bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Protein 4.1 in the Nucleus of Human Cells: Dynamic Rearrangements during Cell Division
Authors: Krauss Sharon Wald , Larabell Carolyn A. , Lockett Stephen , Gascard Philippe , Penman Sheldon , Mohandas Narla , Chasis Joel Anne
Published: July 2002

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.
A Novel Mammalian, Mitotic Spindle–associated Kinase Is Related to Yeast and Fly Chromosome Segregation Regulators
Authors: Gopalan Ganesan , Chan Clarence S.M. , Donovan Peter J.
Published: July 2002

We describe a novel mammalian protein kinase related to two newly identified yeast and fly kinases—Ipl1 and aurora, respectively—mutations in which cause disruption of chromosome segregation. We have designated this kinase as Ipl1- and aurora-related kinase 1 (IAK1). IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle. Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis. The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase. IAK1 protein has a distinct subcellular and temporal pattern of localization. It is first identified on the centrosomes immediately after the duplicated centrosomes have separated. The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis. In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers. A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast. Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells. We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily. The members of this subfamily may be important regulators of chromosome segregation.
Mechanism of the Facilitation of PC2 Maturation by 7B2: Involvement in ProPC2 Transport and Activation but Not Folding
Authors: Muller Laurent , Zhu Xiaorong , Lindberg Iris
Published: July 2002

Among the members of the prohormone convertase (PC) family, PC2 has a unique maturation pattern: it is retained in the ER for a comparatively long time and its propeptide is cleaved in the TGN/ secretory granules rather than in the ER. It is also unique by its association with the neuroendocrine protein 7B2. This interaction results in the facilitation of proPC2 maturation and in the production of activatable proPC2 from CHO cells. In the present study, we have investigated the mechanism of this interaction.

ProPC2 binds 7B2 in the ER, but exits this compartment much more slowly than 7B2. We found that proPC2 was also slow to acquire the capacity to bind 7B2, whereas 7B2 could bind proPC2 rapidly after synthesis. This indicated that proPC2 folding was the limiting step in the formation of the complex. Indeed, sensitivity of native proPC2 to N-glycanase F digestion and inhibition of proPC2 folding supported the notion that 7B2 is not involved in the early steps of proPC2 folding, and that proPC2 must fold before binding 7B2. Under experimental conditions that prevent propeptide cleavage, 7B2 expression increased proPC2 transport to the Golgi. This increase exhibited the same kinetics as the facilitation of the removal of the propeptide. Finally, proPC2 activation could be reconstituted in Golgi- enriched subcellular fractions. In vitro, 7B2 was required for proPC2 activation at an acidic pH.

Taken together, our results demonstrate that rather than promoting proPC2 folding, 7B2 acts as a helper protein involved in proPC2 transport and is required in the proPC2 activation process. We propose, therefore, that 7B2 stabilizes proPC2 in a conformation already competent for these two events.
Targeting of the Synaptic Vesicle Protein Synaptobrevin in the Axon of Cultured Hippocampal Neurons: Evidence for Two Distinct Sorting Steps
Authors: West Anne E. , Neve Rachael L. , Buckley Kathleen M.
Published: July 2002

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10– amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61–70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.
Cytoskeletal Association Is Important for Differential Targeting of Glucose Transporter Isoforms in Leishmania
Authors: Snapp Erik L. , Landfear Scott M.
Published: July 2002

The major glucose transporter of the parasitic protozoan Leishmania enriettii exists in two isoforms, one of which (iso-1) localizes to the flagellar membrane, while the other (iso-2) localizes to the plasma membrane of the cell body, the pellicular membrane. These two isoforms differ only in their cytosolic NH2-terminal domains. Using immunoblots and immunofluorescence microscopy of detergent-extracted cytoskeletons, we have demonstrated that iso-2 associates with the microtubular cytoskeleton that underlies the cell body membrane, whereas the flagellar membrane isoform iso-1 does not associate with the cytoskeleton. Deletion mutants that remove the first 25 or more amino acids from iso-1 are retargeted from the flagellum to the pellicular membrane, suggesting that these deletions remove a signal required for flagellar targeting. Unlike the full-length iso-1 protein, these deletion mutants associate with the cytoskeleton. Our results suggest that cytoskeletal binding serves as an anchor to localize the iso-2 transporter within the pellicular membrane, and that the flagellar targeting signal of iso-1 diverts this transporter into the flagellar membrane and away from the pellicular microtubules.
Identification of Two Regions in Apolipoprotein B100 that are Exposed on the Cytosolic Side of the Endoplasmic Reticulum Membrane
Authors: Du Xiaobo , Daniel Stoops J. , Mertz James R. , Michael Stanley C. , Dixon Joseph L.
Published: July 2002

Protease protection assays of apolipoprotein B100 (apoB) in digitonin-permeabilized HepG2 cells indicated that multiple domains of apoB are exposed to the cytosol through an extensive portion of the secretory pathway. The intracellular orientation of apoB in the secretory pathway was confirmed by immunocytochemistry using antibodies recognizing specific domains of apoB in streptolysin-O (STP-O)– and saponin-permeabilized HepG2 cells. Lumenal epitopes on marker proteins in secretory pathway compartments (p63, p53, and galactosyltransferase) were not stained by antibodies in STP-O–treated cells, but were brightly stained in saponin-treated cells, confirming that internal membranes were not perforated in STP-O–treated cells. An anti-apoB peptide antibody (B4) recognizing amino acids 3221–3240 caused intense staining in close proximity to the nuclear membrane, and less intensely throughout the secretory pathway in STP-O–permeabilized cells. Staining with this antibody was similar in STP-O– and saponin-treated cells, indicating that this epitope in apoB is exposed to the cytosol at the site of apoB synthesis and throughout most of the remaining secretory pathway. Similar results indicating a cytosolic orientation were obtained with monoclonal antibody CC3.4, which recognizes amino acids 690–797 (79–91 kD) in apoB. Two polyclonal antibodies made to human LDL and two monoclonal antibodies recognizing amino acids 1878–2148 (D7.2) and 3214–3506 (B1B6) in apoB did not produce a strong reticular signal for apoB in STP-O–treated cells. The anti-LDL and B1B6 antibodies produced almost identical punctate patterns in STP-O–treated cells that overlapped with LAMP-1, a membrane marker for lysosomes. These observations suggest that the B1B6 epitope of apoB is exposed on the surface of the lysosome. The results identify two specific regions in apoB that are exposed to the cytosol in the secretory pathway.
Agrin Can Mediate Acetylcholine Receptor Gene Expression in Muscle by Aggregation of Muscle-derived Neuregulins
Authors: Meier Thomas , Masciulli Fabrizio , Moore Chris , Schoumacher Fabrice , Eppenberger Urs , Denzer Alain J. , Jones Graham , Brenner Hans Rudolf
Published: July 2002

The neural isoforms of agrin can stimulate transcription of the acetylcholine receptor (AChR) ε subunit gene in electrically active muscle fibers, as does the motor neuron upon the formation of a neuromuscular junction. It is not clear, however, whether this induction involves neuregulins (NRGs), which stimulate AChR subunit gene transcription in vitro by activating ErbB receptors. In this study, we show that agrin- induced induction of AChR ε subunit gene transcription is inhibited in cultured myotubes overexpressing an inactive mutant of the ErbB2 receptor, demonstrating involvement of the NRG/ErbB pathway in agrin- induced AChR expression. Furthermore, salt extracts from the surface of cultured myotubes induce tyrosine phosphorylation of ErbB2 receptors, indicating that muscle cells express biological NRG-like activity on their surface. We further demonstrate by RT-PCR analysis that muscle NRGs have Ig-like domains required for their immobilization at heparan sulfate proteoglycans (HSPGs) of the extracellular matrix. In extrasynaptic regions of innervated muscle fibers in vivo, ectopically expressed neural agrin induces the colocalized accumulation of AChRs, muscle-derived NRGs, and HSPGs. By using overlay and radioligand-binding assays we show that the Ig domain of NRGs bind to the HSPGs agrin and perlecan. These findings show that neural agrin can induce AChR subunit gene transcription by aggregating muscle HSPGs on the muscle fiber surface that then serve as a local sink for focal binding of muscle-derived NRGs to regulate AChR gene expression at the neuromuscular junction.
Dislocation of Type I Membrane Proteins from the ER to the Cytosol Is Sensitive to Changes in Redox Potential
Authors: Tortorella Domenico , Story Craig M. , Huppa Johannes B. , Wiertz Emmanuel J.H.J. , Jones Thomas R. , Ploegh Hidde L.
Published: July 2002

The human cytomegalovirus (HCMV) gene products US2 and US11 dislocate major histocompatibility class I heavy chains from the ER and target them for proteasomal degradation in the cytosol. The dislocation reaction is inhibited by agents that affect intracellular redox potential and/or free thiol status, such as diamide and N-ethylmaleimide. Subcellular fractionation experiments indicate that this inhibition occurs at the stage of discharge from the ER into the cytosol. The T cell receptor α (TCR α) chain is also degraded by a similar set of reactions, yet in a manner independent of virally encoded gene products. Diamide and N-ethylmaleimide likewise inhibit the dislocation of the full-length TCR α chain from the ER, as well as a truncated, mutant version of TCR α chain that lacks cysteine residues. Cytosolic destruction of glycosylated, ER-resident type I membrane proteins, therefore, requires maintenance of a proper redox potential for the initial step of removal of the substrate from the ER environment.
Transcription Sites Are Not Correlated with Chromosome Territories in Wheat Nuclei
Authors: Abranches Rita , Beven Alison F. , Aragón-Alcaide Luis , Shaw Peter J.
Published: July 2002

We have determined the relationship between overall nuclear architecture, chromosome territories, and transcription sites within the nucleus, using three-dimensional confocal microscopy of well preserved tissue sections of wheat roots. Chromosome territories were visualized by GISH using rye genomic probe in wheat/rye translocation and addition lines. The chromosomes appeared as elongated regions and showed a clear centromere–telomere polarization, with the two visualized chromosomes lying approximately parallel to one another across the nucleus. Labeling with probes to telomeres and centromeres confirmed a striking Rabl configuration in all cells, with a clear clustering of the centromeres, and cell files often maintained a common polarity through several division cycles. Transcription sites were detected by BrUTP incorporation in unfixed tissue sections and revealed a pattern of numerous foci uniformly distributed throughout the nucleoplasm, as well as more intensely labeled foci in the nucleoli. It has been suggested that the gene-rich regions in wheat chromosomes are clustered towards the telomeres. However, we found no indication of a difference in concentration of transcription sites between telomere and centromere poles of the nucleus. Neither could we detect any evidence that the transcription sites were preferentially localized with respect to the chromosome territorial boundaries.
imp2, a New Component of the Actin Ring in the Fission Yeast Schizosaccharomyces pombe
Authors: Demeter Janos , Sazer Shelley
Published: July 2002

Cytokinesis is the part of the cell cycle in which the cell is cleaved to form two daughter cells. The unicellular yeast, Schizosaccharomyces pombe is an excellent model organism in which to study cell division, since it shows the general features of eukaryotic cell division and is amenable to genetic analysis. In this manuscript we describe the isolation and characterization of a new protein, imp2, which is required for normal septation in fission yeast. imp2, which colocalizes with the medial ring during septation, is structurally similar to a group of proteins including the S. pombe cdc15 and the mouse PSTPIP that are localized to, and thought to be involved in actin ring organization. Cells in which the imp2 gene is deleted or overexpressed have septation and cell separation defects. An analysis of the actin cytoskeleton shows the lack of a medial ring in septating cells that overexpress imp2, and the appearance of abnormal medial ring structures in septated cells that lack imp2. These observations suggest that imp2 destabilizes the medial ring during septation. imp2 also shows genetic interactions with several, previously characterized septation genes, strengthening the conclusion that it plays a role in normal fission yeast septation.
Roles for Laminin in Embryogenesis: Exencephaly, Syndactyly, and Placentopathy in Mice Lacking the Laminin α5 Chain
Authors: Miner Jeffrey H. , Cunningham Jeanette , Sanes Joshua R.
Published: July 2002

Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are α/β/γ heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development. Here, we show that the laminin α5 chain is required during embryogenesis. The α5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose α5 retain or acquire other α chains. Embryos lacking laminin α5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are α5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin α chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.
The Small GTP-binding Protein Rho Regulates Cortical Activities in Cultured Cells during Division
Authors: O'Connell Christopher B. , Wheatley Sally P. , Ahmed Sohail , Wang Yu-li
Published: July 2002

We have investigated the role of the small GTP-binding protein Rho in cytokinesis by microinjecting an inhibitor, C3 ribosyltransferase, into cultured cells. Microinjection of C3 into prometaphase or metaphase normal rat kidney epithelial cells induced immediate and global cortical movement of actin toward the metaphase plate, without an apparent effect on the mitotic spindle. During anaphase, concentrated cortical actin filaments migrated with separating chromosomes, leaving no apparent concentration of actin filaments along the equator. Myosin II in injected epithelial cells showed a diffuse distribution throughout cell division. All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully. However, cytokinesis became abnormal, generating irregular ingressions and ectopic cleavage sites even when mitosis was blocked with nocodazole. The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis. Poorly adherent HeLa cells showed neither ectopic cleavage nor completion of cytokinesis. Our results indicate that Rho does not simply activate actin–myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.
Sulfotransferases of Two Specificities Function in the Reconstitution of High Endothelial Cell Ligands for L-selectin
Authors: Bistrup Annette , Bhakta Sunil , Lee Jin Kyu , Belov Yevgeniy Y. , Gunn Michael Dee , Zuo Feng-Rong , Huang Chiao-Chain , Kannagi Reiji , Rosen Steven D. , Hemmerich Stefan
Published: July 2002

L-selectin, a lectin-like receptor, mediates rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs by interacting with HEV ligands. These ligands consist of a complex of sialomucins, candidates for which are glycosylation- dependent cell adhesion molecule 1 (GlyCAM-1), CD34, and podocalyxin. The ligands must be sialylated, fucosylated, and sulfated for optimal recognition by L-selectin. Our previous structural characterization of GlyCAM-1 has demonstrated two sulfation modifications, Gal-6-sulfate and GlcNAc-6-sulfate in the context of sialyl Lewis x. We now report the cloning of a Gal-6-sulfotransferase and a GlcNAc-6-sulfotransferase, which can modify GlyCAM-1 and CD34. The Gal-6-sulfotransferase shows a wide tissue distribution. In contrast, the GlcNAc-6-sulfotransferase is highly restricted to HEVs, as revealed by Northern analysis and in situ hybridization. Expression of either enzyme in Chinese hamster ovary cells, along with CD34 and fucosyltransferase VII, results in ligand activity, as detected by binding of an L-selectin/IgM chimera. When coexpressed, the two sulfotransferases synergize to produce strongly enhanced chimera binding.
Combined Biochemical and Electron Microscopic Analyses Reveal the Architecture of the Mammalian U2 snRNP
Authors: Krämer Angela , Grüter Patric , Gröning Karsten , Kastner Berthold
Published: July 2002

The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5′ half of U2 small nuclear RNA, whereas SF3a associates with the 3′ portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.
Insoluble γ-Tubulin–Containing Structures Are Anchored to the Apical Network of Intermediate Filaments in Polarized Caco-2 Epithelial Cells
Authors: Salas Pedro J.I.
Published: July 2002

We have previously shown that a thin (∼1 μm) layer of intermediate filaments located beneath the apical membrane of a variety of simple epithelial cells participates in the organization of apical microfilaments and microtubules. Here, I confirmed the apical distribution of γ-tubulin–containing structures (potential microtubule-organizing centers) in CACO-2 cells and demonstrated perfect colocalization of centrosomes and nearly 50% of noncentrosomal γ-tubulin with apical intermediate filaments, but not with apical F-actin. Furthermore, the antisense-oligonucleotide–mediated downregulation of cytokeratin 19, using two different antisense sequences, was more efficient than anticytoskeletal agents to delocalize centrosomes. Electron microscopy colocalization suggests that binding occurs at the outer boundary of the pericentriolar material. Type I cytokeratins 18 and 19 present in these cells specifically coimmunoprecipitated in multi-protein fragments of the cytoskeleton with γ-tubulin. The size and shape of the fragments, visualized at the EM level, indicate that physical trapping is an unlikely explanation for this result. Drastic changes in the extraction protocol did not affect coimmunoprecipitation. These results from three independent techniques, indicate that insoluble γ-tubulin–containing structures are attached to apical intermediate filaments.
The Tom Core Complex
Authors: Ahting Uwe , Thun Clemens , Hegerl Reiner , Typke Dieter , Nargang Frank E. , Neupert Walter , Nussberger Stephan
Published: July 2002

Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of ∼2.1 nm and a height of ∼7 nm. Tom40 is the key structural element of the TOM core complex.
Duplication and Maintenance of Heterochromatin Domains
Authors: Taddei Angela , Roche Danièle , Sibarita Jean-Baptiste , Turner Bryan M. , Almouzni Geneviève
Published: July 2002

To investigate the mechanisms that assure the maintenance of heterochromatin regions, we took advantage of the fact that clusters of heterochromatin DNA replicate late in S phase and are processed in discrete foci with a characteristic nuclear distribution. At the light microscopy level, within these entities, we followed DNA synthesis, histone H4 acetylation, heterochromatin protein 1 (Hp1α and -β), and chromatin assembly factor 1 (CAF-1). During replication, Hp1α and -β domains of concentration are stably maintained, whereas heterochromatin regions are enriched in both CAF-1 and replication-specific acetylated isoforms of histone H4 (H4Ac 5 and 12). We defined a time window of 20 min for the maintenance of this state. Furthermore, treatment with Trichostatin A (TSA), during and after replication, sustains the H4Ac 5 and 12 state in heterochromatin excluding H4Ac 8 and 16. In comparison, early replication foci, at the same level, did not display any specific enrichment in H4Ac 5 and 12. These data emphasize the specific importance for heterochromatin of the replication-associated H4 isoforms. We propose that perpetuation of heterochromatin involves self-maintenance factors, including local concentration of Hp1α and -β, and that a degree of plasticity is provided by the cycle of H4 acetylation/deacetylation assisted by CAF-1.
Oncogenic Raf-1 Disrupts Epithelial Tight Junctions via Downregulation of Occludin
Authors: Li Danxi , Mrsny Randall J.
Published: July 2002

Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell–cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1–activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1–activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1–induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.
The Mammalian Sec6/8 Complex Interacts with Ca2+ Signaling Complexes and Regulates Their Activity
Authors: Shin Dong Min , Zhao Xiao-Song , Zeng Weizhong , Mozhayeva Marina , Muallem Shmuel
Published: July 2002

The localization of various Ca2+ transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca2+ waves. In the present work, we examined the possible roles of the Sec6/8 complex or the exocyst in polarized Ca2+ signaling in pancreatic acinar cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca2+ signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate βCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP3R3), and the Gβγ subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca2+ pump, the G protein subunits Gαq and Gβγ, the β1 isoform of phospholipase C, and the ER resident IP3R1 from brain microsomal extracts. Antibodies against the various signaling and Ca2+ transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca2+ signaling proteins. Hence, the interaction between the Sec6/8 and Ca2+ signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca2+ signaling at a step upstream of Ca2+ release by IP3. Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca2+ waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca2+ signaling complexes and regulation of their activity.
Ftsz Ring Formation at the Chloroplast Division Site in Plants
Authors: Vitha Stanislav , McAndrew Rosemary S. , Osteryoung Katherine W.
Published: July 2002

Among the events that accompanied the evolution of chloroplasts from their endosymbiotic ancestors was the host cell recruitment of the prokaryotic cell division protein FtsZ to function in chloroplast division. FtsZ, a structural homologue of tubulin, mediates cell division in bacteria by assembling into a ring at the midcell division site. In higher plants, two nuclear-encoded forms of FtsZ, FtsZ1 and FtsZ2, play essential and functionally distinct roles in chloroplast division, but whether this involves ring formation at the division site has not been determined previously. Using immunofluorescence microscopy and expression of green fluorescent protein fusion proteins in Arabidopsis thaliana, we demonstrate here that FtsZ1 and FtsZ2 localize to coaligned rings at the chloroplast midpoint. Antibodies specific for recognition of FtsZ1 or FtsZ2 proteins in Arabidopsis also recognize related polypeptides and detect midplastid rings in pea and tobacco, suggesting that midplastid ring formation by FtsZ1 and FtsZ2 is universal among flowering plants. Perturbation in the level of either protein in transgenic plants is accompanied by plastid division defects and assembly of FtsZ1 and FtsZ2 into filaments and filament networks not observed in wild-type, suggesting that previously described FtsZ-containing cytoskeletal-like networks in chloroplasts may be artifacts of FtsZ overexpression.
Protein kinase B phosphorylates AHNAK and regulates its subcellular localization
Authors: Sussman Joshua , Stokoe David , Ossina Natalya , Shtivelman Emma
Published: July 2002

AHNAK is a ubiquitously expressed giant phosphoprotein that was initially identified as a gene product subject to transcriptional repression in neuroblastoma. AHNAK is predominantly nuclear in cells of nonepithelial origin, but is cytoplasmic or associated with plasma membrane in epithelial cells. In this study we show that the extranuclear localization of AHNAK in epithelial cells depends on the formation of cell–cell contacts. We show that AHNAK is a phosphorylation substrate of protein kinase B (PKB) in vitro and in vivo. Nuclear exclusion of AHNAK is mediated through a nuclear export signal (NES) in a manner that depends on the phosphorylation of serine 5535 of AHNAK by PKB, a process that also plays a major role in determining extranuclear localization of AHNAK. AHNAK is a new PKB substrate whose function, though unknown, is likely to be regulated by its localization, which is in turn regulated by PKB.
MyoD-positive myoblasts are present in mature fetal organs lacking skeletal muscle
Authors: Gerhart Jacquelyn , Bast Brian , Neely Christine , Iem Stephanie , Amegbe Paula , Niewenhuis Robert , Miklasz Steven , Cheng Pei Feng , George-Weinstein Mindy
Published: July 2002

The epiblast of the chick embryo gives rise to the ectoderm, mesoderm, and endoderm during gastrulation. Previous studies revealed that MyoD-positive cells were present throughout the epiblast, suggesting that skeletal muscle precursors would become incorporated into all three germ layers. The focus of the present study was to examine a variety of organs from the chicken fetus for the presence of myogenic cells. RT-PCR and in situ hybridizations demonstrated that MyoD-positive cells were present in the brain, lung, intestine, kidney, spleen, heart, and liver. When these organs were dissociated and placed in culture, a subpopulation of cells differentiated into skeletal muscle. The G8 antibody was used to label those cells that expressed MyoD in vivo and to follow their fate in vitro. Most, if not all, of the muscle that formed in culture arose from cells that expressed MyoD and G8 in vivo. Practically all of the G8-positive cells from the intestine differentiated after purification by FACS®. This population of ectopically located cells appears to be distinct from multipotential stem cells and myofibroblasts. They closely resemble quiescent, stably programmed skeletal myoblasts with the capacity to differentiate when placed in a permissive environment.
Viral Superantigen Drives Extrafollicular and Follicular B Cell Differentiation Leading to Virus-specific Antibody Production
Authors: Luther Sanjiv A. , Gulbranson-Judge Adam , Acha-Orbea Hans , MacLennan Ian C.M.
Published: July 2002

Mouse mammary tumor virus (MMTV[SW]) encodes a superantigen expressed by infected B cells. It evokes an antibody response specific for viral envelope protein, indicating selective activation of antigen-specific B cells. The response to MMTV(SW) in draining lymph nodes was compared with the response to haptenated chicken gamma globulin (NP-CGG) using flow cytometry and immunohistology. T cell priming occurs in both responses, with T cells proliferating in association with interdigitating dendritic cells in the T zone. T cell proliferation continues in the presence of B cells in the outer T zone, and B blasts then undergo exponential growth and differentiation into plasma cells in the medullary cords. Germinal centers develop in both responses, but those induced by MMTV(SW) appear later and are smaller. Most T cells activated in the T zone and germinal centers in the MMTV(SW) response are superantigen specific and these persist for weeks in lymph nodes draining the site MMTV(SW) injection; this contrasts with the selective loss of superantigen-specific T cells from other secondary lymphoid tissues. The results indicate that this viral superantigen, when expressed by professional antigen-presenting cells, drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.
Induction of a CD8+ Cytotoxic T Lymphocyte Response by Cross-priming Requires Cognate CD4+ T Cell Help
Authors: Bennett Sally R.M. , Carbone Francis R. , Karamalis Freda , Miller Jacques F.A.P. , Heath William R.
Published: July 2002

Class I–restricted presentation is usually associated with cytoplasmic degradation of cellular proteins and is often considered inaccessible to exogenous antigens. Nonetheless, certain exogenous elements can gain entry into this so-called endogenous pathway by a mechanism termed cross-presentation. This is known to be effective for class I–restricted cytotoxic T lymphocyte (CTL) cross-priming directed against a variety of exogenous tumor, viral, and minor transplantation antigens. The related effect of cross-tolerance can also effectively eliminate responses to selected self components. In both cases, this presentation appears to require the active involvement of a bone marrow–derived antigen presenting cell (APC). Here, we show that CTL induction by cross-priming with cell-associated ovalbumin requires the active involvement of CD4+ helper T cells. Importantly, this CD4+ population is only effective when both the helper and CTL determinants are recognized on the same APC. Moreover, we would argue that the cognitive nature of this event suggests that the CD4+ T cell actively modifies the APC, converting it into an effective stimulator for the successful priming of the CTL precursor.
In Vitro Translation and Assembly of a Complete T Cell Receptor–CD3 Complex
Authors: Huppa Johannes B. , Ploegh Hidde L.
Published: July 2002

The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven polypeptides: the clonotypic, disulfide-linked α/β heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-γ, -δ, -ε) and ζ, a disulfide-linked homodimer. We achieved the complete assembly of the human TCR in an in vitro transcription/translation system supplemented with dog pancreas microsomes by simultaneous translation of the messenger RNAs encoding the TCR-α, -β and CD3-γ, -δ, -ε, and -ζ subunits. CD3-ε, one of the subunits that initiates the assembly of the TCR in living cells, forms misfolded, disulfide-linked homooligomers when translated alone. However, co-translation of one of its first binding partners in the course of assembly, CD3-γ or -δ, led to the expression of mainly monomeric and correctly folded ε subunits, the only form we could detect as part of a properly assembled TCR complex. In the absence of these subunits, the ER-resident chaperone calnexin interacted with oligomeric, i.e. misfolded, structures of CD3-ε in a glycan-independent manner. A glycan-dependent interaction between CD3-ε and calnexin was mediated by CD3-γ and concerned only monomeric CD3-ε complexed with CD3-γ, but was dispensable for proper folding of CD3-ε. We suggest that in addition to its signaling function, CD3-ε serves as a monitor for proper subunit assembly of the TCR.
Tolerant B Lymphocytes Acquire Resistance to Fas-mediated Apoptosis after Treatment with Interleukin 4 but Not after Treatment with Specific Antigen Unless a Surface Immunoglobulin Threshold Is Exceeded
Authors: Foote Linda C. , Marshak-Rothstein Ann , Rothstein Thomas L.
Published: July 2002

Susceptibility to Fas-mediated apoptosis in nontolerant B cells is regulated in a receptor-specific fashion. To explore the regulation of Fas killing in tolerant, autoreactive B cells, mice doubly transgenic for hen egg lysozyme (HEL)–specific B cell receptors and soluble HEL were examined. Engagement of CD40 led to enhanced Fas expression and acquisition of sensitivity to Fas-mediated apoptosis in tolerant B cells, similar to that observed in nontolerant, receptor transgenic B cells. Engagement of surface immunoglobulin by specific (HEL) antigen failed to induce Fas resistance in tolerant B cells, in contrast to its effect on nontolerant B cells; however, cross-linking of biotinylated HEL with streptavidin induced similar levels of Fas resistance in tolerant and nontolerant B cells, which approximated the degree of Fas resistance produced by anti-Ig. Unlike surface Ig (sIg) engagement, physiological engagement of IL-4 receptors produced similar levels of Fas resistance in tolerant and nontolerant B cells. Thus, tolerant B cells differ from nontolerant B cells in the diminished capacity of surface immunoglobulin engagement to produce Fas resistance; however, tolerant B cells can be induced to become resistant to Fas-mediated apoptosis by IL-4 or by higher order cross-linking of sIg receptors.
The c-kit Ligand, Stem Cell Factor, Can Enhance Innate Immunity Through Effects on Mast Cells
Authors: Maurer Marcus , Echtenacher Bernd , Hültner Lothar , Kollias George , Männel Daniela N. , Langley Keith E. , Galli Stephen J.
Published: July 2002

Mast cells are thought to contribute significantly to the pathology and mortality associated with anaphylaxis and other allergic disorders. However, studies using genetically mast cell–deficient WBB6F1-KitW/KitW-v and congenic wild-type (WBB6F1-+/+) mice indicate that mast cells can also promote health, by participating in natural immune responses to bacterial infection. We previously reported that repetitive administration of the c-kit ligand, stem cell factor (SCF), can increase mast cell numbers in normal mice in vivo. In vitro studies have indicated that SCF can also modulate mast cell effector function. We now report that treatment with SCF can significantly improve the survival of normal C57BL/6 mice in a model of acute bacterial peritonitis, cecal ligation and puncture (CLP). Experiments in mast cell–reconstituted WBB6F1-KitW/KitW-v mice indicate that this effect of SCF treatment reflects, at least in part, the actions of SCF on mast cells. Repetitive administration of SCF also can enhance survival in mice that genetically lack tumor necrosis factor (TNF)-α, demonstrating that the ability of SCF treatment to improve survival after CLP does not solely reflect effects of SCF on mast cell– dependent (or –independent) production of TNF-α. These findings identify c-kit and mast cells as potential therapeutic targets for enhancing innate immune responses.
The Envelope Glycoprotein Ectodomains Determine the Efficiency of CD4+ T Lymphocyte Depletion in Simian– Human Immunodeficiency Virus–Infected Macaques
Authors: Karlsson Gunilla B. , Halloran Matilda , Schenten Dominik , Lee Juliette , Racz Paul , Tenner-Racz Klara , Manola Judith , Gelman Rebecca , Etemad-Moghadam Bijan , Desjardins Elizabeth , Wyatt Richard , Gerard Norma P. , Marcon Luisa , Margolin David , Fanton John , Axthelm Michael K. , Letvin Norman L. , Sodroski Joseph
Published: July 2002

CD4+ T lymphocyte depletion in human immunodeficiency virus type 1 (HIV-1)–infected humans underlies the development of acquired immune deficiency syndrome. Using a model in which rhesus macaques were infected with chimeric simian–human immunodeficiency viruses (SHIVs), we show that both the level of viremia and the structure of the HIV-1 envelope glycoprotein ectodomains individually contributed to the efficiency with which CD4+ T lymphocytes were depleted. The envelope glycoproteins of recombinant SHIVs that efficiently caused loss of CD4+ T lymphocytes exhibited increased chemokine receptor binding and membrane-fusing capacity compared with those of less pathogenic viruses. These studies identify the HIV-1 envelope glycoprotein ectodomains as determinants of CD4+ T lymphocyte loss in vivo and provide a foundation for studying pathogenic mechanisms.
C-Reactive Protein and Complement Are Important Mediators of Tissue Damage in Acute Myocardial Infarction
Authors: Griselli M. , Herbert J. , Hutchinson W.L. , Taylor K.M. , Sohail M. , Krausz T. , Pepys M.B.
Published: July 2002

Myocardial infarction in humans provokes an acute phase response, and C-reactive protein (CRP), the classical acute phase plasma protein, is deposited together with complement within the infarct. The peak plasma CRP value is strongly associated with postinfarct morbidity and mortality. Human CRP binds to damaged cells and activates complement, but rat CRP does not activate complement. Here we show that injection of human CRP into rats after ligation of the coronary artery reproducibly enhanced infarct size by ∼40%. In vivo complement depletion, produced by cobra venom factor, completely abrogated this effect. Complement depletion also markedly reduced infarct size, even when initiated up to 2 h after coronary ligation. These observations demonstrate that human CRP and complement activation are major mediators of ischemic myocardial injury and identify them as therapeutic targets in coronary heart disease.
A Defect in Interleukin 12–Induced Activation and Interferon γ Secretion of Peripheral Natural Killer T Cells in Nonobese Diabetic Mice Suggests New Pathogenic Mechanisms for Insulin-Dependent Diabetes Mellitus
Authors: Falcone Marika , Yeung Brian , Tucker Lee , Rodriguez Enrique , Sarvetnick Nora
Published: July 2002

The function of natural killer T (NKT) cells in the immune system has yet to be determined. There is some evidence that their defect is associated with autoimmunity, but it is still unclear how they play a role in regulating the pathogenesis of T cell–mediated autoimmune diseases. It was originally proposed that NKT cells could control autoimmunity by shifting the cytokine profile of autoimmune T cells toward a protective T helper 2 cell (Th2) type. However, it is now clear that the major function of NKT cells in the immune system is not related to their interleukin (IL)-4 secretion. In fact, NKT cells mainly secrete interferon (IFN)-γ and, activated in the presence of IL-12, acquire a strong inflammatory phenotype and cytotoxic function.