bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.
Authors: Ninomiya Y , Kagawa M , Iyama K , Naito I , Kishiro Y , Seyer J M , Sugimoto M , Oohashi T , Sado Y
Published: May 2004

Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.
Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus.
Authors: Yokota H , van den Engh G , Hearst J E , Sachs R K , Trask B J
Published: May 2004

We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation.
Proenkephalin is a nuclear protein responsive to growth arrest and differentiation signals.
Authors: Böttger A , Spruce B A
Published: May 2004

Neuropeptide precursors are traditionally viewed as molecules destined to be cleaved into bioactive peptides, which are then released from the cell to act on target cell surface receptors. In this report we demonstrate nuclear localization of the enkephalin precursor, proenkephalin, in rodent and human embryonic fibroblasts (Swiss 3T3 and MRC-5 cells) and in rodent myoblasts (C2C12 cells). Nuclear proenkephalin, detected by immunofluorescence with a panel of antiproenkephalin monoclonal antibodies, is distributed predominantly in three patterns. Selective abolition of these patterns with salt, nuclease, or methanol is associated with liberation of immunoprecipitable proenkephalin into the extraction supernatant. Proenkephalin antigenic domains, mapped using phage display libraries and synthetic peptides, are differentially revealed in the three distribution patterns. Selective epitope revelation may reflect different conformational forms of proenkephalin or its existence in complexes with other nuclear proteins, forms which therefore have different biochemical associations with the nuclear substructure. In fibroblast cell populations in transition to growth arrest, nuclear proenkephalin responds promptly to mitogen withdrawal and cell-cell contact by transient, virtually synchronous unmasking of multiple antigenic domains in a fine punctate distribution. A similar phenomenon is observed in myoblasts undergoing differentiation. The acknowledgment of growth arrest and differentiation signals by nuclear proenkephalin suggests its integration with transduction pathways mediating these signals. To begin to address the mechanism of nuclear targeting, we have transfected mutated and nonmutated proenkephalin into COS (African green monkey kidney) cells. Nonmutated proenkephalin is localized exclusively in the cytoplasm; however, proenkephalin mutated at the first ATG codon, or devoid of its signal peptide sequence, is targeted to the nucleus as well as to the cytoplasm. From this we speculate that nuclear proenkephalin arises from a primary translation product that lacks a signal peptide sequence because of initiation at a different site.
NUP82 is an essential yeast nucleoporin required for poly(A)+ RNA export.
Authors: Hurwitz M E , Blobel G
Published: May 2004

We have isolated and characterized the gene encoding a novel essential nucleoporin of 82 kD, termed NUP82. Indirect immunofluorescence of cells containing an epitope tagged copy of the NUP82 localized it to the nuclear pore complex (NPC). Primary structure analysis indicates that the COOH-terminal 195 amino acids contain a putative coiled-coil domain. Deletion of the COOH-terminal 87 amino acids of this domain causes slower cell growth; deletion of the COOH-terminal 108 amino acids results in slower growth at 30 degrees C and lethality at 37 degrees C. Cells in which the last 108 amino acids of NUP82 have been deleted, when shifted to 37 degrees C, do not display any gross morphological defects in their nuclear pore complexes or nuclear envelopes. They do, however, accumulate poly(A)+ RNA in their nuclei at 37 degrees C. We propose that NUP82 acts as a linker to tether nucleoporins directly involved in nuclear transport to pore scaffolding via its coiled-coil domain.
Involvement of the GTP binding protein Rho in constitutive endocytosis in Xenopus laevis oocytes.
Authors: Schmalzing G , Richter H P , Hansen A , Schwarz W , Just I , Aktories K
Published: May 2004

To study an endocytotic role of the GTP-binding protein RhoA in Xenopus oocytes, we have monitored changes in the surface expression of sodium pumps, the surface area of the oocyte and the uptake of the fluid-phase marker inulin. Xenopus oocytes possess intracellular sodium pumps that are continuously exchanged for surface sodium pumps by constitutive endo- and exocytosis. Injection of Clostridium botulinum C3 exoenzyme, which inactivates Rho by ADP-ribosylation, induced a redistribution of virtually all intracellular sodium pumps to the plasma membrane and increased the surface area of the oocytes. The identical effects were caused by injection of ADP-ribosylated recombinant RhoA into oocytes. The C3 exoenzyme acts by blocking constitutive endocytosis in oocytes, as determined using a mAb to the beta 1 subunit of the mouse sodium pump as a reporter molecule and oocytes expressing heterologous sodium pumps. In contrast, an increase in endocytosis and a decrease in the surface area was induced by injection of recombinant Val14-RhoA protein or Val14-rhoA cRNA. PMA stimulated sodium pump endocytosis, an effect that was blocked by a specific inhibitor of protein kinase C (Gö 16) or by ADP-ribosylation of Rho by C3. Similarly, the phorbol ester-induced increase in fluid-phase endocytosis in oocytes was inhibited by Gö 16, C3 transferase, or by injection of ADP-ribosylated RhoA. In contrast to C3 transferase, C. botulinum C2 transferase, which ADP-ribosylates actin, had no effect on sodium pump endocytosis or PMA-stimulated fluid-phase endocytosis. The data suggests that RhoA is an essential component of a presumably clathrin-independent endocytic pathway in Xenopus oocytes which can be regulated by protein kinase C.
Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants.
Authors: Samuels A L , Giddings T H , Staehelin L A
Published: May 2004

Cell plate formation in tobacco root tips and synchronized dividing suspension cultured tobacco BY-2 cells was examined using cryofixation and immunocytochemical methods. Due to the much improved preservation of the cells, many new structural intermediates have been resolved, which has led to a new model of cell plate formation in higher plants. Our electron micrographs demonstrate that cell plate formation consists of the following stages: (1) the arrival of Golgi-derived vesicles in the equatorial plane, (2) the formation of thin (20 +/- 6 nm) tubes that grow out of individual vesicles and fuse with others giving rise to a continuous, interwoven, tubulo-vesicular network, (3) the consolidation of the tubulo-vesicular network into an interwoven smooth tubular network rich in callose and then into a fenestrated plate-like structure, (4) the formation of hundreds of finger-like projections at the margins of the cell plate that fuse with the parent cell membrane, and (5) cell plate maturation that includes closing of the plate fenestrae and cellulose synthesis. Although this is a temporal chain of events, a developing cell plate may be simultaneously involved in all of these stages because cell plate formation starts in the cell center and then progresses centrifugally towards the cell periphery. The "leading edge" of the expanding cell plate is associated with the phragmoplast microtubule domain that becomes concentrically displaced during this process. Thus, cell plate formation can be summarized into two phases: first the formation of a membrane network in association with the phragmoplast microtubule domain; second, cell wall assembly within this network after displacement of the microtubules. The phragmoplast microtubules end in a filamentous matrix that encompasses the delicate tubulo-vesicular networks but not the tubular networks and fenestrated plates. Clathrin-coated buds/vesicles and multivesicular bodies are also typical features of the network stages of cell plate formation, suggesting that excess membrane material may be recycled in a selective manner. Immunolabeling data indicate that callose is the predominant lumenal component of forming cell plates and that it forms a coat-like structure on the membrane surface. We postulate that callose both helps to mechanically stabilize the early delicate membrane networks of forming cell plates, and to create a spreading force that widens the tubules and converts them into plate-like structures. Cellulose is first detected in the late smooth tubular network stage and its appearance seems to coincide with the flattening and stiffening of the cell plate.
Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture.
Authors: de Hoop M , von Poser C , Lange C , Ikonen E , Hunziker W , Dotti C G
Published: May 2004

Certain epithelial cells synthesize the polymeric immunoglobulin receptor (pIgR) to transport immunoglobulins (Igs) A and M into external secretions. In polarized epithelia, newly synthesized receptor is first delivered to the basolateral plasma membrane and is then, after binding the Ig, transcytosed to the apical plasma membrane, where the receptor-ligand complex is released by proteolytic cleavage. In a previous work (Ikonen et al., 1993), we implied the existence of a dendro-axonal transcytotic pathway for the rabbit pIgR expressed in hippocampal neurons via the Semliki Forest Virus (SFV) expression system. By labeling surface-exposed pIgR in live neuronal cells, we now show (a) internalization of the receptor from the dendritic plasma membrane to the dendritic early endosomes, (b) redistribution of the receptor from the dendritic to the axonal domain, (c) inhibition of this movement by brefeldin A (BFA) and (d) stimulation by the activation of protein kinase C (PKC) via phorbol myristate acetate (PMA). In addition, we show that a mutant form of the receptor lacking the epithelial basolateral sorting signal is directly delivered to the axonal domain of hippocampal neurons. Although this mutant is internalized into early endosomes, no transcytosis to the dendrites could be observed. In epithelial Madin-Darby Canine Kidney (MDCK) cells, the mutant receptor could also be internalized into basolaterally derived early endosomes. These results suggest the existence of a dendro-axonal transcytotic pathway in neuronal cells which shares similarities with the basolateral to apical transcytosis in epithelial cells and constitute the basis for the future analysis of its physiological role.
Controlled conversion of an immortalized mesodermal progenitor cell towards osteogenic, chondrogenic, or adipogenic pathways.
Authors: Poliard A , Nifuji A , Lamblin D , Plee E , Forest C , Kellermann O
Published: May 2004

The teratocarcinoma-derived C1 clone behaves as a mesodermal tripotential progenitor cell whose choice of fate, either osteoblast, chondroblast, or adipoblast, is strictly dependent on the spatial organization of the cells and the nature of the induction. In the absence of cell contact before the addition of inducers, the C1 cells maintain a stable undifferentiated phenotype while expressing potential regulators of embryonic mesodermal stem cell fate such a M-twist and Id1. Upon establishment of cell contacts before the induction of differentiation, the early genes characteristic of the three fates become expressed. In the presence of beta glycerophosphate and ascorbate, provided the cells have formed aggregates, 95% of the C1 cells mineralize with a kinetics of gene expression close to that of osteoblasts (Poliard, A., D. Lamblin, P. J. Marie, M. H. Buc, and O. Kellerman. 1993. J. Cell Sci. 106:503-512). With 10(-6)M dexamethasone, 80% of the same aggregates differentiate into foci of chondroblast-like cells. The kinetics of expression of the genes encoding type II, IX, X, and XI collagens, aggrecan and link protein during the conversion toward cartilage hypertrophy resembles that accompanying in vivo chondrogenesis. The synergistic action of dexamethasone and insulin convert most confluent C1 cells into functional adipocytes and induce a pattern of gene expression close to that reported for adipoblast cell lines. The C1 clone with its capacity to differentiate along three alternative pathways with high frequency, therefore appears as a valid in vitro model for deciphering the molecular basis of mesoblast ontogeny.
Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD1).
Authors: Diamond M S , Alon R , Parkos C A , Quinn M T , Springer T A
Published: May 2004

Previous studies have demonstrated that the leukocyte integrin Mac-1 adheres to several cell surface and soluble ligands including intercellular adhesion molecule-1, fibrinogen, iC3b, and factor X. However, experiments with Mac-1-expressing transfectants, purified Mac-1, and mAbs to Mac-1 indicate the existence of additional ligands. In this paper, we demonstrate a direct interaction between Mac-1 and heparan sulfate glycans. Heparin affinity resins immunoprecipitate Mac-1, and neutrophils and transfectant cells that express Mac-1 bind to heparin and heparan sulfate, but not to other sulfated glycosaminoglycans. Inhibition studies with mAbs and chemically modified forms of heparin suggest the I domain as a recognition site on Mac-1 for heparin, and suggest that either N- or O-sulfation is sufficient for heparin to bind efficiently to Mac-1. Under conditions of continuous flow in which heparins and E-selectin are cosubstrates, neutrophils tether to E-selectin and form firm adhesions through a Mac-1-heparin interaction.
Virus-mediated release of endosomal content in vitro: different behavior of adenovirus and rhinovirus serotype 2.
Authors: Prchla E , Plank C , Wagner E , Blaas D , Fuchs R
Published: May 2004

Endosomal penetration by nonenveloped viruses might be accomplished by either local breakdown of the endosomal membrane (e.g., adenovirus) or formation of a membrane-spanning pore by capsid proteins. Uncoating of the nonenveloped virus human rhinovirus serotype 2 (HRV2) has been shown to occur from late endosomes and to be entirely dependent on the acidic pH in this compartment (Prchla, E., E. Kuechler, D. Blaas, and R. Fuchs. 1994. J. Virol. 68: 3713-3723). To investigate further the mechanism of uncoating of HRV2, an in vitro assay was established to test viruses or virus-derived peptides for their capacity to release cointernalized biotin-dextran of different molecular mass (10 and 70 kD) from isolated endosomes. The suitability of the assay was demonstrated by use of a fusogenic peptide derived from influenza virus hemagglutinin (GALA-INF3). Whereas adenovirus induced a low pH-dependent release of up to 46% of the internalized biotin-dextran and did not show any significant size selectivity (as expected for endosome disruption), HRV2 mediated release of 27% of the 10 kD dextran and only traces of the 70-kD dextran. Similarly, GALA-INF3-induced release of biotin-dextran was also size dependent. The potential role of the capsid protein VP1 in HRV2 uncoating in vivo was also substantiated in our in vitro system using an amphipathic, NH2-terminal peptide of VP1. Taken together, these data favor the model of a specific pore-forming mechanism for HRV2 uncoating which is in contrast to the membrane-disrupting mechanism of adenovirus.
Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes.
Authors: Mayer R , Kartenbeck J , Büchler M , Jedlitschky G , Leier I , Keppler D
Published: May 2004

We have previously shown that the multi-drug resistance protein (MRP) mediates the ATP-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes. The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATP-dependent conjugate transport in canalicular membranes from TR- mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transport-deficient TR- mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte.
Myosin is involved in postmitotic cell spreading.
Authors: Cramer L P , Mitchison T J
Published: May 2004

We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time-lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II.
Isolation and characterization of nuclear envelopes from the yeast Saccharomyces.
Authors: Strambio-de-Castillia C , Blobel G , Rout M P
Published: May 2004

We have developed a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE proteins. Thus we found that 20% of endoplasmic reticulum (ER) marker proteins are localized at the NE. Using a novel monospecific mAb made against proteins in the H-NE fraction and found to be directed against the pore membrane protein POM152, we showed that while the majority of POM152 is localized in the NE at the NPC, a proportion of this protein is also present in the ER. This ER pool of POM152 is likely to be involved in the duplication of nuclear pores and NPCs during S-phase. Both the NEs and H-NEs were found to be competent for the in vitro posttranslational translocation of prepro-alpha-factor. They may also be suitable to investigate other ER- and NE-associated functions in cell-free systems.
Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland.
Authors: Yang Y , Spitzer E , Meyer D , Sachs M , Niemann C , Hartmann G , Weidner K M , Birchmeier C , Birchmeier W
Published: May 2004

We have examined the role of two mesenchymal ligands of epithelial tyrosine kinase receptors in mouse mammary gland morphogenesis. In organ cultures of mammary glands, hepatocyte growth factor (HGF, scatter factor) promoted branching of the ductal trees but inhibited the production of secretory proteins. Neuregulin (NRG, neu differentiation factor) stimulated lobulo-alveolar budding and the production of milk proteins. These functional effects are paralleled by the expression of the two factors in vivo: HGF is produced in mesenchymal cells during ductal branching in the virgin animal; NRG is expressed in the mesenchyme during lobulo-alveolar development at pregnancy. The receptors of HGF and NRG (c-met, c-erbB3, and c-erbB4), which are expressed in the epithelial cells, are not regulated. In organ culture, branching morphogenesis and lobulo-alveolar differentiation of the mammary gland could be abolished by blocking expression of endogenous HGF and NRG by the respective antisense oligonucleotides; in antisense oligonucleotide-treated glands, morphogenesis could again be induced by the addition of recombinant HGF and NRG. We thus show that two major postnatal morphogenic periods of mammary gland development are dependent on sequential mesenchymal-epithelial interactions mediated by HGF and NRG.
E-cadherin is functionally involved in the maturation of the erythroid lineage.
Authors: Armeanu S , Bühring H J , Reuss-Borst M , Müller C A , Klein G
Published: May 2004

Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E-cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E-cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E-cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody-inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage.
Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies.
Authors: Carvalho T , Seeler J S , Ohman K , Jordan P , Pettersson U , Akusjärvi G , Carmo-Fonseca M , Dejean A
Published: May 2004

The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.
Parallel secretory pathways to the cell surface in yeast.
Authors: Harsay E , Bretscher A
Published: May 2004

Saccharomyces cerevisiae mutants that have a post-Golgi block in the exocytic pathway accumulate 100-nm vesicles carrying secretory enzymes as well as plasma membrane and cell-wall components. We have separated the vesicle markers into two groups by equilibrium isodensity centrifugation. The major population of vesicles contains Bg12p, an endoglucanase destined to be a cell-wall component, as well as Pma1p, the major plasma membrane ATPase. In addition, Snc1p, a synaptobrevin homologue, copurifies with these vesicles. Another vesicle population contains the periplasmic enzymes invertase and acid phosphatase. Both vesicle populations also contain exoglucanase activity; the major exoglucanase normally secreted from the cell, encoded by EXG1, is carried in the population containing periplasmic enzymes. Electron microscopy shows that both vesicle groups have an average diameter of 100 nm. The late secretory mutants sec1, sec4, and sec6 accumulate both vesicle populations, while neither is detected in wild-type cells, early sec mutants, or a sec13 sec6 double mutant. Moreover, a block in endocytosis does not prevent the accumulation of either vesicle species in an end4 sec6 double mutant, further indicating that both populations are of exocytic origin. The accumulation of two populations of late secretory vesicles indicates the existence of two parallel routes from the Golgi to the plasma membrane.
Yeast SEC16 gene encodes a multidomain vesicle coat protein that interacts with Sec23p.
Authors: Espenshade P , Gimeno R E , Holzmacher E , Teung P , Kaiser C A
Published: May 2004

Temperature-sensitive mutations in the SEC16 gene of Saccharomyces cerevisiae block budding of transport vesicles from the ER. SEC16 was cloned by complementation of the sec16-1 mutation and encodes a 240-kD protein located in the insoluble, particulate component of cell lysates. Sec16p is released from this particulate fraction by high salt, but not by nonionic detergents or urea. Some Sec16p is localized to the ER by immunofluorescence microscopy. Membrane-associated Sec16p is incorporated into transport vesicles derived from the ER that are formed in an in vitro vesicle budding reaction. Sec16p binds to Sec23p, a COPII vesicle coat protein, as shown by the two-hybrid interaction assay and affinity studies in cell extracts. These findings indicate that Sec16p associates with Sec23p as part of the transport vesicle coat structure. Genetic analysis of SEC16 identifies three functionally distinguishable domains. One domain is defined by the five temperature-sensitive mutations clustered in the middle of SEC16. Each of these mutations can be complemented by the central domain of SEC16 expressed alone. The stoichiometry of Sec16p is critical for secretory function since overexpression of Sec16p causes a lethal secretion defect. This lethal function maps to the NH2-terminus of the protein, defining a second functional domain. A separate function for the COOH-terminal domain of Sec16p is shown by its ability to bind Sec23p. Together, these results suggest that Sec16p engages in multiple protein-protein interactions both on the ER membrane and as part of the coat of a completed vesicle.
SED4 encodes a yeast endoplasmic reticulum protein that binds Sec16p and participates in vesicle formation.
Authors: Gimeno R E , Espenshade P , Kaiser C A
Published: May 2004

SEC16 is required for transport vesicle budding from the ER in Saccharomyces cerevisiae, and encodes a large hydrophilic protein found on the ER membrane and as part of the coat of transport vesicles. In a screen to find functionally related genes, we isolated SED4 as a dosage-dependent suppressor of temperature-sensitive SEC16 mutations. Sed4p is an integral ER membrane protein whose cytosolic domain binds to the COOH-terminal domain of Sec16p as shown by two-hybrid assay and coprecipitation. The interaction between Sed4p and Sec16p probably occurs before budding is complete, because Sed4p is not found in budded vesicles. Deletion of SED4 decreases the rate of ER to Golgi transport, and exacerbates mutations defective in vesicle formation, but not those that affect later steps in the secretory pathway. Thus, Sed4p is important, but not necessary, for vesicle formation at the ER. Sec12p, a close homologue of Sed4p, also acts early in the assembly of transport vesicles. However, SEC12 performs a different function than SED4 since Sec12p does not bind Sec16p, and genetic tests show that SEC12 and SED4 are not functionally interchangeable. The importance of Sed4p for vesicle formation is underlined by the isolation of a phenotypically silent mutation, sar1-5, that produces a strong ER to Golgi transport defect when combined with sed4 mutations. Extensive genetic interactions between SAR1, SED4, and SEC16 show close functional links between these proteins and imply that they might function together as a multisubunit complex on the ER membrane.
Envelope glycoprotein interactions in coronavirus assembly.
Authors: Opstelten D J , Raamsman M J , Wolfs K , Horzinek M C , Rottier P J
Published: May 2004

Coronaviruses are assembled by budding into smooth membranes of the intermediate ER-to-Golgi compartment. We have studied the association of the viral membrane glycoproteins M and S in the formation of the virion envelope. Using coimmunoprecipitation analysis we demonstrated that the M and S proteins of mouse hepatitis virus (MHV) interact specifically forming heteromultimeric complexes in infected cells. These could be detected only when the detergents used for their solubilization from cells or virions were carefully chosen: a combination of nonionic (NP-40) and ionic (deoxycholic acid) detergents proved to be optimal. Pulse-chase experiments revealed that newly made M and S proteins engaged in complex formation with different kinetics. Whereas the M protein appeared in complexes immediately after its synthesis, newly synthesized S protein did so only after a lag phase of > 20 min. Newly made M was incorporated into virus particles faster than S, which suggests that it associates with preexisting S molecules. Using the vaccinia virus T7-driven coexpression of M and S we also demonstrate formation of M/S complexes in the absence of other coronaviral proteins. Pulse-chase labelings and coimmunoprecipitation analyses revealed that M and S associate in pre-Golgi membranes because the unglycosylated form of M appeared in M/S complexes rapidly. Since no association of M and S was detected when protein export from the ER was blocked by brefeldin A, stable complexes most likely arise in the ER-to-Golgi intermediate compartment. Sucrose velocity gradient analysis showed the M/S complexes to be heterogeneous and of higher order, suggesting that they are maintained by homo- and heterotypic interactions. M/S complexes colocalized with alpha-mannosidase II, a resident Golgi protein. They acquired Golgi-specific oligosaccharide modifications but were not detected at the cell surface. Thus, the S protein, which on itself was transported to the plasma membrane, was retained in the Golgi complex by its association with the M protein. Because coronaviruses bud at pre-Golgi membranes, this result implies that the envelope glycoprotein complexes do not determine the site of budding. Yet, the self-association of the MHV envelope glycoproteins into higher order complexes is indicative of its role in the sorting of the viral membrane proteins and in driving the formation of the viral lipoprotein coat in virus assembly.
Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba.
Authors: Kelleher J F , Atkinson S J , Pollard T D
Published: May 2004

We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin-binding proteins.
The 78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein.
Authors: King S M , Patel-King R S , Wilkerson C G , Witman G B
Published: May 2004

A previous study (King et al., 1991. J. Biol. Chem. 266:8401-8407) showed that the 78,000-M(r) intermediate chain (IC78) from the Chlamydomonas outer arm dynein is in direct contact with alpha-tubulin in situ, suggesting that this protein may be involved in binding the dynein to the doublet microtubules. Molecular genetic analysis of this chain recently demonstrated that it is a WD repeat protein essential for outer arm assembly (Wilkerson et al., 1995.J. Cell Biol. 129:169-178). We have now transcribed and translated IC78 in vitro, and demonstrate that this molecule binds axonemes and microtubules, whereas a homologous protein (the 69,000-M(r) intermediate chain [IC69] of Chlamydomonas outer arm dynein) does not. Thus, IC78 is a bona fide microtubule-binding protein. Taken together with the previous results, these findings indicate that IC78 is likely to provide at least some of the adhesive force that holds the dynein to the doublet microtubule, and support the general hypothesis that the dynein intermediate chains are involved in targeting different dyneins to the specific cell organelles with which they associate. Analysis of the binding activities of various IC78 deletion constructs translated in vitro identified discrete regions of IC78 that affected the binding to microtubules; two of these regions are specifically missing in IC69. Previous studies also showed that IC78 is in direct contact with IC69; the current work indicates that the region of IC78 that mediates this interaction is coincident with two of IC78's WD repeats. This supports the hypothesis that these repeats are involved in protein-protein interactions within the dynein complex.
Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex.
Authors: McGrail M , Gepner J , Silvanovich A , Ludmann S , Serr M , Hays T S
Published: May 2004

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.
Rapid, widespread, and longlasting induction of nestin contributes to the generation of glial scar tissue after CNS injury.
Authors: Frisén J , Johansson C B , Török C , Risling M , Lendahl U
Published: May 2004

Neuronal regeneration does generally not occur in the central nervous system (CNS) after injury, which has been attributed to the generation of glial scar tissue. In this report we show that the composition of the glial scar after traumatic CNS injury in rat and mouse is more complex than previously assumed: expression of the intermediate filament nestin is induced in reactive astrocytes. Nestin induction occurs within 48 hours in the spinal cord both at the site of lesion and in degenerating tracts and lasts for at least 13 months. Nestin expression is induced with similar kinetics in the crushed optic nerve. In addition to the expression in reactive astrocytes, we also observed nestin induction within 48 hours after injury in cells close to the central canal in the spinal cord, while nestin expressing cells at later timepoints were found progressively further out from the central canal. This dynamic pattern of nestin induction after injury was mimicked by lacZ expressing cells in nestin promoter/lacZ transgenic mice, suggesting that defined nestin regulatory regions mediate the injury response. We discuss the possibility that the spatiotemporal pattern of nestin expression reflects a population of nestin positive cells, which proliferates and migrates from a region close to the central canal to the site of lesion in response to injury.
Protein zero, a nervous system adhesion molecule, triggers epithelial reversion in host carcinoma cells.
Authors: Doyle J P , Stempak J G , Cowin P , Colman D R , D'Urso D
Published: May 2004

Protein zero (P(o)) is the immunoglobulin gene superfamily glycoprotein that mediates the self-adhesion of the Schwann cell plasma membrane that yields compact myelin. HeLa is a poorly differentiated carcinoma cell line that has lost characteristic morphological features of the cervical epithelium from which it originated. Normally, HeLa cells are not self-adherent. However, when P(o) is artificially expressed in this line, cells rapidly aggregate, and P(o) concentrates specifically at cell-cell contact sites. Rows of desmosomes are generated at these interfaces, the plasma membrane localization of cingulin and ZO-1, proteins that have been shown to be associated with tight junctions, is substantially increased, and cytokeratins coalesce into a cohesive intracellular network. Immunofluorescence patterns for the adherens junction proteins N-cadherin, alpha-catenin, and vinculin, and the desmosomal polypeptides desmoplakin, desmocollin, and desmoglein, are also markedly enhanced at the cell surface. Our data demonstrate that obligatory cell-cell adhesion, which in this case is initially brought about by the homophilic association of P(o) molecules across the intercellular cleft, triggers pronounced augmentation of the normally sluggish or sub-basal cell adhesion program in HeLa cells, culminating in suppression of the transformed state and reversion of the monolayer to an epithelioid phenotype. Furthermore, this response is apparently accompanied by an increase in mRNA and protein levels for desmoplakin and N-cadherin which are normally associated with epithelial junctions. Our conclusions are supported by analyses of ten proteins we examined immunochemically (P(o), cingulin, ZO-1, desmoplakin, desmoglein, desmocollin, N-cadherin, alpha-catenin, vinculin, and cytokeratin-18), and by quantitative polymerase chain reactions to measure relative amounts of desmoplakin and N-cadherin mRNAs. P(o) has no known signaling properties; the dramatic phenotypic changes we observed are highly likely to have developed in direct response to P(o)-induced cell adhesion. More generally, the ability of this "foreign" membrane adhesion protein to stimulate desmosome and adherens junction formation by augmenting well-studied cadherin-based adhesion mechanisms raises the possibility that perhaps any bona fide cell adhesion molecule, when functionally expressed, can engage common intracellular pathways and trigger reversion of a carcinoma to an epithelial-like phenotype.
Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis.
Authors: Roach H I , Erenpreisa J , Aigner T
Published: May 2004

We have investigated the early cellular events that take place during the change in lineage commitment from hypertrophic chondrocytes to osteoblast-like cells. We have induced this osteogenic differentiation by cutting through the hypertrophic cartilage of embryonic chick femurs and culturing the explants. Immunocytochemical characterization, [3H]thymidine pulse-chase labeling, in situ nick translation or end labeling of DNA breaks were combined with ultrastructural studies to characterize the changing pattern of differentiation. The first responses to the cutting, seen after 2 d, were upregulation of alkaline phosphatase activity, synthesis of type I collagen and single-stranded DNA breaks, probably indicating a metastable state. Associated with the change from chondrogenic to osteogenic commitment was an asymmetric cell division with diverging fates of the two daughter cells, where one daughter cell remained viable and the other one died. The available evidence suggests that the viable daughter cell then divided and generated osteogenic cells, while the other daughter cell died by apoptosis. The results suggest a new concept of how changes in lineage commitment of differentiated cells may occur. The concepts also reconcile previously opposing views of the fate of the hypertrophic chondrocyte.