bluuebunny/crossref_metadata_2025 huggingface dataset:
Rho stimulates tyrosine phosphorylation of focal adhesion kinase, p130 and paxillin
Authors: Flinn Helen M. , Ridley Anne J.
Published: April 2021

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
The casein kinase 1α gene of Drosophila melanogaster is developmentally regulated and the kinase activity of the protein induced by DNA damage
Authors: Santos João A. , Logarinho Elsa , Tapia Claudio , Allende Catherine C. , Allende Jorge E. , Sunkel Claudio E.
Published: April 2021

We report the molecular cloning and characterisation of the first CK1(casein kinase) gene of Drosophila melanogaster (dmCK1). The protein sequence (DMCK1) shares significant homology with other mammalian CK1 protein kinases of the α sub-class. The dmCK1 gene is expressed only in adult females and during early embryonic development as a single transcript. Western blot analysis of total protein extracts of different stages of development show that the gene product is likewise present during early embryogenesis and in adult females. Kinase activity studies show that DMCK1 is active when in vitro translated but inactive when immunoprecipitated from total early embryo extracts. However, after dephosphorylation treatment the immunoprecipitates show high kinase activity. More significantly, DMCK1 kinase activity present in the immunoprecipitates can be specifically activated by γ-irradiation of early embryos. Also, when DMCK1 is immunoprecipitated after irradiation it appears to undergo phosphorylation. Immunolocalization of DMCK1 in early embryos shows that the protein is predominantly cytoplasmic but after irradiation there is a significant relocalization to the interphase nucleus. The results suggest a possible requirement of the Drosophila CK1α for mechanisms associated with DNA repair during early embryogenesis.
Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium
Authors: Fewell Joseph W. , Kuff Edward L.
Published: April 2021

Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells. Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations. Ku is generally regarded as a nuclear component. However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities. The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes. The immunostaining pattern of sparsely grown cells could be converted to the ‘confluent’ configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells. The confluent antigen pattern could also be induced in sparse cells within 15–30 minutes by exposure of the cells to serum- or Ca2+-free medium or overnight with 2 mM hydroxyurea. Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation on Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity. However, somatostatin did not alter the expected immunostaining patterns of p86. Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-β1 in the culture medium by the rapid appearance of nuclear immunoreactivity. TGF-α had no apparent effect. These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other.
The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells
Authors: Nilsson Tommy , Rabouille Catherine , Hui Norman , Watson Rose , Warren Graham
Published: April 2021

Using a series of chimeric and truncated N-acetylglucosaminyltransferase I (NAGT I) molecules we have shown that part of the lumenal stalk region is both necessary and sufficient for kin recognition of mannosidase II and retention in the Golgi stack. The membrane-spanning domain was not required for retention, but replacing part or all of this domain with leucine residues did have a dramatic effect on Golgi morphology. In stable cell lines, stacked cisternae were replaced by tubulo-vesicular clusters containing the mutated NAGT I. The loss of stacked cisternae was proportional to the number of leucines used to replace the membrane-spanning domain.
Role of laminin-1 and TGF-β3 in acinar differentiation of a human submandibular gland cell line (HSG)
Authors: Hoffman Matthew P. , Kibbey Maura C. , Letterio John J. , Kleinman Hynda K.
Published: April 2021

Previous studies show that culturing an immortalized human submandibular gland cell line (HSG) on Matrigel, a basement membrane extract, induces cytodifferentiation. We have further defined this model system and identified factors involved in HSG cell acinar development and cytodifferentiation. Acinar development is marked by cell migration into multi-cellular spherical structures, cell proliferation and apoptosis of the centrally localized cells. In addition, functional differentiation was determined by indirect immunofluorescence and immunoblot analysis for cystatin, a salivary gland acinar cell-specific protein found to be produced by differentiated HSG cells. Matrigel contains multiple extracellular matrix proteins, however, laminin-1 was identified as the major matrix component that induced HSG cell acinar development and cytodifferentiation. Antibodies against specific components of Matrigel and against cell surface adhesion molecules were added to cells in culture to identify components important for HSG cell acinar differentiation. Immunostaining of HSG cell acini identified TGF-β2 and β3 as the predominant isoforms within the cells. Neutralizing antibodies directed against TGF-β3 significantly decreased (P≤0.0002) the size of acini formed. These results indicate that multiple components, including laminin-1 and TGF-β3, contribute to HSG cell acinar development. This model system will be useful to study acinar differentiation and salivary glandspecific protein expression in vitro.
Actin filament bundles are required for microtubule reorientation during growth cone turning to avoid an inhibitory guidance cue
Authors: Challacombe Jean F. , Snow Diane M. , Letourneau Paul C.
Published: April 2021

The extracellular matrix through which growth cones navigate contains molecules, such as chondroitin sulfate proteoglycan, that can inhibit growth cone advance and induce branching and turning. Growth cone turning is accompanied by rearrangement of the cytoskeleton. To identify changes in the organization of actin filaments and microtubules that occur as growth cones turn, we used time-lapse phase contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones at a substratum border between fibronectin and chondroitin sulfate proteoglycan, in the presence and absence of cytochalasin B. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and dynamic and stable microtubules.

Our results suggest that microtubules are rearranged within growth cones to accomplish turning to avoid chondroitin sulfate proteoglycan. Compared to growth cones migrating on fibronectin, turning growth cones were more narrow, and they contained dynamic microtubules that were closer to the leading edge and were more bundled. Cytochalasin B-treated growth cones sidestepped laterally along the border instead of turning, and in sidestepping growth cones, microtubules were not bundled and aligned. We conclude that actin filament bundles are required for microtubule reorientation and growth cone turning to avoid chondroitin sulfate proteoglycan.
Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging
Authors: Wilson Keith M. , Morrison Ian E. G. , Smith Patricia R. , Fernandez Nelson , Cherry Richard J.
Published: April 2021

The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were obtained by sequential imaging over a period of typically 30 minutes. Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200-800 nm. Diffusion coefficients for random diffusion were in the range 1×10−13-5×10−12 cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.
The envelope of vaccinia virus reveals an unusual phospholipid in Golgi complex membranes
Authors: Cluett Edward B. , Machamer Carolyn E.
Published: April 2021

We isolated two forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself in the membranes of the cis-Golgi network. A fraction of these virions then acquires a second envelope by enwrapping trans-Golgi network membranes to form the intracellular enveloped virus. Lipids were analyzed by high performance thin layer chromatography and digital densitometry to establish a steady-state lipid profile of viral membranes, which should reflect the compositions of the cis-Golgi network and trans-Golgi network. Phosphatidylinositol was slightly enriched in the cis-Golgi network of HeLa cells, whereas the trans-Golgi network showed a minor increase in phosphatidylserine and sphingomyelin. Similarly, cholesterol was only slightly more abundant in the trans-Golgi compared to the cis-Golgi. An unusual lipid, semilysobisphosphatidic acid, was present in significant amounts in vaccinia envelopes. Semilysobisphosphatidic acid was present in similar levels in infected and uninfected cells, and was therefore not induced by vaccinia infection. Subcellular fractionation of HeLa cells indicated that the recovery of semilysobisphosphatidic acid paralleled the recovery of a Golgi marker. Furthermore, a lipid species that comigrated with semilysobisphosphatidic acid was also present in lipids extracted from highly purified, intact Golgi complexes from rat liver. Together, these results suggest that semilysobisphosphatidic acid is a normal component of Golgi membranes.
Contrasting migratory response of astrocytoma cells to tenascin mediated by different integrins
Authors: Giese Alf , Loo Melinda A. , Norman Sylvia A. , Treasurywala Sherri , Berens Michael E.
Published: April 2021

Tenascin, an extracellular matrix protein, is expressed in human gliomas in vitro and in vivo. The distribution of tenascin at the invasive edge of these tumors, even surrounding solitary invading cells, suggests a role for this protein as a regulator of glioma cell migration. We tested whether purified tenascin, passively deposited on surfaces, influenced the adhesion or migration of a human gliomaderived cell line, SF-767. Adhesion of glioma cells to tenascin increased in a dose-dependent fashion up to a coating concentration of 10 µg/ml. Higher coating concentrations resulted in progressively fewer cells attaching. Cell adhesion could be blocked to basal levels using anti-β1 integrin antibodies. In contrast, when anti-αv antibodies were added to the medium of cells on tenascin, cell adhesion was enhanced slightly. Using a microliter scale migration assay, we found that cell motility on tenascin was dose dependently stimulated at coating concentrations of 1 and 3 µg/ml, but migration was inhibited below levels of nonspecific motility when tested at coating concentrations of 30 and 100 µg/ml. Migration on permissive concentrations of tenascin could be reversibly inhibited with anti-β1, while treatment with anti-αv antibodies increased migration rates. We conclude that SF-767 glioma cells express two separate integrin receptors that mediate contrasting adhesive and migratory responses to tenascin.
The fission yeast STS5+ gene is required for maintenance of growth polarity and functionally interacts with protein kinase C and an osmosensing MAP-kinase pathway
Authors: Toda Takashi , Niwa Hajime , Nemoto Takeshi , Dhut Susheela , Eddison Mark , Matsusaka Takahiro , Yanagida Mitsuhiro , Hirata Dai
Published: April 2021

Cell morphogenesis is a fundamental phenomenon that involves understanding a number of biological processes including the developmental program, polarity and cell division. Fission yeast sts5 mutant cells are round rather than cylindrical with cortical actin randomly dispersed. Genetic analyses demonstrate that the sts5+ gene is required for maintenance of cell shape during interphase when the cell normally exhibits polarised growth. The sts5 mutant is not defective in cell wall integrity. Deletion of ppe1+, which encodes a type 2A-like protein phosphatase, shows similar phenotypes to the sts5 mutant and these two mutations are synthetically lethal. Multicopy plasmids containing either the protein kinase C-like gene pck1+ or the protein tyrosine phosphatase pyp1+, an inhibitor of an osmosensing Sty1/Spc1 MAP-kinase, are capable of suppressing the sts5 mutation. Consistent with this, we have found that the wis1 mutation, which is defective in a MAP-kinase kinase of the pathway, suppresses the sts5 mutation. The predicted sts5+ gene product exhibits sequence similarity to two yeast proteins, Dis3 and Ssd1 and a nematode protein, F46E8.6, where the former two yeast proteins have been shown to be involved in cell cycle control and cell morphogenesis. The sts5+ gene is not essential for cell viability, but is absolutely required for polarised growth as the gene disruption showed the same phenotypes as those of the original mutants. Overexpression of the sts5+ gene resulted in altered cell morphology and, cortical actin in these overproducing cells was also abnormal, fainter and often dispersed. Anti-Sts5 antibody specifically detected a 130 kDa protein by western blotting. A green fluorescent protein-Sts5 fusion protein localised in the cytoplasm with a discrete punctate pattern, suggesting that the Sts5 protein is a component of a novel structure. These results have indicated that the Sts5 protein is a crucial determinant of polarised growth and that it functionally interacts with the serine/threonine phosphatase, protein kinase C, and an osmosensing MAP-kinase to maintain cell morphology.
Endo-exonuclease of human leukaemic cells: Evidence for a role in apoptosis
Authors: Fraser Murray J. , Tynan Stephen J. , Papaioannou Arthur , Ireland Christine M. , Pittman Sally M.
Published: April 2021

Inactive forms of endo-exonuclease, activated in vitro by treatment with trypsin, have been identified in human leukaemic CEM and MOLT-4 cells. They comprise over 95% of the total single-strand DNase activity in nuclei and are mainly bound to chromatin and the nuclear matrix. The activated enzyme had Mg2+(Mn2+)-dependent, Ca2+-stimulated activities with single- and double-strand DNAs and RNA (polyriboadenylic acid) and other properties characteristic of endo-exonucleases previously described. At least twice as much inactive endo-exonuclease has also been localised in extranuclear compartments of CEM and MOLT-4 cells, 85% bound to the membranes of the endoplasmic reticulum and 15% free in the cytosol. The soluble cytosolic trypsin-activatable endo-exonuclease was immunoprecipitated by antibodies raised independently to both Neurospora and monkey CV-1 cell endo-exonucleases. The free and bound enzymes of both nuclear and extranuclear compartments also cross-reacted on immunoblots with the antibody raised to Neurospora endo-exonuclease to reveal multiple polypeptides ranging in size from 18 to 145 kDa, many of which exhibited activity on DNA gels. The major species bound to the chromatin/matrix were in the 55-63 kDa range. Limited proteolysis of the large polypeptides to those of 18 to 46 kDa accompanied spontaneous chromatin DNA fragmentation to form DNA ‘ladders’ in an isolated nuclei/cytosol system.

When the leukaemic cells were treated in culture with either etoposide or podophyllotoxin to induce apoptosis, the largest polypeptides disappeared and smaller endo-exonuclease-related polypeptides of 18 to 46 kDa were detected in the nuclear extracts. The appearance of these polypeptides also correlated with extensive chromatin DNA fragmentation. In addition, there were correlations between the depletion of the major 55-63 kDa species bound to the membranes of the endoplasmic reticulum, depletion of the extranuclear trypsin-activatable activity and the onset and extent of chromatin DNA fragmentation in both cell lines. The extranuclear 55-63 kDa species may be precursors of the chromatin/matrix bound endo-exonuclease. The results indicate that endo-exonuclease plays a role in chromatin DNA degradation in mammalian cells during apoptosis.
Quality control in protein biogenesis: Thiol-mediated retention monitors the redox state of proteins in the endoplasmic reticulum
Authors: Brewer Joseph W. , Corley Ronald B.
Published: April 2021

There is accumulating evidence that proteins can be retained in the endoplasmic reticulum by a mechanism that is believed to monitor the oxidation status of one or more cysteines in their sequences. For example, a single cysteine residue critical for retention of secretory IgM assembly intermediates has been mapped to the C-terminal cysteine, Cys575, of the secretory µ chain. Little is known concerning the mechanism responsible for this system of quality control, which has been termed thiolmediated retention. In particular, it is not known if the mechanism monitors the redox state of the important cysteine residue in the secretory µ protein itself or within the context of higher-order IgM complexes. To address this question, we evaluated the fidelity of retention of secretory IgM and determined the redox status of cysteines in secretory µ proteins in polymers and polymer intermediates at various stages of maturation. We demonstrate that all secreting B cells and B cell lines secrete assembly intermediates in addition to completed, covalent pentameric and hexameric IgM polymers. A fraction of assembly intermediates exit the endoplasmic reticulum as individual components, mature through the Golgi without undergoing further assembly, and most, if not all, are secreted. While the majority of IgM assembly intermediates have exposed thiols and are contained within the endoplasmic reticulum where they can be utilized for oligomerization, maturing assembly intermediates found in the Golgi and extracellular space are completely oxidized. Thus, while the retention of unpolymerized IgM is highly efficient, the retention system lacks the ability to distinguish fully oxidized assembly intermediates from fully oxidized completed polymers. The molecular mechanisms that may contribute to this aspect of IgM biogenesis and their implications for the concept of thiolmediated retention are discussed.
Transport of protein kinase C α into the nucleus requires intact cytoskeleton while the transport of a protein containing a canonical nuclear localization signal does not
Authors: Schmalz Dirk , Kalkbrenner Frank , Hucho Ferdinand , Buchner Klaus
Published: April 2021

Protein kinase C undergoes a redistribution from the cytosol into the nucleus upon various stimuli. Since protein kinase C does not contain any known nuclear localization signal, the exact pathway and mechanism of the translocation into the nucleus is not known.

We used immunofluorescence microscopy to investigate the role of the cytoskeleton in this process, and to detect the subcellular distribution of protein kinase C α in NIH 3T3 fibroblasts. In these cells protein kinase C α is translocated into the nucleus after stimulation with phorbol ester. We observed that cells treated with the cytoskeleton disrupting agents cytochalasin B or colchicine do not show the nuclear translocation of protein kinase C α after stimulation. In contrast, the nuclear accumulation of a nuclear localization signal containing reporter protein in an in vitro nuclear transport assay is not affected by these drugs. This observation has been confirmed for intact cells by microinjection experiments: cells which have been incubated with cytochalasin B or colchicine prior to microinjection of the reporter protein show the same accumulation in the nucleus as untreated cells.

Our data show that intact cytoskeleton plays an important role in the translocation of protein kinase C α into the nucleus but not in the nuclear import of a karyophilic reporter protein.
Regulation of the acquisition of meiotic competence in the mouse: Changes in the subcellular localization of cdc2, cyclin B1, cdc25C and weel, and in the concentration of these proteins and their transcripts
Authors: Mitra Jayashree , Schultz Richard M.
Published: April 2021

During their development, mammalian oocytes acquire the ability to resume meiosis. We demonstrate that the concentration of p34cdc2 increases during the acquisition of meiotic competence, as determined by immunoblotting, whereas the concentration of cyclin B1 decreases. Laserscanning confocal microscopy corroborated these changes and furthermore indicate that an increase occurs in the nuclear concentration of each protein. Results of immunoblotting experiments demonstrate that associated with the acquisition of meiotic competence is an increase in the concentration of cdc25C, an activator of p34cdc2/cyclin B kinase, and a decrease in wee1, an inhibitor of cdc2/cyclin B kinase. These changes were again corroborated by laserscanning confocal microscopy, which also indicates that an increase in the nuclear concentration of wee1 occurs. The concentration of the transcripts encoding these proteins, however, is essentially similar in meiotically incompetent and competent oocytes. Thus, these changes in protein concentration that occur during oocyte development likely reflect changes in the translational efficiency of their mRNAs. Consistent with this is that the relative rate of synthesis of p34cdc2 in meiotically competent oocytes is ∼3 times greater than that in meiotically incompetent oocytes, whereas the stability of newly synthesized p34cdc2 is essentially the same in each cell type.