bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutations in the fission yeast silencing factors clr4
+ and rik1
+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function
Authors: Ekwall Karl , Nimmo Elaine R. , Javerzat Jean-Paul , Borgstrøm Britta , Egel Richard , Cranston Gwen , Allshire Robin 
Published: April 2021

ABSTRACT
Transcriptional silencing is known to occur at centromeres, telomeres and the mating type region in the nucleus of fission yeast, Schizosaccharomyces pombe. Mating-type silencing factors have previously been shown also to affect transcriptional repression within centromeres and to some extent at telomeres. Mutations in the clr4+, rik1+ and swi6+ genes dramatically reduce silencing at certain centromeric regions and cause elevated chromosome loss rates. Recently, Swi6p was found to co-localise with the three silent chromosomal regions. Here the involvement of clr4+, rik1+ and swi6+ in centromere function is investigated in further detail. Fluorescence in situ hybridisation (FISH) was used to show that, as in swi6 mutant cells, centromeres lag on late anaphase spindles in clr4 and rik1 mutant cells. This phenotype is consistent with a role for these three gene products in fission yeast centromere function. The Swi6 protein was found to be delocalised from all three silent chromosomal regions, and dispersed within the nucleus, in both clr4 and rik1 mutant cells. The phenotypic similarity observed in all three mutants is consistent with the products of both the clr4+ and rik1+ genes being required to recruit Swi6p to the centromere and other silent regions. Mutations in clr4, rik1 and swi6 also result in elevated sensitivity to reagents which destabilise microtubules and show a synergistic interaction with a mutation in the β-tubulin gene (nda3). These observations suggest that clr4+ and rik1+ must play a role in the assembly of Swi6p into a transcriptionally silent, inaccessible chromatin structure at fission yeast centromeres which is required to facilitate interactions with spindle microtubules and to ensure normal chromosome segregation.
Cell surface expression of a functional rubella virus E1 glycoprotein by addition of a GPI anchor
Authors: Bernasconi Eric , Fasel Nicolas , Wittek Riccardo 
Published: April 2021

ABSTRACT
Rubella virus (RV) envelope glycoproteins E1 and E2 are targeted to the Golgi as heterodimers. While E2 contains a transmembrane Golgi retention signal, E1 is arrested in a pre-Golgi compartment in the absence of E2, and appears to require heterodimerization in order to reach the Golgi. Various forms of E1 with deletions in the ectodomain or lacking the cytoplasmic (CT) and transmembrane (TM) domains, as well as the 29 C-terminal amino acid residues of the ectodomain were also retained intracellularly. We therefore investigated the possibility of targetting E1 to the plasma membrane by addition of a glycosylphosphatidylinositol (GPI) anchor. We found that E1GPI was transported to the cell surface where it retained the hem-adsorption activity characteristic of the wild-type E1/E2 heterodimer. Furthermore, coexpression of a mammalian GPI-specific phospholipase D (GPI-PLD) resulted in the release of E1GPI and in constitutive expression of a soluble form of E1. This study thus demonstrates that the GPI anchor has a dominant effect over the E1 pre-Golgi retention signal and that E1 is sufficient for hemadsorption.
The osteoclast generation: an in vitro and in vivo study with a genetically labelled avian monocytic cell line
Authors: Solari F. , Flamant F. , Cherel Y. , Wyers M. , Jurdic P. 
Published: April 2021

ABSTRACT
Osteoclasts are multinucleate giant cells responsible for bone resorption. Osteoclast precursors are hematopoietic mononucleate cells, which give rise to osteoclasts after fusion. Nevertheless, the precise stage of differentiation where osteoclast precursors diverge from other hematopoi-etic lineages is still debated. We describe here both in vitro and in vivo approaches to the study of the osteoclast differ-entiation pathway.
We used cells of the BM2 avian monocytic cell line, which are able to differentiate into macrophages both in vitro and in vivo. In order to follow the progeny of BM2 monocytes, we have derived a BM2 cell clone expressing the nlslacZ gene (BM2nlslacZ) which has still retained the main features of the parental cell line.
In vitro, when BM2nlslacZ cells were triggered toward macrophages, they participated in the formation of multi-nucleate osteoclast-like cells as seen by their blue nuclei. Furthermore, when BM2nlslacZ cells were injected into the blood stream of chicken embryos, they could give rise to blue nucleate macrophages in the bone marrow, as well as to osteoclasts with blue nuclei in bone.
Finally, we have shown that fusion of tagged mononucleate precursor cells not only occurs with other mononucleate precursor cells but also with mature multinucleate osteoclasts.
This work shows that cells already engaged in the late stages of the monocytic differentiation pathway are able to differentiate into osteoclasts and that osteoclast divergence takes place after the monocyte stage.
Localisation of the Ki-67 antigen within the nucleolus: Evidence for a fibrillarin-deficient region of the dense fibrillar component
Authors: Kill Ian R. 
Published: April 2021

ABSTRACT
The Ki-67 antigen is detected in proliferating cells in all phases of the cell division cycle. Throughout most of inter-phase, the Ki-67 antigen is localised within the nucleolus. To learn more about the relationship between the Ki-67 antigen and the nucleolus, we have compared the distribution of Ki-67 antibodies with that of a panel of antibodies reacting with nucleolar components by confocal laser scanning microscopy of normal human dermal fibroblasts in interphase stained in a double indirect immunofluor-escence assay. During early G1, the Ki-67 antigen is detected at a large number of discrete foci throughout the nucleoplasm, extending to the nuclear envelope. During S-phase and G2, the antigen is located in the nucleolus. Double indirect immunofluorescence studies have revealed that during early to mid G1 the Ki-67 antigen is associated with reforming nucleoli within discrete domains which are distinct from domains containing two of the major nucleolar antigens fibrillarin and RNA polymerase I.
Within mature nucleoli the Ki-67 antigen is absent from regions containing RNA polymerase I and displays only partial co-localisation within domains containing either fib-rillarin or B23/nucleophosmin. Following disruption of nucleolar structure, induced by treatment of cells with the drug 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole or with actinomycin D, the Ki-67 antigen translocates to nucleoplasmic foci which are associated with neither fib-rillarin nor RNA polymerase I. However, in treated cells the Ki-67 Ag remains associated with, but not co-localised to, regions containing B23/nucleophosmin. Our observa-tions suggest that the Ki-67 antigen associates with a fib-rillarin-deficient region of the dense fibrillar component of the nucleolus. Integrity of this region is lost following either nucleolar dispersal or nucleolar segregation.
Mutations which block the binding of calmodulin to Spc110p cause multiple mitotic defects
Authors: Stirling Douglas A. , Rayner Timothy F. , Prescott Alan R. , Stark Michael J. R. 
Published: April 2021

ABSTRACT
We have generated three temperature-sensitive alleles of SPC110, which encodes the 110 kDa component of the yeast spindle pole body (SPB). Each of these alleles carries point mutations within the calmodulin (CaM) binding site of Spc110p which affect CaM binding in vitro; two of the mutant proteins fail to bind CaM detectably (spc110-111, spc110-118) while binding to the third (spc110-124) is tem-perature-sensitive. All three alleles are suppressed to a greater or lesser extent by elevated dosage of the CaM gene (CMD1), suggesting that disruption of CaM binding is the primary defect in each instance. To determine the consequences on Spc110p function of loss of effective CaM binding, we have therefore examined in detail the progression of synchronous cultures through the cell division cycle at the restrictive temperature. In each case, cells replicate their DNA but then lose viability. In spc110-124, most cells duplicate and partially separate the SPBs but fail to generate a functional mitotic spindle, a phenotype which we term ‘abnormal metaphase’. Conversely, spc110-111 cells initially produce nuclear microtubules which appear well-organised but on entry into mitosis accumulate cells with ‘broken spindles’, where one SPB has become completely detached from the nuclear DNA. In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.
Fibronectin regulates calvarial osteoblast differentiation
Authors: Moursi Amr M. , Damsky Caroline H. , Lull Jonathan , Zimmerman Deborah , Doty Stephen B. , Aota Shin-ichi , Globus Ruth K. 
Published: April 2021

ABSTRACT
The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of α5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.
Differential expression and cell envelope incorporation of small proline-rich protein 1 in different cornified epithelia
Authors: Jarnik Michal , Kartasova Tonja , Steinert Peter M. , Lichti Ulrike , Steven Alasdair C. 
Published: April 2021

ABSTRACT
In the final stages of terminal differentiation in the epidermis and other squamous epithelia, a ∼15 nm thick protein layer called the cornified cell envelope (CE) assembles on the keratinocytes’ inner surface. Its constituent proteins are covalently crosslinked by the action of transglutaminases. Recent studies have indicated that the expression of CE precursor proteins may vary in different tissues. To investigate such variations further, we have studied the CEs of two different keratinizing epithelia of mouse: epidermis and forestomach, with particular focus on their contents of loricrin and the small proline-rich proteins (SPRs). To this end, we have applied electron microscopic immunocytochemistry and estimated the CE protein compositions by mathematical modeling of their amino acid compositions. Ultrastructurally, forestomach resembles the epidermis in having well defined cornified and granular layers. Minor but significant differences are: in forestomach, striated material resembling lamellar granules is intercalated between the cornified squames; and in forestomach granular layer cells, loricrin-containing L-granules are more abundant, and filaggrin-containing F-granules less abundant than in epidermis.
In forestomach, dense labeling with anti-SPR1 antibody was observed at the margin of cornified layer cells; and in the granular layer, diffuse but positive labeling of both cytoplasm and nucleus. In contrast, epidermis was uniformly negative. Isolated forestomach CEs (but not epidermal CEs), labeled positively on the cytoplasmic side, consistent with the presence of covalently crosslinked SPR1. Our compositional analysis predicts the content of loricrin in forestomach CEs to be very high (∼65%), as in the epidermis, and accompanied by ∼18% content of total SPRs. Of these, a substantial proportion should be SPR1, according to our immunolabeling data. In contrast, epidermal CEs are calculated to have a much lower amount of SPRs or SPR-like proteins (∼8%), with a negligible content of SPR1. Thus both kinds of CEs have loricrin as their major constituent but differ in their respective complements of SPRs, which are thought to inter-connect loricrin molecules in the final phase of CE assembly. Applying a basic concept of materials science, it may be that the observed differences in their SPR contents reflect differences in the mechanical and chemical properties required for the function of the respective CEs.
Active RNA polymerases are localized within discrete transcription ‘factories’ in human nuclei
Authors: Iborra Francisco J. , Pombo Ana , Jackson Dean A. , Cook Peter R. 
Published: April 2021

ABSTRACT
Nascent transcripts in permeabilized HeLa cells were elongated by ∼30-2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in ∼2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription ‘factories’. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.
Dynamics of calcium regulation in Paramecium and possible morphogenetic implication
Authors: Prajer Malgorzata , Fleury Anne , Laurent Michel 
Published: April 2021

ABSTRACT
This paper is the first report of the use of a fluorescent indicator (Dextran-coupled calcium green-1) for imaging of cytosolic free calcium in ciliate cells. Using this technique in Paramecium, we show that a very transient increase in the mean intracellular calcium concentration accompanied exocytosis. It has long been postulated based on indirect experimental evidence, that a calcium wave which would spread across the cortex at the time of cell division, would be the primary event that triggers morphogenesis in these species. We theoretically show that a unifying interpretation can be given for the possible occurrence of a single wave and that of multiple oscillations of cytosolic calcium: both of which correspond to two different behaviors of the same dynamic system. Experimental conditions allowing the visualization of possible calcium periodicities in the interphase Paramecium cell are much more easily fulfilled than those permitting the observation of a single wave at the time of cell division. Hence, experiments were performed on interphase cells. After microinjection of calcium indicator into a mutant strain which is defective in exocytosis, we observed Ca2+ oscillations with a period close to 2 minutes. Hence, we conclude that Paramecium possesses all the dynamic elements required to generate, at the time of cell division, a morphogenetic calcium wave.
Cyclic changes in the organization of cell adhesions and the associated cytoskeleton, induced by stimulation of tyrosine phosphorylation in bovine aortic endothelial cells
Authors: Ayalon Oran , Geiger Benjamin 
Published: April 2021

ABSTRACT
In this study we have investigated the relationships between the stimulation of tyrosine-specific protein phosphorylation and the state of assembly of cell-cell and cell-matrix adherens-type junctions. Bovine aortic endothelial (BAE) cells were treated with either the phosphotyrosine phosphatase inhibitor pervanadate or with epidermal growth factor (EGF), and the effect of the treatment on the organization of cell contacts and the actin cytoskeleton was evaluated by digital immunomicroscopy. We show here that pervanadate induced a dramatic (about 40-fold) increase in the level of phosphotyrosine labeling of cell-cell junctions, which reached maximal values following 20 minutes of incubation. Concomitantly, the junctional levels of vinculin, actin and plakoglobin increased, followed by a slower recruitment of cadherins to these sites. Upon longer incubation cell-cell junctions deteriorated and stress fibers and focal adhesions were formed. EGF stimulation of serum-starved BAE cells induced a rapid ‘wave’ of junctional tyrosine phosphorylation, followed by cyclic changes in the local levels of phosphotyrosine labeling. Periodic changes were also found in the intensity of labeling of junctional actin, vinculin and cadherins.
These results suggest that tyrosine phosphorylation and the assembly of cell-cell adherens junctions are interdependent processes, and raise the possibility that the cross-talk between the two is responsible both for the regulation of junction formation and for adhesion-mediated signaling.
Localization of fibronectin matrix assembly sites on fibroblasts and endothelial cells
Authors: Christopher Renee A. , Kowalczyk Andrew P. , McKeown-Longo Paula J. 
Published: April 2021

ABSTRACT
Polymerization of soluble fibronectin into extracellular matrix fibers occurs through the interaction between the amino terminus of fibronectin contained within a 70 kDa fragment and ‘matrix assembly sites’ on the cell surface. The present studies were performed to localize the ‘matrix assembly sites’ (defined by 70 kDa binding sites) on newly adherent cells and on cells containing preformed fibronectin matrix. Matrix nucleation sites on newly spread cells were visualized using Texas Red conjugated 70 kDa fragment and were found to colocalize with vinculin and substrate fibronectin fibrils. Cells plated onto vitronectin coated coverslips did not exhibit any 70 kDa binding sites although these cells were well-spread with fully developed focal adhesions. Time course studies indicated that 70 kDa binding sites could be detected on newly adherent cells within 30-40 minutes following cell plating onto fibronectin coated coverslips, prior to the reorganization of substrate fibronectin into fibrils. Similarly, exogenous fibronectin conjugated with Texas Red was also colocalized with vinculin when added to newly adherent cells. The disruption of actin filaments with cytochalasin D both prevented the expression of 70 kDa binding sites and also resulted in the loss of established 70 kDa binding sites on newly spread cells. After 3 days in culture, cells organized an extensive fibronectin matrix and 70 kDa was colocalized with two distinct types of matrix fibronectin fibers: fine linear cell-associated fibers which co-stained with the β1integrin and coarse extracellular fibers which did not stain for the β1integrin. There was also a third type of fibronectin fiber which was organized into a meshwork structure. There was no localization of either β1or 70 kDa to these structures. Treatment of 3-day cells with cytochalasin D resulted in the disruption of cell-matrix fibers and cell-associated 70 kDa binding sites. In contrast, the coarse extracellular matrix fibers as well as the meshwork fibers were unaffected by cytochalasin. In the presence of cytochalasin D, 70 kDa bound to sites which colocalized with the coarse extracellular matrix fibers. These data suggest that de novo assembly of fibronectin matrix occurs at sites of focal adhesion and as fibronectin polymerization proceeds, matrix nucleation sites colocalize along cell associated fibronectin fibers. At later times 70 kDa is localized to a subset of more mature fibronectin-containing fibers. These results suggest that there are at least three morphologically distinct 70 kDa binding sites on adherent cells: one which colocalizes with β1to focal adhesions, a second which colocalizes with β1and fibronectin in matrix contacts, and a third which localizes to extracellular matrix fibers.
Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts
Authors: Sato Takuya , Ovejero Maria del Carmen , Hou Peng , Heegaard Anne-Marie , Kumegawa Masayoshi , Tækker Foged Niels , Delaissé Jean-Marie 
Published: April 2021

ABSTRACT
The osteoclasts are the cells responsible for bone resorption. Matrix metalloproteinases (MMPs) appear crucial for this process. To identify possible MMP expression in osteoclasts, we amplified osteoclast cDNA fragments having homology with MMP genes, and used them as a probe to screen a rabbit osteoclast cDNA library. We obtained a cDNA of 1,972 bp encoding a polypeptide of 582 amino acids that showed more than 92% identity to human, mouse, and rat membrane-type 1 MMP (MT1-MMP), a cell surface proteinase believed to trigger cancer cell invasion. By northern blotting, MT1-MMP was found to be highly expressed in purified osteoclasts when compared with alveolar macrophages and bone stromal cells, as well as with various tissues. In situ hybridization on bone sections showed that MT1-MMP is expressed also in osteoclasts in vivo. Antibodies recognizing MT1-MMP reacted with specific plasma membrane areas corresponding to lamellipodia and podosomes involved, respectively, in migratory and attachment activities of the osteoclasts. These observations highlight how cells might bring MT1-MMP into contact with focal points of the extracellular matrix, and are compatible with a role of MT1-MMP in migratory and attachment activities of the osteoclast.
Proteinase-activated receptor-2: expression by human neutrophils
Authors: Howells Gareth L. , Macey Marion G. , Chinni Carla , Hou Linda , Fox Mark T. , Harriott Patrick , Stone Stuart R. 
Published: April 2021

ABSTRACT
Neutrophils were shown to express the proteinaseactivated receptor-2 (PAR-2), a seven transmembrane domain receptor, which is activated by cleavage by trypsin. Granulocytes from 14 donors stained positively for PAR-2 with affinity-purified rabbit antibodies raised against a peptide corresponding to the trypsin cleavage site of human PAR-2. Neutrophil activation in response to a receptor activating peptide (RAP) varied between donors. RAP (Ser-Leu-Ile-Gly-Lys-Val-NH2) alone induced an increase in the forward and side light scatter after 5-10 minutes and a small increase in the expression of the activation molecule CD11b. The increased expression of CD11b induced by RAP was markedly enhanced by priming the neutrophils with a low concentration (1 nM) of formyl-Leu-Met-Phe. Trypsin and RAP also induced an increase in intracellular calcium, but there were large variations in the magnitude of responses between donors also in this assay. The effects of RAP in the different assays were specific; acetylated RAP was completely without activity.