bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor.
Authors: Albini A , Allavena G , Melchiori A , Giancotti F , Richter H , Comoglio P M , Parodi S , Martin G R , Tarone G
Published: May 2004

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.
Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits.
Authors: Wayner E A , Carter W G
Published: May 2004

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)
The platelet glycoprotein IIb/IIIa-like protein in human endothelial cells promotes adhesion but not initial attachment to extracellular matrix.
Authors: Chen C S , Thiagarajan P , Schwartz S M , Harlan J M , Heimark R L
Published: May 2004

On platelets the membrane glycoprotein IIb/IIIa complex (GPIIb/IIIa) functions in adhesive interactions with fibrinogen, von Willebrand factor, and fibronectin. However, the function of GPIIb/IIIa-like proteins on endothelial cells, as well as the ligand(s) the complex binds, is unknown. Using a highly specific polyclonal antibody we have explored the function of GPIIb/IIIa-like proteins on human umbilical vein endothelial cells (HUVE). Analysis by immunoblotting shows that this antiserum recognizes the endothelial GPIIIa-like protein of the complex. The IgG fraction of the polyclonal antiserum and its Fab' fragments detach confluent and subconfluent HUVE from extracellular substrata. The effect of the anti-GPIIb/IIIa IgG is not toxic as the detached cells maintain their viability after trypsinization and replating. Anti-GPIIb/IIIa IgG does not inhibit HUVE binding to extracellular matrix or purified fibronectin in an attachment assay despite the presence of intact GPIIb/IIIa on HUVE detached from substrate by various methods. Apparently, the GPIIb/IIIa-like protein on HUVE is important in normal HUVE adhesion to the extracellular matrix, but it is not required in the initial attachment of HUVE to extracellular matrix.
Myelin-associated glycoprotein, a member of the L2/HNK-1 family of neural cell adhesion molecules, is involved in neuron-oligodendrocyte and oligodendrocyte-oligodendrocyte interaction.
Authors: Poltorak M , Sadoul R , Keilhauer G , Landa C , Fahrig T , Schachner M
Published: May 2004

A monoclonal antibody to the myelin-associated glycoprotein (MAG) was prepared and characterized to probe for the involvement of MAG in cell surface interactions among neural cells in vitro. The antibody reacts specifically with oligodendrocyte cell surface and myelin-rich brain regions as expected from previous investigations. Not all O4 antigen-positive oligodendrocytes express MAG in vitro. Fab fragments of the antibody interfere with neuron to oligodendrocyte and oligodendrocyte to oligodendrocyte adhesion, but not with oligodendrocyte to astrocyte adhesion. MAG-containing liposomes bind to the cell surfaces of the appropriate target cells by a mechanism that is specifically inhibitable by Fab fragments of monoclonal MAG antibodies, demonstrating that MAG is a neural cell adhesion molecule.
Renin, a secretory glycoprotein, acquires phosphomannosyl residues.
Authors: Faust P L , Chirgwin J M , Kornfeld S
Published: May 2004

Renin is an aspartyl protease which is highly homologous to the lysosomal aspartyl protease cathepsin D. During its biosynthesis, cathepsin D acquires phosphomannosyl residues that enable it to bind to the mannose 6-phosphate (Man-6-P) receptor and to be targeted to lysosomes. The phosphorylation of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase (phosphotransferase) occurs by recognition of a protein domain that is thought to be present only on lysosomal enzymes. In order to determine whether renin, being structurally similar to cathepsin D, also acquires phosphomannosyl residues, human renin was expressed from cloned DNA in Xenopus oocytes and a mouse L cell line and its biosynthesis and posttranslational modifications were characterized. In Xenopus oocytes, the majority of the renin remained intracellular and underwent a proteolytic cleavage which removed the propiece. Most of the renin synthesized by oocytes was able to bind to a Man-6-P receptor affinity column (53%, 57%, and 90%, in different experiments), indicating the presence of phosphomannosyl residues. In the L cells, the majority of the renin was secreted but 5-6% of the renin molecules contained phosphomannosyl residues as demonstrated by binding of [35S]methionine-labeled renin to the Man-6-P receptor as well as direct analysis of [2-3H]mannose-labeled oligosaccharides. Although the level of renin phosphorylation differed greatly between the two cell types examined, these results demonstrate that renin is recognized by the phosphotransferase and suggest that renin contains at least part of the lysosomal protein recognition domain.
Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers.
Authors: Doms R W , Keller D S , Helenius A , Balch W E
Published: May 2004

We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.
Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran.
Authors: Preston R A , Murphy R F , Jones E W
Published: May 2004

Concurrent with Riezman's report (Riezman, H. 1985, Cell. 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M. 1985. EMBO [Eur. Mol. Biol. Organ.] J. 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole. Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD. Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds. Dialysis was ineffective for removing the contaminants. After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles. Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography. In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 micrograms/ml) was an effective labeling agent for vacuoles. The rapid kinetics (0.28 pmol/min per 10(6) cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis. In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution.
Clathrin assembly involves a light chain-binding region.
Authors: Blank G S , Brodsky F M
Published: May 2004

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.
Antibodies to rat pancreas Golgi subfractions: identification of a 58-kD cis-Golgi protein.
Authors: Saraste J , Palade G E , Farquhar M G
Published: May 2004

A 58-kD cis-Golgi protein has been identified by generating polyclonal antibodies against heavy (cis) Golgi subfractions. Total microsomes isolated from rat pancreatic homogenates were subfractionated to yield a rough microsomal fraction (B1) and three smooth membrane subfractions (B2-B4) enriched in cis-, middle, and trans-Golgi elements, respectively. The heavy (cis) subfraction, B2 (d = 1.17 g/ml), was fractionated by Triton X-114 phase separation, and the proteins recovered in the detergent phase were used to immunize rabbits. One of the anti-B2 antibodies obtained gave a "Golgi"-staining pattern when screened by immunofluorescence on normal rat kidney cells and mouse RPC 5.4 myeloma cells. In rat pancreatic exocrine cells the antibody reacted with the plasmalemma as well as elements in the Golgi region. By immunoelectron microscopy, the antigen recognized by anti-B2 IgG was found to be restricted to cis-Golgi elements in myeloma cells where it was concentrated in the fenestrated cis-most cisterna and in some of the tubules and vesicles located along the cis face of the Golgi complex. By immunoprecipitation and immunoblotting, the anti-B2 IgG exclusively recognized a 58-kD protein in myeloma cells. The anti-B2 IgG reacted with several proteins in solubilized pancreatic B2 membranes, including a 58-kD protein, but affinity-purified anti-58-kD IgG reacted exclusively with the 58-kD protein. These results suggest that the 58-kD protein is a specific component of cis-Golgi membranes.
Changes in heterogeneous nuclear RNP core polypeptide complements during the cell cycle.
Authors: Leser G P , Martin T E
Published: May 2004

Mammalian heterogeneous nuclear RNP (hnRNP) subcomplexes are shown to be comprised of 14-17 basic A and B core group polypeptides (chrp) when subjected to two-dimensional immunoblot analysis. These proteins are normally confined to the nucleus but are distributed throughout the cell during mitosis. However, not all of the 17 protein spots are observed for all stages of the cell cycle. HeLa cell populations have been synchronized and the basic hnRNP core protein complement examined during S, G2, mitosis, and G1. During cell division several distinct chrp polypeptide species at 35 and 37 kD appear, while another of 37 kD and a chrp of 38 kD are diminished. These altered chrp complements are not due to any effects induced by thymidine treatment but appear to be physiological changes in the chrp polypeptide modification state. The new charge isomers found during mitosis are not the result of selective phosphorylation of the chrp polypeptides. However the nature of the modifications has yet to be determined. The mitosis-specific modified forms of the chrp polypeptides are found in the cytoplasmic fraction derived from mitotic cell populations. When this fraction is centrifuged upon sucrose density gradients the modified chrp polypeptides sediment from 30-200S in a distribution similar to that of hnRNP complexes isolated from the nuclei of randomly dividing cell populations. RNase digestion experiments indicate that the general substructure of the RNA/protein complexes in mitotic cell cytoplasm is similar to that of nuclear hnRNP isolated from unsynchronized cells or tissue.
Drosophilia spectrin. I. Characterization of the purified protein.
Authors: Dubreuil R , Byers T J , Branton D , Goldstein L S , Kiehart D P
Published: May 2004

We purified a protein from Drosophila S3 tissue culture cells that has many of the diagnostic features of spectrin from vertebrate organisms: (a) The protein consists of two equimolar subunits (Mr = 234 and 226 kD) that can be reversibly cross-linked into a complex composed of equal amounts of the two subunits. (b) Electron microscopy of the native molecule reveals two intertwined, elongated strands with a contour length of 180 nm. (c) Antibodies directed against vertebrate spectrin react with the Drosophila protein and, similarly, antibodies to the Drosophila protein react with vertebrate spectrins. One monoclonal antibody has been found to react with both of the Drosophila subunits and with both subunits of vertebrate brain spectrin. (d) The Drosophila protein exhibits both actin-binding and calcium-dependent calmodulin-binding activities. Based on the above criteria, this protein appears to be a bona fide member of the spectrin family of proteins.
Determination of cell division axes in the early embryogenesis of Caenorhabditis elegans.
Authors: Hyman A A , White J G
Published: May 2004

The establishment of cell division axes was examined in the early embryonic divisions of Caenorhabditis elegans. It has been shown previously that there are two different patterns of cleavage during early embryogenesis. In one set of cells, which undergo predominantly determinative divisions, the division axes are established successively in the same orientation, while division axes in the other set, which divide mainly proliferatively, have an orthogonal pattern of division. We have investigated the establishment of these axes by following the movement of the centrosomes. Centrosome separation follows a reproducible pattern in all cells, and this pattern by itself results in an orthogonal pattern of cleavage. In those cells that divide on the same axis, there is an additional directed rotation of pairs of centrosomes together with the nucleus through well-defined angles. Intact microtubules are required for rotation; rotation is prevented by inhibitors of polymerization and depolymerization of microtubules. We have examined the distribution of microtubules in fixed embryos during rotation. From these and other data we infer that microtubules running from the centrosome to the cortex have a central role in aligning the centrosome-nuclear complex.
Free calcium increases during anaphase in stamen hair cells of Tradescantia.
Authors: Hepler P K , Callaham D A
Published: May 2004

Changes in free calcium concentration [( Ca]) have been detected during anaphase in stamen hair cells of Tradescantia. Cells have been injected iontophoretically with the calcium sensitive metallochromic dye arsenazo III and changes in differential absorbance have been measured using a spinning wheel microspectrophotometer. The results obtained on single cells progressing from midmetaphase through to cytokinesis show that the free [Ca] first begins in increase after the initial separation of the sister chromosomes marking the onset of anaphase. The increase continues for 10-15 min while the chromosomes move to the poles; thereafter the [Ca] declines with the cell plate appearing about the time that the ion returns to its basal level. The close temporal correlation firstly between the rise in [Ca] and the breakdown of spindle microtubules (MTs) during anaphase and secondly, between the subsequent fall in [Ca] and the emergence of the MT-containing phragmoplast provides evidence consistent with the idea that endogenous fluctuations in [Ca] control the disassembly/assembly of MTs during mitosis.
Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells.
Authors: Malgaroli A , Milani D , Meldolesi J , Pozzan T
Published: May 2004

The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.
In vivo coassembly of a divergent beta-tubulin subunit (c beta 6) into microtubules of different function.
Authors: Joshi H C , Yen T J , Cleveland D W
Published: May 2004

alpha- and beta-Tubulin are encoded in vertebrate genomes by a family of approximately 6-7 functional genes whose polypeptide products differ in amino acid sequence. In the chicken, one beta-tubulin isotype (c beta 6) has previously been found to be expressed only in thrombocytes and erythroid cells, where it is assembled into a circumferential ring of marginal band microtubules. In light of its unique in vivo utilization and its divergent assembly properties in vitro, we used DNA transfection to test whether this isotype could be assembled in vivo into microtubules of divergent functions. Using an antibody specific to c beta 6, we have found that upon transfection this polypeptide is freely coassembled into an extensive array of interphase cytoplasmic microtubules and into astral and pole-to-chromosome or pole-to-pole microtubules during mitosis. Further, examination of developing chicken erythrocytes reveals that both beta-tubulins that are expressed in these cells (c beta 6 and c beta 3) are found as co-polymers of the two isoforms. These results, in conjunction with efforts that have localized various other beta-tubulin isotypes, demonstrate that to the resolution limit afforded by light microscopy in vivo microtubules in vertebrates are random copolymers of available isotypes. Although these findings are consistent with functional interchangeability of beta-tubulin isotypes, we have also found that in vivo microtubules enriched in c beta 3 polypeptides are more sensitive to cold depolymerization than those enriched in c beta 6. This differential quantitative utilization of the two endogenous isotypes documents that some in vivo functional differences between isotypes do exist.
The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments.
Authors: Horowits R , Podolsky R J
Published: May 2004

Electron microscopy was used to study the positional stability of thick filaments in isometrically contracting skinned rabbit psoas muscle as a function of sarcomere length at 7 degrees C. After calcium activation at a sarcomere length of 2.6 micron, where resting stiffness is low, sarcomeres become nonuniform in length. The dispersion in sarcomere length is complete by the time maximum tension is reached. A-bands generally move from their central position and continue moving toward one of the Z-discs after tension has reached a plateau at its maximum level. The lengths of the thick and thin filaments remain constant during this movement. The extent of A-band movement during contraction depends on the final length of the individual sarcomere. After prolonged activation, all sarcomeres between 1.9 and 2.5 micron long exhibit A-bands that are adjacent to a Z-disc, with no intervening I-band. Sarcomeres 2.6 or 2.7 micron long exhibit a partial movement of A-bands. At longer sarcomere lengths, where the resting stiffness exceeds the slope of the active tension-length relation, the A-bands remain perfectly centered during contraction. Sarcomere symmetry and length uniformity are restored upon relaxation. These results indicate that the central position of the thick filaments in the resting sarcomere becomes unstable upon activation. In addition, they provide evidence that the elastic titin filaments, which join thick filaments to Z-discs, produce almost all of the resting tension in skinned rabbit psoas fibers and act to resist the movement of thick filaments away from the center of the sarcomere during contraction.
Rigor crossbridges are double-headed in fast muscle from crayfish.
Authors: Bard F , Franzini-Armstrong C , Ip W
Published: May 2004

The structure of rigor crossbridges was examined by comparing rigor crossbridges in fast muscle fibers from glycerol-extracted abdominal flexor muscle of crayfish with those in "natively decorated" thin filaments from the same muscle. Natively decorated thin filaments were obtained by dissociating the backbone of the myosin filaments of rigor myofibrils in 0.6 M KCl. Intact fibers were freeze-fractured, deep-etched, and rotary shadowed; isolated filaments were either negatively stained or freeze dried and rotary shadowed. The crossbridges on the natively decorated actin maintain the original spacing and the disposition in chevrons and double chevrons for several hours, indicating that no rearrangement of the actomyosin interactions occurs. Thus the crossbridges of the natively decorated filaments were formed within the geometrical constraints of the intact myofibril. The majority of crossbridges in the intact muscle have a triangular shape indicative of double-headed crossbridge. The triangular shape is maintained in the isolated filaments and negative staining resolves two heads in a single crossbridge. In the isolated filaments, crossbridges are attached at uniform acute angles. Unlike those in insect flight muscle (Taylor et al., 1984), lead and rear elements of the double chevron may be both double-headed. Deep-etched images reveal a twisted arrangement of subfilaments in the backbone of the thick filament.
A transient intracellular coupling explains the facilitation of responses in the bioluminescent system of scale worms.
Authors: Bassot J M
Published: May 2004

Isolated elytra of polynoid worms emit a flash of bioluminescence when stimulated by an electric shock. With repeated stimulation, hundreds of flashes can be elicited which, in typical series, exhibit large and progressive variations. The amount of luminescence emitted by each flash first increases during a period of facilitation and then decreases exponentially during a longer period of decay. Through a microscope and image intensifier, the activity of individual microsources or photosomes was observed, using their fluorescence as a natural probe, in that its intensity is a function of the amount of luminescence previously emitted. Sequential observation showed a progressive and basically intracellular recruitment that correlated with facilitation. Facilitation and/or recruitment depended on the frequency of the stimulation. Recruitment proceeded among the photosomes of each photocyte, beginning with those of the cell periphery and progressing to those of the center. When the repetitive stimulation was interrupted and then resumed, the refacilitation was a function of the duration of the pause, and the pathway of recruitment duplicated that of the preceding sequence. It therefore appears that, within a given cell, individual photosomes can be either coupled and respond to stimulation or uncoupled and quiescent, that the coupled state has a basic lifetime of about 1 s which can be lengthened by reinforcement, and that this state must be established in a matter of milliseconds as a result of the stimulation. In preparing an increased response to a forthcoming stimulation, coupling acts as a short-term memory.
Cone outer segment morphogenesis: taper change and distal invaginations.
Authors: Eckmiller M S
Published: May 2004

Because cone outer segments (COS) are now known to be continually renewed, I reexamined COS morphogenesis in retinas of Xenopus tadpoles (prepared by standard histologic techniques and viewed by light and electron microscopy) to clarify how COS incorporate new membrane. I observed that developing COS underwent an unexpected shape change: they were always conical, but their taper (width divided by length) continually decreased. Ultrastructural examination revealed that many of the membrane foldings within distal COS were partial or incomplete, not extending across the full COS width but ending at variable distances from the ciliary side. Because these partial folds represented infoldings of the plasma membrane of an existing lamella, and they occurred at all COS levels except the base, I have termed them distal invaginations (DI). The completion of each DI increased COS length by one lamella but caused no noticeable change in local COS width; thus the formation of many DI throughout the distal COS presumably resulted in the observed decrease in overall COS taper. Based on these findings, I suggest that DI indicate growing membrane fronts and may represent sites where newly synthesized membrane is incorporated into COS. Because DI occur in developing and adult COS of various vertebrate species, I propose that DI formation plays an important role in the generation of COS taper during development and the remodeling of COS taper in mature cones after tip shedding.
Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii.
Authors: Pasquale S M , Goodenough U W
Published: May 2004

When Chlamydomonas reinhardtii gametes of opposite mating type are mixed together, they adhere by a flagella-mediated agglutination that triggers three rapid mating responses: flagellar tip activation, cell wall loss, and mating structure activation accompanied by actin polymerization. Here we show that a transient 10-fold elevation of intracellular cAMP levels is also triggered by sexual agglutination. We further show that gametes of a single mating type can be induced to undergo all three mating responses when presented with exogenous dibutyryl-cAMP (db-cAMP). These events are also induced by cyclic nucleotide phosphodiesterase inhibitors, which elevate endogenous cAMP levels and act synergistically with db-cAMP. Non-agglutinating mutants of opposite mating type will fuse efficiently in the presence of db-cAMP. No activation of mating events is induced by calcium plus ionophores, 8-bromo-cGMP, dibutyryl-cGMP, nigericin at alkaline pH, phorbol esters, or forskolin. H-8, an inhibitor of cyclic nucleotide-dependent protein kinase, inhibits mating events in agglutinating cells and antagonizes the effects of cAMP on non-agglutinating cells. Adenylate cyclase activity was detected in both the gamete cell body and flagella, with the highest specific activity displayed in flagellar membrane fractions. The flagellar membrane adenylate cyclase is preferentially stimulated by Mn++, unresponsive to NaF, GTP, GTP gamma S, AlF4-, and forskolin, and is inhibited by trifluoperazine. Cyclic nucleotide phosphodiesterase activity is also present in flagella. Our observations indicate that cAMP is a sufficient initial signal for all of the known mating reaction events in C. reinhardtii, and suggest that the flagellar cyclase and/or phosphodiesterase may be important loci of control for the agglutination-stimulated production of this signal.
Differential effects of temperature on cAMP-induced excitation, adaptation, and deadaptation of adenylate and guanylate cyclase in Dictyostelium discoideum.
Authors: Van Haastert P J
Published: May 2004

Extracellular cAMP induces excitation of adenylate and guanylate cyclase in Dictyostelium discoideum. Continuous stimulation with cAMP leads to adaptation, while cells deadapt upon removal of the cAMP stimulus. Excitation of guanylate cyclase by cAMP has a lag time of approximately 1 s; excitation of adenylate cyclase is much slower with a lag time of 30 s. Excitation of both enzyme activities is less than twofold slower at 0 degrees C than at 20 degrees C. Adaptation of guanylate cyclase is very fast (t1/2 = 2.4 s at 20 degrees C), and virtually absent at 0 degrees C. Adaptation of adenylate cyclase is much slower (t1/2 = 110 s at 20 degrees C) but not very temperature sensitive (t1/2 = 290 s at 0 degrees C). At 20 degrees C, deadaptation of adenylate cyclase is about twofold slower than deadaptation of guanylate cyclase (t1/2 = 190 and 95 s, respectively). Deadaptation of adenylate cyclase is absent at 0 degrees C, while that of guanylate cyclase proceeds slowly (t1/2 = 975 s). The results show that excitation, adaptation, and deadaptation of guanylate cyclase have different kinetics and temperature sensitivities than those of adenylate cyclase, and therefore are probably independent processes.
Morphology of astroglial cells is controlled by beta-adrenergic receptors.
Authors: Shain W , Forman D S , Madelian V , Turner J N
Published: May 2004

Astroglial cells in vivo and in vitro respond to hormones, growth factors, and neurotransmitters by changing from an epithelial-like to stellate morphology. We have studied the temporal relationship between receptor activation, second messenger mobilization, and morphological changes using LRM55 astroglial cells. Maintenance of an altered morphology required continuous beta-adrenergic receptor activation. These changes appeared to be mediated by cAMP since they were elicited by its analogue, dibutyryl cAMP, and by forskolin, a direct activator of adenylate cyclase. Changes in cell morphology may require a relatively small increase in intracellular cAMP, since receptor-stimulated changes in cAMP levels were transient and peaked approximately 5 min after receptor activation while changes in morphology took at least 30 min to reach a new steady state. Time-lapse videomicroscopy and high voltage electron microscopy indicated that receptor activation resulted in a sequence of morphological events. Time-lapse observations revealed the development and enlargement of openings through the cytoplasm associated with cytoplasmic withdrawal to the perinuclear region and process formation. Higher resolution high voltage electron microscopy indicated that the transition to a stellate morphology was preceded by the appearance of two distinct cytoplasmic domains. One contained an open network of filaments and organelles. The other was characterized by short broad cytoplasmic filaments. The first domain was similar to cytoplasm in control cells while the second was associated with the development and enlargement of openings through the cytoplasm and regions of obvious cytoplasmic withdrawal.
Interactions of a neuronal cell line (PC12) with laminin, collagen IV, and fibronectin: identification of integrin-related glycoproteins involved in attachment and process outgrowth.
Authors: Tomaselli K J , Damsky C H , Reichardt L F
Published: May 2004

Neuronal responses to extracellular matrix (ECM) constituents are likely to play an important role in nervous system development and regeneration. We have studied the interactions of a neuron-like rat pheochromocytoma cell line, PC12, with ECM protein-coated substrates. Using a quantitative cell attachment assay, PC12 cells were shown to adhere readily to laminin (LN) or collagen IV (Col IV) but poorly to fibronectin (FN). The specificity of attachment to these ECM proteins was demonstrated using ligand-specific antibodies and synthetic peptides. To identify PC12 cell surface proteins that mediate interactions with LN, Col IV, and FN, two different antisera to putative ECM receptors purified from mammalian cells were tested for their effects on PC12 cell adhesion and neuritic process outgrowth. Antibodies to a 140-kD FN receptor heterodimer purified from Chinese hamster ovarian cells (anti-FNR; Brown, P. J., and R. L. Juliano, 1986, J. Cell Biol., 103:1595-1603) inhibited attachment to LN and FN but not to Col IV. Antibodies to an ECM receptor preparation purified from baby hamster kidney fibroblastic cells (anti-ECMR; Knudsen, K. A., P. E. Rao, C. H. Damsky, and C. A. Buck, 1981, Proc. Natl. Acad. Sci. USA., 78:6071-6075) inhibited attachment to LN, FN, and Col IV, but did not prevent attachment to other adhesive substrates. In addition to its effects on adhesion, the anti-ECMR serum inhibited both PC12 cell and sympathetic neuronal process outgrowth on LN substrates. Immunoprecipitation of surface-iodinated or [3H]glucosamine-labeled PC12 cells with either the anti-FNR or anti-ECMR serum identified three prominent cell surface glycoproteins of 120, 140, and 180 kD under nonreducing conditions. The 120-kD glycoprotein, which could be labeled with 32P-orthophosphate and appeared to be noncovalently associated with the 140- and 180-kD proteins, cross reacted with antibodies to the beta-subunit (band 3) of the avian integrin complex, itself a receptor or receptors for the ECM constituents LN, FN, and some collagens.
Nucleated assembly of Chlamydomonas and Volvox cell walls.
Authors: Adair W S , Steinmetz S A , Mattson D M , Goodenough U W , Heuser J E
Published: May 2004

The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria.
Type III collagen can be present on banded collagen fibrils regardless of fibril diameter.
Authors: Keene D R , Sakai L Y , Bächinger H P , Burgeson R E
Published: May 2004

Monoclonal antibodies that recognize an epitope within the triple helix of type III collagen have been used to examine the distribution of that collagen type in human skin, cornea, amnion, aorta, and tendon. Ultrastructural examination of those tissues indicates antibody binding to collagen fibrils in skin, amnion, aorta, and tendon regardless of the diameter of the fibril. The antibody distribution is unchanged with donor age, site of biopsy, or region of tissue examined. In contrast, antibody applied to adult human cornea localizes to isolated fibrils, which appear randomly throughout the matrix. These studies indicate that type III collagen remains associated with collagen fibrils after removal of the amino and carboxyl propeptides, and suggests that fibrils of skin, tendon, and amnion (and presumably many other tissues that contain both types I and III collagens) are copolymers of at least types I and III collagens.
The tandemly repeated sequences of cartilage link protein contain the sites for interaction with hyaluronic acid.
Authors: Goetinck P F , Stirpe N S , Tsonis P A , Carlone D
Published: May 2004

The ternary complex involving link protein (LP), proteoglycan monomer, and hyaluronic acid (HA) is an important component of the extracellular matrix of cartilage. LP contains tandemly repeated sequences that were tested for their ability to interact with HA. A solid-phase assay was developed in which LP could specifically bind to immobilized HA. Detection of LP was by means of an antiserum directed against a peptide from the NH2-terminal half of LP. LP binding to HA could be inhibited with mAb 8A4 (Caterson, B., J. R. Baker, J. E. Christner, Y. Lee, and M. Lentz. 1985. J. Biol. Chem. 260:11348-11356). Using synthetic peptides that correspond to specific amino acid sequences of chicken LP (Deák, F., I. Kiss, K. J. Sparks, W. S. Argraves, G. Hampikian, and P. F. Goetinck. 1986. Proc. Natl. Acad. Sci. USA. 83:3766-3770) the epitopes for mAb 8A4 were determined to reside in peptides Gly217-Pro226 and Arg316-Arg325. These two peptides were also capable of inhibiting the interaction between LP and HA. The peptide Trp242-Val251 and Pro339-Val348 could also inhibit the interaction between LP and HA. All four peptides reside in the tandemly repeated domains of LP and they contain clusters of positively charged amino acids. Polylysine could not inhibit the interaction of LP with HA. The results indicate that the sites for interaction with HA are in the tandemly repeated sequences of LP and that there are four potential sites available for that interaction.
Nonrandom chromosome arrangements in germ line nuclei of Sciara coprophila males: the basis for nonrandom chromosome segregation on the meiosis I spindle.
Authors: Kubai D F
Published: May 2004

Meiosis I in males of the Dipteran Sciara coprophila results in the nonrandom distribution of maternally and paternally derived chromosome sets to the two division products. Based on an earlier study (Kubai, D.F. 1982. J. Cell Biol. 93:655-669), I suggested that the meiosis I spindle does not play a direct role in the nonrandom sorting of chromosomes but that, instead, haploid sets are already separated in prophase nuclei well before the onset of spindle formation. Here I report more direct evidence that this hypothesis is true; this evidence was gained from ultrastructural reconstruction analyses of the arrangement of chromosomes in germ line nuclei (prophase nuclei in spermatogonia and spermatocytes) of males heterozygous for an X-autosome chromosome translocation. Because of this translocation, the maternal and paternal chromosome sets are distinguishable, so it is possible to demonstrate that (a) the two haploid chromosome sets occupy distinct maternal and paternal nuclear compartments and that (b) nuclei are oriented so that the two haploid chromosome sets have consistent relationships to a well-defined cellular axis. The consequences of such nonrandom aspects of nuclear structure for chromosome behavior on premeiotic and meiotic spindles are discussed.
Biosynthesis, membrane association, and release of N-CAM-120, a phosphatidylinositol-linked form of the neural cell adhesion molecule.
Authors: He H T , Finne J , Goridis C
Published: May 2004

The neural cell adhesion molecule (N-CAM) of rodents comprises three distinct proteins of Mr 180,000, 140,000, and 120,000 (designated N-CAM-180, -140, and -120). They are expressed in different proportions by different tissues and cell types. but the individual contribution of each form to cell adhesion is presently unknown. Previous studies have shown that the two N-CAM species of higher relative molecular mass span the membrane whereas N-CAM-120 lacks a transmembrane domain and can be released from the cell surface by phosphatidylinositol-specific phospholipase C. In this report, we provided evidence that N-CAM-120 contained covalently bound phosphatidylinositol and studied N-CAM-120 from its biosynthesis to its membrane insertion and finally to its release from the cell surface. Evidence was presented showing that the lipid tail of N-CAM-120 contained ethanolamine as is the case for other lipid-linked molecules. The phospholipid anchor was attached to the protein during the first minutes after completion of the polypeptide chain. This process took place in the endoplasmic reticulum as judged from endoglycosidase H digestion experiments. Immediately after a 2-min pulse with [35S]methionine, we detected also a short-lived precursor that had not yet acquired the lipid tail. Pulse-chase studies established that N-CAM-120 was transported to the cell surface from which it was slowly released into the extracellular milieu. The molecules recovered in the incubation medium appeared to have lost all of their bound fatty acid but only around half of the ethanolamine. Upon fractionation of brain tissue, approximately 75% of N-CAM-120 was recovered with a membrane fraction and approximately 25% in a membrane-free supernatant. A small proportion (approximately 6%) was found to be resistant to extraction by non-ionic detergent. A major posttranslational modification of N-CAM is polysialylation. Our results showed that also N-CAM-120 was polysialylated in the young postnatal brain and released in this form from cultured cerebellar cells. The presence of N-CAM in a form that can be released from the cell surface and accumulates in the extracellular fluid suggests a novel mechanism by which N-CAM-mediated adhesion may be modulated.