-1 and Nr-CAM perturbed the formation of contacts between neurites and peripheral glial cells. Together, these results implicate a binding between axonin-1 of the neuritic and Nr-CAM of the glial cell membrane in the early phase of axon ensheathment in the peripheral nervous system.
Testicular development involves the spatiotemporal control of PDGFs and PDGF receptors gene expression and action.
Authors: Gnessi L , Emidi A , Jannini E A , Carosa E , Maroder M , Arizzi M , Ulisse S , Spera G
Published: May 2004

Platelet-derived growth factors (PDGFs) are growth-regulatory molecules that stimulate chemotaxis, proliferation and metabolism primarily of cells of mesenchymal origin. In this study, we found high levels of PDGFs and PDGFs receptors (PDGFRs) mRNAs, and specific immunostaining for the corresponding proteins in the rat testis. PDGFs and PDGFRs expression was shown to be developmentally regulated and tissue specific. Expression of PDGFs and PDGFRs genes was observed in whole testis RNA 2 d before birth, increased through postnatal day 5 and fell to low levels in adult. The predominant cell population expressing transcripts of the PDGFs and PDGFRs genes during prenatal and early postnatal periods were Sertoli cells and peritubular myoid cells (PMC) or their precursors, respectively, while in adult animals PDGFs and PDGFRs were confined in Leydig cells. We also found that early postnatal Sertoli cells produce PDGF-like substances and that this production is inhibited dose dependently by follicle-stimulating hormone (FSH). The expression of PDGFRs by PMC and of PDGFs by Sertoli cells corresponds in temporal sequence to the developmental period of PMC proliferation and migration from the interstitium to the peritubulum. Moreover, we observed that all the PDGF isoforms and the medium conditioned by early postnatal Sertoli cells show a strong chemotactic activity for PMC which is inhibited by anti-PDGF antibodies. These data indicate that, through the spatiotemporal pattern of PDGF ligands and receptors expression, PDGF may play a role in testicular development and homeostasis.
Saccharomyces cerevisiae cells execute a default pathway to select a mate in the absence of pheromone gradients.
Authors: Dorer R , Pryciak P M , Hartwell L H
Published: May 2004

During conjugation, haploid S. cerevisiae cells find one another by polarizing their growth toward each other along gradients of pheromone (chemotropism). We demonstrate that yeast cells exhibit a second mating behavior: when their receptors are saturated with pheromone, wild-type a cells execute a default pathway and select a mate at random. These matings are less efficient than chemotropic matings, are induced by the same dose of pheromone that induces shmoo formation, and appear to use a site near the incipient bud site for polarization. We show that the SPA2 gene is specifically required for the default pathway: spa2 delta mutants cannot mate if pheromone concentrations are high and gradients are absent, but can mate if gradients are present. ste2 delta, sst2 delta, and far1 delta mutants are chemotropism-defective and therefore must choose a mate by using a default pathway; consistent with this deduction, these strains require SPA2 to mate. In addition, our results suggest that far1 mutants are chemotropism-defective because their mating polarity is fixed at the incipient bud site, suggesting that the FAR1 gene is required for inhibiting the use of the incipient bud site during chemotropic mating. These observations reveal a molecular relationship between the mating and budding polarity pathways.
Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment.
Authors: Lussier M , Sdicu A M , Ketela T , Bussey H
Published: May 2004

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.
Successful histocompatible myoblast transplantation in dystrophin-deficient mdx mouse despite the production of antibodies against dystrophin.
Authors: Vilquin J T , Wagner E , Kinoshita I , Roy R , Tremblay J P
Published: May 2004

Myoblast transplantation has been considered a potential treatment for some muscular disorders. It has proven very successful, however, only in immunodeficient or immunosuppressed mice. In this study, myoblasts from C57BL10J +/+ mice were transplanted, with no immunosuppressive treatment, in the tibialis anterior of fully histocompatible but dystrophin-deficient C57BL10J mdx/mdx mice. One to 9 months after transplantation, the success of the graft was evaluated by immunohistochemistry. All the transplanted mice (n = 24) developed dystrophin-positive fibers following transplantation. Depending on myoblast cultures, transplantations, and time of analysis, the mice presented 15 to 80% of dystrophin-positive fibers in transplanted muscles. These fibers were correctly oriented and they were either from donor or hybrid origin. The dystrophin-positive fibers remained stable up to 9 months. Possible humoral and cellular immune responses were investigated after grafting. Antibodies directed against dystrophin and/or muscle membrane were developed by 58% of the mice as demonstrated by immunohistochemistry and Western blotting. Despite the presence of these antibodies, dystrophin-positive fibers were still present in grafted muscles 9 months after transplantation. Moreover, the muscles did not show massive infiltration by CD4 cells, CD8 cells, or macrophages, as already described in myoblast allotransplantations. This lack of rejection was attributed to the sequestrated nature of dystrophin after fiber formation. These results indicate that myoblast transplantation leads to fiber formation when immunocompetent but fully histocompatible donors and recipients are used and that dystrophin incompatibility alone is not sufficient to induce an immunological rejection reaction.