bluuebunny/crossref_metadata_2025 huggingface dataset:
The characteristic which makes the cell-coded HSV-inducible U90 distinctive in transformed cells is its greatly increased half-life
Authors: Grassie M. , McNab D. , Macnab J. C. M.
Published: April 2021

A set of polypeptides detected in transformed cells of Mr values 90,000 (a doublet) 40,000 and 32,000 that are recognised by immunoprecipitation of L-[35S]methionine-labelled tumour cell lysates, with sera from tumour-bearing rats and with antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, were previously reported (Macnab et al., 1985, 1992; Hewitt et al., 1991). These proteins are cell-coded and cannot be pre-cipitated from similarly radiolabelled control cells. Two of these polypeptides are significantly induced by HSV-2 infection (Hewitt et al., 1991; Macnab et al., 1992). This paper further characterises one of these polypeptides, U90, and addresses which properties distinguish the behaviour of U90 in tumour cells, whether there is an equivalent in control cells and whether U90 can be induced without HSV replication. U90 can be immunoprecipitated from radiolabelled human cells which are capable of extended passage in culture, as well as from human tumour cells, rodent tumour cells and cells of different lineages, e.g. epithelial, fibroblastic and lymphoid. Purification of U90 and the subsequent production of monospecific antibodies revealed, by western blotting, a homologue of 90 kDa in control cells. Western blotting shows that HSV can increase the amount of the U90 homologue in these normal cells.

The absence of an immunoprecipitate of the U90 homologue from control cells is a result of the very short half-life (i.e. high turnover) of the protein in these cells (32.9 minutes) as opposed to tumour cells (about 13 hours). Induction of U90 is an early event after HSV infection, occurring in the absence of viral DNA synthesis, but requiring cellular RNA synthesis.

Infection with HSV-2 is not in itself sufficient to convert the homologue of U90 in control cells to a protein with a similar half-life to that of U90 in tumour cells, showing that a function other than HSV infection is involved in raising the half-life of the U90 homologue to tumour cell levels.
Expression of a mitochondrial stress protein in the protozoan parasite Leishmania major
Authors: Searle Susan , McCrossan Maria V. , Smith Deborah F.
Published: April 2021

The DNA sequence has been determined of a gene from Leishmania major that shares sequence identity with members of the eukaryotic heat shock protein (hsp) 70 gene family. The deduced open reading frame for translation shares a number of features common to hsp70 stress proteins, including conserved amino acids implicated in ATP binding and a putative calmodulin-binding site. In addition, the protein has an N-terminal sequence characteristic of a mitochondrial targeting signal. Specific antibodies to this protein, generated by the use of recombinant fusion peptides, recognise a 65 kDa molecule of pI 6.7. This molecule is constitutively expressed and localises to the mitochondrion in all stages of the parasite life cycle. These features suggest a role for this protein as a molecular chaperone in Leishmania.
Formation of the annexin II2p112 complex upon differentiation of F9 teratocarcinoma cells
Authors: Harder Thomas , Thiel Carsten , Gerke Volker
Published: April 2021

Murine teratocarcinoma F9 cells, which remain undifferentiated under standard cell culture conditions, can form cellular layers resembling early embryonic tissues upon induction of differentiation by retinoic acid and cyclic AMP. We have employed a combination of Northern and Western blot analyses to elucidate the regulation of expression of the tyrosine kinase substrate annexin II and its cellular ligand p11 during this differentiation process. Interestingly, the synthesis of the two subunits of the annexin II2p112 complex is not co-regulated during F9 differentiation. Annexin II, which is only very weakly expressed in undifferentiated F9 cells, shows a strong increase in the amount of transcript and protein once the differentiated phenotype is established. The level of this induction does not depend on the type of F9 differentiation. In contrast to the regulated synthesis of annexin II, a significant amount of p11 mRNA and protein is already present in the undifferentiated cells and remains constant during the differentiation of F9 cells. Immunofluorescence analysis reveals that annexin II and p11 are concentrated in the submembranous region of the differentiated F9 cells. In contrast, p11 is uniformly distributed throughout the cytoplasm of undifferentiated cells. p11 is translocated to the submembranous region of the undifferentiated F9 cells upon coexpression of an exogenous annexin II introduced by transient transfection. Thus the localization of annexin II and p11 to the submembranous cytoskeleton depends on the formation of the tight annexin II 2p112 complex.
Calmodulin and the contractile vacuole complex in mitotic cells of Dictyostelium discoideum
Authors: Zhu Qianlong , Liu Tongyao , Clarke Margaret
Published: April 2021

In amoebae of the eukaryotic microorganism Dictyostelium discoideum, calsmodulin is greatly enriched on membranes of the contractile vacuole complex, an osmoregulatory organelle. Antibodies specific for Dictyostelium calmodulin were used in the present study to immunolocalize the contractile vacuole complex in relation to the Golgi complex (detected with wheat germ agglutinin) and the microtubule organizing center (MTOC, detected with anti-tubulin antibodies). Cells were examined throughout the cell cycle. Double-staining experiments indicated that the contractile vacuole complex extended to the MTOC in interphase cells, usually, but not always, overlapping the Golgi complex. In metaphase and anaphase cells, the Golgi staining became diffuse, suggesting dispersal of Golgi membranes. In the same mitotic cells, anti-calmodulin antibodies labeled numerous small cortical vacuoles, indicating that the contractile vacuole complex had also become dispersed. When living mitotic cells were examined, the small cortical vacuoles were seen to be active, implying that all parts of the Dictyostelium contractile vacuole complex possess the ability to accumulate fluid and fuse with the plasma membrane. In contrast to observations reported for other types of cells, anti-calmodulin antibodies did not label the mitotic spindle in Dictyostelium. Despite this difference in localization, it is possible that vacuole-associated calmodulin in Dictyostelium cells and spindle-associated calmodulin in larger eukaryotic cells might perform a similar function, namely, regulating calcium levels.
Effect of shear stress upon localization of the Golgi apparatus and microtubule organizing center in isolated cultured endothelial cells
Authors: Coan Daniel E. , Wechezak Arlene R. , Viggers Robert F. , Sauvage Lester R.
Published: April 2021

Despite substantial evidence to suggest that directed cell migration is dependent upon positioning of the Golgi apparatus (GA) and the microtubule organizing center (MTOC), some controversy exists about whether such a relationship is relevant to endothelial cells under flow. The present study was undertaken to provide an indepth investigation of the relationship between shear stress, GA/MTOC localization, cell migration and nuclear position. Bovine carotid endothelial cells were exposed to 22 or 88 dynes/cm2 for 0.5, 2, 8 or 24 h, and localization of their GA/MTOCs was determined relative to the direction of flow. In no-flow control specimens, (0, 0.5, 2, 8 and 24 h) there was no change in the equally distributed GA/MTOCs. In contrast, during the first 8 h at 88 dynes/cm2 and by 2 h at 22 dynes/cm2 there was a significant increase in the number of cells with GA/MTOCs localized upstream to flow direction. The effect was temporary, however, and by 24 h there was no significant difference between the no-flow, 22 and 88 dynes/cm2 specimens. Analysis of GA/MTOC localization with respect to the direction of cell migration determined that 72.5% of no-flow cells possessed GA/MTOCs localized to the sides of nuclei nearest the direction of migration. In contrast, 64% of the specimens shear stressed over the same time period had GA/MTOCs localized to the sides of nuclei opposite the direction of migration. These results suggest that positioning of the GA/MTOC in endothelial cells is not dependent completely upon the direction of migration. Also, analysis of GA/MTOC localization with respect to nuclear positioning determined that restrictions within the perinuclear space are not solely responsible for GA/MTOC localization.
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

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

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.
Gamma-tubulin in differentiated cell types: localization in the vicinity of basal bodies in retinal photoreceptors and ciliated epithelia
Authors: Muresan V. , Joshi H.C. , Besharse J.C.
Published: April 2021

gamma-Tubulin, a newly discovered member of the tubulin superfamily required for microtubule nucleation, is associated with the centrosome(s) throughout the vertebrate cell cycle. We have used a polyclonal antibody, generated against a highly conserved segment of gamma-tubulin, to localize this protein in postmitotic, ciliated cells, in which the major microtubule organizing centers are the basal bodies. Single-cilium photoreceptor cells from bovine retina contained a strongly immunoreactive species, with molecular characteristics of gamma-tubulin, in association with a detergent-resistant, cytoskeletal fraction devoid of cytoplasmic microtubules. gamma-Tubulin was discretely localized throughout the basal body region, extending opposite to the axonemal shaft, in mechanically detached rod outer segments and whole-mounted, connecting cilium-derived axonemes. In multiciliated epithelia from bovine trachea and oviduct, gamma-tubulin immunoreactivity was detected at the base of the cilia, where basal bodies are located. These results suggest that this key centrosomal protein of mitotically active cells is also an integral component of microtubule organizing centers, required for the generation of the microtubule network in terminally differentiated cells.
Mechanical properties of ciliary axonemes and membranes as shown by paddle cilia
Authors: Deiner Michael , Tamm Sidney L. , Tamm Signhild
Published: April 2021

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

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

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

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

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

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

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

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

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

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.
An in vitro model for the analysis of intestinal brush border assembly I. Ultrastructural analysis of cell contact-induced brush border assembly in Caco-2BBe cells
Authors: Peterson Michelle D. , Mooseker Mark S.
Published: April 2021

Intestinal epithelial cells assemble and maintain a polarized, highly organized membrane-cytoskeleton array, the brush border. We describe an in vitro, cell contact-induced brush border assembly model using the Caco-2BBe clones. Subconfluent cells were ‘depolarized’ by brief passage through suspension culture in the presence of cytochalasin D and re-plated on filters at high density in low-Ca2+ medium. Upon return to regular medium, these small, rounded cells with bleb-like protrusions formed, over the course of 19 days, a polarized monolayer of tall, columnar cells with a well-defined brush border. Ultrastructural changes were documented by both transmission and scanning electron microscopy. The earliest events of microvillar assembly coincided with a short period of cell aggregation. Inter-cellular cysts were occasionally observed within these aggregates, and junction formation between cells which had no contact with the filter was also observed. Monolayer formation was completed within 48 hours, and cell height steadily increased approximately 3.5-fold over 19 days. Concurrent with monolayer formation and the increase in cell height, sparse microvilli with a few actin core filaments gradually became more dense and better organized. By the third day, the actin core bundles had begun to extend into the subjacent cytoplasm, while terminal web assembly was underway at five days. The mature morphology of the brush border was first observed at nine days, although cell height and microvillar density continued to increase during the subsequent ten days. Microvillar density rose approximately nine-fold throughout brush border assembly in the Caco-2BBe cells. With the exception of the formation of cellular aggregates at the onset of the time course, this sequence of morphological changes is comparable to that observed during brush border assembly in embryonic intestinal epithelial cells. The Caco-2BBe assembly model provides a useful system in which to investigate various molecular aspects of brush border assembly.
Inhibition of protein synthesis separates autophagic sequestration from the delivery of lysosomal enzymes
Authors: Lawrence B. Paige , Brown William J.
Published: April 2021

To investigate the role of newly synthesized proteins during autophagic sequestration and degradation, the effects of protein synthesis inhibition on autophagic vacuole (AV) formation and degradation were analyzed. The inhibition of protein synthesis was found to separate autophagic sequestration from the delivery of lysosomal enzymes to (AVs). Pretreatment with cycloheximide for ≥ 3 h caused a drastic inhibition of autophagy-induced degradation. Surprisingly, morphological analyses showed that the inhibition of protein synthesis for up to 12 h did not block the formation of nascent AVs; however, it did prevent their conversion into degradative AVs. Using immunoperoxidase cytochemistry with an antibody against cathepsin D and labeling of lysosomes with endocytosed colloidal gold, we found that the nascent AVs that formed during prolonged cycloheximide pretreatment had not received lysosomal markers. The inhibition of autophagic degradation and lysosomal enzyme delivery were rapidly reversed following the removal of cycloheximide. These results suggest that there is a fairly rapid turnover of protein(s) that are necessary for lysosomal fusion, but that the initial formation of AVs is independent of new protein synthesis for a long period of time.
The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain
Authors: Boschelli Frank , Uptain Susan M. , Lightbody James J.
Published: April 2021

The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints. Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine. Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed. The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain. Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity. These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.
Transitions between in situ and isolated chromatin
Authors: Giannasca P. J. , Horowitz R. A. , Woodcock C. L.
Published: April 2021

We show that the mechanism by which chromatin displaying higher-order structure is usually isolated from nuclei involves a transition to an extended nucleosomal arrangement. After being released from nuclei, chromatin must refold in order to produce the typical chromatin fibers observed in solution. For starfish sperm chromatin with a long nucleosome repeat (222 bp), isolated fibers are significantly wider than those in the nucleus, indicating that the refolding process does not regenerate the native higher-order structure. We also propose that for typical eukaryotic nuclei, the concept that the native state of the (inactive) bulk of the genome is a chromatin fiber with defined architecture be reconsidered.
Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic map located at the spindle pole and characterized as a homologous protein to NuMA
Authors: Maekawa Takami , Kuriyama Ryoko
Published: April 2021

Using a human autoantibody, SP-H, we identified a 200-230 kDa mitotic MAP in a variety of mammalian cell lines which shows affinity for the minus end of microtubules and also becomes associated with the spindle pole during mitosis. To examine the detailed structure and functional organization of the protein, the gene coding for the end-specific MAP was isolated and characterized by screening a human placenta gt11 expression library using SP-H as a probe. Overlapping cDNA clones, which covered the entire length of the coding region of the SP-H antigen, were obtained. Polyclonal antibodies raised against fusion proteins generated from non-overlapping cDNA fragments stained the HeLa SP-H antigen in interphase and mitotic cells, and recognized a single 215 kDa band on immunoblots, as did the original SP-H antibody. Analysis of the nucleotide sequence revealed a 7,091 nucleotide sequence with an open reading frame of 6,345 nucleotides encoding a 2,115 amino acid polypeptide with a calculated molecular mass of 238,376 Da. The predicted amino acid sequence showed the protein to be composed of an -helical domain, flanked by globular domains located at the amino and carboxy termini. The sequence contained five repeats of the hypothetical leucine zipper motif: one is in the N-terminal globular domain, and four are in the central -helical stalk. Comparison with other sequences in the database shows that the SP-H antigen is identical to the NuMA protein reported by Yang et al. (1992)J. Cell Biol. 116, 1303-1317, but there are differences between the SP-H antigen and NuMA sequence reported by Compton et al. (1992)J. Cell Biol. 116, 1395-1408. cDNA inserts of the truncated SP-H antigen were expressed in both insect Sf9 cells and in cultured mammalian cells. The recombinant protein corresponding to the C-terminal half of the protein was restricted to the nucleus, whereas the N-terminal half of the protein was localized in the cytoplasm, suggesting the presence of a nuclear translocation signal(s) in the C-terminal domain. The C-terminal polypeptide expressed in mitotic COS cells was shown to specifically localize at the spindle pole. Microtubule-binding assays using in vitro transcribed/translated polypeptide products from different domains of the SP-H antigen further suggested that the SP-H antigen interacts with microtubules through the globular domain at the C-terminus.
Avian pluripotent haemopoietic progenitor cells: detection and enrichment from the para-aortic region of the early embryo
Authors: Cormier Françoise
Published: April 2021

In the avian embryo, the wall of the aorta is a site where haemopoiesis occurs in large diffuse foci from day 3 to day 10. In contrast to haemopoiesis in other organs of the embryo, para-aortic haemopoiesis is sustained by stem cells, which emerge in situ. Previous studies have demonstrated that the para-aortic region from the day-4 chick embryo harbours committed myeloid progenitors and committed erythroid progenitors. The present paper reports the in vitro development of para-aortic progenitors with both myelomonocytic and erythroid potentialities. Three types of myelo-erythroid progenitors were observed, giving rise to erythroblasts and monocytes, to erythroblasts and granulocytes, or to erythroblasts, monocytes and granulocytes. Their frequency in the para-aortic cell suspension was 1 per 10,000 cells. In cell sorting experiments, they co-sorted with committed progenitors in the cell population that immunolabeled with the VI-A2 monoclonal antibody, which is specific for chicken haemopoietic cells. Cell sorting also demonstrated that these multipotential progenitors did not express the BEN cell surface molecule, in contrast to late myeloid progenitors. The BEN molecule belongs to the immunoglobulin superfamily and is expressed by haemopoietic progenitors from bone marrow, selective sets of neurons and epithelial cells from the bursa of Fabricius. The myelo-erythroid progenitors were enriched 4 times in the VI-A2-positive cell population, and 2 to 5 times in the BEN-negative population. These results represent the first in vitro demonstration of avian normal myelo-erythroid progenitors.
α3β1 Integrin is moved into focal contacts in kidney mesangial cells
Authors: Grenz Harald , Carbonetto Salvatore , Goodman Simon L.
Published: April 2021

The movement of integrins into focal adhesive structures accompanies cell attachment to extracellular matrix. The kinetics of incorporation of integrins into focal contacts was studied during attachment to matrix of mesangial cells of the kidney glomerulus. On collagen, fibronectin, laminin and vitronectin, the number and intensity of talin-focal contacts increased with time. Talin-containing focal contacts were present in mesangial cells within 2 h of plating and in control cells (HT1080 and Rugli) within 1 h. Integrin α-chains colocalized with talin, dependent on the matrix substrate. The attachment, spreading and organization of integrin into focal contacts was not affected when endogenous protein synthesis was suppressed with cycloheximide.

In Rugli, α1 β1 organized into focal contacts on collagen and laminin, while in HT1080 α2 β1 organized on collagen type I, α5 β1 on fibronectin, α6 β1 on laminin, and α3 β1 and α4 β1 were diffusely distributed on all substrates. These distributions mirrored the usage and expression patterns previously established for integrins in these cells and was as predicted from the literature. In mesangial cells, however, α3 β1 was also organized into prominent focal contact arrays on collagen, fibronectin, EHS and human placental laminins, but not on vitronectin, while α6 β1 was not organized.

Initial attachment and spreading of mesangial cells was absolutely dependent on divalent cations. Mg2+ and Mn2+ supported attachment on all substrates, while Ca2+ stimulated attachment on laminin (E8), fibronectin and vitronectin. The data suggest that the functional integrins on mesangial cells include α1 β1 (on collagen and laminin) α2 β1 (on collagen), α5 β1 (on fibronectin) and αV β3 (on vitronectin). However, mesangial cells do not use α6 β1 on laminin, and the data support a role for α3 β1 as putative receptor for fibronectin, collagen and laminin.
Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D
Authors: Friederich E. , Kreis T. E. , Louvard D.
Published: April 2021

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca2+-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the ‘random’ nucleation of actin filaments throughout the cytoplasm.

On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the prefered end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxyterminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalain D in living cells can be modulated by actin-binding proteins.