bluuebunny/crossref_metadata_2025_split huggingface dataset:
Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes
Authors: Dirks R. W. , Rijke F. M. van de , Fujishita S. , Ploeg M. van der , Raap A. K. 
Published: April 2021

ABSTRACT
We have determined optimal conditions for the detection of mRNA sequences in cultured cells by non-radioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein.
The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.
A γ-tubulin-related protein associated with the microtubule arrays of higher plants in a cell cycle-dependent manner
Authors: Liu B. , Marc J. , Joshi H. C. , Palevitz B. A. 
Published: April 2021

ABSTRACT
An antibody specific for a conserved γ-tubulin peptide identifies a plant polypeptide of 58 kDa. γ-Tubulin antibody affinity purified from this polypeptide recognizes the centrosome in mammalian cells. Using immunofluorescence microscopy, we determined the distribution of thistubulin-related polypeptide during the complex changes in microtubule arrays that occur throughout the plant cell cycle. We report a punctate association of γ-tubulin-related polypeptide with the cortical microtubule array and the preprophase band. As cells enter prophase, γ-tubulin-related polypeptide accumulates around the nucleus and forms a polar cap from which early spindle microtubules radiate. During metaphase and anaphase, γ-tubulin-related polypeptide preferentially associates with kinetochore fibers and eventually accumulates at the poles. In telophase, localization occurs over the phragmoplast. Tubulin-related polypeptide appears to be excluded from the plus ends of microtubules at the metaphase plate and cell plate. Its distribution during the cell cycle may be significant in light of differences in the behavior and organization of plant microtubules. The identification of-tubulinrelated polypeptide could help characterize microtubule organizing centers in these organisms.
Mechanical properties of ciliary axonemes and membranes as shown by paddle cilia
Authors: Deiner Michael , Tamm Sidney L. , Tamm Signhild 
Published: April 2021

ABSTRACT
Cilia with a distal membrane expansion enclosing a coiled end of the axoneme (paddle cilia or discocilia) have been commonly reported in marine invertebrates. We recently showed that paddle cilia in molluscan veligers are artifacts of non-physiological conditions. Here we investigated the possible mechanisms of formation of paddle cilia under hypotonic conditions; particularly, whether a helical conformational change of doublet microtubules induced by Ca or proton flux is responsible. Typical paddle cilia are induced by hypotonic Ca-free solutions at normal or low pH, showing that axonemal coiling does not require Ca influx or proton efflux. In addition, Triton-demembranated straight axonemes do not coil in high Ca solutions. Most decisively, complete removal of paddle ciliary membranes with detergents, but not mere permeabilization, causes immediate uncoiling and straightening of the axonemes to approximately their original length before hypotonic treatment. These findings and other data show that axonemal coiling in paddles is due to membrane tensile stress acting on an elastic axoneme. Light and electron microscopy of paddles show that axonemes coil uniformly toward the direction of the effective stroke (doublets nos 5–6), even when beating is inhibited by sodium azide or glutaraldehyde before hypotonic treatment. This indicates that axonemes possess an intrinsic asymmetry of stiffness within the beat plane, independent of active microtubule sliding. Paddle cilia thus reveal important mechanical properties of ciliary axonemes and membranes that should be useful for understanding ciliary function.
GPI-anchored proteins associate to form microdomains during their intracellular transport in Caco-2 cells
Authors: Garcia Martine , Mirre Christian , Quaroni Andrea , Reggio Hubert , Le Bivic André 
Published: April 2021

ABSTRACT
In this study, we have investigated the possibility that glycosyl-phosphatidylinositol (GPI)-anchored proteins form insoluble membrane complexes in Caco-2 cells and that transmembrane proteins are associated with these complexes. GPI-anchored proteins were mainly resistant to Triton X-100 (TX-100) extraction at 4°C but fully soluble in n-octyl-glucoside. Resistance to Triton X-100 extraction was not observed in the endoplasmic reticulum but appeared during transport through the Golgi complex. It was not dependent upon N-glycosylation processing, or pH variation from 6.5 to 8.5, and was not affected by sterol-binding agents. Other apical or baso-lateral transmembrane proteins were well solubilized in TX-100, with the exception of sucrase-isomaltase, which was partly insoluble. We isolated a membrane fraction from Caco-2 cells that contained GPI-anchored proteins and sucrase-isomaltase but no antigen 525, a basolateral marker, or dipeptidylpeptidase IV, an apical one. These data suggest that GPI-anchored proteins cluster to form membrane microdomains together with an apical trans-membrane protein, providing a possible apical sorting mechanism for intestinal cells in vitro that might be related to apical sorting in MDCK cells, and that other mechanisms might exist to sort proteins to the apical membrane.
Odd chromosome movement and inaccurate chromosome distribution in mitosis and meiosis after treatment with protein kinase inhibitors
Authors: Nicklas R. Bruce , Krawitz Lawrence E. , Ward Suzanne C. 
Published: April 2021

ABSTRACT
Errors in chromosome orientation in mitosis and meiosis are inevitable, but normally they are quickly corrected. We find that such errors usually are not corrected in cells treated with protein kinase inhibitors. Highly inaccurate chromosome distribution is the result. When grasshopper spermatocytes were treated with the kinase inhibitor 6-dimethylaminopurine (DMAP), 84% of maloriented chromosomes failed to reorient; in anaphase, both partner chromosomes were distributed to the same daughter cell. These chromosomes were observed for a total of over 60 h, and not a single reorientation was seen. In contrast, in untreated cells, maloriented chromosomes invariably reoriented, and quickly: in 10 min, on average. A second protein kinase inhibitor, genistein, had exactly the same effect as DMAP. DMAP affected PtK1 cells in mitosis as it did spermatocytes in meiosis: improper chromosome orientations persisted, leading to frequent errors in distribution. We micromanipulated chromosomes in spermatocytes treated with DMAP to learn why maloriented chromosomes often fail to reorient. Reorientation requires the loss of improper microtubule attachments and the acquisition of new, properly directed kinetochore microtubules. Micromanipulation experiments disclose that neither the loss of old nor the acquisition of new microtubules is sufficiently affected by DMAP to account for the indefinite persistence of malorientations. Drug treatment causes a novel form of chromosome movement in which one kinetochore moves toward another kinetochore. Two kinetochores in the same chromosome or in different chromosomes can participate, producing varied, dance-like movements executed by one or two chromosomes. These kinetochore-kinetochore interactions evidently are at the expense of kinetochore-spindle interactions. We propose that malorientations persist in treated cells because the kinetochores have numerous, short microtubules with a free end that can be captured by a second kinetochore. Kinetochores capture each other’s kinetochore microtubules, leaving too few sites available for the efficient capture of spindle microtubules. Since the efficient capture of spindle microtubules is essential for the correction of errors, failure of capture allows malorientations to persist. Whether the effects of DMAP actually are due to protein kinase inhibition remains to be seen. In any case, DMAP reveals interactions of one kinetochore with another, which, though ordinarily suppressed, have implications for normal mitosis.
Identification of the epiplasmins, a new set of cortical proteins of the membrane cytoskeleton in Paramecium
Authors: Nahon Pierre , Coffe Gérard , Le Guyader Hervé , Darmanaden-Delorme Julienne , Jeanmaire-Wolf Rachel , Clérot Jean-Claude , Adoutte André 
Published: April 2021

ABSTRACT
In most ciliates, the epiplasm, a superficial cytoskeletal layer of variable thickness, both surrounds basal bodies and interacts tightly with adjacent membrane networks; it constitutes the predominant structure in Paramecium cell ghosts. Previous indirect data suggested several cortical proteins as potential constituents of the epiplasm. New sharp monoclonal antibodies presented in this paper, positive both on immunotransfers and in immunocytochemical tests carried out on permeabilized cells and ultrathin sections, definitively identify the epiplasmins: a set of about twenty protein bands ranging from 45 to 33 kDa and making up the bulk of the epiplasmic layer. The complete epiplasmin pattern characterized from gradient-purified cortex is also present in unfractionated whole cells, confirming that the pattern is not generated artifactually. Comparative one-step extractions, performed either in 1 M KI or in 4 M urea, solubilize the epiplasmins as a whole, indicating that all of them share very similar biochemical properties. Two-dimensional electrophoresis shows the great complexity of this epiplasmin group. Epiplasmin solu-bilization properties are discussed with respect to other models of membrane-cytoskeleton interaction developed among protists and metazoans and also to intermediate filaments, specially lamins. Immunofluorescent labelling combined with confocal microscopy permits a more detailed study of epiplasm formation at the level of the fission furrow, with new insights into two successive steps of epiplasm growth. A first series of interspecific reactions has been carried out with one of the anti-epiplasmin antibodies, yielding results which are discussed in an evolutionary framework.
Interphotoreceptor retinoid-binding protein (IRBP), a major 124 kDa glycoprotein in the interphotoreceptor matrix of Xenopus laevis Characterization, molecular cloning and biosynthesis
Authors: Gonzalez-Fernandez Federico , Kittredge Karen L. , Rayborn Mary E. , Hollyfield Joe G. , Landers Robert A. , Saha Margaret , Grainger Robert M. 
Published: April 2021

ABSTRACT
We have demonstrated that the neural retina of Xeno -pus laevis secretes into the extracellular matrix surrounding the inner and outer segments of its photoreceptors a glycoprotein containing hydrophobic domains conserved in mammalian interphotoreceptor retinoid-binding proteins (IRBPs). The soluble extract of the interphotoreceptor matrix contains a 124 kDa protein that crossreacts with anti-bovine IRBP immunoglobulins. In vitro [3H]fucose incorporation studies combined with in vivo light and electron microscopic autoradi-ographic analysis, showed that the IRBP-like glycoprotein is synthesized by the neural retina and secreted into the interphotoreceptor matrix. A 1.2 kb Xenopus IRBP cDNA was isolated by screening a stage 42 (swimming tadpole) AZap II library with a human IRBP cDNA under low-stringency conditions. The cDNA hybridizes with a 4.2 kb mRNA in adult Xenopus neural retina, tadpole heads as well as a less-abundant mRNA of the same size in brain. During development, IRBP and opsin mRNA expression correlates with photoreceptor differ-entiation. The translated amino acid sequence of the Xenopus IRBP clone has an overall 70% identity with the fourth repeat of the human protein. Sequence align-ment with the four repeats of human IRBP showed three highly conserved regions, rich in hydrophobic residues. This focal conservation predicts domains important to the protein’s function, which presumably is to facilitate the exchange of 11-cis retinal and all-trans retinol between the pigment epithelium and photoreceptors, and to the transport of fatty acids through the hydrophilic interphotoreceptor matrix.
Desmoyokin, a 680 kDa keratinocyte plasma membrane-associated protein, is homologous to the protein encoded by human gene AHNAK
Authors: Hashimoto Takashi , Amagai Masayuki , Parry David A. D. , Dixon Tony W. , Tsukita Sachiko , Tsukita Shoichiro , Miki Keizaburo , Sakai Kosuke , Inokuchi Yoshio , Kudoh Jun , Shimizu Nobuyoshi , Nishikawa Takeji 
Published: April 2021

ABSTRACT
We have obtained a monoclonal antibody (33A-3D) that specifically recognize desmoyokin, a 680 kDa desmosomal plaque protein that is well characterized in bovine muzzle epidermis. A cDNA clone (DY6, 3693 bp) was isolated by immunoscreening a mouse keratinocyte expression library with 33A-3D, and it was confirmed that DY6 has a partial coding sequence for desmoyokin. DY6 consists of highly homologous repeats about 128 residues long. Furthermore, the 128-residue repeats exhibit a quasi seven-residue substructure, which we believe will adopt an antiparallel β-sheet structure. Surprisingly, the amino acid sequence showed a significant homology with AHNAK, a newly identified human gene encoding a 700 kDa protein, which was suggested to be down-regulated in neuroblastoma. From its extensive homology, the similarity in both size and structure, and the identical patterns on Southern blot analysis of genomic DNAs, desmoyokin and AHNAK protein are thought to be identical. Although the desmoyokin/AHNAK protein is detected in a variety of cell types at both protein and mRNA levels, its distribution in keratinocytes (associated closely with cell membrane) is quite different from that in cells other than keratinocytes (distributed diffusely in the cytoplasm). These findings suggest that the desmoyokin/AHNAK protein is a ubiquitous molecule with a unique structure and appears to have different distributions (and probably different functions) among different cells.
Polarized distribution of Na,K-ATPase in honeybee photoreceptors is maintained by interaction with glial cells
Authors: Baumann Otto , Takeyasu Kunio 
Published: April 2021

ABSTRACT
Arthropod photoreceptors are polarized cells displaying distinct surface domains. The distribution of the Na,K-ATPase (sodium pump) over these domains was examined in the honeybee photoreceptor using a monoclonal antibody that specifically recognizes the sodium pump -subunit (∼100 kDa). We find that the sodium pump is restricted to sites of the nonreceptive photoreceptor surface closely juxtaposed to glial cells; no sodium pumps were detected on the glia-free domains of the nonreceptive surface and on the light-sensitive microvillar membranes. In order to determine the role of photoreceptor-glia contact in maintaining this polarized pump distribution, we assayed the distribution of the Na,K-ATPase after experimentally influencing photoreceptor-glia contact. Sodium pumps were present on the entire nonreceptive photoreceptor surface when photoreceptor-glia contact was removed by isolating the photoreceptors. Remodeling photoreceptor-glia contact by incubation in hyperosmotic saline caused a redistribution of sodium pumps on the photoreceptor surface corresponding to the redistribution of glial cells. We show, further, that both photoreceptor-glia contact and Na,K-ATPase distribution are independent of extracellular Ca2+. No junctional structures were observed at the borders between Na,K-ATPase-positive and Na,K-ATPase-negative membrane domains. Together, these results suggest that adhesion of glial cells to the photoreceptors plays a crucial role in the maintenance of the polarized distribution of Na,K-ATPase in the honeybee photoreceptors. The Ca2+-independent adhesion of glial cells to the photoreceptor surface may trap the pump molecules at the sites of photoreceptor-glia contact.
Distribution of snRNPs, splicing factor SC-35 and actin in interphase nuclei: immunocytochemical evidence for differential distribution during changes in functional states
Authors: Sahlas Demetrios J. , Milankov Kosta , Park Paul C. , Boni Umberto De 
Published: April 2021

ABSTRACT
Small nuclear ribonucleoproteins (snRNPs) play an integral role in the processing of pre-mRNA in eukaryotic nuclei. snRNPs often occur in a speckled intranuclear distribution, together with the non-snRNP splicing factor SC-35. snRNPs have also been shown to be associated with actin in the nuclear matrix, suggesting that both actin and snRNPs may be involved in the processing and transport of transcripts.
The work reported here was undertaken to compare the spatial relationship of snRNPs, SC-35, and intra-nuclear actin in neuronal and non-neuronal cell types. In undifferentiated PC12 cells and in non-neuronal cells growing in association with dorsal root ganglion neurons, confocal immunocytochemistry revealed a typical, speckled distribution of snRNP aggregates, which colocalized with the SC-35 splicing factor.
In contrast, a unique snRNP distribution was observed in dorsal root ganglion neurons in vitro and in PC12 cells differentiated by nerve growth factor. In nuclei of these cells, snRNPs were predominantly located at the periphery where they formed a spherical shell apposed to the nuclear envelope. Ultrastructural immunogold labelling of snRNPs in dorsal root ganglion neurons in vitro confirmed this distribution. In contrast, SC-35 remained distributed in a speckled pattern throughout nuclei of dorsal root ganglion neurons and PC12 cells, even in cases where snRNPs were almost exclusively positioned at the nuclear periphery.
In non-neuronal cells in dorsal root ganglion cultures and in undifferentiated PC12 cells, snRNP aggregates were frequently associated with actin aggregates, as determined by Nearest Neighbor Analyses. In PC12 cells, this spatial relationship was altered during nerve growth factor-induced differentiation, prior to the time at which these cells showed morphological evidence of differentiation. Specifically, Nearest Neighbor Analyses between snRNP and actin aggregates in PC12 cells exposed to nerve growth factor for 4 hours revealed that snRNP and actin aggregates exhibited a closer association than in undifferentiated cells.
These results suggest that sites of pre-mRNA processing and transcription may differ between cell types, and that the functions of snRNPs and actin within interphase nuclei may be related. The results also indicate that the distribution of snRNPs is dynamic and that it may depend upon the functional state of the cell as well as upon its state of differentiation.
Monolayer and aggregate cultures of rainbow trout hepatocytes: long-term and stable liver-specific expression in aggregates
Authors: Flouriot G. , Vaillant C. , Salbert G. , Pelissero C. , Guiraud J. M. , Valotaire Y. 
Published: April 2021

ABSTRACT
An aggregate culture system for rainbow trout hepatocytes was developed to study liver-specific mRNA expression. Maintenance of differentiated functions and morphology of hepatocytes were examined using both monolayer and aggregate systems. The rainbow trout estrogen receptor and vitellogenin genes were induced by estradiol and their mRNAs used as markers of cell differentiation during cell culture.
In monolayer culture, rainbow trout hepatocytes established very few cell-cell contacts in minimal media. The use of more complete media promotes cell-cell contacts and cell islet formation. Hepatocyte response to estradiol stimulation was generally lower than in vivo but a correlation between the degree of cellular organization and the intensity of the hormonal response was observed. However, in this system hepatocytes progressively lost their specific hormonal response between 5 and 10 days.
In aggregates with DMEM/F12 and Ultroser SF, cell-cell contacts were maximized and stabilized during at least one month. The levels of rainbow trout estrogen receptor and vitellogenin mRNAs induced by estradiol were stable and maintained at a level comparable to in vivo levels; vitellogenin synthesis and secretion remained fully functional for the duration of the culture.
A significant soluble keratin fraction in ‘simple’ epithelial cells Lack of an apparent phosphorylation and glycosylation role in keratin solubility
Authors: Chou Chih-Fong , Riopel Carrie L. , Rott Lusijah S. , Omary M. Bishr 
Published: April 2021

ABSTRACT
We studied the solubility of keratin polypeptides 8 and 18 (K8/18), which are the predominant intermediate filaments in the human colonic epithelial cell line HT29. We find that asynchronously growing cells (G0/G1 stage of the cell cycle) have a substantial pool of soluble keratin that constitutes approx. 5% of total cellular keratin. This soluble keratin pool was observed after immunoprecipitation of K8/18 from the cytosolic fraction of cells disrupted using three detergent-free methods. Several other cell lines showed a similar significant soluble cytosolic K8/18 pool. Arrest of HT29 cells in G2/M stage of the cell cycle was associated with a concurrent increase in keratin solubility. Comparison of K8/18 obtained from the soluble cytosolic fraction and the insoluble high-speed pellet fraction showed similar levels of phosphorylation and glycosylation and similar tryptic radiolabeled phospho- and glycopeptide patterns. Soluble K8/18 can form characteristic 10 nm filaments in vitro as determined by electron microscopy. Cross-linking of soluble K8/18 followed by immunoprecipitation resulted in dimeric and tetrameric forms, based on migration in SDS-polyacrylamide gels. In addition, cross-linked and native soluble K8/18 showed similar migration on nondenaturing gels and similar sedimentation after sucrose density gradient centrifugation. Our results indicate that simple epithelial keratins are appreciably more soluble than previously recognized. The soluble keratin form is assembly competent and appears to be primarily tetrameric. Although K8/18 solubility was found to increase during mitotic arrest, glycosylation and phosphorylation did not play an obvious role in generating the soluble fraction, suggesting an alternate mechanism for keratin solubility.