bluuebunny/crossref_metadata_2025 huggingface dataset:
Two type XII-like collagens localize to the surface of banded collagen fibrils.
Authors: Keene D R , Lunstrum G P , Morris N P , Stoddard D W , Burgeson R E
Published: May 2004

Two recently identified collagen molecules, termed twelve-like A and twelve-like B (TL-A and TL-B) have properties similar to type XII collagen. These molecules have been localized in human and calf tissues by immunoelectron microscopy. The observations strongly suggest that both molecules are located along the surface of banded collagen fibers. The epitopes recognized by the antibodies are contained in large, nontriple-helical domains at one end of the collagen helix. The epitopes are visualized at a distance from the surface of the banded fibers roughly equal to the length of the nonhelical domains, suggesting that the nonhelical domains extend from the fibril, while the triple-helical domains are likely to bind directly to the fibril surface. Occasionally, both TL-A and TL-B demonstrate periodic distribution along the fibril surface. The period corresponds to the primary interband distance of the banded fibrils. Not all fibrils in a fiber bundle are labeled, nor is the labeling continuous along the length of labeled fibrils. Simultaneous labeling of TL-A and type VI collagen only rarely shows colocalization, suggesting that TL-A and TL-B do not mediate interactions between the type VI collagen beaded filaments and banded collagen fibrils. Also, interfibrillar distances are approximately equivalent in the presence and absence of these type XII-like molecules. While the results do not directly indicate a specific function for these molecules, the localization at the fibril surface suggests that they mediate interactions between the fibrils and other matrix macromolecules or with cells.
PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A.
Authors: Ktistakis N T , Roth M G , Bloom G S
Published: May 2004

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug.
Motility of bile canaliculi in the living animal: implications for bile flow.
Authors: Watanabe N , Tsukada N , Smith C R , Phillips M J
Published: May 2004

Modern fluorescence microscopic techniques were used to image the bile canalicular system in the intact rat liver, in vivo. By combining the use of sodium fluorescein secretion into bile, with digitally enhanced fluorescence microscopy and time-lapse video, it was possible to capture and record the canalicular motility events that accompany the secretion of bile in life. Active bile canalicular contractions were found predominantly in zone 1 (periportal) hepatocytes of the liver. The contractile movements were repetitive, forceful, and appeared unidirectional moving bile in a direction towards the portal bile ducts. Contractions were not seen in the network of canaliculi on the surface of the liver. Cytochalasin B administration resulted in reduced canalicular motility, progressive dilation of zone 1 canaliculi, and impairment of bile flow. Canalicular dilations invariably involved the branch points of the canalicular network. The findings add substantively to previous in vitro studies using couplets, and suggest that canalicular contractions contribute physiologically to bile flow in the liver.
The centromere-kinetochore complex: a repeat subunit model.
Authors: Zinkowski R P , Meyne J , Brinkley B R
Published: May 2004

The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.
Sorting out IF networks: consequences of domain swapping on IF recognition and assembly.
Authors: McCormick M B , Coulombe P A , Fuchs E
Published: May 2004

Vimentin and keratin are coexpressed in many cells, but they segregate into two distinct intermediate filament (IF) networks. To understand the molecular basis for the sorting out of these IF subunits, we genetically engineered cDNAs encoding hybrid IF proteins composed of part vimentin and part type I keratin. When these cDNAs were transiently expressed in cells containing vimentin, keratin, or both IFs, the hybrid IF proteins all recognized one or the other or both networks. The ability to distinguish networks was dependent upon which segments of IF proteins were present in each construct. Constructs containing sequences encoding either helix 1B or helix 2B seemed to be the most critical in conferring IF recognition. At least for type I keratins, recognition was exerted at the level of dimer formation with wild-type type II keratin, as demonstrated by anion exchange chromatography. Interestingly, despite the fact that swapping of helical domains was not as deleterious to IF structure/function as deletion of helical domains, keratin/vimentin hybrids still caused structural aberrations in one or more of the cytoplasmic IF network. Thus, sequence diversity among IF proteins seems to influence not only coiled-coil but also higher ordered associations leading to 10-nm filament formation and/or IF interactions with other cellular organelles/proteins.
BIP associates with newly synthesized subunits of the mouse muscle nicotinic receptor.
Authors: Blount P , Merlie J P
Published: May 2004

A slow conformational change in newly synthesized acetylcholine receptor subunits is thought to be a requisite step in the biogenesis of this multi-subunit transmembrane glycoprotein. Previously, we demonstrated that this early conformational change within the alpha-subunit was inefficient and dependent upon disulfide bond formation (Blount, P. and J.P. Merlie. 1990. J. Cell Biol. 111:2613-2622). Here we show that newly synthesized acetylcholine receptor subunits and subunit complexes in the muscle-like cell line, BC3H-1, are associated with Bip, a ubiquitous binding protein of the endoplasmic reticulum. Characterization of the Bip/alpha-subunit complex in stably transfected fibroblasts revealed that Bip associates with newly synthesized unassembled alpha-subunit and some alpha gamma and alpha delta subunit complexes. Significantly, Bip does not associate well with the more mature form of the alpha-subunit containing an intramolecular disulfide bridge. Hence, Bip may play an important role in the conformational maturation and/or editing of unassembled AChR subunits and subunit complexes in vivo.
Retinoic acid stimulates the differentiation of PC12 cells that are deficient in cAMP-dependent protein kinase.
Authors: Scheibe R J , Ginty D D , Wagner J A
Published: May 2004

Retinoic acid (RA) induced neuronal differentiation in A126-1B2 cells and 123.7 cells, two mutant lines of PC12 that are deficient in cAMP-dependent protein kinase, but not in the parental PC12 cell line. A single exposure to RA was sufficient to cause neurite formation and inhibit cell division for a period of greater than 3 wk, suggesting that RA may cause a long-term, stable change in the state of these cells. In A126-1B2 cells, RA also induced the expression of other markers of differentiation including acetylcholinesterase and the mRNAs for neurofilament (NF-M) and GAP-43 as effectively as nerve growth factor (NGF). Neither NGF nor RA stimulated an increase in the expression of smg-25A in A126-1B2 cells, suggesting that the cAMP-dependent protein kinases may be required for an increase in the expression of this marker. RA also caused a rapid increase in the expression of the early response gene, c-fos, but did not effect the expression of egr-1. RA equivalently inhibited the division of A126-1B2 cells, 123.7 cells and parental PC12 cells, so RA induced differentiation is not an indirect response to growth arrest. In contrast, the levels of retinoic acid receptors (RAR alpha and RAR beta), and retinoic acid binding protein (CRABP) mRNA were strikingly higher in both A126-1B2 cells and 123.7 cells than in the parental PC12 cells. The deficiencies in cAMP-dependent protein kinase may increase the expression of CRABP and the RARs; and, thus, cAMP may indirectly regulate the ability of RA to control neurite formation and neural differentiation. Thus, RA appears to regulate division and differentiation of PC12 cells by a biochemical mechanism that is quite distinct from those used by peptide growth factors.
Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor.
Authors: Mignatti P , Mazzieri R , Rifkin D B
Published: May 2004

Basic fibroblast growth factor, a potent angiogenesis inducer, stimulates urokinase (uPA) production by vascular endothelial cells. In both basic fibroblast growth factor-stimulated and -nonstimulated bovine capillary endothelial and human umbilical vein endothelial cells single-chain uPA binding is mediated by a membrane protein with a Mr of 42,000. Exposure of bovine capillary or endothelial human umbilical vein endothelial cells to pmolar concentrations of basic fibroblast growth factor results in a dose-dependent, protein synthesis-dependent increase in the number of membrane receptors for uPA (19,500-187,000) and in a parallel decrease in their affinity (KD = 0.144-0.790 nM). With both cells, single-chain uPA binding is competed by synthetic peptides whose sequence corresponds to the receptor-binding sequence in the NH2-terminal domain of uPA. Exposure of bovine capillary endothelial cells to transforming growth factor beta 1, which inhibits uPA production and upregulates type 1 plasminogen activator inhibitor, the major endothelial cell plasminogen activator inhibitor, has no effect on uPA receptor levels. These results show that basic fibroblast growth factor, besides stimulating uPA production by vascular endothelial cells, also increases the production of receptors, which modulates their capacity to focalize this enzyme on the cell surface. This effect may be important in the degradative processes that occur during angiogenesis.
A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity.
Authors: Stochaj U , Osborne M , Kurihara T , Silver P
Published: May 2004

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.
Inhibition of autophagic-lysosomal delivery and autophagic lactolysis by asparagine.
Authors: Høyvik H , Gordon P B , Berg T O , Strømhaug P E , Seglen P O
Published: May 2004

Overall autophagy was measured in isolated hepatocytes as the sequestration and lysosomal hydrolysis of electroinjected [14C]lactose, using HPLC to separate the degradation product [14C]glucose from undegraded lactose. In addition, the sequestration step was measured separately as the transfer from cytosol to sedimentable cell structures of electroinjected [3H]raffinose or endogenous lactate dehydrogenase (LDH; in the presence of leupeptin to inhibit lysosomal proteolysis). Inhibitor effects at postsequestrational steps could be detected as the accumulation of autophaged lactose (which otherwise is degraded intralysosomally), or of LDH in the absence of leupeptin. Asparagine, previously shown to inhibit autophagic but not endocytic protein breakdown, strongly suppressed the autophagic hydrolysis of electroinjected lactose. Vinblastine, which inhibits both types of degradation, likewise suppressed lactose hydrolysis. Asparagine had little or no effect on sequestration, but caused an accumulation of autophaged LDH and lactose, indicating inhibition at a postsequestrational step. Neither asparagine nor vinblastine affected the degradation of intralysosomal lactose preaccumulated in the presence of the reversible lysosome inhibitor propylamine. However, if lactose was preaccumulated in the presence of asparagine, both asparagine and vinblastine suppressed its subsequent degradation. The data thus indicate that autophagic-lysosomal delivery, i.e., the transfer of autophaged material from prelysosomal vacuoles to lysosomes, is inhibited selectively by asparagine and non-selectively by vinblastine.
The intercentriolar linkage is critical for the ability of heterologous centrosomes to induce parthenogenesis in Xenopus.
Authors: Tournier F , Komesli S , Paintrand M , Job D , Bornens M
Published: May 2004

Centrosomes isolated from various sources, including human cells, have the capacity to induce parthenogenetic development when injected into unfertilized amphibian eggs. We recently isolated calf thymus centrosomes and showed that they differ structurally and functionally from previously isolated centrosomes of KE37 cells, in that the two centrioles in calf thymocytes are linearly associated by their proximal ends through a mass of electron dense material and nucleate few microtubules from their distal ends (Komesli, S., F. Tournier, M. Paintrand, R. Margolis, D. Job, and M. Bornens. 1989. J. Cell Biol. 109:2869-2878). We report here that these centrosomes are also unable to induce egg cleavage and examine the various possibilities which could account for this lack of competence. The results show that: (a) the kinetics of microtubule assembly on calf thymus centrosomes in Xenopus extracts are comparable to those of KE37 centrosomes; (b) centrosomes isolated from thymus of calves raised under controlled conditions (without anabolic agents) also lack competence; (c) centrosomes isolated from bovine cells of other tissues are competent; (d) centrosomes isolated from thymus of three other species (rat, mouse, and human) are competent. Since the lack of activity of calf thymus centrosomes apparently was not linked to species or tissue differences, we compared the ultrastructure of the centrosomes in the various centrosome preparations. The results show a strict correlation between the linear arrangement of centrioles and the lack of activity of the centrosomes. They suggest that the centrosome cycle can be blocked when the centrioles are prevented from separating into a nonlinear configuration, a step which might be critical for the initiation of procentriole budding. They also indicate that the centrosome may be involved in the G0-G1 transition.
Analysis of early events in acetylcholine receptor assembly.
Authors: Paulson H L , Ross A F , Green W N , Claudio T
Published: May 2004

Mammalian cell lines expressing nicotinic acetylcholine receptor (AChR) subunit cDNAs from Torpedo californica were used to study early events in AChR assembly. To test the hypothesis that individual subunits form homooligomeric intermediates before assembling into alpha 2 beta gamma delta pentamers, we analyzed the sedimentation on sucrose density gradients of each subunit expressed separately in cell lines. We have shown previously that the acute temperature sensitivity of Torpedo AChR subunit assembly is due, in part, to misfolding of the polypeptide chains (Paulson, H.L., and T. Claudio. 1990. J. Cell Biol. 110:1705-1717). We use this phenomenon to further analyze putative assembly-competent intermediates. In nonionic detergent at an assembly-permissive temperature, the majority of alpha, beta, gamma, and delta subunits sediment neither as 3-4S monomers nor as 9S complexes, but rather as 6S species whether synthesized in fibroblasts, myoblasts, or differentiated myosyncytia. Several results indicate that the 6S species are complexes comprised predominantly of incorrectly folded subunit polypeptides. The complexes represent homoaggregates which form rapidly within the cell, are stable to mild SDS treatment and, in the case of alpha, contain some disulfide-linked subunits. The coprecipitation of alpha subunit with BiP or GRP78, a resident protein of the ER, further indicates that at least some of these internally sequestered subunits also associated with an endogenous protein implicated in protein folding. The majority of subunits expressed in these cell lines appear to be aggregates of subunits which are not assembly intermediates and are not assembly-competent. The portion which migrates as monomer, in contrast, appears to be the fraction which is assembly competent. This fraction increases at temperatures more permissive for assembly, further indicating the importance of the monomer as the precursor to assembly of alpha 2 beta gamma delta pentamers.
Integration site-dependent expression of a transgene reveals specialized features of cells associated with neuromuscular junctions.
Authors: Weis J , Fine S M , David C , Savarirayan S , Sanes J R
Published: May 2004

After skeletal muscle is denervated, fibroblasts near neuromuscular junctions proliferate more than fibroblasts distant from synaptic sites, and they accumulate adhesive molecules such as tenascin (Gatchalian, C. L., M. Schachner, and J. R. Sanes. 1989. J. Cell Biol. 108:1873-1890). This response could reflect signals that arise perisynaptically after denervation, preexisting differences between perisynaptic and extrasynaptic fibroblasts, or both. Here, we describe a line of transgenic mice in which patterns of transgene expression provide direct evidence for differences between perisynaptic and extrasynaptic fibroblasts in normal muscle. Transgenic mice were generated using regulatory elements from a major histocompatibility complex (MHC) class I gene linked to the Escherichia coli beta-galactosidase (lacZ) gene. Expression of lacZ was detected histochemically. In each of eight lines, lacZ was detected in different subsets of cells, none of which included lymphocytes. In contrast, endogenous MHC is expressed in most tissues and at high levels in lymphocytes. Thus, the MHC gene sequences appeared inactive in the transgene, and lacZ expression was apparently controlled by genomic regulatory elements that were specific for the insertion site. In one line, cells close to the neuromuscular junction were lacZ positive in embryonic and young postnatal mice. Electron microscopy identified these cells as fibroblasts and Schwann cells associated with motor nerve terminals, as well as endoneurial fibroblasts, perineurial cells, and Schwann cells in the distal branches of motor nerves. No intramuscular cells greater than 200 microns from synaptic sites were lacZ positive. These results indicate that there are molecular differences between perisynaptic and extrasynaptic fibroblasts even in normal muscle and that diverse perisynaptic cell types share a specific pattern of gene expression.
Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells.
Authors: Schleef R R , Loskutoff D J , Podor T J
Published: May 2004

Immunogold EM was employed to compare the distribution of type 1 plasminogen activator inhibitor (PAI-1) on the surface of agonist-activated human umbilical vein endothelial cells (HUVECs) with that of control, unactivated cells. As previously observed, (Schleef, R.R., T.J. Podor, E. Dunne, J. Mimuro, and D.J. Loskutoff. J. Cell Biol. 110:155-163), analysis of cross-sections of nonpermeabilized control HUVEC monolayers stained first with affinity-purified rabbit antibodies to PAI-1 and then with gold-conjugated goat anti-rabbit IgG, revealed the presence of relatively few gold particles (less than 1-2% of the total) on the apical cell surface. The majority of gold particles were detected primarily in the extracellular matrix between the culture substratum and the cell membrane. In contrast, treatment of HUVECs with tumor necrosis factor alpha (TNF alpha; 200 U/ml, 24 h) or with lipopolysaccharide (LPS; 10 micrograms/ml, 24 h) resulted in an increased staining of PAI-1 not only in the extracellular matrix, but also on the apical cell surface (10-fold increase). Immunoabsorption of the rabbit anti-PAI-1 with purified PAI-1, or treatment of HUVECs with tissue-type plasminogen activator (2.5 micrograms/ml, 2 h, 4 degrees C) reduced the amount of staining both on the apical surface and in the extracellular matrix of agonist-activated HUVECs by 80-95%. The topographical location of PAI-1 on the cell surface was examined further by coupling immunogold staining with high resolution surface replication. Transmission EM of surface replicas from TNF alpha- or LPS-activated HUVECs revealed a general increase in PAI-1 staining both on planar regions and within indentations of the apical cell surface. Nonactivated HUVECs revealed PAI-1-specific immunogold particles only in areas of exposed extracellular matrix between the cells and occasionally at regions of cell-cell contacts. Analysis of activated bovine aortic endothelial cells by immuno-electron microscopy, immunologic assays, and flow cytometry revealed similar increases in surface PAI-1. These increases in surface PAI-1 could be detected by 3 h and continued over a 24-h period. The expression of PAI-1 on the luminal surface of endothelial cells during immune or inflammatory reactions could reduce endothelial fibrinolytic activity, thus, promoting the localized, pathologic formation of intravascular thrombi.
Identification of the fibronectin sequences required for assembly of a fibrillar matrix.
Authors: Schwarzbauer J E
Published: May 2004

During extracellular matrix assembly, fibronectin (FN) binds to cell surface receptors and initiates fibrillogenesis. As described in this report, matrix assembly has been dissected using recombinant FN polypeptides (recFNs) expressed in mammalian cells via retroviral vectors. RecFNs were assayed for incorporation into the detergent-insoluble cell matrix fraction and for formation of fibrils at the cell surface as detected by indirect immunofluorescence. Biochemical and immunocytochemical data are presented defining the minimum domain requirements for FN fibrillogenesis. The smallest functional recFN is half the size of native FN and contains intact amino- and carboxy-terminal regions with a large internal deletion spanning the collagen binding domain and the first seven type III repeats. Five type I repeats at the amino terminus are required for assembly and have FN binding activity. The dimer structure mediated by the carboxy-terminal interchain disulfide bonds is also essential. Surprisingly, recFNs lacking the RGDS cell binding site formed a significant fibrillar matrix. Therefore, FN-FN interactions and dimeric structure appear to be the major determinants of fibrillogenesis.
CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae.
Authors: Valdivieso M H , Mol P C , Shaw J A , Cabib E , Durán A
Published: May 2004

The CAL1 gene was cloned by complementation of the defect in Calcofluor-resistant calR1 mutants of Saccharomyces cerevisiae. Transformation of the mutants with a plasmid carrying the appropriate insert restored Calcofluor sensitivity, wild-type chitin levels and normal spore maturation. Southern blots using the DNA fragment as a probe showed hybridization to a single locus. Allelic tests indicated that the cloned gene corresponded to the calR1 locus. The DNA insert contains a single open-reading frame encoding a protein of 1,099 amino acids with a molecular mass of 124 kD. The predicted amino acid sequence shows several regions of homology with those of chitin synthases 1 and 2 from S. cerevisiae and chitin synthase 1 from Candida albicans. calR1 mutants have been found to be defective in chitin synthase 3, a trypsin-independent synthase. Transformation of the mutants with a plasmid carrying CAL1 restored chitin synthase 3 activity; however, overexpression of the enzyme was not achieved even with a high copy number plasmid. Since Calcofluor-resistance mutations different from calR1 also result in reduced levels of chitin synthase 3, it is postulated that the products of some of these CAL genes may be limiting for expression of the enzymatic activity. Disruption of the CAL1 gene was not lethal, indicating that chitin synthase 3 is not an essential enzyme for S. cerevisiae.
ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein.
Authors: Kellaris K V , Bowen S , Gilmore R
Published: May 2004

We have used the homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER. Translocation intermediates were assembled by supplementing cell free translations of truncated mRNAs with the signal recognition particle (SRP) and microsomal membrane vesicles. Two prominent cross-linked products of 45 and 64 kD were detected. The 64-kD product was obtained when the cell free translation contained SRP, while formation of the 45-kD product required both SRP and translocation competent microsomal membrane vesicles. In agreement with previous investigators, we suggest that the 64-kD product arises by cross-linking of the nascent polypeptide to the 54-kD subunit of SRP. The 45-kD product resists alkaline extraction from the membrane, so we conclude that the 11-kD nascent polypeptide has been crosslinked to an integral membrane protein of approximately 34 kD (imp34). The cross-linked product does not bind to ConA Sepharose, nor is it sensitive to endoglycosidase H digestion; hence imp34 is not identical to the alpha or beta subunits of the signal sequence receptor (SSR). We propose that imp34 functions in concert with SSR to form a translocation site through which nascent polypeptides pass in traversing the membrane bilayer of the rough endoplasmic reticulum.
Synthesis of abnormal immunoglobulins by hybridomas from autoimmune "viable motheaten" mutant mice.
Authors: Schweitzer P A , Taylor S E , Shultz L D
Published: May 2004

Secretory defects in abnormal plasma cells, called Mott cells, that appear in lymphoid tissues of spontaneously autoimmune, "viable motheaten" (mev/mev) mice lead to deposition of immunoglobulin in RER-bound vesicles. Such vesicles have been termed Russel bodies. Cells with Russel bodies can also be observed rarely in normal animals, usually as a result of extreme antigenic loads or pathologic states. To understand why these abnormal cells appear commonly in mev/mev mice, we have established a panel of hybridomas that contain Russell bodies. Using immunochemical analysis and immunoelectron microscopy, we have characterized the secretory defects. Although these hybridoma cells synthesize a normal size heavy chain and it associates with light chain, the Russell bodies have many characteristics of inclusion bodies, which commonly appear in cells synthesizing mutant proteins and often are associated with incompletely or abnormally folded proteins. Pulse-chase experiments showed that immunoglobulins synthesized by these hybridomas accumulate rapidly into insoluble complexes and have an intracellular half life approximately 10 time greater than normal immunoglobulins. The defect affected only the immunoglobulin derived from the mev/mev mice and did not affect the secretion of normal immunoglobulin produced by an IgG1-secreting fusion partner. In addition to accumulating intracellular immunoglobulins, many mutant cell lines also secreted immunoglobulin. Endoglycosidase H digestion was used to determine the state of processing of the N-linked carbohydrates on the immunoglobulin molecules. This analysis demonstrated that the N-linked carbohydrates on the secreted immunoglobulin were resistant to endoglycosidase H digestion, indicating that they were processed normally. The insoluble IgM molecules were sensitive to endoglycosidase H, which is consistent with their localization to the RER. We propose several models by which these abnormal immunoglobulin-secreting cells commonly appear in this autoimmune mutant mouse.
Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil.
Authors: Wiche G , Becker B , Luber K , Weitzer G , Castañon M J , Hauptmann R , Stratowa C , Stewart M
Published: May 2004

We have determined the complete cDNA sequence of rat plectin from a number of well-characterized overlapping lambda gt11 clones. The 4,140-residue predicted amino acid sequence (466,481 D) is consistent with a three-domain structural model in which a long central rod domain, having mainly an alpha-helical coiled coil conformation, is flanked by globular NH2- and COOH-terminal domains. The plectin sequence has a number of repeating motifs. The rod domain has five subregions approximately 200-residues long in which there is a strong repeat in the charged amino acids at 10.4 residues that may be involved in association between plectin molecules. The globular COOH-terminal domain has a prominent six-fold tandem repeat, with each repeat having a strongly conserved central region based on nine tandem repeats of a 19-residue motif. The plectin sequence has several marked similarities to that of desmoplakin (Green, K. J., D. A. D. Parry, P. M. Steinert, M. L. A. Virata, R. M. Wagner, B. D. Angst, and L.A. Nilles. 1990. J. Biol. Chem. 265:2,603-2,612), which has a shorter coiled-coil rod domain with a similar 10.4 residue charge periodicity and a COOH-terminal globular domain with three tandem repeats homologous to the six found in plectin. The plectin sequence also has homologies to that of the bullous pemphigoid antigen. Northern blot analysis indicated that there is a significant degree of conservation of plectin genes between rat, human, and chicken and that, as shown previously at the protein level, plectin has a wide tissue distribution. There appeared to be a single rat plectin gene that gave rise to a 15-kb message. Expression of polypeptides encoded by defined fragments of plectin cDNA in E. coli has also been used to localize the epitopes of a range of monoclonal and serum antibodies. This enabled us to tentatively map a sequence involved in plectin-vimentin and plectin-lamin B interactions to a restricted region of the rod domain.
A chicken red cell inhibitor of transcription associated with the terminally differentiated state.
Authors: Walmsley M E , Buckle R S , Allan J , Patient R K
Published: May 2004

When a red cell nuclear extract (RCE) from adult chickens was injected into Xenopus oocytes along with the chicken beta globin gene, transcript levels were dramatically reduced compared to injection of DNA alone. The inhibitory action of the RCE was not specific to the beta globin gene since the Herpes thymidine kinase and Xenopus 5S RNA gene transcript levels were similarly reduced. Transcriptional repression was observed even after passage of the RCE through oocyte cytoplasm to the nucleus. The inhibitory activity binds to DNA cellulose, which suggests that the inhibitor either binds to DNA or associates with DNA-binding proteins. Nuclease digestion of the chromatin assembled on injected beta globin DNA revealed that inhibition was not associated with local changes in chromatin structure. Extracts from 9-d chicken embryonic erythroid cells, in which the endogenous beta globin gene is actively expressed, did not inhibit transcription. The inhibitory activity is, therefore, restricted to transcriptionally quiescent, adult erythrocytes. Since the inhibitory effects were seen with both polymerase II and III directed genes, we speculate that the activity may be part of the extreme transcriptional repression which occurs in the terminally differentiated erythrocyte.
Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles.
Authors: Rexach M F , Schekman R W
Published: May 2004

The transport of pro-alpha-factor from the ER to the Golgi apparatus in gently lysed yeast spheroplasts is mediated by diffusible vesicles. These transport vesicles contain core-glycosylated pro-alpha-factor and are physically separable from donor ER and target Golgi compartments. The formation of diffusible vesicles from the ER requires ATP, Sec12p, Sec23p, and GTP hydrolysis. The vesicles produced are functionally distinct from the ER: they transfer pro-alpha-factor to the Golgi apparatus faster and more efficiently than the ER, they do not require Sec12p or Sec23p to complete transfer, and transfer is resistant to GTP gamma S. Targeting of vesicles to the Golgi apparatus requires Ypt1p and Sec18p. Fusion of vesicles that have targeted requires calcium and ATP.
Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes.
Authors: Otto E , Kunimoto M , McLaughlin T , Bennett V
Published: May 2004

Ankyrins are a family of membrane-associated proteins that can be divided into two immunologically distinct groups: (a) erythrocyte-related isoforms (ankyrinR) that have polarized distributions in particular cell types; and (b) brain-related isoforms (ankyrinB) that display a broader distribution. In this paper, we report the isolation and sequences of cDNAs related to two ankyrinB isoforms, human brain ankyrin 1 and 2, and show that these isoforms are produced from alternatively spliced mRNAs of a single gene. Human brain ankyrin 1 and 2 share a common NH2-terminus that is similar to human erythrocyte ankyrins, with the most striking conservation occurring between areas composed of a repeated 33-amino acid motif and between areas corresponding to the central portion of the spectrin-binding domain. In contrast, COOH-terminal sequences of brain ankyrin 1 and 2 are distinct from one another and from human erythrocyte ankyrins, and thus are candidates to mediate protein interactions that distinguish these isoforms. The brain ankyrin 2 cDNA sequence includes a stop codon and encodes a polypeptide with a predicted molecular mass of 202 kD, which is similar to the Mr of the major form of ankyrin in adult bovine brain membranes. Moreover, an antibody raised against the conserved NH2-terminal domain of brain ankyrin cross-reacts with a single Mr = 220 kD polypeptide in adult human brain. These results strongly suggest that the amino acid sequence of brain ankyrin 2 determined in this report represents the complete coding sequence of the major form of ankyrin in adult human brain. In contrast, the brain ankyrin 1 cDNAs encode only part of a larger isoform. An immunoreactive polypeptide of Mr = 440 kD, which is evident in brain tissue of young rats, is a candidate to be encoded by brain ankyrin 1 mRNA. The COOH-terminal portion of brain ankyrin 1 includes 15 contiguous copies of a novel 12-amino acid repeat. Analysis of DNA from a panel of human/rodent cell hybrids linked this human brain ankyrin gene to chromosome 4. This result, coupled with previous reports assigning the human erythrocyte ankyrin gene to chromosome 8, demonstrates that human brain and erythrocyte ankyrins are encoded by distinct members of a multigene family.
An increase or a decrease in myosin II phosphorylation inhibits macrophage motility.
Authors: Wilson A K , Gorgas G , Claypool W D , de Lanerolle P
Published: May 2004

Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin-independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells.
Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes.
Authors: Wordeman L , Steuer E R , Sheetz M P , Mitchison T
Published: May 2004

We have used indirect immunofluorescence in combination with correlative EM to subdivide the mammalian kinetochore into two domains based on the localization of specific antigens. We demonstrate here that the fibrous corona on the distal face of the kinetochore plate contains tubulin (previously shown by Mitchison, T. J., and M. W. Kirschner. 1985. J. Cell Biol. 101:755-765) and the minus end-directed, ATP-dependent microtubule motor protein, dynein; whereas a 50-kD CREST antigen is located internal to these components in the kinetochore. Tubulin and dynein can be extracted from the kinetochore by 150 mM KI, leaving other, as yet uncharacterized, components of the kinetochore corona intact. Microtubules and tubulin subunits will associate with kinetochores in vitro after extraction with 150 mM KI, suggesting that other functionally significant, corona-associated molecules remain unextracted. Our results suggest that the corona region of the kinetochore contains the machinery for chromosome translocation along microtubules.
Kinesin associates with anterogradely transported membranous organelles in vivo.
Authors: Hirokawa N , Sato-Yoshitake R , Kobayashi N , Pfister K K , Bloom G S , Brady S T
Published: May 2004

Biochemical, pharmacological and immunocytochemical studies have implicated the microtubule-activated ATPase, kinesin, in the movement of membrane bounded organelles in fast axonal transport. In vitro studies suggested that kinesin moves organelles preferentially in the anterograde direction, but data about the function and precise localization of kinesin in the living axon were lacking. The current study was undertaken to establish whether kinesin associates with anterograde or retrograde moving organelles in vivo. Peripheral nerves were ligated to produce accumulations of organelles moving in defined directions. Regions proximal (anterograde) and distal (retrograde) to the ligation were analyzed for kinesin localization by immunofluorescence, and by immunogold electron microscopy using ultracryomicrotomy. Substantial amounts of kinesin were associated with anterograde moving organelles on the proximal side, while significantly less kinesin was detected distally. Statistical analyses indicated that kinesin was mostly associated with membrane-bounded organelles. These observations indicate that axonal kinesin is primarily associated with anterograde moving organelles in vivo.
The ryanodine receptor/junctional channel complex is regulated by growth factors in a myogenic cell line.
Authors: Marks A R , Taubman M B , Saito A , Dai Y , Fleischer S
Published: May 2004

The ryanodine receptor/junctional channel complex (JCC) forms the calcium release channel and foot structures of the sarcoplasmic reticulum. The JCC and the dihydropyridine (DHP) receptor in the transverse tubule are two of the major components involved in excitation-contraction (E-C) coupling in skeletal muscle. The DHP receptor is believed to serve as the voltage sensor in E-C coupling. Both the JCC and DHP receptor, as well as many skeletal muscle-specific contractile protein genes, are expressed in the BC3H1 muscle cell line. In the present study, we find that during differentiation of BC3H1 cells, induced by mitogen withdrawal, induction of the JCC and DHP receptor mRNAs is temporally similar to that of the skeletal muscle contractile protein genes alpha-tropomyosin and alpha-actin. Our data suggest that there is coordinate regulation of both the contractile protein genes (which have been studied in detail previously) and the genes encoding the calcium channels involved in E-C coupling. Induction of both calcium channels is accompanied by profound changes in BC3H1 cell morphology including the development of many components of mature skeletal muscle cells, despite lack of myoblast fusion. Visualized by electron microscopy, the JCC appears as "foot structures" located in the dyad junction between the plasmalemma and the sarcoplasmic reticulum of the BC3H1 cells. Development of foot structures is concomitant with JCC mRNA expression. Expression of the JCC and DHP receptor mRNAs and formation of the foot structures are inhibited specifically by fibroblast growth factor.
Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation.
Authors: Panet R , Atlan H
Published: May 2004

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl- cotransport in the mitogenic signal of human skin fibroblast proliferation. The Na+/K+/Cl- cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent G0/G1 human skin fibroblasts. The following mitogens, FGF, PDGF, alpha-thrombin, insulin-like growth factor-1, transforming growth factor-alpha, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, all stimulated the Na+/K+/Cl- cotransport. In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the G0/G1 phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis. In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport plays a major role in the mitogenic signaling pathway of the human fibroblasts.