bluuebunny/crossref_metadata_2025 huggingface dataset:
100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies
Authors: Robinson MS
Published: May 2004

Proteins with molecular weights of around 100,000 (designated 100K) are found in all coated vesicles. Five monoclonal antibodies have been raised against the major 100K proteins of bovine brain coated vesicles, which migrate on SDS gels as three closely spaced bands. One antibody stains the middle band (band B), two stain both upper and lower bands (bands A and C), and two stain the lower band (band C) only. Thus, the polypeptides in bands A and C are related (but not identical), a result confirmed by NH2-terminal sequencing. Other tissues were found to express proteins corresponding to, and co-migrating with, bands B and C but not band A. Only the two antibodies that recognize both A and C stained fixed and permeabilized tissue culture cells; they both showed a punctate pattern in the plane of the plasma membrane. Double labeling with anti-clathrin antibodies confirmed that the dots correspond to coated pits and vesicles. However, perinuclear staining seen with anti-clathrin, corresponding to Golgi-derived coated vesicles, was conspicuously absent with the two monoclonal antibodies. Affinity-purified polyclonal antisera against the 100K proteins, reported earlier, gave perinuclear as well as punctate staining; these included one antiserum which gave mainly perinuclear staining (Robinson, M. S., and B. M. F. Pearse, 1986, J. Cell Biol., 102:48-54). Thus, different 100K proteins appear to be found in different membrane compartments. Since the 100K proteins are thought to lie between clathrin and the membrane proteins of the vesicle, these results may help to explain how different membrane proteins can be sorted into coated vesicles in different parts of the cell.
Mapping two functional domains of clathrin light chains with monoclonal antibodies
Authors: Kohtz DS , Georgieva-Hanson V , Kohtz JD , Schook WJ , Puszkin S
Published: May 2004

The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F. M., 1985, J. Cell Biol., 101:2047-2054; Kirchhausen, T., S. C. Harrison, P. Parham, and F. M. Brodsky, 1983, Proc. Natl. Acad. Sci. USA, 80:2481-2485). Four new anti-CAP mAbs are reported here: two, C-7H12 and C-6C1, react with both forms; two others, C-10B2 and C-4E5, react only with the lower form. Sandwich ELISAs indicated that C-10B2, C-4E5, C-6C1, and C-7H12 react with distinct epitopes. Monoclonal antibodies C-10B2 and C-4E5 immunoprecipitate clathrin-coated vesicles (CCVs) and react with CAP2 epitopes accessible to chymotrypsin on the vesicle. These mAbs inhibit phosphorylation of CAP2 by endogenous CCV casein kinase II. In contrast, C-6C1 and C-7H12 react with epitopes that are relatively insensitive to chymotrypsin. CAP peptide fragments containing these epitopes remain bound to reassembled cages or CCVs after digestion. Immunoprecipitation and ELISAs demonstrate that C-7H12 and C-6C1 react with unbound CAPs but not with CAPs bound to triskelions or CCVs. The data indicate that the CAPs consist of at least two discernible structural domains: a nonconserved, accessible domain that is relevant to the phosphorylation of CAP2 and a conserved, inaccessible domain that mediates the binding of CAPs to CCVs.
Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm
Authors: Cowan AE , Myles DG , Koppel DE
Published: May 2004

PH-20 protein on the plasma membrane (PH-20PM) is restricted to the posterior head of acrosome-intact guinea pig sperm. During the exocytotic acrosome reaction the inner acrosomal membrane (IAM) becomes continuous with the posterior head plasma membrane, and PH-20PM migrates to the IAM. There it joins a second population of PH-20 protein localized to this region of the acrosomal membrane (PH-20AM) (Cowan, A.E., P. Primakoff, and D.G. Myles, 1986, J. Cell Biol. 103:1289-1297). To investigate how the localized distributions of PH-20 protein are maintained, the lateral mobility of PH-20 protein on these different membrane domains was determined using fluorescence redistribution after photobleaching. PH-20PM on the posterior head of acrosome-intact sperm was found to be mobile, with a diffusion coefficient and percent recovery typical of integral membrane proteins (D = 1.8 X 10(-10) cm2/s; %R = 73). This value of D was some 50-fold lower than that found for the lipid probe 1,1-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (C14diI) in the same region (D = 8.9 X 10(-9) cm2/s). After migration to the IAM of acrosome-reacted sperm, this same population of molecules (PH-20PM) exhibited a 30-fold increase in diffusion rate (D = 4.9 X 10(-9) cm2/s; %R = 78). This rate was similar to diffusion of the lipid probe C14diI in the IAM (D = 5.4 X 10(-9) cm2/s). The finding of free diffusion of PH-20PM in the IAM of acrosome-reacted sperm supports the proposal that PH-20 is maintained within the IAM by a barrier to diffusion at the domain boundary. The slower diffusion of PH-20PM on the posterior head of acrosome-intact sperm is also consistent with localization by barriers to diffusion, but does not rule out alternative mechanisms.
Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils
Authors: Godfrey RW , Manzi RM , Clark MA , Hoffstein ST
Published: May 2004

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.
Movement of the actin filament bundle in Mytilus sperm: a new mechanism is proposed
Authors: Tilney LG , Fukui Y , DeRosier DJ
Published: May 2004

An actin filament bundle approximately 2-5 microns in length is present in the sperm of the blue mussel, Mytilus. In unfired sperm this bundle extends from the midpiece through a canal in the center of the nucleus to terminate on the membrane limiting the inside of the cone-shaped acrosomal vacuole. The bundle is composed of 45-65 actin filaments which are hexagonally packed and regularly cross-bridged together to form an actin paracrystal so well ordered that it has six nearly equal faces. Upon induction of the acrosomal reaction, a needle-like process is formed in a few seconds. Within this process is the actin filament bundle which appears unchanged in filament number and packing as determined by optical diffraction methods. Using fluorescein-conjugated phalloidin we were able to establish that the bundle does not change length but instead is projected anteriorly out of the midpiece and nuclear canal like an arrow. Existing mechanisms to explain this extension cannot apply. Specifically, the bundle does not increase in length (no polymerization), does not change its organization (no change in actin twist), does not change filament number (no filament sliding), and cannot move by myosin (wrong polarity). Thus we are forced to look elsewhere for a mechanism and have postulated that at least a component of this movement, or cell elongation, is the interaction of the actin filament bundle with the plasma membrane.
Sea urchin maternal and embryonic U1 RNAs are spatially segregated in early embryos.
Authors: Nash M A , Kozak S E , Angerer L M , Angerer R C , Schatten H , Schatten G , Marzluff W F
Published: May 2004

We have used in situ hybridization and cell fractionation methods to follow the distribution of U1 RNA and immunofluorescence microscopy to follow the distribution of snRNP proteins in oocytes, eggs, and embryos of several sea urchin species. U1 RNA and U1-specific snRNP antigens are concentrated in germinal vesicles of oocytes. Both appear to relocate after oocyte maturation because they are found primarily, if not exclusively, in the cytoplasm of mature unfertilized eggs. This cytoplasmic residence is maintained during early cleavage and U1 RNA is first detectable in nuclei of micromeres at the 16-cell stage. Between morula and gastrula stages the steady-state concentrations of both RNA and antigens gradually increase in nuclei and decrease in cytoplasm. Surprisingly, analysis of the distribution of newly synthesized U1 RNA shows that it does not equilibrate with the maternal pool. Instead new transcripts are confined to nuclei, while cytoplasmic U1 RNAs are of maternal origin. This lack of equilibration and the conversion of maternal U1 RNAs from nuclear species in oocytes to cytoplasmic in embryos suggests that these RNPs (or RNAs) are structurally altered when released to the cytoplasm at oocyte maturation.
Monoclonal antibodies identify a group of nuclear pore complex glycoproteins.
Authors: Snow C M , Senior A , Gerace L
Published: May 2004

Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.
Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells.
Authors: Corless C L , Matzuk M M , Ramabhadran T V , Krichevsky A , Boime I
Published: May 2004

The glycoprotein hormones lutropin (LH) and chorionic gonadotropin (CG) share a common structure consisting of an identical alpha subunit noncovalently linked to a hormone-specific beta subunit. While LH is produced in the anterior pituitary, CG is synthesized in placenta. To compare the assembly, processing, and secretion of human LH and CG in the same cell type, we have expressed their subunits, individually and together, in mouse C-127 mammary tumor cells. Analysis of transfected clones revealed an unexpected difference in the secretion of individually expressed subunits. Whereas alpha and CG beta subunits were rapidly and quantitatively secreted, only 10% of newly synthesized LH beta subunit reached the medium. The remaining subunit was found in an intracellular, endoglycosidase H (endo H)-sensitive pool that had a turnover rate of approximately 8 h. Coexpression with alpha subunit resulted in "rescue" of LH beta subunit by formation of LH dimer, which was efficiently secreted. However, combination of LH beta with alpha was slow, with an overall efficiency of only 50% despite the presence of excess alpha. In contrast, CG beta was rapidly assembled with the alpha subunit after synthesis. The two beta subunits also differed in their influence on the N-linked oligosaccharide processing of combined alpha. The oligosaccharides of LH dimer were endo H resistant, while those of CG dimer remained partially endo H sensitive. Thus, despite a high degree of homology between LH beta and CG beta, the two subunits differ in their secretion as free subunits, their rate of assembly with alpha subunit, and in their effect on the N-linked oligosaccharide processing of combined alpha.
A signal sequence domain essential for processing, but not import, of mitochondrial pre-ornithine carbamyl transferase.
Authors: Nguyen M , Argan C , Sheffield W P , Bell A W , Shields D , Shore G C
Published: May 2004

Studies using deletion mutagenesis indicate that a processing recognition site lies proximal to the normal cleavage position between gln32 and ser33 of pre-ornithine carbamyl transferase (pOCT). pOCT cDNA was manipulated to delete codons specifying amino acids 22-30 of the signal sequence. The mutant precursor, designated pOCT delta 22-30, was imported to the matrix compartment by purified mitochondria, but remained largely unprocessed; the low level of processing that was observed did not involve the normal cleavage site. Several manipulations performed downstream of the normal pOCT processing site (deletion, substitution, and hybrid protein constructions) affected neither import nor correct processing. Our data suggest that domains specifying import and processing site recognition may be functionally segregated within the signal peptide; that processing is not requisite for import of pOCT; and that a proximal region, not involving the normal signal peptide cleavage site, is required for processing site recognition.
The posttranslational processing of sucrase-isomaltase in HT-29 cells is a function of their state of enterocytic differentiation.
Authors: Trugnan G , Rousset M , Chantret I , Barbat A , Zweibaum A
Published: May 2004

The biosynthesis of sucrase-isomaltase was compared in enterocyte-like differentiated (i.e., grown in the absence of glucose) and undifferentiated (i.e., grown in the presence of glucose) HT-29 cells. Unlike differentiated cells, in which the enzyme is easily detectable and active, undifferentiated cells display almost no enzyme activity and the protein cannot be detected by means of cell surface immunofluorescence or immunodetection in membrane-enriched fractions or cell homogenates. Pulse experiments with L-[35S]-methionine show that the enzyme is, however, synthesized in these undifferentiated cells. As compared with the corresponding molecular forms in differentiated cells, the high-mannose form of the enzyme in undifferentiated cells is similarly synthesized and has the same apparent Mr. However, its complex form is less labeled and has a lower apparent Mr. Pulse-chase experiments with L-[35S]methionine show that, although the enzyme is synthesized to the same extent in both situations, the high-mannose and complex forms are rapidly degraded in undifferentiated cells, with an apparent half-life of 6 h, in contrast to differentiated cells in which the enzyme is stable for at least 48 h. A comparison of the processing of the enzyme in both situations shows that the conversion of the high-mannose to the complex form is markedly decreased in undifferentiated cells. These results indicate that the absence of sucrase-isomaltase expression in undifferentiated cells is not the consequence of an absence of biosynthesis but rather the result of both an impaired glycosylation and a rapid degradation of the enzyme.
Tubular lysosomes accompany stimulated pinocytosis in macrophages.
Authors: Swanson J , Burke E , Silverstein S C
Published: May 2004

A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.
Redistribution of microtubules and pericentriolar material during the development of polarity in mouse blastomeres.
Authors: Houliston E , Pickering S J , Maro B
Published: May 2004

The distribution of microtubules and microtubule organizing centers (MTOCs) during the development of cell polarity in eight-cell mouse blastomeres was studied by immunofluorescence and immunoelectron microscopy using monoclonal anti-tubulin antibodies and an anti-pericentriolar material (PCM) serum. In early eight-cell blastomeres microtubules were found mainly around the nucleus and in the cell cortex, whereas PCM foci were observed dispersed in the cytoplasm. During the eight-cell stage, microtubules disappeared from the area adjacent to the zone of intercellular contact and accumulated in the apical part of the cell while their number decreased in the basal domain. The PCM also relocalized to the apical domain of the cell, but this occurred after the redistribution of the microtubules by a mechanism that involved the microtubule network. The possible roles of both MTOCs and microtubules in establishing cell polarity are discussed.
Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle.
Authors: Jorgensen A O , Jones L R
Published: May 2004

The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2+-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was approximately 35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be three- to fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogold-labeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.
Adhesion molecules during somitogenesis in the avian embryo.
Authors: Duband J L , Dufour S , Hatta K , Takeichi M , Edelman G M , Thiery J P
Published: May 2004

In avian embryos, somites constitute the morphological unit of the metameric pattern. Somites are epithelia formed from a mesenchyme, the segmental plate, and are subsequently reorganized into dermatome, myotome, and sclerotome. In this study, we used somitogenesis as a basis to examine tissue remodeling during early vertebrate morphogenesis. Particular emphasis was put on the distribution and possible complementary roles of adhesion-promoting molecules, neural cell adhesion molecule (N-CAM), N-cadherin, fibronectin, and laminin. Both segmental plate and somitic cells exhibited in vitro calcium-dependent and calcium-independent systems of cell aggregation that could be inhibited respectively by anti-N-cadherin and anti-N-CAM antibodies. In vivo, the spatio-temporal expression of N-cadherin was closely associated with both the formation and local disruption of the somites. In contrast, changes in the prevalence of N-CAM did not strictly accompany the remodeling of the somitic epithelium into dermamyotome and sclerotome. It was also observed that fibronectin and laminin were reorganized secondarily in the extracellular spaces after CAM-mediated contacts were modulated. In an in vitro culture system of somites, N-cadherin was lost on individual cells released from somite explants and was reexpressed when these cells reached confluence and established intercellular contacts. In an assay of tissue dissociation in vitro, antibodies to N-cadherin or medium devoid of calcium strongly and reversibly dissociated explants of segmental plates and somites. Antibodies to N-CAM exhibited a smaller disrupting effect only on segmental plate explants. In contrast, antibodies to fibronectin and laminin did not perturb the cohesion of cells within the explants. These results emphasize the possible role of cell surface modulation of CAMs during the formation and remodeling of some transient embryonic epithelia. It is suggested that N-cadherin plays a major role in the control of tissue remodeling, a process in which N-CAM is also involved but to a lesser extent. The substratum adhesion molecules, fibronectin and laminin, do not appear to play a primary role in the regulation of these processes but may participate in cell positioning and in the stabilization of the epithelial structures.
Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro.
Authors: Dejana E , Colella S , Languino L R , Balconi G , Corbascio G C , Marchisio P C
Published: May 2004

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.
Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy.
Authors: Galvin N J , Vance P M , Dixit V M , Fink B , Frazier W A
Published: May 2004

Binding of thrombospondin (TSP) to types I-V collagen was examined by direct binding assays using 125I-TSP and by visualization of rotary-shadowed intermolecular complexes in the electron microscope. The binding of TSP was highest to type V collagen in the absence of Ca, while lower but significant levels of binding were observed to all other collagen types in the presence or absence of Ca. Unlike intact TSP, the trimeric collagen-binding domain of TSP composed of 70-kD chains showed no Ca dependence in its binding to type V collagen. Further evidence for binding of TSP to types I and III collagen was obtained by competition studies in which these soluble collagens effectively inhibited binding of 125I-TSP to immobilized type V collagen. The binding of TSP to type V collagen was inhibited by heparin and fucoidin, both high-affinity ligands of TSP's heparin-binding domain. mAb A6.1, which binds to the 70-kD domain of TSP, is also the best of a panel of anti-TSP mAbs at inhibiting the TSP-collagen interaction. Electron microscopy of rotary-shadowed replicas of TSP-collagen complexes revealed that all five types of collagen examined had a binding site for TSP at one end of the pepsinized, triple helical molecule. The specificity of this site was tested by examining the ability of BSA to form a complex with the end of the pepsinized collagens. Rotary-shadowed replicas revealed a low frequency of apparent BSA-collagen complexes, and histograms of these data showed no evidence for the preferential association of BSA with the end of the collagen molecules. In addition to the specific end site, type V collagen had an internal binding site for TSP located about two-thirds of the distance along the length of the collagen molecule from the end site. The internal binding site for TSP on type V collagen is apparently the site responsible for the higher affinity binding of TSP to that protein observed in direct binding assays. The trimeric 70-kD collagen-binding domain of TSP bound to the same sites on the collagens as did intact TSP.
Three-dimensional organization of Drosophila melanogaster interphase nuclei. II. Chromosome spatial organization and gene regulation.
Authors: Hochstrasser M , Sedat J W
Published: May 2004

In the preceding article we compared the general organization of polytene chromosomes in four different Drosophila melanogaster cell types. Here we describe experiments aimed at testing for a potential role of three-dimensional chromosome folding and positioning in modulating gene expression and examining specific chromosome interactions with different nuclear structures. By charting the configurations of salivary gland chromosomes as the cells undergo functional changes, it is shown that loci are not repositioned within the nucleus when the pattern of transcription changes. Heterologous loci show no evidence of specific physical interactions with one another in any of the cell types. However, a specific subset of chromosomal loci is attached to the nuclear envelope, and this subset is extremely similar in at least two tissues. In contrast, no specific interactions between any locus and the nucleolus are found, but the base of the X chromosome, containing the nucleolar organizer, is closely linked to this organelle. These results are used to evaluate models of gene regulation that involve the specific intranuclear positioning of gene sequences. Finally, data are presented on an unusual class of nuclear envelope structures, filled with large, electron-dense particles, that are usually associated with chromosomes.
Tetrahymena contain two distinct and unusual high mobility group (HMG)-like proteins.
Authors: Schulman I G , Cook R G , Richman R , Allis C D
Published: May 2004

Previous studies have described the existence of high mobility group (HMG)-like proteins in macronuclei of the ciliated protozoan, Tetrahymena thermophila (Hamana, K., and K. Iwai, 1979, J. Biochem. [Tokyo], 69:1097-1111; Levy-Wilson, B., M. S. Denker, and E. Ito, 1983, Biochemistry, 22:1715-1721). In this report, two of these proteins, LG-1 and LG-2, have been further characterized. Polyclonal antibodies raised against LG-1 and LG-2 fail to cross react with each other or any other macronuclear polypeptide in immunoblotting analyses. As well, LG-1 and LG-2 antibodies do not react with calf thymus, chicken, or yeast HMG proteins. Consistent with these results, a 47 amino-terminal sequence of LG-1 has been determined that shows limited homology to both calf thymus HMGs 1 and 2 and HMGs 14 and 17. Two internal sequences of V8 protease-generated peptides from LG-2 have been determined, and these do not share any homology to the LG-1 sequence or any other sequenced HMG proteins. Comparison of the partial sequences of LG-1 and LG-2 with the complete amino acid sequence of the Tetrahymena histone H1 (Wu, M., C. D. Allis, R. Richman, R. G. Cook, and M. A. Gorovsky, 1986, Proc. Natl. Acad. Sci. USA, 83:8674-8678) rules out the possibility that LG-1 and LG-2 are proteolytically derived from H1, the other major macronuclear perchloric acid-soluble protein. Interestingly, however, both LG-1 and LG-2 are efficiently extracted from macronuclei by elutive intercalation (Schröter, H., G. Maier, H. Ponsting, and A. Nordheim, 1985, Embo (Eur. Mol. Biol. Organ.) J., 4:3867-3872), suggesting that both may share yet undetermined properties with HMGs 14 and 17 of higher eukaryotes. Examination of the pattern of LG-1 and LG-2 synthesis during the sexual phase of the life cycle, conjugation, demonstrates that the synthesis of LG-1 and LG-2 is coordinately increased from basal levels during the differentiation of new macronuclei (7-13 h), suggesting that both of these proteins play a role in determining a macronuclear phenotype. However, a specific induction of LG-2 synthesis is detected in early stages of conjugation (meiotic prophase, 1-4 h), leading to maximal synthesis of LG-2 at 3 h. Interestingly, the early induction of LG-2 synthesis closely parallels the hyperphosphorylation of histone H1. Taken together, these data suggest that LG-1 and LG-2 are not strongly related to each other or to higher eukaryotic HMG proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
The Dictyostelium discoideum 30,000-dalton protein is an actin filament-bundling protein that is selectively present in filopodia.
Authors: Fechheimer M
Published: May 2004

The interaction with actin and intracellular localization of the 30,000-D actin-binding protein from the cellular slime mold Dictyostelium discoideum have been investigated to analyze the potential contributions of this protein to cell structure and movement. The formation of anisotropic cross-linked filament networks (bundles) containing actin and the 30,000-D protein has been observed by electron microscopy, light scattering, viscometry, and polarization microscopy. Cosedimentation experiments indicate that a maximum of one molecule of the 30,000-D protein can bind to 10 actin monomers in filaments with an apparent association constant of 1 X 10(7) liters/mol. Inhibition of the interaction of the 30,000-D protein with actin by either magnesium or calcium was observed by viscometry, light scattering, polarization microscopy, and direct binding assays. However, the concentration of magnesium required to diminish the interaction is greater than 100 times greater than that of calcium. The association constant of the 30,000-D protein for actin is 4.2 X 10(6) liters/mol, or less than 1 X 10(5) liters/mol in the presence of increased concentrations of either Mg2+ or Ca2+, respectively. Enzyme-linked immunoassays indicate that the 30,000-D protein comprises 0.04% of the protein in D. discoideum. Extensive interaction of the 30,000-D protein with actin in cytoplasm is predicted from these measurements of the concentration of this protein and its affinity for actin. The distribution of the 30,000-D protein was analyzed by immunofluorescence microscopy using mono-specific affinity-purified polyclonal antibody. The 30,000-D protein exhibits a diffuse distribution in cytoplasm, is excluded from prominent organelles, and is quite prominent in fine extensions protruding from the cell surface. The number, length, and distribution of these extensions containing the 30,000-D protein are similar to those of filopodia observed by scanning electron microscopy. To analyze the effects of cell thickness and the distribution of organelles on the immunofluorescence localization, fluorescein-labeled BSA was incorporated into the cytoplasm of living cells before fixation and staining using a sonication loading technique. The results indicate that the 30,000-D protein is selectively incorporated into filopodia. These results provide a clear distinction between the multiple actin-cross-linking proteins present in D. discoideum, and suggest that the 30,000-D protein contributes to organization of bundles of actin filaments in filopodia.
Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration.
Authors: Antonicek H , Persohn E , Schachner M
Published: May 2004

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.
Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection.
Authors: Heumann R , Korsching S , Bandtlow C , Thoenen H
Published: May 2004

The intact sciatic nerve contains levels of nerve growth factor (NGF) that are comparable to those of densely innervated peripheral target tissues of NGF-responsive (sympathetic and sensory) neurons. There, the high NGF levels are reflected by correspondingly high mRNANGF levels. In the intact sciatic nerve, mRNANGF levels were very low, thus indicating that the contribution of locally synthesized NGF by nonneuronal cells is small. However, after transection an increase of up to 15-fold in mRNANGF was measured in 4-mm segments collected both proximally and distally to the transection site. Distally to the transection site, augmented mRNANGF levels occurred in all three 4-mm segments from 6 h to 2 wk after transection, the longest time period investigated. The augmented local NGF synthesis after transection was accompanied by a reexpression of NGF receptors by Schwann cells (NGF receptors normally disappear shortly after birth). Proximal to the transection site, the augmented NGF synthesis was restricted to the very end of the nerve stump that acts as a "substitute target organ" for the regenerating NGF-responsive nerve fibers. While the mRNANGF levels in the nerve stump correspond to those of a densely innervated peripheral organ, the volume is too small to fully replace the lacking supply from the periphery. This is reflected by the fact that in the more proximal part of the transected sciatic nerve, where mRNANGF remained unchanged, the NGF levels reached only 40% of control values. In situ hybridization experiments demonstrated that after transection all nonneuronal cells express mRNANGF and not only those ensheathing the nerve fibers of NGF-responsive neurons.
A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle.
Authors: Froehner S C , Murnane A A , Tobler M , Peng H B , Sealock R
Published: May 2004

In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.
Ligand- and weak base-induced redistribution of asialoglycoprotein receptors in hepatoma cells.
Authors: Zijderhand-Bleekemolen J E , Schwartz A L , Slot J W , Strous G J , Geuze H J
Published: May 2004

The receptor for asialoglycoproteins (ASGPR) was localized in human hepatoma Hep G2 cells by means of quantitative immunoelectron microscopy. Without ligand added to the culture medium, we found 34% of the total cellular receptors on the plasma membrane, 37% in compartment of uncoupling receptor and ligand (CURL), and 21% in a trans-Golgi reticulum (TGR) that was defined by the presence of albumin after immuno-double labeling. A small percent of the ASGPR was associated with coated pits, the Golgi stacks, and lysosomes. After incubation of the cells with saturating concentrations of the ligand asialo-orosomucoid (ASOR), the number of cell surface receptors decreased to 20% of total cellular receptors, whereas the receptor content of CURL increased by a corresponding amount to 50%. The ASGPR content of TGR remained constant. In contrast, after treatment of the cells with 300 microM of the weak base primaquine (PMQ), cell surface ASGPR had decreased dramatically to only 4% of total cellular receptors whereas label in the TGR had increased to 42%. ASGPR labeling of CURL increased only to 47%. The labeling of other organelles remained unchanged. This affect of PMQ was independent of the presence of additional ASOR. Implications for the intracellular pathway of the ASGPR are discussed.
The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering.
Authors: Isenberg W M , McEver R P , Phillips D R , Shuman M A , Bainton D F
Published: May 2004

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.
Platelet proteins, including platelet-derived growth factor, specifically depress a subset of the multiple components of the response elicited by glutathione in Hydra.
Authors: Hanai K , Kato H , Matsuhashi S , Morita H , Raines E W , Ross R
Published: May 2004

Human serum more strongly depressed the feeding response of Hydra (ball formation) elicited by S-methylglutathione than plasma. On the basis of the effect of several proteins released by platelets, at least five apparent components of the response (R1-R5) were suggested. Each of the platelet proteins examined specifically depressed a subset of these components. Among the platelet proteins examined, platelet-derived growth factor (PDGF) specifically depressed the R2 response (the concentration at which the depressing effect was 50% of the maximum [ED50] was 0.17 pM), and basic fibroblast growth factor depressed the R3 and R5 responses (ED50 0.50 aM) and the R2 response (ED50 0.55 pM). With respect to the depression of the R2 response by PDGF, addition of an anti-PDGF IgG or chemical reduction of PDGF, both of which prevent PDGF from binding to its cell surface receptor on responsive cells, eliminated the depressing effect of PDGF on the hydra response. The implications of these observations are discussed.
Interaction of small dermatan sulfate proteoglycan from fibroblasts with fibronectin.
Authors: Schmidt G , Robenek H , Harrach B , Glössl J , Nolte V , Hörmann H , Richter H , Kresse H
Published: May 2004

Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.
Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum.
Authors: Jingami H , Brown M S , Goldstein J L , Anderson R G , Luskey K L
Published: May 2004

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic NH2-terminal domain that contains seven apparent membrane-spanning regions and a single N-linked carbohydrate chain. The catalytic domain, which includes the COOH-terminal two-thirds of the protein, extends into the cytoplasm. The enzyme is normally degraded with a rapid half-life (2 h), but when cells are depleted of cholesterol, its half-life is prolonged to 11 h. Addition of sterols accelerates degradation by fivefold. To explore the requirements for regulated degradation, we prepared expressible reductase cDNAs from which we either deleted two contiguous membrane-spanning regions (numbers 4 and 5) or abolished the single site for N-linked glycosylation. When expressed in hamster cells after transfection, both enzymes retained catalytic activity. The deletion-bearing enzyme continued to be degraded with a rapid half-life in the presence of sterols, but it no longer was stabilized when sterols were depleted. The glycosylation-minus enzyme was degraded at a normal rate and was stabilized normally by sterol deprivation. When cells were induced to overexpress the deletion-bearing enzyme, they did not incorporate it into neatly arranged crystalloid ER tubules, as occurred with the normal and carbohydrate-minus enzymes. Rather, the deletion-bearing enzyme was incorporated into hypertrophied but disordered sheets of ER membrane. We conclude that the carbohydrate component of HMG CoA reductase is not required for proper subcellular localization or regulated degradation. In contrast, the native structure of the transmembrane component is required to form a normal crystalloid ER and to allow the enzyme to undergo regulated degradation by sterols.