bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple levels of control of the stage- and region-specific expression of rat intestinal lactase.
Authors: Duluc I , Jost B , Freund J N
Published: May 2004

To elucidate the mechanisms leading to the functional regionalization of the digestive epithelium, lactase expression was analyzed at the protein, mRNA and gene levels, along the intestinal tract at various stages of the rat postnatal development. In the colon of neonates, the transient expression of mRNA and enzyme correlated well with gene transcription. In contrast to the colon, complex patterns were observed in the small intestine. In suckling animals, the mRNA was present at a high level despite the progressive decline of enzyme activity. Crypts were devoid of mRNA and the transcript mainly accumulated in the lower half of the villi. From weaning onwards, a functional regionalization of the epithelium was defined, characterized by the modification of the longitudinal distribution of lactase mRNA. Indeed the transcript remained abundant in the distal duodenum, jejunum and proximal ileum, but decreased in the proximal duodenum and became virtually absent in the distal ileum. Concomitantly, the mRNA and enzyme distribution along the villi changed in the different segments of the small intestine. Patterns similar to those described in sucklings were retained in the adult jejunum. In contrast, mRNA and enzyme could no longer be detected in the distal ileum, while mosaicism appeared in the proximal duodenum. In vitro transcription assays carried out with isolated nuclei suggested that the decay of lactase mRNA in the proximal duodenum at weaning was associated with a decreasing rate of transcription of the gene. However active gene transcription was retained in the nuclei of the adult jejunum and ileum. The loss of mRNA in the adult distal ileum despite the maintenance of active transcription did not result from an intragenic block of pre-RNA elongation, as shown by transcription assays carried out at various positions of the lactase gene. In addition, we looked for the ontogenic decline of lactase protein despite the maintenance of a high amount of mRNA in the jejunum, and it became evident that the fraction of mRNA present in polysomes was constant with age. Taken together, these data indicate that lactase constitutes an unusual marker of development and of functional regionalization of the intestinal tract which exhibits a complex time- and space-specific pattern of gene, mRNA, and protein expression. The distinct patterns occurring in the duodenum, jejunum, ileum, and the colon of pre- and postweaned rats depend on a combination of transcriptional, posttranscriptional, and posttranslational levels of regulation. and are associated with a different mRNA distribution along villi in each intestinal segment.
Differential effects of over-expressed neural cell adhesion molecule isoforms on myoblast fusion.
Authors: Peck D , Walsh F S
Published: May 2004

We have used a transfection based approach to analyze the role of neural cell adhesion molecule (NCAM) in myogenesis at the stage of myoblast fusion to form multinucleate myotubes. Stable cell lines of myogenic C2 cells were isolated that express the transmembrane 140- or 180-kD NCAM isoforms or the glycosylphosphatidylinositol (GPI) linked isoforms of 120 or 125 kD. We found that expression of the 140-kD transmembrane isoform led to a potent enhancement of myoblast fusion. The 125-kD GPI-linked NCAM also enhanced the rate of fusion but less so when a direct comparison of cell surface levels of the 140-kD transmembrane form was carried out. While the 180-kD transmembrane NCAM isoform was effective in promoting C2 cell fusion similar to the 140-kD isoform, the 120-kD isoform did not have an effect on fusion parameters. It is possible that these alterations in cell fusion are associated with cis NCAM interactions in the plane of the membrane. While all of the transfected human NCAMs (the transmembrane 140- and 180-kD isoforms and the 125- and 120-kD GPI isoforms) could be clustered in the plane of the plasma membrane by species-specific antibodies there was a concomitant clustering of the endogenous mouse NCAM protein in all cases except with the 120-kD human isoform. These studies show that different isoforms of NCAM can undergo specific interactions in the plasma membrane which are likely to be important in fusion. While the transmembrane and the 125-kD GPI-anchored NCAMs are capable of enhancing fusion the 120-kD GPI NCAM is not. Thus it is likely that interactions associated with NCAM intracellular domains and also the muscle specific domain (MSD) region in the extracellular domain of the GPI-linked 125-kD NCAM are important. In particular this is the first role ascribed to the O-linked carbohydrate containing MSD region which is specifically expressed in skeletal muscle.
Targeted deletion of beta 1 integrins in F9 embryonal carcinoma cells affects morphological differentiation but not tissue-specific gene expression.
Authors: Stephens L E , Sonne J E , Fitzgerald M L , Damsky C H
Published: May 2004

The integrin superfamily of heterodimeric transmembrane adhesion receptors mediates many cell-cell and cell-matrix interactions whose functions are believed to be critical for normal morphogenesis and differentiation. By eliminating the beta 1 integrin gene through homologous recombination, we have assessed the role of the beta 1 integrin family in the F9 embryonal carcinoma model for endodermal differentiation. F9 cells were unexpectedly found to maintain three copies of the beta 1 gene and complete elimination required three sequential rounds of targeting to generate triple knockout lines (beta 1 TKO). Elimination of the beta 1 integrin family of adhesion receptors from F9 cells resulted in reduced adhesion to fibronectin, laminin and collagen, but strongly enhanced adhesion to vitronectin. The absence of beta 1 integrins did not promote significant compensatory upregulation of either beta 3 or beta 5 subunits, both of which are known to act as vitronectin receptors when associated with alpha v. The loss of beta 1 integrins severely affected morphological differentiation when the beta 1-deficient cells were induced to differentiate to either parietal or visceral endoderm. Parietal endoderm derived from beta 1-deficient cells retained a rounded morphology and migrated poorly on both fibronectin and vitronectin. Visceral endoderm derived from beta 1-deficient cells were also unable to form a normal, confluent epithelial monolayer; instead, a non-contiguous layer containing clumps of disorganized cells was observed. However, loss of beta 1 integrins did not interfere with induction by differentiating agents of tissue-specific gene products for either visceral or parietal endoderm. These results suggest that beta 1 integrins mediate morphological differentiation (migration and epithelial formation) but not tissue-specific gene expression in induced F9 cells, and that these two processes are not necessarily linked in this system.
SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family.
Authors: Strunnikov A V , Larionov V L , Koshland D
Published: May 2004

The smc1-1 mutant was identified initially as a mutant of Saccharomyces cerevisiae that had an elevated rate of minichromosome nondisjunction. We have cloned the wild-type SMC1 gene. The sequence of the SMC1 gene predicts that its product (Smc1p) is a 141-kD protein, and antibodies against Smc1 protein detect a protein with mobility of 165 kD. Analysis of the primary and putative secondary structure of Smc1p suggests that it contains two central coiled-coil regions flanked by an amino-terminal nucleoside triphosphate (NTP)-binding head and a conserved carboxy-terminal tail. These analyses also indicate that Smc1p is an evolutionary conserved protein and is a member of a new family of proteins ubiquitous among prokaryotes and eukaryotes. The SMC1 gene is essential for viability. Several phenotypic characteristics of the mutant alleles of smc1 gene indicate that its product is involved in some aspects of nuclear metabolism, most likely in chromosome segregation. The smc1-1 and smc1-2 mutants have a dramatic increase in mitotic loss of a chromosome fragment and chromosome III, respectively, but have no increase in mitotic recombination. Depletion of SMC1 function in the ts mutant, smc1-2, causes a dramatic mitosis-related lethality. Smc1p-depleted cells have a defect in nuclear division as evidenced by the absence of anaphase cells. This phenotype of the smc1-2 mutant is not RAD9 dependent. Based upon the facts that Smc1p is a member of a ubiquitous family, and it is essential for yeast nuclear division, we propose that Smc1p and Smc1p-like proteins function in a fundamental aspect of prokaryotic and eukaryotic cell division.
Lamin B distribution and association with peripheral chromatin revealed by optical sectioning and electron microscopy tomography.
Authors: Belmont A S , Zhai Y , Thilenius A
Published: May 2004

We have used a combination of immunogold staining, optical sectioning light microscopy, intermediate voltage electron microscopy, and EM tomography to examine the distribution of lamin B over the nuclear envelope of CHO cells. Apparent inconsistencies between previously published light and electron microscopy studies of nuclear lamin staining were resolved. At light microscopy resolution, an apparent open fibrillar network is visualized. Colocalization of lamin B and nuclear pores demonstrates that these apparent fibrils, separated by roughly 0.5 micron, are anti-correlated with the surface distribution of nuclear pores; pore clusters lie between or adjacent to regions of heavy lamin B staining. Examination at higher, EM resolution reveals that this apparent lamin B network does not correspond to an actual network of widely spaced, discrete bundles of lamin filaments. Rather it reflects a quantitative variation in lamin staining over a roughly 0.5-micron size scale, superimposed on a more continuous but still complex distribution of lamin filaments, spatially heterogeneous on a 0.1-0.2-micron size scale. Interestingly, lamin B staining at this higher resolution is highly correlated to the underlying chromatin distribution. Heavy concentrations of lamin B directly "cap" the surface of envelope associated, large-scale chromatin domains.
Intracellular routing of GLcNAc-bearing molecules in thyrocytes: selective recycling through the Golgi apparatus.
Authors: Miquelis R , Courageot J , Jacq A , Blanck O , Perrin C , Bastiani P
Published: May 2004

Previous experiments led us to speculate that thyrocytes contain a recycling system for GlcNAc-bearing immature thyroglobulin molecules which prevents these molecules from lysosomal degradation (Miquelis, R., C. Alquier, and M. Monsigny. 1987. J. Biol. Chem. 262:15291-15298). To confirm this hypothesis, the fate of GlcNAc-bearing proteins after internalization by thyrocytes was monitored and compared to that of fluid phase markers. Kinetic internalization studies were performed using 125I-GlcNAc-BSA and 131I-Man-BSA. We observed that the apparent intake rate as well as the amount of hydrolyzed GlcNAc-BSA are smaller than the corresponding values for Man-BSA. These differences were reduced by GlcNAc competitors (thyroglobulin and ovomucoid) or a weak base (chloroquine). Part of the internalized GlcNAc-BSA was released into the extracellular milieu at a higher rate and shorter half life (t1/2 = approximately 30 min) than the Man-BSA (t1/2 = approximately 8 h). Subcellular homing was first studied by cell fractionation after internalization using 125I-ovomucoid and 131I-BSA. During Percoll density gradient fractionation, endogenous thyroperoxidase was used to separate subsets of organelles involved in the biosynthetic exocytotic pathway. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation give rise to a shift in the density of organelles containing 3.5 times more ovomucoid than BSA. Discontinuous sucrose gradient showed that: (a) thyroperoxidase was colocalized with galactosyltransferase-contraining organelles in Golgi-rich subfractions; and (b) that at every time studied from 10 to 100 min, the ovomucoid/BSA ratio was higher in these organelles than in other subfractions. Finally we also observed that: (a) ovomucoid sequestered in the Golgi-rich subfraction incorporated [3H]galactose; and (b) that part of internalized ovomucoid was localized on the Golgi stacks as well as elements of the trans-Golgi, as revealed by immunogold labeling on ultrathin cryosections. These data prove that in thyrocytes GlcNAc accessible sugar moieties on soluble internalized molecules are sufficient to trigger their recycling via the Golgi apparatus.
Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast.
Authors: Tan P K , Davis N G , Sprague G F , Payne G S
Published: May 2004

The role of clathrin in endocytosis of the yeast phermone receptors was examined using strains expressing a temperature-sensitive clathrin heavy chain. The yeast phermone receptors belong to the family of seven transmembrane segment, G-protein-coupled receptors. A rapid and reversible defect in uptake of radiolabeled alpha-factor pheromone occurred when the cells were transferred to the nonpermissive temperature. Constitutive, pheromone-independent internalization of newly synthesized a-factor phermone receptor was also rapidly inhibited in mutant strains at the nonpermissive temperature. In both cases residual endocytosis, 30-50% of wild-type levels, was detected in the absence of functional clathrin heavy chain. Once internalized, the a-factor receptor was delivered to the vacuole at comparable rates in chc1-ts and wild-type cells at the nonpermissive temperature. Clathrin heavy chain was also required for maximal uptake of a mutant a-factor receptor which is dependent on pheromone for internalization. In the presence of a-factor, the internalization rate of the mutant receptor in chc1-ts cells at the nonpermissive temperature was 2.5 times slower than the rate observed for endocytosis of the mutant receptor in wild-type cells. These experiments provide in vivo evidence that clathrin plays an important role in the endocytosis of the seven trans-membrane segment pheromone receptors in yeast.
Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.
Authors: Jaunin P , Jaisser F , Beggah A T , Takeyasu K , Mangeat P , Rossier B C , Horisberger J D , Geering K
Published: May 2004

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K-ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K-pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K-pumps.
WIF-B cells: an in vitro model for studies of hepatocyte polarity.
Authors: Ihrke G , Neufeld E B , Meads T , Shanks M R , Cassio D , Laurent M , Schroer T A , Pagano R E , Hubbard A L
Published: May 2004

We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction-associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.
Occludin: a novel integral membrane protein localizing at tight junctions.
Authors: Furuse M , Hirase T , Itoh M , Nagafuchi A , Yonemura S , Tsukita S , Tsukita S
Published: May 2004

Recently, we found that ZO-1, a tight junction-associated protein, was concentrated in the so called isolated adherens junction fraction from the liver (Itoh, M., A. Nagafuchi, S. Yonemura, T. Kitani-Yasuda, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 121:491-502). Using this fraction derived from chick liver as an antigen, we obtained three monoclonal antibodies specific for a approximately 65-kD protein in rats. This antigen was not extractable from plasma membranes without detergent, suggesting that it is an integral membrane protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this approximately 65-kD membrane protein was exclusively localized at tight junctions of both epithelial and endothelial cells: at the electron microscopic level, the labels were detected directly over the points of membrane contact in tight junctions. To further clarify the nature and structure of this membrane protein, we cloned and sequenced its cDNA. We found that the cDNA encoded a 504-amino acid polypeptide with 55.9 kDa. A search of the data base identified no proteins with significant homology to this membrane protein. A most striking feature of its primary structure was revealed by a hydrophilicity plot: four putative membrane-spanning segments were included in the NH2-terminal half. This hydrophilicity plot was very similar to that of connexin, an integral membrane protein in gap junctions. These findings revealed that an integral membrane protein localizing at tight junctions is now identified, which we designated as "occludin."
Activation of actin polymerization by phosphatidic acid derived from phosphatidylcholine in IIC9 fibroblasts.
Authors: Ha K S , Exton J H
Published: May 2004

alpha-Thrombin induced a change in the cell morphology of IIC9 fibroblasts from a semiround to an elongated form, accompanied by an increase in stress fibers. Incubation of the cells with phospholipase D (PLD) from Streptomyces chromofuscus and exogenous phosphatidic acid (PA) caused similar morphological changes, whereas platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) induced different changes, e.g., disruption of stress fibers and cell rounding. alpha-Thrombin, PDGF, and exogenous PLD increased PA by 20-40%, and PMA produced a smaller increase. alpha-Thrombin and exogenous PLD produced rapid increases in the amount of filamentous actin (F-actin) that were sustained for at least 60 min. However, PDGF produced a transient increase of F-actin at 1 min and PMA caused no significant change. Dioctanoylglycerol was ineffective except at 50 micrograms/ml. Phospholipase C from Bacillus cereus, which increased diacylglycerol (DAG) but not PA, did not change F-actin content. Down-regulation of protein kinase C (PKC) did not block actin polymerization induced by alpha-thrombin. H-7 was also ineffective. Exogenous PA activated actin polymerization with a significant effect at 0.01 microgram/ml and a maximal increase at 1 microgram/ml. No other phospholipids tested, including polyphosphoinositides, significantly activated actin polymerization. PDGF partially inhibited PA-induced actin polymerization after an initial increase at 1 min. PMA completely or largely blocked actin polymerization induced by PA or PLD. These results show that PC-derived PA, but not DAG or PKC, activates actin polymerization in IIC9 fibroblasts, and indicate that PDGF and PMA have inhibitory effects on PA-induced actin polymerization.
Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain.
Authors: Rodionov V I , Gyoeva F K , Tanaka E , Bershadsky A D , Vasiliev J M , Gelfand V I
Published: May 2004

One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself.
The SLT2 (MPK1) MAP kinase homolog is involved in polarized cell growth in Saccharomyces cerevisiae.
Authors: Mazzoni C , Zarov P , Rambourg A , Mann C
Published: May 2004

Bud emergence, spindle pole body duplication and DNA replication are all dependent on the activation of the CDC28 protein kinase at the Start point in the G1 phase of the cell cycle. Bud emergence requires polarization of the cytoskeleton and secretory vesicles to a specific site on the cell surface. Cdc28p activated by G1-cyclins triggers polarization of actin to the site of bud emergence and favors apical bud growth (Lew, D. J., and S. I. Reed. 1993. J. Cell Biol. 120:1305-1320). We isolated slt2-1 as a mutation that enhances the division defect of cdc28 mutants with defects at Start. Slt2p(Mpk1p) is a member of the MAP kinase family (Lee, K. S., K. Irie, Y. Gotoh, Y. Watanabe, H. Araki, E. Nishida, K. Matsumoto, and D. E. Levin. 1993. Mol. Cell. Biol. 13:3067-3075). We show that slt2 mutants exhibit phenotypes similar to those shown by mutants of the yeast actin cytoskeleton, including delocalization of chitin deposition and of actin cortical spots and the accumulation of secretory pathway membranes and vesicles. Furthermore, slt2::HIS3 act1-1 and slt2::HIS3 myo2-66 double mutants are inviable. We suggest that Slt2p functions downstream or in parallel with Cdc28p in promoting bud formation and apical growth.
Interferon-alpha induces the expression of the L-selectin homing receptor in human B lymphoid cells.
Authors: Evans S S , Collea R P , Appenheimer M M , Gollnick S O
Published: May 2004

The L-selectin homing receptor expressed by lymphocytes mediates the initial attachment of these cells to high endothelial venules within peripheral lymph nodes. This adhesive interaction is required for the migration of B and T lymphocytes from the blood into peripheral lymph nodes. There is currently little information regarding the nature of the factors involved in the regulation of the synthesis and expression of L-selectin by lymphocytes. In this report, the immunomodulatory cytokine interferon-alpha (IFN-alpha) was shown to markedly upregulate the surface density of L-selectin in the established human B lymphoid Daudi cell line and in a subpopulation of tissue-derived human B lymphoid cells. Other cytokines such as IFN-gamma, tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-2, IL-4, IL-6, and low molecular weight B cell growth factor did not affect L-selectin surface expression in the model Daudi B cell line. Upregulation of L-selectin surface density in IFN-alpha-treated Daudi B cells correlated directly with an increase in L-selectin mRNA steady state levels and enhanced L-selectin-dependent binding to a carbohydrate-based ligand, phosphomonoester core polysaccharide. Regulation of L-selectin mRNA by IFN-alpha had characteristics similar to that of classical IFN-stimulated genes including rapid kinetics of induction, protein-synthesis-independent induction, and sensitivity to tyrosine-kinase inhibitors. IFN-alpha did not upregulate L-selectin mRNA levels or surface expression in an IFN-resistant Daudi subclone which exhibits a defect in the signal transduction pathway required for the transcriptional induction of IFN-stimulated genes. These data demonstrate a fundamental role for IFN-alpha in regulating L-selectin synthesis and expression in human B lymphoid cells and suggest a mechanism whereby this cytokine regulates the regional trafficking of B cells to peripheral lymph nodes.
The dynamic structure of the pericellular matrix on living cells.
Authors: Lee G M , Johnstone B , Jacobson K , Caterson B
Published: May 2004

Although up to several microns thick, the pericellular matrix is an elusive structure due to its invisibility with phase contrast or DIC microscopy. This matrix, which is readily visualized by the exclusion of large particles such as fixed red blood cells is important in embryonic development and in maintenance of cartilage. While it is known that the pericellular matrix which surrounds chondrocytes and a variety of other cells consists primarily of proteoglycans and hyaluronan with the latter binding to cell surface receptors, the macromolecular organization is still speculative. The macromolecular organization previously could not be determined because of the collapse of the cell coat with conventional fixation and dehydration techniques. Until now, there has been no way to study the dynamic arrangement of hyaluronan with its aggregated proteoglycans on living cells. In this study, the arrangement and mobility of hyaluronan-aggrecan complexes were directly observed in the pericellular matrix of living cells isolated from bovine articular cartilage. The complexes were labeled with 30- to 40-nm colloidal gold conjugated to 5-D-4, an antibody to keratan sulfate, and visualized with video-enhanced light microscopy. From our observations of the motion of pericellular matrix macromolecules, we report that the chondrocyte pericellular matrix is a dynamic structure consisting of individual tethered molecular complexes which project outward from the cell surface. These complexes undergo restricted rotation or wobbling. When the cells were cultured with ascorbic acid, which promotes production of matrix components, the size of the cell coat and the position of the gold probes relative to the plasma membrane were not changed. However, the rapidity and extent of the tethered motion were reduced. Treatment with Streptomyces hyaluronidase removed the molecules that displayed the tethered motion. Addition of hyaluronan and aggrecan to hyaluronidase-treated cells yielded the same labeling pattern and tethered motion observed with native cell coats. To determine if aggrecan was responsible for the extended configuration of the complexes, only hyaluronan was added to the hyaluronidase-treated cells. The position and mobility of the hyaluronan was detected using biotinylated hyaluronan binding region (b-HABR) and gold streptavidin. The gold-labeled b-HABR was found only near the cell surface. Based on these observations, the hyaluronan-aggrecan complexes composing the cell coat are proposed to be extended in a brush-like configuration in an analogous manner to that previously described for high density, grafted polymers in good solvents.
A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells
Authors: Zahraoui A , Joberty G , Arpin M , Fontaine JJ , Hellio R , Tavitian A , Louvard D
Published: May 2004

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.
Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton
Authors: Crawford AW , Pino JD , Beckerle MC
Published: May 2004

LIM domains are present in a number of proteins including transcription factors, a proto-oncogene product, and the adhesion plaque protein zyxin. The LIM domain exhibits a characteristic arrangement of cysteine and histidine residues and represents a novel zinc binding sequence (Michelsen et al., 1993). Previously, we reported the identification of a 23-kD protein that interacts with zyxin in vitro (Sadler et al., 1992). In this report, we describe the purification and characterization of this 23-kD zyxin-binding protein from avian smooth muscle. Isolation of a cDNA encoding the 23-kD protein has revealed that it consists of 192 amino acids and exhibits two copies of the LIM motif. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP); therefore we refer to it as the chicken cysteine-rich protein (cCRP). Examination of a number of chick embryonic tissues by Western immunoblot analysis reveals that cCRP exhibits tissue-specific expression. cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. We have performed quantitative Western immunoblot analysis of cCRP, zyxin, and alpha-actinin levels during embryogenesis. By this approach, we have demonstrated that the expression of cCRP is developmentally regulated.
In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells
Authors: Yamakita Y , Yamashiro S , Matsumura F
Published: May 2004

Phosphorylation of the regulatory light chain of myosin II (MLC) controls the contractility of actomyosin in nonmuscle and muscle cells. It has been reported that cdc2 phosphorylates MLC in vitro at Ser-1 or Ser-2 and Thr-9 which protein kinase C phosphorylates (Satterwhite, L. L., M. J. Lohka, K. L. Wilson, T. Y. Scherson, L. K. Cisek, J. L. Corden, and T. D. Pollard. 1992 J. Cell Biol. 118:595-605). We have examined in vivo phosphorylation of MLC during mitosis and after the release of mitotic arrest. Phosphate incorporation of MLC in mitotic cells is found to be 6-12 times greater than that in nonmitotic cells. Phosphopeptide maps have revealed that the MLC from mitotic cells is phosphorylated at Ser-1 and/or Ser-2 (Ser-1/2), but not at Thr-9. MLC is also phosphorylated to a much lesser extent at Ser-19 which myosin light chain kinase phosphorylates. On the other hand, MLC of nonmitotic cells is phosphorylated at Ser-19 but not at Ser-1/2. The extent of phosphate incorporation is doubled at 30 min after the release of mitotic arrest when some cells start cytokinesis. Phosphopeptide analyses have revealed that the phosphorylation at Ser-19 is increased 20 times, while the phosphorylation at Ser-1/2 is decreased by half. This high extent of MLC phosphorylation at Ser-19 is maintained for another 30 min and gradually decreased to near the level of interphase cells as cells complete spreading at 180 min. On the other hand, phosphorylation at Ser-1/2 is decreased to 18% at 60 min, and is practically undetectable at 180 min after the release of mitotic arrest. The stoichiometry of MLC phosphorylation has been determined by quantitation of phosphorylated and unphosphorylated forms of MLC separated on 2D gels. The molar ratio of phosphorylated MLC to total MLC is found to be 0.16 +/- 0.06 and 0.31 +/- 0.05 in interphase and mitotic cells, respectively. The ratio is increased to 0.49 +/- 0.05 at 30 min after the release of mitotic arrest. These results suggest that the change in the phosphorylation site from Ser-1/2 to Ser-19 plays an important role in signaling cytokinesis.
Sodium channels aggregate at former synaptic sites in innervated and denervated regenerating muscles
Authors: Lupa MT , Caldwell JH
Published: May 2004

The role of innervation in the establishment and regulation of the synaptic density of voltage-activated Na channels (NaChs) was investigated at regenerating neuromuscular junctions. Rat muscles were induced to degenerate after injection of the Australian tiger snake toxin, notexin. The loose-patch voltage clamp technique was used to measure the density and distribution of NaChs on muscle fibers regenerating with or without innervation. In either case, new myofibers formed within the original basal lamina sheaths, and, NaChs became concentrated at regenerating endplates nearly as soon as they formed. The subsequent increase in synaptic NaCh density followed a time course similar to postnatal muscles. Neuromuscular endplates regenerating after denervation, with no nerve terminals present, had NaCh densities not significantly different from endplates regenerating in the presence of nerve terminals. The results show that the nerve terminal is not required for the development of an enriched NaCh density at regenerating neuromuscular synapses and implicate Schwann cells or basal lamina as the origin of the signal for NaCh aggregation. In contrast, the change in expression from the immature to the mature form of the NaCh isoform that normally accompanies development occurred only partially on muscles regenerating in the absence of innervation. This aspect of NaCh regulation is thus dependent upon innervation.
Cerebroglycan: an integral membrane heparan sulfate proteoglycan that is unique to the developing nervous system and expressed specifically during neuronal differentiation
Authors: Stipp CS , Litwack ED , Lander AD
Published: May 2004

Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. We report here the cloning and pattern of expression of cerebroglycan, a glycosylphosphatidylinositol (GPI)-anchored HSPG that is found in the developing rat brain (previously referred to as HSPG M13; Herndon, M. E., and A. D. Lander. 1990. Neuron. 4:949-961). The cerebroglycan core protein has a predicted molecular mass of 58.6 kD and five potential heparan sulfate attachment sites. Together with glypican (David, G., V. Lories, B. Decock, P. Marynen, J.-J. Cassiman, and H. Van den Berghe. 1990. J. Cell Biol. 111:3165-3176), it defines a family of integral membrane HSPGs characterized by GPI linkage and conserved structural motifs, including a pattern of 14 cysteine residues that is absolutely conserved. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is expressed in only one tissue: the nervous system. In situ hybridization experiments at several developmental stages strongly suggest that cerebroglycan message is widely and transiently expressed by immature neurons, appearing around the time of final mitosis and disappearing after cell migration and axon outgrowth have been completed. These results suggest that cerebroglycan may fulfill a function related to the motile behaviors of developing neurons.
Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein
Authors: Taipale J , Miyazono K , Heldin CH , Keski-Oja J
Published: May 2004

The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse-chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.
Matrix-bound thrombospondin promotes angiogenesis in vitro
Authors: Nicosia RF , Tuszynski GP
Published: May 2004

Thrombospondin (TSP) is a multidomain adhesive protein postulated to play an important role in the biological activity of the extracellular matrix. To test this hypothesis, TSP-containing fibrin and collagen matrices were evaluated for their capacity to support angiogenesis and cell growth from explants of rat aorta. This serum-free model allowed us to study the angiogenic effect of TSP without the interference of attachment and growth factors present in serum. TSP promoted dose-dependent growth of microvessels and fibroblast-like cells. The number of microvessels in TSP-containing collagen and fibrin gels increased by 136 and 94%, respectively. The TSP effect was due in part to cell proliferation since a 97% increase in [3H]thymidine incorporation by the aortic culture was observed. The effect was TSP-specific because TSP preparations adsorbed with anti-TSP antibody showed no activity. TSP did not promote angiogenesis directly since no TSP-dependent growth of isolated endothelial cells could be demonstrated. Rather TSP directly stimulated the growth of aortic culture-derived myofibroblasts which in turn promoted microvessel formation when cocultured with the aortic explants. Angiogenesis was also stimulated by myofibroblast-conditioned medium. Partial characterization of the conditioned medium suggests that the angiogenic activity is due to heparin-binding protein(s) with molecular weight > 30 kD. These results indicate that matrix-bound TSP can indirectly promote microvessel formation through growth-promoting effects on myofibroblasts and that TSP may be an important stimulator of angiogenesis and wound healing in vivo.
Differential trafficking of soluble and integral membrane secretory granule-associated proteins
Authors: Milgram SL , Eipper BA , Mains RE
Published: May 2004

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.
Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis
Authors: Jena BP , Gumkowski FD , Konieczko EM , von Mollard GF , Jahn R , Jamieson JD
Published: May 2004

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.