bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular matrix-specific induction of elastogenic differentiation and maintenance of phenotypic stability in bovine ligament fibroblasts.
Authors: Mecham R P , Madaras J G , Senior R M
Published: May 2004

We studied the process of elastogenic differentiation in the bovine ligamentum nuchae to assess the mechanisms that regulate elastin gene expression during development. Undifferentiated ( nonelastin -producing) ligament cells from early gestation animals initiate elastin synthesis when grown on an extracellular matrix (ECM) substratum prepared from late gestation ligamentum nuchae. ECM from ligaments of fetal calves younger than the time when elastin production occurs spontaneously in situ (i.e., beginning the last developmental trimester at approximately 180 d of gestation) does not stimulate elastin production in undifferentiated cells. Matrix-induced differentiation requires direct cell matrix interaction, is dependent upon cell proliferation after cell-matrix contact, and can be blocked selectively by incorporation of bromodeoxyuridine into the DNA of undifferentiated cells before (but not after) contact with inducing matrix. Quantitative analysis of elastin synthesis in young cells after matrix-induced differentiation indicates that the entire cell population is competent to respond to the matrix inducer, and continued synthesis of elastin after young cells are removed from the ECM substratum indicates that the phenotypic transition to elastin synthesis is stable and heritable. Although ligament cells do not require continuous contact with ECM to express the elastin phenotype, elastin synthesis is increased substantially when elastin-producing cells are grown on ligament matrix, suggesting that elastogenic differentiation is stabilized by ECM. The matrix substratum was also found to alter the distribution of tropoelastin between the medium and matrix cell layer. When grown on tissue culture plastic, ligament cells secrete greater than 80% of newly synthesized tropoelastin into the culture medium. When cultured on ECM, however, 50-70% of the newly synthesized tropoelastin remains associated with the cell layer and is cross-linked to form insoluble elastin as shown by the incorporation of radiolabeled lysine into desmosine.
Endogenous glycosyltransferases glucosylate lipids in flagella of Euglena.
Authors: Chen S J , Bouck G B
Published: May 2004

Flagella, intact deflagellated cells and isolated cell surfaces of the unicell , Euglena were separately assayed for glycosyltransferase activity by incubating these fractions with uridine diphosphate-[3H]glucose and isolating radiolabeled products. Most of the label was incorporated into lipophilic products, soluble in chloroform/methanol, which could be separated via thin layer chromatography or LH-60 chromatography into four distinct classes. The most polar of these products was extracted from flagella and purified by column chromatography for use as an in vitro substrate to identify flagella-associated glycosyltransferases. After flagella were treated with the detergent CHAPS , a soluble fraction was removed that was capable of glycosylation in solution. The glycosyltransferase(s) responsible for this activity were further enriched on sucrose or fructose gradients and ultimately identified on acrylamide gels through the combined use of nondenaturing gels, dial-[3H]uridine diphosphate binding, and fluorography. The enzyme had an apparent monomer molecular weight of 32,000 and consisted of four or fewer subunits. The occurrence of endogenous glycosyltransferase(s) in flagella suggests that modifications and/or assembly of the flagella surface can take place in situ in this organism.
Structure of (Na+,K+)-ATPase as revealed by electron microscopy and image processing.
Authors: Mohraz M , Smith P R
Published: May 2004

(Na+,K+)-ATPase was studied by electron microscopy and image processing of negatively stained and freeze-dried and shadowed crystalline sheets induced by a number of inorganic salts. Extensive experiments have identified new conditions for optimum crystal formation. Two crystal forms have been observed, one with a monomer and the other with a dimer, in the unit cell. Both show the same structure for the enzyme monomer. The enzyme can also be crystallized after partial proteolysis of its alpha subunit by trypsin. The proteolysed enzyme crystallizes under the same conditions as the whole enzyme. Comparison of the mass distributions in the images of the intact and proteolysed enzyme has allowed the tentative identification of the location of the alpha subunit within the monomer. The relationship between the structure of the crystallized enzyme and that of the enzyme in its native form is discussed, as is its apparent close structural relationship to the calcium-ATPase.
Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis.
Authors: Chiquet M , Fambrough D M
Published: May 2004

Extracellular matrix components are likely to be involved in the interaction of muscle with nonmuscle cells during morphogenesis and in adult skeletal muscle. With the aim of identifying relevant molecules, we generated monoclonal antibodies that react with the endomysium, i.e., the extracellular matrix on the surface of single muscle fibers. Antibody M1, which is described here, specifically labeled the endomysium of chick anterior latissimus dorsi muscle (but neither the perimysium nor, with the exception of blood vessels and perineurium, the epimysium ). Endomysium labeling was restricted to proximal and distal portions of muscle fibers near their insertion points to tendon, but absent from medial regions of the muscle. Myotendinous junctions and tendon fascicles were intensely labeled by M1 antibody. In chick embryos, " myotendinous antigen" (as we tentatively call the epitope recognized by M1 antibody) appeared first in the perichondrium of vertebrae and limb cartilage elements, from where it gradually extended to the premuscle masses. Around day 6, tendon primordia were clearly labeled. The other structures labeled by M1 antibody in chick embryos were developing smooth muscle tissues, especially aorta, gizzard, and lung buds. In general, tissues labeled with M1 antibody appeared to be a subset of the ones accumulating fibronectin. In cell cultures, M1 antibody binds to fuzzy, fibrillar material on the substrate and cell surfaces of living fibroblast and myogenic cells, which confirms an extracellular location of the antigenic site. The appearance of myotendinous antigen during limb morphogenesis and its distribution in adult muscle and tendon are compatible with the idea that it might be involved in attaching muscle fibers to tendon fascicles. Its biochemical characterization is described in the accompanying paper ( Chiquet , M., and D. Fambrough , 1984, J. Cell Biol. 98:1937-1946).
Chick myotendinous antigen. II. A novel extracellular glycoprotein complex consisting of large disulfide-linked subunits.
Authors: Chiquet M , Fambrough D M
Published: May 2004

This report describes the biochemical characterization of a novel extracellular matrix component, " myotendinous antigen," which appears early in chick limb morphogenesis at sites connecting developing muscle fibers, tendons, and bone ( Chiquet , M., and D. Fambrough , 1984; J. Cell Biol., 98:1926-1936). This extracellular matrix antigen is a major component of the secretory proteins released into the medium by fibroblast and muscle cultures; the soluble form is characterized here. This form of myotendinous antigen is a large glycoprotein complex consisting of several disulfide linked subunits (Mr approximately 150,000-240,000). The differently sized antigen subunits are related, since they yielded very similar proteolytic cleavage patterns. M1 antibody can bind to the denatured subunits. The antigen subunits, as well as a Mr approximately 80,000 pepsin-resistant antigenic domain derived from them, are resistant to bacterial collagenase. Despite possessing subunits similar in size to fibronectin, myotendinous antigen appears to be both structurally and antigenically unrelated to fibronectin or to other known extracellular matrix components. About seven times more M1 antigen per cell nucleus was released into the medium in fibroblast as compared to muscle cultures. In muscle conditioned medium, myotendinous antigen is noncovalently complexed to very high molecular weight material that could be heavily labeled by [3H]glucosamine and [35S]sulfate. This material is sensitive to chondroitinase ABC and hence appears to contain sulfated glycosaminoglycans. We speculate that myotendinous antigen might interact with proteoglycans on the surface of muscle fibers, thereby acting as a link to tendons.
Distributions of vimentin and desmin in developing chick myotubes in vivo. I. Immunofluorescence study.
Authors: Tokuyasu K T , Maher P A , Singer S J
Published: May 2004

Antibodies against chicken erythrocyte vimentin and gizzard desmin were affinity purified and then cross-absorbed with the heterologous antigen. They were used to study the in vivo distributions of these proteins in developing and mature myotubes by immunofluorescence microscopy of 0.5-2-micron frozen sections of iliotibialis muscle in 7-21-day chick embryos, neonatal and 1-d postnatal chicks, and adult chickens. The distributions of vimentin and desmin were coincidental throughout the development of myotubes, but the concentration of vimentin was gradually reduced as the myotubes matured and became largely undetectable at the time of hatching. The process of confining these proteins to the level of Z line from the initial uniform distribution occurred subsequent to the process of bringing myofibrils into lateral registry: in-register lateral association of several myofibrils was occasionally seen as early as in 7-11-d embryos, whereas the cross-striated immunofluorescence pattern of desmin and vimentin was only vaguely discerned in myotubes of 17-d embryos, just 4 d before hatching. In some myotubes of 21-d embryos, myofibrils were in lateral registry as precisely as in adult myofibers but desmin was still widely distributed around Z line in an irregular manner. Nevertheless, in many other myotubes of prenatal or neonatal chicks, desmin became confined to the level of Z line in a manner similar to that seen in adult myofibers, thus essentially completing its redistribution to the confined state of adult myofibers in coincidence with the time of hatching. In extracts from iliotibialis and posterior latissimus dorsi muscles of adult chickens, we detected a hitherto unidentified protein that was very similar to vimentin in molecular weight but did not react with our antivimentin antibody. We discuss the possibility that this protein was confused with vimentin in the past.
Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.
Authors: Fey E G , Wan K M , Penman S
Published: May 2004

Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization. We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis. First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer. The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely unaltered by detergent extraction. Residual desmosomes, as can be seen in thin sections, were also well-preserved. The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation. The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders. This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts. Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold. Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders. The 52,000-mol-wt nuclear matrix protein was confined to the chromatin-depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions. The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold.
Platelet activation and cytoskeletal reorganization: high voltage electron microscopic examination of intact and Triton-extracted whole mounts.
Authors: Loftus J C , Choate J , Albrecht R M
Published: May 2004

The sequential changes in the three-dimensional organization of the filamentous components of human platelets following surface activation were investigated in whole-mount preparations. Examination of intact and Triton-extracted platelets by high voltage electron microscopy provides morphological evidence of increased polymerization of actin into the filamentous form and an increased organization of the cytoskeletal elements after activation. The structure of resting platelets consists of the circumferential band of microtubules and a small number of microfilaments randomly arranged throughout a dense cytoplasmic matrix. Increased spreading is accompanied by cytoskeletal reorganization resulting in the development of distinct ultrastructural zones including the peripheral web, the outer filamentous zone, the "trabecular-like" inner filamentous zone, and the granulomere . These zones are present only in well-spread platelets during the late stages of surface activation and are retained following Triton extraction. Extraction of the less stable cytoplasmic components provides additional information about the underlying structure and filament interactions within each zone.
Mutant strains of Chlamydomonas reinhardtii that move backwards only.
Authors: Segal R A , Huang B , Ramanis Z , Luck D J
Published: May 2004

Mutations at three independent loci in Chlamydomonas reinhardtii result in a striking alteration of cell motility. Mutant cells representing the three mbo loci move backwards only, propelled by a symmetrical "flagellar" type of bending pattern. The characteristic asymmetric "ciliary" type of flagellar bend pattern responsible for forward movement that predominates in wild-type cells is seldom seen in the mutants. This defect in motility was found to be a property of the mutant axonemes themselves: the isolated axonemes, reactivated by addition of ATP, showed exclusively the symmetrical wave form, and the protein composition of these axonemes differed from the wild-type composition. Axonemes obtained from mbo1 , mbo2 , and mbo3 cells were found to be deficient in six polypeptides regularly present in wild type. The mbo2 axonemes were deficient in two additional polypeptides. The polypeptides were identified in autoradiograms of two-dimensional SDS polyacrylamide gel electrophoretograms of 35S- or 32P-labeled axonemes. One of the six polypeptides has previously been identified; it is a component missing in a mutant deficient for inner dynein arms. Of the five axonemal polypeptides newly identified by the mbo mutants, four were shown to be present as phosphoproteins in wild-type axonemes. One of the additional polypeptides deficient in mbo2 axonemes was also shown to be phosphorylated in wild-type axonemes. Detailed ultrastructural analysis of the mbo1 flagella and the mbo1 , mbo2A , and mbo3 axonemes revealed that the mutants specifically lack the beak-like projections found within the B-tubules of outer doublets 5 and 6.
Endogenous and monoclonal antibodies to the rat pancreatic acinar cell Golgi complex.
Authors: Smith Z D , D'Eugenio-Gumkowski F , Yanagisawa K , Jamieson J D
Published: May 2004

Normal, unimmunized mouse serum from several strains (BALB/c, C57/b, DBA/2, NZB, SJL, CD/1) contains an endogenous IgG antibody that localizes to the Golgi complex of rat pancreatic acinar cells. Treatment of pancreatic acini with 5 microM monensin resulted in the swelling and vacuolization of the Golgi cisternae, and in a corresponding annular staining by the mouse serum as observed by immunofluorescence, suggesting that the antigen recognized is on the Golgi complex cisternal membrane. The antiserum did not react with pancreatic secretory proteins, and its binding to smooth microsomal membranes was retained following sodium carbonate washing, supporting a Golgi membrane localization. Advantage was taken of the existence of the endogenous murine antibody for the isolation of monoclonal antibodies directed to the Golgi complex of the rat pancreas. Two antibodies, antiGolgi 1 and antiGolgi 2, are described. Both antibodies are IgMs that recognize integral membrane proteins of the trans-Golgi cisternae, with lighter and patchy staining of the pancreatic lumen membrane, as observed both by light and electron microscopy. AntiGolgi 1 recognizes predominately a protein of molecular weight 103,000-108,000, whereas antiGolgi 2 shows a strong reaction to a 180-kd band as well as the 103-108-kd protein.
Ultrastructural localization of the mannose 6-phosphate receptor in rat liver.
Authors: Geuze H J , Slot J W , Strous G J , Hasilik A , Von Figura K
Published: May 2004

An affinity-purified rabbit antibody against rat liver mannose 6-phosphate receptor (MP-R) was prepared. The antibody was directed against a 215 kd-polypeptide and it recognized both ligand-occupied and free receptor. Anti-MP-R was used for immunofluorescence and immunoelectron microscopy of cryosections from rat liver. MP-R was demonstrated in all parenchymal liver cells, but not in endothelial lining cells. MP-R labeling was found at the entire plasma membrane, in coated pits and coated vesicles, in the compartment of uncoupling receptor and ligand, and in the Golgi complex. Lysosomes showed only scarce MP-R label. In double-labeling immunoelectron microscopy, MP-R co-localized with albumin in the Golgi cisternae and in secretory vesicles with lipoprotein particles. Cathepsin D was associated with MP-R in the Golgi cisternae. This finding indicates that MP-R/cathepsin D complexes traverse the Golgi complex on their way to the lysosomes. The possible involvement of CURL in lysosomal enzyme targeting is discussed.
Exclusion of erythrocyte-specific membrane proteins from clathrin-coated pits during differentiation of human erythroleukemic cells.
Authors: Marshall L M , Thureson-Klein A , Hunt R C
Published: May 2004

When human erythroleukemic cells are induced to differentiate, they produce globin and redistribute glycophorin and spectrin to one pole of the cell. This process was accompanied by an alteration in the clathrin-coated pits at the cell surface. In nondifferentiating cells, receptors for Concanavalin A have been shown, using electron microscopy, to be concentrated into coated pits and rapidly internalized. Glycophorin was also internalized via coated pits, but was not greatly concentrated into these portions of the surface membrane. Ligands attached to glycophorin were, therefore, cleared from the cell surface more slowly than Concanavalin A. In nondifferentiating cells, immunoelectron microscopy showed that spectrin is largely excluded from coated pits. After erythroid differentiation proceeded for several days, glycophorin was totally excluded from the coated pits along with spectrin. This did not reflect a general cessation of endocytosis, however, because Concanavalin A receptors continued to be internalized. It is possible that the specific exclusion of glycophorin from coated pits is part of the remodeling process that occurs when the precursor cell membrane differentiates into that of the mature erythrocyte.
Monomer-polymer equilibria in the axon: direct measurement of tubulin and actin as polymer and monomer in axoplasm.
Authors: Morris J R , Lasek R J
Published: May 2004

The monomer-polymer equilibria for tubulin and actin were analyzed for the cytoskeleton of the squid giant axon. Two methods were evaluated for measuring the concentrations of monomer, soluble (equilibrium) polymer, and stable polymer in extruded axoplasm. One method, the Kinetic Equilibration Paradigm ( KEP ), employs the basic principles of diffusion to distinguish freely diffusible monomer from proteins that are present in the form of polymer. The other method is pharmacological and employs either taxol or phalloidin to stabilize the microtubules and microfilaments, respectively. The results of the two methods agree and demonstrate that 22-36% of the tubulin and 41-47% of the actin are monomeric. The in vivo concentration of monomeric actin and tubulin were two to three times higher than the concentration required to polymerize these proteins in vitro, suggesting that assembly of these proteins is regulated by additional mechanisms in the axon. A significant fraction of the polymerized actin and tubulin in the axoplasm was stable microtubules and microfilaments, which suggests that the dissociation reaction is blocked at both ends of these polymers. These results are discussed in relationship to the axonal transport of the cytoskeleton and with regard to the ability of axons to change their shape in response to environmental stimuli.
Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells.
Authors: Beckerle M C
Published: May 2004

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.
Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics.
Authors: Dunn W A , Hubbard A L
Published: May 2004

We have used biochemical and morphological techniques to demonstrate that hepatocytes in the perfused liver bind, internalize, and degrade substantial amounts of murine epidermal growth factor (EGF) via a receptor-mediated process. Before ligand exposure, about 300,000 high-affinity receptors were detectable per cell, displayed no latency, and co-distributed with conventional plasma membrane markers. Cytochemical localization using EGF coupled to horseradish peroxidase (EGF-HRP) revealed that the receptors were distributed along the entire sinusoidal and lateral surfaces of hepatocytes. When saturating concentrations of EGF were perfused through a liver at 35 degrees C, ligand clearance was biphasic with a rapid primary phase of 20,000 molecules/min per cell that dramatically changed at 15-20 min to a slower secondary phase of 2,500 molecules/min per cell. During the primary phase of uptake, approximately 250,000 molecules of EGF and 80% of the total functional receptors were internalized into endocytic vesicles which could be separated from enzyme markers for plasma membranes and lysosomes on sucrose gradients. The ligand pathway was visualized cytochemically 2-25 min after EGF-HRP internalization and a rapid transport from endosomes at the periphery to those in the Golgi apparatus-lysosome region was observed (t 1/2 approximately equal to 7 min). However, no 125I-EGF degradation was detected for at least 20 min. Within 30 min after EGF addition, a steady state was reached which lasted up to 4 h such that (a) the rate of EGF clearance equaled the rate of ligand degradation (2,500 molecules/min per cell); (b) a constant pool of undegraded ligand was maintained in endosomes; and (c) the number of accessible (i.e., cell surface) receptors remained constant at 20% of initial values. By 4 h hepatocytes had internalized and degraded 3 and 2.3 times more EGF, respectively, than the initial number of available receptors, even in the presence of cycloheximide and without substantial loss of receptors. All of these results suggest that EGF receptors are internalized and that their rate of recycling to the surface from intracellular sites is governed by the rate of entry of ligand and/or receptor into lysosomes.
Cyclic AMP-dependent phosphorylation of the precursor to beta subunit of mitochondrial F1-ATPase: a physiological mistake?
Authors: Steinberg R A
Published: May 2004

By using the purified rat liver protein for reference in electrophoresis and peptide mapping experiments, I have identified the beta subunit of mitochondrial F1-ATPase and its cytoplasmic precursor in two-dimensional gel patterns of proteins from S49 mouse lymphoma cells. The beta subunit precursor is a substrate for cAMP-dependent phosphorylation during its synthesis. Normally, both nonphosphorylated and phosphorylated forms of beta subunit precursor are processed rapidly to the smaller, more acidic forms of mature beta subunit. When processing is inhibited with valinomycin, both nonphosphorylated and phosphorylated forms of beta subunit precursor are stabilized. Nonphosphorylated beta subunit is one of the most stable of cellular proteins, but the phosphorylated form is eliminated within minutes of processing. This suggests that phosphorylated beta subunit is recognized as aberrant and excluded from assembly into the ATPase complex. These results argue that cAMP-dependent phosphorylation of the beta subunit precursor is a physiological mistake that is remedied after mitochondrial import and processing.
Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate.
Authors: Vicker M G , Schill W , Drescher K
Published: May 2004

Myxamoebae of the morphogenetic cellular slime mold Dictyostelium discoideum are thought to be able to accurately read and respond to directional information in spatial gradients of cyclic AMP. We examined the spatial and temporal mechanisms proposed for chemotaxis by comparing the behavior of spreading or evenly distributed cell populations after exposure to well-defined spatial gradients. The effects of gradient generation on cells were avoided by using predeveloped gradients. Qualitatively different responses were obtained using (a) isotropic, (b) static spatial, or (c) temporal (impulse) gradients in a simple chamber of penetrable micropore filters. We simulated models of chemotaxis and chemokinesis to aid our interpretations. The attractive and locomotory responses of populations were maximally stimulated by 0.05 microM cyclic AMP, provided that cellular phosphodiesterase was inhibited. But a single impulse of cyclic AMP during gradient development caused a greater and qualitatively different attraction. Attraction in spatial gradients was only transient, in that populations eventually developed a random distribution when confined to a narrow territory. Populations never accumulated nor lost their random distribution even in extremely steep spatial gradients. Attraction in spatial gradients was inducible only in spreading populations, not randomly distributed ones. Thus, spatial gradients effect biased-random locomotion: i.e., chemokinesis without adaptation. Cells cannot read gradients; the reaction of the cells is stochastic. Spatial gradients do not cause chemotaxis, which probably requires a sharp stimulant concentration increase (a temporal gradient) as a pulse or impulse. The results also bear on concepts of how embryonic cells might be able to decipher the positional information in a morphogen spatial gradient during development.
Centriole distribution during tripolar mitosis in Chinese hamster ovary cells.
Authors: Keryer G , Ris H , Borisy G G
Published: May 2004

During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole.
Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies.
Authors: Sealock R , Wray B E , Froehner S C
Published: May 2004

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.
Co-translational excision of alpha-glucose and alpha-mannose in nascent vesicular stomatitis virus G protein.
Authors: Atkinson P H , Lee J T
Published: May 2004

Membrane bound polysomes were prepared from HeLa cells infected with vesicular stomatitis virus (VSV), after pulse labeling with [3H]mannose for various times from 15 to 90 min. Oligosaccharides on nascent chains were released from peptides by treatment with endoglycosidase H and sized by high resolution Biogel P4 chromatography. Processing on some nascent chains proceeded to the removal of all three types of alpha-linked glucose and one alpha-1,2-mannose from the Glc3Man9GlcNAc precursor showing that the enzymes responsible were not only active on nascent chains but were present in the rough endoplasmic reticulum (RER). Incubation of cells for various times in cycloheximide, where chain elongation had ceased, made no difference to the profile of oligosaccharides on the nascent chains, and trimming proceeded no further than Man8GlcNAc2Asn . Carbonyl cyanide m-chlorophenylhydrazone (CCCP), an energy inhibitor reportedly able to block the transfer of glycoproteins from the RER, increases the amount of Man8-oligosaccharides on the nascent chains and also the amount of Glc3Man9GlcNAc precursor. On completed G protein in the RER fraction from which membrane bound polysomes were prepared, processing occurred to Man6 - but not to Man5GlcNAc sized oligosaccharides in the CCCP-treated cells. By contrast, processing to Man5GlcNAc oligosaccharides was observed in unfractionated control cells.
Genetics of microtubule systems.
Authors: Raff E C
Published: May 2004

In most eucaryotes the tubulin genes comprise small multigene families with approximately equal numbers of genes for alpha- and beta-tubulin, the structural proteins of microtubules. The recent isolation of tubulin mutations in several species is proving to be a powerful tool for examining the structure and function of specific sets of microtubules. In Drosophila melanogaster, genetic analysis of a testis-specific beta-tubulin gene has shown that a single tubulin gene product may fulfill a number of different microtubule functions. In addition to tubulin mutations, mutations in other genes whose products are involved in the regulation or structure of specific microtubule arrays have also been isolated. The combination of analysis of both classes of mutations is beginning to allow a molecular description of the construction and function of three-dimensional cellular structures. In addition, such studies may also shed light on the evolutionary pressures that gave rise to and serve to maintain small families of genes encoding very similar proteins.
Specific localization of the alpha-latrotoxin receptor in the nerve terminal plasma membrane.
Authors: Valtorta F , Madeddu L , Meldolesi J , Ceccarelli B
Published: May 2004

The receptor for alpha-latrotoxin, the major protein component of the black widow spider venom, was investigated by the use of the purified toxin and of polyclonal, monospecific anti-alpha-latrotoxin antibodies. Experiments on rat brain synaptosomes (where the existence of alpha-latrotoxin receptors was known from previous studies) demonstrated that the toxin-receptor complex is made stable by glutaraldehyde fixation. At saturation, each such complex was found to bind on the average five antitoxin antibody molecules. In frog cutaneous pectoris muscles, the existence of a finite number of high-affinity receptors was revealed by binding experiments with 125I-alpha-latrotoxin (Kd = 5 X 10(-10) M; bmax = 1.36 +/- 0.16 [SE] X 10(9) sites/mg tissue, dry weight). Nonpermeabilized muscles were first treated with alpha-latrotoxin, and then washed, fixed, dissociated into individual fibers, and treated with anti-alpha-latrotoxin antibodies and finally with rhodamine-conjugated sheep anti-rabbit antibodies. In these preparations, muscle fibers and unmyelinated preterminal nerve branches were consistently negative, whereas bright specific fluorescent images, indicative of concentrated alpha-latrotoxin binding sites, appeared in the junctional region. These images closely correspond in size, shape, and localization to endplates decorated by the acetylcholinesterase reaction. The presynaptic localization of the specific fluorescence found at frog neuromuscular junctions is supported by two sets of findings: (a) fluorescent endplate images were not seen in muscles that had been denervated; and (b) the distribution of fluorescence in many fibers treated with alpha-latrotoxin at room temperature was the one expected from swollen terminal branches. Swelling of terminals is a known morphological change induced by alpha-latrotoxin in this preparation. When muscles were treated with either proteolytic enzymes (trypsin, collagenase) or detergents (Triton X-100) before exposure to alpha-latrotoxin, the specific fluorescent endplate images failed to appear. Taken together these findings indicate that the alpha-latrotoxin receptor is an externally exposed protein highly concentrated in the nerve terminal plasma membrane. Its density (number per unit area) at the frog neuromuscular junction can be calculated to be approximately 2,400/micron2.
Transient N-acetylglucosamine in the biosynthesis of phytohemagglutinin: attachment in the Golgi apparatus and removal in protein bodies.
Authors: Vitale A , Chrispeels M J
Published: May 2004

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the lectin phytohemagglutinin (PHA) during seed development. The polypeptides of PHA are synthesized by endoplasmic reticulum-bound polysomes and co-translationally glycosylated, pass through the Golgi complex, and accumulate in protein bodies, which constitute the lysosomal compartment in these cells. Some of the high-mannose sidechains of PHA are modified in the Golgi complex, and in mature PHA they contain N-acetylglucosamine, mannose, fucose, and xylose in the molar ratios 2, 3.8, 0.6, and 0.5. The results reported here show that the Golgi complex is also the site of additional N-acetylglucosamine incorporation into the modified sidechains. When developing cotyledons are labeled with [3H]glucosamine and glycopeptides of PHA present in the Golgi complex isolated, the radioactivity can be released as [3H]N-acetylglucosamine by digestion of the glycopeptides with beta-N-acetylglucosaminidase, indicating that the residues are in a terminal position. Arrival of PHA in the protein bodies is followed by the slow removal of these terminal N-acetylglucosamine residues, resulting in a decrease in the Mr of the modified sidechains. The biosynthetic intermediates of the glycoproteins destined for the lysosomal compartments of animal cells contain high-mannose sidechains modified by phosphate groups covered by N-acetylglucosamine that is labile to mild acid treatment. When cotyledons are labeled with [32P]orthophosphate, there is no radioactivity in PHA obtained from any of the subcellular fractions. There is also no release of radioactivity when [3H]glucosamine-labeled glycopeptides obtained from PHA in the Golgi complex are subjected to mild acid hydrolysis. These results indicate that the sorting-signals and posttranslational processing steps for proteins that are transported to the lysosomal compartment are different in plant cells and animal cells.
Solvent properties of ground substance studied by cryomicrodissection and intracellular reference-phase techniques.
Authors: Horowitz S B , Miller D S
Published: May 2004

Water, sodium, potassium, ATP, amino acids, and sugars are not uniformly distributed in Rana pipiens oocytes. Concentration differences exist between nucleus (germinal vesicle) and ooplasm and between animal and vegetal ooplasmic regions. The mechanisms responsible for these differences were investigated using intracellular reference-phase (iRP) analysis. The iRP is an artificial "organelle" that has the solvent properties of a dilute salt solution and is in diffusional equilibrium with water and solutes present in other cellular compartments. Ooplasm/iRP solute distributions show that ooplasm differs from ordinary aqueous solutions--exhibiting both solute exclusion and solute binding. Yolk platelets are an important cause of this behavior, largely because their proteins are present as hydrate crystals, which are rich in anionic sites and which interact intensely with associated water. Because of yolk's abundance, it obscures the solvent and binding properties of ooplasmic ground substance. The oocyte nucleus is yolk and organelle free and the nuclear envelope is readily permeable. Consequently, nucleus/iRP solute concentration differences reflect the binding and solvent properties of nuclear ground substance. Nucleoplasm binds approximately 19 meq of potassium. Furthermore, the monosaccharides, 3-O-methylglucose, L-glucose, and D-xylose, are selectively excluded, their nucleus/iRP concentration ratios averaging about 0.7; ratios for other solutes studied are unity. We interpret monosaccharide exclusion to mean that nuclear ground substance water is different in its "instantaneous" structure from ordinary saline water. Because of this difference, hydrogen bond interaction between nuclear water and certain sterically restricted solutes, of which ringed monosaccharides are examples, is reduced. Some implications of modified ground substance water and selective solute exclusion are discussed.
A reevaluation of the structure of purified tubulin in solution: evidence for the prevalence of oligomers over dimers at room temperature.
Authors: Kravit N G , Regula C S , Berlin R D
Published: May 2004

We studied the molecular form of tubulin in solution by ultrafiltration, nondenaturing electrophoresis, and chemical cross-linking. Our results are not consistent with the generally-held belief that tubulin in solution is a 110,000-mol-wt dimer. Rather, tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. We first compared the filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC-tubulin) with aldolase (142,000 mol wt). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperature and at a concentration of 0.5 mg/ml was only 0.12, whereas under the same conditions the filterability of aldolase was 0.60. We determined the average effective molecular weight of tubulin from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective molecular weight of approximately 300,000. This molecular weight was significantly reduced at 10 degrees C, indicating that oligomers dissociated at low temperatures. Oligomers were also demonstrated by chemical cross-linking using glutaraldehyde, dimethyl suberimidate, and bis[2-(succinimidooxycarbonyoxy)ethyl] sulfone. In addition, PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE system at alkaline pH. Quantitative examination of the mobilities of these bands and of standard proteins revealed that the bands represented a series of oligomeric forms. Similar electrophoretic patterns were observed in solutions of tubulin containing microtubule-associated proteins (MAPs) but with a shift to a greater proportion of higher oligomers. Nondenaturing PAGE at pH 8.3 showed that a shift towards higher oligomers also occurred in the absence of MAPs as the concentration of tubulin was increased. This concentration-dependence of oligomerization at room temperature was further demonstrated by ultrafiltration. When solutions of PC-tubulin at concentrations less than 0.25 mg/ml were ultrafiltered, filterability increased as concentration decreased. Quantitative studies of filterability following progressive dilution or concentration showed that this process was completely and rapidly reversible. A diffuse pattern of PC-tubulin on nondenaturing PAGE at pH 7 was observed and is consistent with a mixture of oligomers in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)
Epithelial structure revealed by chemical dissection and unembedded electron microscopy.
Authors: Fey E G , Capco D G , Krochmalnic G , Penman S
Published: May 2004

Cytoskeletal structures obtained after extraction of Madin-Darby canine kidney epithelial cell monolayers with Triton X-100 were examined in transmission electron micrographs of cell whole mounts and unembedded thick sections. The cytoskeleton, an ordered structure consisting of a peripheral plasma lamina, a complex network of filaments, and chromatin-containing nuclei, was revealed after extraction of intact cells with a nearly physiological buffer containing Triton X-100. The cytoskeleton was further fractionated by extraction with (NH4)2SO4, which left a structure enriched in intermediate filaments and desmosomes around the nuclei. A further digestion with nuclease and elution with (NH4)2SO4 removed the chromatin. The stable structure that remained after this procedure retained much of the epithelial morphology and contained essentially all of the cytokeratin filaments and desmosomes and the chromatin-depleted nuclear matrices. This structural network may serve as a scaffold for epithelial organization. The cytoskeleton and the underlying nuclear matrix intermediate filament scaffold, when examined in both conventional embedded thin sections and in unembedded whole mounts and thick sections, showed the retention of many of the detailed morphological aspects of the intact cells, which suggests a structural continuum linking the nuclear matrix, the intermediate filament network, and the intercellular desmosomal junctions. Most importantly, the protein composition of each of the four fractions obtained by this sequential procedure was essentially unique. Thus, the proteins constituting the soluble fraction, the cytoskeleton, the chromatin fraction, and the underlying nuclear matrix-intermediate filament scaffold are biochemically distinct.
Beta 2-microglobulin induces intracellular transport of human class I transplantation antigen heavy chains in Xenopus laevis oocytes.
Authors: Severinsson L , Peterson P A
Published: May 2004

Human class I transplantation antigens are cell-surface-expressed molecules composed of one glycosylated, membrane-integrated heavy chain and one nonglycosylated, water-soluble subunit, beta 2-microglobulin (beta 2m). We have examined the intracellular transport of the two subunits by microinjecting mRNA into Xenopus laevis oocytes. Beta 2m, translated in oocytes, was transported and secreted into the medium in the absence of heavy chains whereas heavy chains were retained in the endoplasmic reticulum if not cotranslated with beta 2m. In the presence of beta 2m, heavy chains resisted digestion by endoglycosidase H (Endo H), suggesting that beta 2m promotes the transport of heavy chains from endoplasmic reticulum to the Golgi compartment. Pulse-chase experiments confirmed this notion. The possibility that heavy chains aggregate irreversibly when synthesized in the absence of beta 2m was ruled out and it is demonstrated that performed heavy chains will become transported once beta 2m is available. It is suggested that intracellular transport is controlled by structural features that are part of the transported polypeptide. If so, beta 2m but not heavy chains may possess such features.