bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rho stimulates tyrosine phosphorylation of focal adhesion kinase, p130 and paxillin
Authors: Flinn Helen M. , Ridley Anne J. 
Published: April 2021

ABSTRACT
The small GTP-binding protein Rho rapidly stimulates the formation of focal adhesions and actin stress fibres when microinjected into serumstarved Swiss 3T3 fibroblasts. This response is inhibited by tyrosine kinase inhibitors. Addition of growth factors such as lysophosphatidic acid and bombesin to Swiss 3T3 cells stimulates a similar response, which is dependent on endogenous Rho proteins. To investigate signalling events regulated by Rho, we have scrape loaded Rho into serumstarved cells. Activated Rho stimulates the tyrosine phosphorylation of a number of proteins, including three proteins known to localise to focal adhesions, pp125FAK, p130 and paxillin. Rhoinduced phosphorylation of pp125FAK, p130 and paxillin is observed in the absence of stress fibre formation and is, therefore, independent of Rhoinduced actin polymerisation. We propose that the tyrosine kinase, pp125FAK, and the putative adapter proteins, paxillin and p130, are components of a Rhoregulated signal transduction pathway, and that these protein tyrosine phosphorylation events are likely to be important for the regulation of focal adhesion formation.
Establishment and characterization of conditionally immortalized cells from the mouse urogenital ridge
Authors: Capel Blanche , Hawkins J. R. , Hirst Elizabeth , Kioussis Dimitris , Badge Robin Lovell- 
Published: April 2021

ABSTRACT
Cell cultures from the urogenital ridge have been established to facilitate the study of the regulation and downstream interactions of Sry in mammalian sex determination. Cells have been explanted from transgenic mice carrying a temperature sensitive SV40 large T-antigen, and established in ongoing cultures. Analysis of the cells in these cultures at the electron microscope level reveals multiple cell types that compare to the cell types found in vivo during this period of development. Primordial germ cells, that are simultaneously explanted in the course of these experiments, also survive in culture. The explants undergo a morphogenetic organization into branching cordlike structures when cells are trypsinized and plated in extracellular matrix (Matrigel). We analyzed the expression of a number of molecular markers of the fetal gonad during monolayer culture, during in vitro morphogenesis in Matrigel, and in clonal lines derived from the complex explants. This analysis included Sry which is found to be expressed in some cultures from XY urogenital ridges that have been maintained for as long as 8 months.
Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines
Authors: Noel Josette , Roux Danièle , Pouysségur Jacques 
Published: April 2021

ABSTRACT
Na+/H+ exchangers (NHEs) are transporters that exchange sodium and proton ions across the plasma membrane at the expense of their chemical gradient. In higher eukaryotes these transporters exist as multiple specialized isoforms. For example, NHE1, the ubiquitously expressed form is a major pH-regulating system whereas the epithelial NHE3 isoform is specialized in transepithelial Na+ transport. NHE1 and NHE3 can be very well distinguished pharmacologically with the HOE694 specific inhibitor and immunologically with specific polyclonal and monoclonal antibodies. With these molecular tools we investigated the specific steady state expression of the two NHE isoforms in polarized epithelial cells in culture.
Endogenous NHE3 in OK cells or NHE3-VSVG transfected in either OK or MDCK cells showed an exclusive expression of the transporter at the apical membrane. Overexpression of NHE3 did not result in any spill over on the basal lateral side. These results obtained by functional measurement of NHE3 activity were fully consistent with its detection only at the apical side by immunofluorescence and confocal microscopy. By contrast, using the same cells, the same culture conditions and the same detection methods, we clearly detected NHE1 at both specialized membranes of four different polarized epithelial cell lines. Furthermore, biotinylation of cell surface proteins of MDCK, OK and HT-29 cells followed by immunoprecipitation of NHE1 revealed expression of the transporter at both sides of the polarized epithelial cells. Interestingly, the cell surface expression correlated well with the corresponding NHE1 activities. In addition, immunodetection by fluorescence microscopy was found to be qualitatively consistent with the abovereported results.
We therefore conclude that the epithelial and more specialized NHE3 isoform is exclusively restricted to the apical side of epithelial cells. In marked contrast, both endogenous or ectopically expressed NHE1 isoform, have the capacity to be expressed in both the apical and basal lateral membranes of polarized cells in cultures.
The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A
Authors: Brown Kevin D. , Wood Kenneth W. , Cleveland Don W. 
Published: April 2021

ABSTRACT
The kinesinlike protein CENP-E transiently associates with kinetochores following nuclear envelope breakdown in late prophase, remains bound throughout metaphase, but sometime after anaphase onset it releases and by telophase becomes bound to interzonal microtubules of the mitotic spindle. Inhibition of poleward chromosome movement in vitro by CENP-E antibodies and association of CENP-E with minusend directed microtubule motility in vitro have combined to suggest a key role for CENP-E as an anaphase chromosome motor. For this to be plausible in vivo depends on whether CENP-E remains kinetochore associated during anaphase. Using Indian muntjac cells whose seven chromosomes have large, easily tracked kinetochores, we now show that CENP-E is kinetochoreassociated throughout the entirety of anaphase-A (poleward chromosome movement), relocating gradually during spindle elongation (anaphase-B) to the interzonal microtubules. These observations support roles for CENP-E not only in the initial alignment of chromosomes at metaphase and in spindle elongation in anaphase-B, but also in poleward chromosome movement in anaphase-A.
Evidence that the endogenous histone H1 phosphatase in HeLa mitotic chromosomes is protein phosphatase 1, not protein phosphatase 2A
Authors: Paulson James R. , Patzlaff Jason S. , Vallis Amy J. 
Published: April 2021

ABSTRACT
Histone H1 is highly phosphorylated in mitotic HeLa cells, but is quickly dephosphorylated in vivo at the end of mitosis and in vitro following cell lysis. We show here that okadaic acid and microcystin-LR block the in vitro dephosphorylation of H1 and that they do so directly by inhibiting the histone H1 phosphatase rather than by some indirect mechanism. The concentrations of microcystin and okadaic acid required for inhibition strongly suggest that the histone H1 phosphatase is either PP1 or an unknown protein phos-phatase with okadaic acid-sensitivity similar to PP1. The histone H1 phosphatase is predominantly located in chromosomes with at most one copy for every 86 nucleosomes. This tends to support its identification as PP1, since localization in mitotic chromosomes is a characteristic of PP1 but not of the other known okadaic acid-sensitive protein phosphatases.
We also show that treatment of metaphase-arrested HeLa cells with staurosporine and olomoucine, inhibitors of p34cdc2 and other protein kinases, rapidly induces reassembly of interphase nuclei and dephosphorylation of histone H1 without chromosome segregation. This result indicates that protein kinase activity must remain elevated to maintain a mitotic block. Using this as a model system for the M-to G1-phase transition, we present evidence from inhibitor studies suggesting that the in vivo histone H1 phosphatase may be either PP1 or another phosphatase with similar okadaic acid-sensitivity, but not PP2A.
The thermolability of nuclear protein import in tsBN2 cells is suppressed by microinjected Ran-GTP or Ran-GDP, but not by RanQ69L or RanT24N
Authors: Dickmanns Achim , Bischoff F. Ralf , Marshallsay C. , Lührmann R. , Ponstingl Herwig , Fanning Ellen 
Published: April 2021

ABSTRACT
The nuclear protein regulator of chromosome condensation 1 (RCC1) stimulates guanine nucleotide exchange on a protein, Ran, that is required for nuclear protein import. In the present report, we confirm that RCC1 is also required for nuclear protein import in tsBN2 hamster cells in vivo. The thermolability of nuclear protein import in tsBN2 cells was suppressed by microinjection of purified Ran-GTP into the cytoplasm, but Ran-GDP also relieved the import deficiency, suggesting either that both forms of Ran are active in import in vivo or that tsBN2 cells at restrictive temperature retain a mechanism to convert Ran-GDP to Ran-GTP. To distinguish between these possibilities, nuclear protein import in tsBN2 cells was tested in the presence of Ran mutants, one deficient in GTP hydrolysis (RanQ69L), and one with weak binding to GDP and little or no binding to GTP (RanT24N). Microinjection of the mutant RanQ69L inhibited import in vivo in either the GTP- or GDP-bound form at both the permissive and nonpermissive temperatures. RanT24N-GDP inhibited import in vivo at the permissive temperature and failed to stimulate nuclear protein import at the nonpermissive temperature. The implications of these results for the roles of RCC1 and Ran in nuclear protein import in vivo are discussed.
Accelerated neuronal differentiation induced by p53 suppression
Authors: Ferreira Adriana , Kosik Kenneth S. 
Published: April 2021

ABSTRACT
p53, a tumor suppressor gene product, has been implicated in the control of cell growth and malignant trans-formation in different cell types. Here we studied the role of p53 in normal central nervous system development. We show that p53 is expressed in neuroblasts and is down regulated when migrating neurons reach their destination. The suppression of p53 either by the addition of antisense oligonucleotides to culture medium or by the culture of neurons from p53−/− mice accelerated their differentiation. This effect is accompanied by an early induction of MAP1b and a premature dephosphorylation of tau. p53 suppression also reduced levels of p21. Taken collectively these results suggest that the expression of p53 in neuroblasts might prevent neuronal terminal differentiation.
Human osteoclast-like cells selectively recognize laminin isoforms, an event that induces migration and activates Ca2+ mediated signals
Authors: Colucci Silvia , Giannelli G. , Grano M. , Faccio R. , Quaranta V. , Zallone Alberta Zambonin 
Published: April 2021

ABSTRACT
Osteoclast precursors are chemotactically attracted to sites of bone resorption via migration pathways that include transendothelial crossing in blood capillaries. Transendothelial migration involves poorly understood interactions with basal lamina molecules, including laminins. To investigate osteoclast-laminin interactions, we used human osteoclast-like cell lines obtained from giant cell tumors of bone (GCT 23 and GCT 24). These cell lines are a well-characterized model for osteoclast functions, such as bone resorption and the behaviour of osteoclast precursors. Both GCT cell lines adhered to laminin-2 (merosin) coated wells in standard adhesion assays, but failed to adhere to laminin-1 (EHS-laminin). By light microscopy, GCT cells on laminin-2 were partially spread, with a motile morphology. None of the anti-integrin anti-bodies tested inhibited GCT cells adhesion to laminin-2. Peptides containing the integrin adhesion site RGD or the laminin adhesion sequence IKVAV did not inhibit GCT cell adhesion to laminin-2. By immunofluorescence, β1 integrins were organized in focal adhesions. However, in the presence of monensin this reorganization of β1 integrins was abolished, indicating that it was probably due to secretion of fibronectin by GCT cells subsequent to adhesion to laminin-2. GCT cells transmigrated through membranes coated with laminin-2, much more efficiently than through membranes coated with collagen. Migration was induced by osteocalcin, as a chemoattractant, in a dose-dependent manner. At low osteocalcin concentrations, transmigration was detectable on laminin-2 but not collagen. In cells loaded with fura-2, a sharp increase in intracellular Ca2+ was detected upon addition of soluble laminin-2, but not laminin-1, due to release from thapsigargin-dependent intracellular stores. In summary, osteo-clasts may recognize laminin isoforms differentially. Initial adhesion to laminin-2 appears to be due to integrin-independent mechanisms. Such adhesion, though, may trigger secretion of fibronectin that could then support spreading and efficient chemotactic migration. These mechanisms may play an important role in facilitating chemotactic migration of osteoclast precursors toward the bone surface.
Cellular phosphorylation of tau by GSK-3β influences tau binding to microtubules and microtubule organisation
Authors: Wagner Uta , Utton Michelle , Gallo Jean-Marc , Miller Christopher C. J. 
Published: April 2021

ABSTRACT
Tau is a neuronal microtubule-associated protein that appears to function in the formation and maintenance of axons by influencing microtubule organisation. Tau is a phosphoprotein and is more heavily phosphorylated in fetal than in adult brain, and is also hyperphosphorylated in Alzheimer’s disease where it forms the major component of paired helical filaments (PHFs). Tau phosphorylation probably modulates microtubule dynamics since in vitro, phosphorylated tau has a reduced affinity for microtubules and is less potent at promoting microtubule assembly. In order to understand how phosphorylation effects cellular microtubule organisation, we studied 3T3 and CHO cells transfected with tau and the tau kinase GSK-3β. Tau transfected cells displayed prominent bundles of microtubules that did not appear to be nucleated by a microtubule-organising centre. Co-transfection of tau with GSK-3β led to increased phosphorylation of tau and also to a reduction in microtubule bundling such that the microtubule network in many of the tau/GSK-3β transfected cells appeared similar to non-transfected interphase cells. Transfection of a mutant tau, in which five of the known GSK-3β targeted phosphorylation sites were mutated to alanine so as to preclude phosphorylation, also induced microtubule bundling. However, co-transfection of this mutant with GSK-3β did not diminish the bundling effect. Biochemical analyses of microtubule and cytosolic fractions from the transfected cells demonstrated that GSK-3β-mediated phosphorylation of tau reduced its affinity for microtubules. These results suggest that phosphorylation of tau by GSK-3β modulates its ability to organise microtubules into ordered arrays such as are found in axons.
Mechanical strain induces constitutive and regulated secretion of glycosaminoglycans and proteoglycans in fetal lung cells
Authors: Xu Jing , Liu Mingyao , Liu Jason , Caniggia Isabella , Post Martin 
Published: April 2021

ABSTRACT
We have previously shown that an intermittent strain regimen, which simulates fetal breathing movements, enhanced mixed fetal rat lung cell proliferation in organotypic culture. As glycosaminoglycans (GAGs) and proteoglycans (PGs) may modulate growth factor activities, we investigated the effect of intermittent strain on the formation and secretion of GAGs and PGs. Mechanical strain increased the incorporation of [3H]glucosamine and 35SO4 into GAGs and promoted the release of GAGs into the medium. The composition of the individual GAG molecules was not altered by strain. Mixed fetal lung cells subjected to strain secreted more [35S]biglycan into the medium than static controls but biglycan mRNA expression was not significantly altered. As mechanical strain primarily affected the secretion of GAGs and PGs, we then investigated which secretory pathways were stimulated by strain. Fetal lung cells secreted GAGs mainly through a constitutive (basal) pathway which was stimulated by strain. In contrast to static cultures, strain-induced constitutive secretion was partially blocked by the cytoskeletal disruptors colchicine and cytochalasin B, but not by the small G-protein inhibitors N-acetyl-S-farnesyl-L-cysteine and perillic acid. This result suggests that strain-induced constitutive export of GAGs depends on the functional integrity of the cytoskeleton. Strain also triggered the regulated secretion of GAGs. The strain-induced regulatory pathway in fetal lung cells was blocked by ionomycin, BAPTA/AM and gadolinium, suggesting that strain stimulated the regulatory pathway by inducing a rapid calcium influx via a stretch-activated ion channel. We conclude that mechanical strain of mixed fetal lung cells stimulates GAG and PG exocytosis via activation of both the regulated and constitutive pathways.
Destabilization of plasma membrane structure by prevention of actin polymerization: Microtubule-dependent tubulation of the plasma membrane
Authors: van Deurs B. , von Bülow F. , Vilhardt F. , Holm P. Kaae , Sandvig K. 
Published: April 2021

ABSTRACT
Electron microscopy of thick (0.2-1.0 μm) sections of cytochalasin D-treated cells fixed in the presence of Ruthenium red revealed an extensive, surface-connected tubular compartment in HEp-2 cells. The tubules measured 120-220 nm in diameter and at least up to 6 μm in length. Morphometric analysis showed that in control cells about 0.2% of the total plasma membrane area (defined as all Ruthenium red-labeled membrane) appeared as vesicular or tubular profiles beneath the cell surface. However, after 15-30 minutes of cytochalasin D incubation about 4% of the total plasma membrane area is tubulated, and after 60-105 minutes as much as about 15% of the total plasma membrane appears as tubules. Clathrincoated pits and caveolae-like structures were occasionally associated with the tubular membrane. Moreover, immunogold labeling showed that the tubular membrane contained transferrin receptors at about the same density as the nontubulated plasma membrane. Examination of cells in which endosomes and lysosomes were labeled with horseradish peroxidase before or after exposure to cytochalasin D showed that these organelles remained spherical, and that no horseradish peroxidase was present in the tubules. Moreover, the surface to volume ratio remained constant with increasing time of cytochalasin D incubation. Accordingly, the surface-connected tubules were not derived from endocytic structures but were formed by invagination of the plasma membrane. The tubule formation is reversible. When microtubules are depolymerized by nocodazole or colchicine treatment before the cells are exposed to cytochalasine D, tubule formation is strongly inhibited. Hence, the cytochalasin D-induced plasma membrane tubulation depends on intact microtubules.
The Drosophila genes grauzone and cortex are necessary for proper female meiosis
Authors: Page Andrea W. , Orr-Weaver Terry L. 
Published: April 2021

ABSTRACT
In Drosophila, normal female meiosis arrests at metaphase I. After meiotic arrest is released by egg activation, the two meiotic divisions are rapidly completed, even in unfertilized eggs. Since little is known about the regulation of the meiotic cell cycle after the meiotic arrest, we screened for mutants that arrest in meiosis. Here we describe the phenotype of eggs laid by sterile mothers mutant for either grauzone or cortex. These eggs arrest in metaphase of meiosis II, and although they can enter into an aberrant anaphase II, they never exit meiosis. Prolonged sister-chromatid cohesion is not the cause of this arrest, since a premature release of sister cohesion does not rescue the meiotic arrest of cortex eggs. Aberrant chromosome segregation at meiosis I was the earliest observable defect, suggesting that grauzone and cortex are first required immediately after egg activation. The cortical microtubules are also defective, remaining in a pre-activated state in activated mutant eggs. The mutations had no observable effect on either male meiosis or mitosis. We believe these genes will provide insight into the developmental regulation of meiosis in a genetically tractable organism.
IL-3, GM-CSF and CSF-1 modulate c-fms mRNA more rapidly in human early monocytic progenitors than in mature or transformed monocytic cells
Authors: Panterne Béatrice , Hatzfeld Antoinette , Sansilvestri Patricia , Cardoso Angelo , Monier Marie-Noëlle , Batard Pascal , Hatzfeld Jacques 
Published: April 2021

ABSTRACT
We have previously shown that a low concentration of CSF-1 (1 U/ml) can trigger human immature monocytic progenitor proliferation in the presence of low concentrations of IL3 (1.7 U/ml). No c-fms down-regulation was observed during this early cell activation. In contrast, 20 U/ml of CSF-1, active on late monocytic cell growth, downregulated c-fms mRNA expression in immature progenitors and monocytes derived from bone marrow CD34+ cells in culture. We have now extended this study to include the effects of various concentrations of GM-CSF, IL3 and G-CSF on c-fms expression. We observed that high doses of GM-CSF or IL3 down-modulated c-fms mRNA, whereas low doses of GM-CSF or IL3, which were active on early monocytic growth, had no such effect. Similar results were observed at the protein level. In contrast, whatever the concentration, G-CSF had no effect on c-fms mRNA or protein levels. We further observed that the more immature the c-fms expressing progenitors, the faster the down-modulation of this receptor. This was observed within less than 1 hour for immature bone marrow cells, 6 hours for peripheral blood monocytes and even longer for transformed monocytic cells. These results suggest that oncogene expression can be regulated much more rapidly in immature progenitors than was previously observed in mature cells or transformed cell lines.
A biochemical and immunological comparison of nuclear and cytoplasmic pore complexes
Authors: Ewald Andrea , Kossner Ursula , Scheer Ulrich , Dabauvalle Marie-Christine 
Published: April 2021

ABSTRACT
Pore complexes are not confined to the nuclear envelope but can also be found in the cytoplasm of numerous cell types in the form of annulate lamellae (AL). We have induced formation of AL by exposure of rat cells (line RV) to sublethal doses of the antimitotic drug vinblastine sulfate, and compared the distribution of several nuclear pore complex proteins (nucleoporins) in the nuclear envelope and AL by immunocytochemistry, cytochemical lectin binding studies and immunoblot analyses of nuclear and AL-enriched fractions. All the antibodies used yielded punctate nuclear surface staining in immunofluorescence microscopy which is characteristic for nuclear pore complex components. When we applied antibodies against the nucleoporin p62, AL were visualized as numerous cytoplasmic dot-like structures. Immunogold electron microscopy confirmed the correspondence of the cytoplasmic bodies with stacks of AL. Antibodies to constituents of the cytoplasmic (nup180) and nucleoplasmic (nup153) filaments extending from both sides of nuclear pore complexes also stained the AL, indicating that pore complexes are intrinsically asymmetric assemblies independent of their specific intracellular topology. By contrast, AL were negative with five different antibodies against the transmembrane nuclear pore glycoprotein gp210 and the lectin concanavalin A (ConA) known to bind to the oligosaccharide side chains of gp210. Similarly, there was no staining of the AL with antibodies to the other nuclear pore membrane protein so far known in higher eukaryotes, POM121. Immunoblot analyses confirmed the presence of p62, nup180 and nup153 in both the nuclear and AL fractions and the absence of gp210 and POM121 from AL. Our results do not support the generally held view that gp210 and POM121 function in anchoring the pore complex scaffold to the pore membrane. Rather, they point to a role for these proteins in transport processes through the nuclear pore complexes. Since AL are not involved in nucleocytoplasmic transport processes they may lack components of the transport machinery.