bluuebunny/crossref_metadata_2025 huggingface dataset:
Rearrangements of desmosomal and cytoskeletal proteins during the transition from epithelial to fibroblastoid organization in cultured rat bladder carcinoma cells.
Authors: Boyer B , Tucker G C , Vallés A M , Franke W W , Thiery J P
Published: May 2004

Changes of cell morphology and the state of differentiation are known to play important roles in embryogenesis as well as in carcinogenesis. Examples of particularly profound changes are the conversions of epithelial to mesenchymal cells; i.e., the dissociation of some or all polygonal, polar epithelial cells and their transformation into elongate, fibroblastoid cells of high motility. As an in vitro model system for such changes in cell morphology, we have used cell cultures of the rat bladder carcinoma-derived cell line NBT-II which, on exposure to inducing medium containing a commercial serum substitute (Ultroser G), show an extensive change in their organization (epithelial-mesenchymal transition): the junctions between the epithelial cells are split, the epithelial cell organization is lost, and the resulting individual cells become motile and assume a spindle-like fibroblastoid appearance. Using immunofluorescence microscopy and biochemical protein characterization techniques, we show that this change is accompanied by a redistribution of desmosomal plaque proteins (desmoplakins, desmoglein, plakoglobin) and by a reorganization of the cytokeratin and the actin-fodrin filament systems. Moreover, intermediate-sized filaments of the vimentin type are formed in the fibroblastoid cells. We demonstrate that the modulation of desmosomal proteins, specifically an increase in soluble desmoplakins, is a relatively early event in cell dissociation and in epithelial-mesenchymal transition. In this process, a latent period of 5 h upon addition of inducing medium precedes the removal of these desmosomal components from the plasma membrane. The transition, which is reversible, is dependent on continued protein synthesis and phosphorylation but not on the presence of the inducing medium beyond the initial 2-h period. We discuss the value of this experimental system as a physiologically relevant approach for studying the regulation of the assembly and disassembly of desmosomes and other intercellular adhesion structures, and as a model of the conversion of cells from one state of differentiation into another.
A new high molecular mass protein showing unique localization in desmosomal plaque.
Authors: Hieda Y , Tsukita S , Tsukita S
Published: May 2004

A high molecular mass protein of 680 kD was identified and purified from the isolated desmosomes in bovine muzzle epidermal cells. This protein, called "desmoyokin" (from the English, yoke) here, showed no binding ability with keratin filaments in vitro, and its molecule had a characteristic dumbell shape approximately 170 nm in length. We have succeeded in obtaining one monoclonal antibody specific to desmoyokin. By the use of this monoclonal antibody and antidesmoplakin monoclonal antibody, desmoyokin was shown to be colocalized with desmoplakin at the immunofluorescence microscopic level; desmoyokin occurred only in the stratified epithelium, not in the simple epithelium nor in the other tissues. At the electron microscopic level, these two proteins were clearly seen to be sorted out in the plaque of desmosomes with desmoyokin at the periphery and desmoplakin at the center of the disk-shaped desmosomal plaque, suggesting that these two plaque proteins play distinct roles in forming and maintaining the desmosomes in stratified epithelium.
The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.
Authors: Sinard J H , Stafford W F , Pollard T D
Published: May 2004

We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.
Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study.
Authors: Okabe S , Hirokawa N
Published: May 2004

We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers. By 60 min after injection, stress fibers were labeled uniformly. We also analyzed the actin incorporation into polygonal nets of actin bundles. Circular dense foci, where actin bundles radiate, were stable structures, and actin filaments around the foci incorporated biotin-actin the slowest among the actin-containing structures within the injected cells. These results indicate that the rate and pattern of actin subunit incorporation differ in different regions of the cytoplasm and suggest the possible role of rapid actin polymerization at the leading margin on the protrusive movement of fibroblastic cells.
A marginal band-associated protein has properties of both microtubule- and microfilament-associated proteins.
Authors: Birgbauer E , Solomon F
Published: May 2004

The marginal band of nucleated erythrocytes is a microtubule organelle under rigorous quantitative and spatial control, with properties quite different from those of the microtubule organelles of cultured cells. Previous results suggest that proteins other than tubulin may participate in organizing the marginal band, and may interact with elements of the erythrocyte cytoskeleton in addition to microtubules. To identify such species, we raised mAbs against the proteins that assemble from chicken brain homogenates with tubulin. One such antibody binds to a single protein in chicken erythrocytes, and produces an immunofluorescence pattern colocalizing with marginal band microtubules. Several properties of this protein are identical to those of ezrin, a protein isolated from brush border and localized to motile elements of cultured cells. A significant proportion of the antigen is not soluble in erythrocytes, as determined by extraction with nonionic detergent. This cytoskeleton-associated fraction is unaffected by treatments that solubilize the marginal band microtubules. The protein has properties of both microtubule- and microfilament-associated proteins. In the accompanying manuscript (Goslin, K., E. Birgbauer, G. Banker, and F. Solomon. 1989. J. Cell Biol. 109:1621-1631), we show that the same antibody recognizes a component of growth cones with a similar dual nature. In early embryonic red blood cells, the antigen is dispersed throughout the cell and does not colocalize with assembled tubulin. Its confinement to the marginal band during development follows rather than precedes that of microtubules. These results, along with previous work, suggest models for the formation of the marginal band.
The role of cytoskeleton in organizing growth cones: a microfilament-associated growth cone component depends upon microtubules for its localization.
Authors: Goslin K , Birgbauer E , Banker G , Solomon F
Published: May 2004

We are interested in the relationship between the cytoskeleton and the organization of polarized cell morphology. We show here that the growth cones of hippocampal neurons in culture are specifically stained by a monoclonal antibody called 13H9. In other systems, the antigen recognized by 13H9 is associated with marginal bands of chicken erythrocytes and shows properties of both microtubule-and microfilament-associated proteins (Birgbauer, E., and F. Solomon. 1989 J. Cell Biol. 109:1609-1620). This dual nature is manifest in hippocampal neurons as well. At early stages after plating, the antibody stains the circumferential lamellipodia that mediate initial cell spreading. As processes emerge, 13H9 staining is heavily concentrated in the distal regions of growth cones, particularly in lamellipodial fans. In these cells, the 13H9 staining is complementary to the localization of assembled microtubules. It colocalizes partially, but not entirely, with phalloidin staining of assembled actin. Incubation with nocodazole rapidly induces microtubule depolymerization, which proceeds in the distal-to-proximal direction in the processes. At the same time, a rapid and dramatic redistribution of the 13H9 staining occurs; it delocalizes along the axon shaft, becoming clearly distinct from the phalloidin staining and always remaining distal to the receding front of assembled microtubules. After longer times without assembled microtubules, no staining of 13H9 can be detected. Removal of the nocodazole allows the microtubules to reform, in an ordered proximal-to-distal fashion. The 13H9 immunoreactivity also reappears, but only in the growth cones, not in any intermediate positions along the axon, and only after the reformation of microtubules is complete. The results indicate that the antigen recognized by 13H9 is highly concentrated in growth cones, closely associated with polymerized actin, and that its proper localization depends upon intact microtubules.
Tau protein becomes long and stiff upon phosphorylation: correlation between paracrystalline structure and degree of phosphorylation.
Authors: Hagestedt T , Lichtenberg B , Wille H , Mandelkow E M , Mandelkow E
Published: May 2004

In a previous report we have shown that microtubule-associated protein tau can be induced to form paracrystals (Lichtenberg, B., E.-M. Mandelkow, T. Hagestedt, and E. Mandelkow. 1988. Nature [Lond.]. 334:359-362). A striking feature was the high degree of elasticity of the molecules. We now report that this property is related to the state of phosphorylation. When tau is dephosphorylated by alkaline phosphatase, it becomes shorter and more elastic; when it is phosphorylated by Ca++/calmodulin-dependent kinase, it becomes longer and stiffer. This may provide a model for the control of structural properties of tau-like molecules by phosphorylation.
Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.
Authors: Tilney M S , Tilney L G , Stephens R E , Merte C , Drenckhahn D , Cotanche D A , Bretscher A
Published: May 2004

The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57- and 65-kD bands present in the sensory epithelium and the cytoskeleton. It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57- and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125 A transverse stripping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.
Localization of light-harvesting complex II to the occluded surfaces of photosynthetic membranes.
Authors: Hinshaw J E , Miller K R
Published: May 2004

The photosynthetic membranes of green plants are organized into stacked regions interconnected by nonstacked regions that have been shown to be biochemically and structurally distinct. Because the stacking process occludes the surfaces of appressed membranes, it has been impossible to conduct structural or biochemical studies of the outer surfaces of the photosynthetic membrane in regions of membrane stacking. Although stacking is mediated at this surface, it has not been possible to determine whether membrane components implicated in the stacking process, including a major light-harvesting complex (LHC-II), are in fact exposed at the membrane surface. We have been able to expose this surface for study in the electron microscope and directly label it with antibodies to determine protein exposure. The appearance of the newly exposed outer stacked surface highlights the extreme lateral heterogeneity of the photosynthetic membrane. The surface is smooth in contrast to the neighboring nonstacked surface that is covered with distinct particles. Although some investigators have suggested the existence of a cytochrome b6/f-rich boundary region between stacked and nonstacked membranes, our results provide no structural support for this concept. To explore the biochemical nature of the occluded membrane surface, we have used an mAb against the amino terminal region of the LHC-II. This mAb clearly labels the newly exposed outer stacked surface but does not label the inner surface or the outer nonstacked surface. These experimental results confirm the presence of the amino terminal region of this complex at the outer surface of the membrane in stacked regions, and also show that this complex is largely absent from nonstacked membranes.
A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle.
Authors: Carr C , Fischbach G D , Cohen J B
Published: May 2004

To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.
J1-160 and J1-180 are oligodendrocyte-secreted nonpermissive substrates for cell adhesion.
Authors: Pesheva P , Spiess E , Schachner M
Published: May 2004

The glia-derived J1 extracellular matrix glycoproteins have been referred to as J1-160/J1-180 (the developmentally late appearing lower molecular weight group) and J1-200/J1-220 (the developmentally early appearing higher molecular group immunochemically related to tenascin). Members of the two groups show distinct cross-reactivities. To characterize the structural and functional differences between these J1 glycoproteins, two monoclonal antibodies were generated which recognize only the members of the lower molecular weight group. The two antibodies detect immunochemical similarities among the members of the lower molecular weight group, but do not react with J1/tenascin. J1-160 and J1-180 are specifically expressed by differentiated oligodendrocytes in culture and by myelin of the central nervous system and have not been found in the peripheral nervous system nor in any other organ of the adult mice tested. Electron microscopic examination of rotary-shadowed J1-160 and J1-180 reveals, respectively, dimeric and trimeric (tribrachion) kink-armed rodlike structures, which are linked by disulfide bridges. J1-160/J1-180 are nonpermissive substrates for the attachment and spreading of early postnatal small cerebellar neurons, astrocytes, and fibroblasts. In a mixture with laminin, J1-160/J1-180 are nonpermissive substrates for neurons, but not for astrocytes or fibroblasts. The repulsive effect toward neurons can be neutralized by one of the monoclonal antibodies, but not by the other. These observations are discussed in the context of cell interactions during regeneration in the mammalian nervous system.
Involvement of cell surface phosphatidylinositol-anchored glycoproteins in cell-cell adhesion of chick embryo myoblasts.
Authors: Knudsen K A , Smith L , McElwee S
Published: May 2004

During myogenesis myoblasts fuse to form multinucleate cells that express muscle-specific proteins. A specific cell-cell adhesion process precedes lipid bilayer union during myoblast fusion (Knudsen, K. A., and A. F. Horwitz. 1977. Dev. Biol. 58:328-338) and is mediated by cell surface glycoproteins (Knudsen, K. A., 1985. J. Cell Biol. 101:891-897). In this paper we show that myoblast adhesion and myotube formation are inhibited by treating fusion-competent myoblasts with phosphatidylinositol-specific phospholipase C (PI-PLC). The effect of PI-PLC on myoblast adhesion is dose dependent and inhibited by D-myo-inositol 1-monophosphate and the effect on myotube formation is reversible, suggesting a specific, nontoxic effect on myogenesis by the enzyme. A soluble form of adhesion-related glycoproteins is released from fusion-competent myoblasts by treatment with PI-PLC as evidenced by (a) the ability of phospholipase C (PLC)-released material to block the adhesion-perturbing activity of a polyclonal antiserum to intact myoblasts; and (b) the ability of PLC-released glycoprotein to stimulate adhesion-perturbing antisera when injected into mice. PI-PLC treatment of fusion-competent myoblasts releases an isoform of N-CAM into the supernate, suggesting that N-CAM may participate in mediating myoblast interaction during myogenesis.
Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues.
Authors: Shimoyama Y , Yoshida T , Terada M , Shimosato Y , Abe O , Hirohashi S
Published: May 2004

P-cadherin is a subclass of Ca2+-dependent cell-cell adhesion molecules present in mouse placenta, where its localization suggests a function of connecting the embryo to the uterus (Nose, A., and M. Takeichi. 1986. J. Cell Biol. 103:2649-2658). We recently identified a human cadherin detected by an mAb capable of disrupting cell-cell adhesion of A-431 cells, and found that it was closely related immunochemically to mouse P-cadherin. Curiously, this cadherin was undetectable in human placenta by immunohistochemical examination (Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, and O. Abe. 1989. Cancer Res. 49:2128-2133). We here report the cloning and sequencing of cDNA clone encoding the human homologue of mouse P-cadherin. The deduced amino acid sequence of the human P-cadherin consists of 829 amino acid and shows striking homology with mouse P-cadherin. On Northern blot analysis, human P-cadherin was scarcely expressed in human placenta in contrast to mouse P-cadherin, which was abundantly expressed in mouse placenta throughout pregnancy, and it was shown that E-cadherin, but not P-cadherin, was the major cadherin molecule in human placenta. Moreover, NIH3T3 cells transfected with human P-cadherin cDNA expressed the functional cadherin molecule, which was identical to the cadherin we had previously identified using the mAb, showing that this molecule really does mediate cell-cell adhesion and that the cadherin we detected immunochemically is undoubtedly human P-cadherin. The results obtained in this study support the idea that P-cadherin plays little role, if any, in Ca2+-dependent cell-cell binding in human placental tissue at least after several weeks of pregnancy.
Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures.
Authors: Kaufmann R , Frösch D , Westphal C , Weber L , Klein C E
Published: May 2004

The integrin VLA-3 is a cell surface receptor, which binds to fibronectin, laminin, collagen type I and VI (Takada, Y., E. A. Wayner, W. G. Carter, and M. E. Hemler. 1988. J. Cell. Biochem. 37:385-393) and is highly expressed in substrate adherent cultures of almost all human cell types. The ligand specificity of VLA-3 and the inhibition of cell adhesion by anti-VLA-3 monoclonal antibodies suggest its involvement in cell-substrate interaction. In normal tissues, VLA-3 is restricted to few cell types, notably the kidney glomeruli and basal cells of the epidermis. In the epidermis, VLA-3 is generally strongly expressed on the entire plasma membrane of basal cells and is not polarized towards the basement membrane (Klein, C. E., C. Cardon-Cardo, R. Soehnchen, R. J. Cote, H. F. Oettgen, M. Eisinger, and L. J. Old. 1987. J. Invest. Dermatol. 89:500-507). Based on this finding we speculated that, in addition to a role of VLA-3 for adhesion of cells to substrate, it could also be relevant for cell-cell interaction. To investigate this, we ultrastructurally localized VLA-3 on the surface of cultured cells by immunoelectron microscopy. In accordance with our concept, we found VLA-3 strongly associated with intercellular contact sites. Interestingly, very little immunoreactivity was detected at the under-surface of cells which had been cultured for 18-32 h. This observation was unexpected but is consistent with previous findings (Kantor, R. R. S., M. J. Mattes, K. D. Lloyd, L. J. Old, and A. P. Albino. 1987. J. Biol. Chem. 262:15158-15165) which suggest that the association of VLA-3 with the basal surface of substrate adherent tumor cells is a late event occurring after days of culture under confluent conditions. However, we cannot formally rule out VLA-3 expression at the undersurface of cells under our experimental conditions, since VLA-3 molecules at this location could be inaccessible for in situ labeling of unfixed cells because of spatial interferences. In conclusion, our results demonstrate the expression of VLA-3 at intercellular contact sites of cultured cells supporting the concept that it may be relevant for intercellular interactions also.
Origin and deposition of basement membrane heparan sulfate proteoglycan in the developing intestine.
Authors: Simon-Assmann P , Bouziges F , Vigny M , Kedinger M
Published: May 2004

The deposition of intestinal heparan sulfate proteoglycan (HSPG) at the epithelial-mesenchymal interface and its cellular source have been studied by immunocytochemistry at various developmental stages and in rat/chick interspecies hybrid intestines. Polyclonal heparan sulfate antibodies were produced by immunizing rabbits with HSPG purified from the Engelbreth-Holm-Swarm mouse tumor; these antibodies stained rat intestinal basement membranes. A monoclonal antibody (mAb 4C1) produced against lens capsule of 11-d-old chick embryo reacted with embryonic or adult chick basement membranes, but did not stain that of rat tissues. Immunoprecipitation experiments indicated that mAb 4C1 recognized the chicken basement membrane HSPG. Immunofluorescent staining with these antibodies allowed us to demonstrate that distribution of HSPG at the epithelial-mesenchymal interface varied with the stages of intestinal development, suggesting that remodeling of this proteoglycan is essential for regulating cell behavior during morphogenesis. The immunofluorescence pattern obtained with the two species-specific HSPG antibodies in rat/chick epithelial/mesenchymal hybrid intestines developed as grafts (into the coelomic cavity of chick embryos or under the kidney capsule of adult mice) led to the conclusion that HSPG molecules located in the basement membrane of the developing intestine were produced exclusively by the epithelial cells. These data emphasize the notion already gained from previous studies, in which type IV collagen has been shown to be produced by mesenchymal cells (Simon-Assmann, P., F. Bouziges, C. Arnold, K. Haffen, and M. Kedinger. 1988. Development (Camb.). 102:339-347), that epithelial-mesenchymal interactions play an important role in the formation of a complete basement membrane.
Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage.
Authors: Kwan A P , Dickson I R , Freemont A J , Grant M E
Published: May 2004

The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis.
Rapid effect of heat shock on two heterogeneous nuclear ribonucleoprotein-associated antigens in HeLa cells.
Authors: Mähl P , Lutz Y , Puvion E , Fuchs J P
Published: May 2004

During severe heat shock, which known to interrupt both splicing of RNA transcripts and nucleocytoplasmic transport, it is to be expected that the substructure of heterogeneous nuclear ribonucleoproteins (hnRNP) is altered in some way. Recently, we have shown that such a stress actually induces rapid alterations at the level of individual proteins (Lutz, Y., M. Jacob, and J.-P. Fuchs. 1988 Exp. Cell Res. 175:109-124). Here we report further investigations on two related 72.5-74-kD hnRNP proteins whose behavior is also rapidly modified by a heat shock at 45 degrees C, whereas no effect is observed at 42 degrees C. Using a monoclonal antibody, we show that in situ the antigens are available only when the cells are heat shocked at 45 degrees C. Subcellular fractionation shows that in normal cells the antigens are associated with the bulk of hnRNP (50-200S). During heat shock, whereas the overall characteristics of the bulk of preexisting hnRNP are unchanged, these antigens rapidly switch to a subpopulation of hnRNP with larger average size (50 to less than 300S) and increased stability. Structural analysis of the associated hnRNP in normal and stressed cells shows that in both cases the antigens are associated with the nuclear matrix subcomplex of hnRNP, which in situ is part of the internal nuclear matrix. Such hnRNP antigens, which are rapidly redistributed during a heat shock at the upper temperature range of the stress response, might well be involved in splicing and/or transport control.
A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite.
Authors: Masumoto H , Masukata H , Muro Y , Nozaki N , Okazaki T
Published: May 2004

We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170-bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.
High-mobility group protein HMG-I localizes to G/Q- and C-bands of human and mouse chromosomes.
Authors: Disney J E , Johnson K R , Magnuson N S , Sylvester S R , Reeves R
Published: May 2004

Mammalian metaphase chromosomes can be identified by their characteristic banding pattern when stained with Giemsa dye after brief proteolytic digestion. The resulting G-bands are known to contain regions of DNA enriched in A/T residues and to be the principal location for the L1 (or Kpn 1) family of long interspersed repetitive sequences in human chromosomes. Here we report that antibodies raised against a highly purified and biochemically well characterized nonhistone "High-Mobility Group" protein, HMG-I, specifically localize this protein to the G-bands in mammalian metaphase chromosomes. In some preparations in which chromosomes are highly condensed, HMG-I appears to be located at the centromere and/or telomere regions of mammalian chromosomes as well. To our knowledge, this is the first well-characterized mammalian protein that localizes primarily to G-band regions of chromosomes.
Nucleus-specific and temporally restricted localization of proteins in Tetrahymena macronuclei and micronuclei.
Authors: White E M , Allis C D , Goldfarb D S , Srivastva A , Weir J W , Gorovsky M A
Published: May 2004

Labeled nuclear proteins were microinjected into the cytoplasm of Tetrahymena thermophila. Macronuclear H1, calf thymus H1, and the SV40 large T antigen nuclear localization signal linked to BSA accumulated specifically in macronuclei, even if cells were in micronuclear S phase or were nonreplicating. The way in which histone H4 localized to either the macronucleus or the micronucleus suggested that it accumulates in whichever nucleus is replicating. The inability of the micronucleus to accumulate Tetrahymena H1 or heterologous nuclear proteins, even at a period in the cell cycle when it is accumulating H4, suggests that it has a specialized transport system. These studies demonstrate that although the mechanism for localizing proteins to nuclei is highly conserved among eukaryotes, it can differ between two porecontaining nuclei lying in the same cytoplasm.
Cellular and adenovirus dl312 DNA metabolism in cycling or mitotic human cultures exposed to supralethal gamma radiation.
Authors: Ross P M
Published: May 2004

Cellular repair of DNA damage due to lethal gamma irradiation was studied to reveal differences between strains and cell cycle stages that are otherwise difficult to detect. Cycling and metaphase-blocked cultures of normal fibroblasts and carcinoma cells were compared for repair of gamma sites (gamma radiation-induced nicks, breaks, and alkalilabile sites in DNA) at supralethal exposures ranging from 7 to 150 krad 137Cs radiation and at postirradiation incubations of 20-180 min. Fibroblasts from normal human skin or lung repaired gamma sites efficiently when cycling but did not repair them when blocked at mitosis. Bladder (253J) or lung (A549) carcinoma cells, unlike normal fibroblasts, repaired gamma sites efficiently even when blocked at mitosis. HeLa cells degraded their DNA soon after exposure at all doses tested, regardless of mitotic arrest. Whether the above differences in DNA repair between cell cycle stages and between strains result from differences in chromatin structure (cis effects) or from differences in the nuclear enzymatic environment (trans effects) could be resolved by placing an inert, extrachromosomal DNA molecule in the cell nucleus. Specifically, cis effects should be confined to the host chromosomes and would not be detected in the inert probe whereas trans effects should be detected in host chromosomes and inert probe DNA alike. Indeed, we found a suitable DNA molecule in the adenovirus deletion mutant dl312, which does not proliferate in the absence of E1A complementation. Gamma sites in 32P-labeled adenovirus dl312 DNA were repaired efficiently in all hosts, regardless of mitotic arrest. Failure of mitosis-arrested fibroblasts to repair gamma sites was therefore due to a cis effect of chromatin organization rather than to a trans effect such as repair enzyme insufficiency. In sharp contrast, chromosomes of mitotic carcinoma cells remained accessible to repair enzymes and nucleases alike. By means of these new tools, we should get a better understanding of higher-order chromatin management in normal and cancer cells.
The conserved carboxy-terminal cysteine of nuclear lamins is essential for lamin association with the nuclear envelope.
Authors: Krohne G , Waizenegger I , Höger T H
Published: May 2004

We have analyzed the interaction of soluble nuclear lamins with the nuclear envelope by microinjection of normal and mutated lamins into the cytoplasm of Xenopus laevis oocytes. Our results demonstrate that the conserved cysteine of the carboxy-terminal tetrapeptide Cys Ala/Ser Ile Met of lamins is essential for their association with the nuclear envelope. Removal of this sequence or replacement of the cysteine by serine resulted in Xenopus lamin L1 remaining in a soluble, non-envelope-associated state within the nucleus. Similar mutations of Xenopus lamin A resulted in only partial reduction of nuclear envelope association, indicating that lamin A contains additional signals that can partially compensate for the lack of the cysteine. Mammalian lamin C lacks this tetrapeptide and is not associated with the nuclear envelope in our experimental system. Cloning of the tetrapeptide Cys Ala Ile Met to the carboxy terminus of human lamin C resulted in lamin being found in a nuclear envelope-associated form in oocytes. Mutations at the amino terminus and in the alpha-helical region of lamin L1 revealed that the carboxy terminus mediates the association of lamins with the nuclear envelope; however, this alone is insufficient for maintenance of a stable association with the nuclear envelope.
Structural requirements for membrane assembly of proteins spanning the membrane several times.
Authors: Lipp J , Flint N , Haeuptle M T , Dobberstein B
Published: May 2004

We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the opposite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.
Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation.
Authors: Parks G D , Hull J D , Lamb R A
Published: May 2004

The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.
Accessibility to proteases of the cytoplasmic G protein domain of vesicular stomatitis virus is increased during intracellular transport.
Authors: Mack D , Kluxen B , Kruppa J
Published: May 2004

G1 and G2 are two forms of the membrane-integrated G protein of vesicular stomatitis virus that migrate differently in gel electrophoresis because G1 is modified by high-mannose and G2 by complex-type oligosaccharide side chains. The cytoplasmic domain in G1 is less exposed to cleavage by several proteases than in G2 molecules. Acylation by palmitic acid as well as inhibition of carbohydrate processing by swainsonine and deoxynojirimycin resulted in the same pattern of proteolytic sensitivity of both glycoproteins as in untreated cells. In contrast, accessibility of the cytoplasmic domain to proteases did not change when the intracellular transport of the G protein was blocked in carbonyl cyanide m-chlorophenylhydrazone- or monensin-treated BHK-21 cells, respectively. The results suggest that the increase in accessibility of the cytoplasmic tail of the G protein occurs after the monensin block in the trans-Golgi and might reflect a conformational change of functional significance--i.e., making the cytoplasmic domain of the viral spike protein competent for its interaction with the viral core, inducing thereby the formation of the budding virus particle.
Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy.
Authors: Pagano R E , Sepanski M A , Martin O C
Published: May 2004

We have previously shown that a fluorescent derivative of ceramide, N-(epsilon-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-eryth ro-sphingosin e (C6-NBD-Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6-NBD-Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6-NBD-Cer at 2 degrees C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24 degrees C removed excess C6-NBD-Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland, 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6-NBD-Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6-NBD-Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6-NBD-Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.
The response of the Golgi complex to microtubule alterations: the roles of metabolic energy and membrane traffic in Golgi complex organization.
Authors: Turner J R , Tartakoff A M
Published: May 2004

A striking example of the interrelation between the Golgi complex (GC) and microtubules is the reversible fragmentation and dispersal of the GC which occurs upon microtubule depolymerization. We have characterized dispersal of the GC after nocodazole treatment as well as its recovery from the dispersed state by immunofluorescent localization of beta 1, 4-galactosyltransferase in Madin-Darby bovine kidney cells. Immunofluorescent anti-tubulin staining allowed simultaneous examination of the microtubule array. Based on our results, dispersal can be divided into a three-step process: microtubule depolymerization, GC fragmentation, and fragment dispersal. In cells treated with metabolic inhibitors after microtubule depolymerization, neither fragmentation nor dispersal occur, despite the absence of assembled microtubules. Thus, fragmentation is energy dependent and not tightly linked to microtubule depolymerization. The slowing of fragmentation and dispersal by monensin or ammonium chloride, as well as progressive inhibition at less than 34 degrees C, suggest that ongoing membrane traffic is required for these processes. Similarly, recovery may be separated into four steps: microtubule depolymerization, GC fragment centralization, fragment coalescence, and polarization of the reticular GC network. Fragment centralization and coalescence were arrested by metabolic inhibitors, despite the presence of microtubules. Neither monensin nor ammonium choride inhibited GC recovery. Partial inhibition of recovery at reduced temperatures paralleled the extent of microtubule assembly. These data demonstrate that dispersal and recovery are multi-step operations, and that the individual steps differ in temperature dependence, energy dependence, and sensitivity to ionic perturbation. GC distribution and microtubule status have also been clearly dissociate, thereby proving that organization of the GC is an active process that is not simply determined by microtubule binding. Furthermore, the results indicate that ongoing intra-GC membrane traffic may participate in fragmentation and dispersal.