bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear translocation and aggregate formation of heat shock cognate protein 70 (Hsc70) in oxidative stress and apoptosis
Authors: Dastoor Zubin , Dreyer Jean-Luc
Published: April 2021

Recent evidence has shown a role for the heat shock cognate protein Hsc70 in the response to oxidative stress. We have investigated the subcellular distribution of Hsc70 by means of laser scanning confocal microscopy in neuroblastoma NB41A3 cells, in fibroblasts R6 cells and in R6-Bcl-2, an apoptosis-resistant cell line, and its function in oxidative stress and in apoptosis has been evaluated. Endogenous Hsc70 is localised predominantly in the cytoplasm in unstressed cells, whereas oxidative stress but not apoptosis induces its translocation into the nucleus. In transfected cells overexpressing Hsc70 increased nuclear translocation and aggregation of Hsc70 in intracellular speckles is observed after oxidative stress and, to a lesser degree, after exposure to apoptotic agents. Bcl-2 did not influence the movement of Hsc70 nor the formation of Hsc70-containing speckles. Nuclear translocation of Hsc70 can be modulated by the expression of components from a previously described plasma membrane oxidoreductase involved in the cellular response against oxidative stress. Our data may suggest a correlation between differential translocation of Hsc70 with specific functions in apoptosis and a potential role in the protection against reactive oxygen species.
Signaling specificities of fibroblast growth factor receptors in early Xenopus embryo
Authors: Umbhauer Muriel , Penzo-Méndez Alfredo , Clavilier Léa , Boucaut Jean-Claude , Riou Jean-François
Published: April 2021

Formation of mesoderm and posterior structures in early Xenopus embryos is dependent on fibroblast growth factor (FGF) signaling. Although several FGF receptors (FGFRs) are expressed in the early embryo, their respective role in these processes remains poorly understood. We provide evidence that FGFR-1 and FGFR-4 signals elicit distinct responses both in naive and neuralized ectodermal cells. We show that naive ectodermal cells expressing a constitutively active chimeric torso-FGFR-1 (t-R1) are converted into mesoderm in a Ras-dependent manner, while those expressing torso-FGFR-4 (t-R4) differentiate into epidermis without significant activation of Erk-1. In neuralized ectoderm, expression of t-R4 causes the up- regulation of the midbrain markers En-2 and Wnt-1, but not of the hindbrain nor the spinal cord markers Krox20 and Hoxb9. Mutation of tyr776 in the phospholipase C-γ binding consensus sequence YLDL of t-R4 completely abolishes En-2 and Wnt-1 induction. In contrast to t-R4, platelet derived growth factor (PDGF)-dependent FGFR-1 activation in neuralized ectodermal cells expressing a chimeric PDGFR-FGFR-1 receptor results in the expression of Krox20 and Hoxb9. A similar effect is observed when an inducible form of oncogenic Raf is expressed, therefore implicating FGFR-1 and Raf in the transduction of FGF-caudalizing signals in neural tissue. Our results suggest that FGFR-1 and FGFR-4 transduce distinct signals in embryonic cells, and mainly differ in their ability to activate the Ras/MAPK pathway.
The conserved amphipatic α-helical core motif of RARγ and RARα activating domains is indispensable for RA-induced differentiation of F9 cells
Authors: Plassat Jean-Luc , Penna Lucia , Chambon Pierre , Rochette-Egly Cécile
Published: April 2021

In monolayers cultures, retinoic acid (RA) induces the differentiation of F9 embryonal carcinomal (EC) cells into primitive endoderm-like cells, while a combination of RA and dibutyryl cAMP leads to parietal endoderm-like differentiation. Knock out of all RARγ isoforms (RARγ−/− line) drastically impairs primitive and subsequent parietal endodermal differentiation and affects the induction of many endogenous RA-responsive genes. Using lines that reexpress RARγ2 or overexpress RARα1 lacking their AF-2AD core (RARγΔAF2 and RARαΔAF2, respectively), we show that this conserved amphipatic α-helical motif (helix 12) of the ligand binding domain, and therefore the activation function AF-2 of both receptors, is required for the induction of differentiation and target gene expression upon RA treatment of F9 EC cells. We also show that these deletion mutants behave as dominant negatives.
Mouse keratinocytes immortalized with large T antigen acquire α3β1 integrin-dependent secretion of MMP-9/gelatinase B
Authors: DiPersio C. Michael , Shao Michael , Costanzo Lara Di , Kreidberg Jordan A. , Hynes Richard O.
Published: April 2021

Remodeling of the extracellular matrix during tissue development, wound repair and tumor cell invasion depends on the coordinated regulation of cell adhesion receptors, matrix proteins and enzymes that proteolyse the extracellular matrix. Integrin α3β1 is a major receptor on epidermal keratinocytes for laminin-5 in the cutaneous basement membrane and is required for normal basement membrane organization during skin development. α3β1 is also expressed at high levels in the majority of adherent transformed cells and in most tumors, and it could have similar roles in extracellular matrix remodeling during tumorigenesis and cell invasion. In the present study, we show that α3β1 expression is required in immortalized mouse keratinocytes (MK) for the production of the matrix metalloproteinase MMP-9/gelatinase B, an MMP that is coexpressed with α3β1 in epithelial cell carcinomas and during wound healing, and contributes to the invasive potential of some tumor cells. MMP-9 was expressed in MK cells derived from wild-type mice, but not in MK cells derived from α3-null mice. Reconstitution of α3β1 expression in α3-null MK cells through transfection with the α3 subunit restored MMP-9 secretion, indicating an α3β1-dependent pathway for MMP-9 production. α3β1- dependent expression of MMP-9 was associated with the immortalized phenotype, since nonimmortalized, primary keratinocytes required soluble growth factors, but not α3β1, for efficient expression of MMP-9. Our results suggest that an α3β1-independent pathway(s) for MMP-9 production is suppressed in keratinocytes immortalized with large T antigen, and that an α3β1-dependent pathway is required for sustained production of MMP-9 in the absence of other pathways.
The carboxy terminus of Tub4p is required for γ-tubulin function in budding yeast
Authors: Vogel Jacalyn , Snyder Michael
Published: April 2021

The role of γ-tubulin in microtubule nucleation is well established, however, its function in other aspects of microtubule organization is unknown. The carboxy termini of α/β-tubulins influence the assembly and stability of microtubules. We investigated the role of the carboxy terminus of yeast γ-tubulin (Tub4p) in microtubule organization. This region consists of a conserved domain (DSYLD), and acidic tail. Cells expressing truncations lacking the DSYLD domain, tail or both regions are temperature sensitive for growth. Growth defects of tub4 mutants lacking either or both carboxy-terminal domains are suppressed by the microtubule destabilizing drug benomyl. tub4 carboxy-terminal mutants arrest as large budded cells with short bipolar spindles positioned at the bud neck. Electron microscopic analysis of wild-type and CTR mutant cells reveals that SPBs are tightly associated with the bud neck/cortex by cytoplasmic microtubules in mutants lacking the tail region (tub4-Δ444, tub4-Δ448). Mutants lacking the DSYLD residues (tub4-Δ444, tub4-ΔDSYLD) form many cytoplasmic microtubules. We propose that the carboxy terminus of Tub4p is required for re-organization of the microtubules upon completion of nuclear migration, and facilitates spindle elongation into the bud.
ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis
Authors: Norton John D.
Published: April 2021

The ubiquitously expressed family of ID helix-loop-helix (HLH) proteins function as dominant negative regulators of basic HLH (bHLH) transcriptional regulators that drive cell lineage commitment and differentiation in metazoa. Recent data from cell line and in vivo studies have implicated the functions of ID proteins in other cellular processes besides negative regulation of cell differentiation. ID proteins play key roles in the regulation of lineage commitment, cell fate decisions and in the timing of differentiation during neurogenesis, lymphopoiesis and neovascularisation (angiogenesis). They are essential for embryogenesis and for cell cycle progression, and they function as positive regulators of cell proliferation. ID proteins also possess pro-apoptotic properties in a variety of cell types and function as cooperating or dominant oncoproteins in immortalisation of rodent and human cells and in tumour induction in Id-transgenic mice. In several human tumour types, the expression of ID proteins is deregulated, and loss- and gain-of-function studies implicate ID functions in the regulation of tumour growth, vascularisation, invasiveness and metastasis. More recent biochemical studies have also revealed an emerging ‘molecular promiscuity’ of mammalian ID proteins: they directly interact with and modulate the activities of several other families of transcriptional regulator, besides bHLH proteins.
Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap
Authors: Infante A. S. , Stein M. S. , Zhai Y. , Borisy G. G. , Gundersen G. G.
Published: April 2021

Many cell types contain a subset of long-lived, ‘stable’ microtubules that differ from dynamic microtubules in that they are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). Elevated Glu tubulin does not stabilize the microtubules and the mechanism for the stability of Glu microtubules is not known. We used detergent-extracted cell models to investigate the nature of Glu microtubule stability. In these cell models, Glu microtubules did not incorporate exogenously added tubulin subunits on their distal ends, while >70% of the bulk microtubules did. Ca2+-generated fragments of Glu microtubules incorporated tubulin, showing that Glu microtubule ends are capped. Consistent with this, Glu microtubules in cell models were resistant to dilution-induced breakdown. Known microtubule end-associated proteins (EB1, APC, p150Glued and vinculin focal adhesions) were not localized on Glu microtubule ends. ATP, but not nonhydrolyzable analogues, induced depolymerization of Glu microtubules in cell models. Timelapse and photobleaching studies showed that ATP triggered subunit loss from the plus end. ATP breakdown of Glu microtubules was inhibited by AMP-PNP and vanadate, but not by kinase or other inhibitors. Additional experiments showed that conventional kinesin or kif3 were not involved in Glu microtubule capping. We conclude that Glu microtubules are stabilized by a plus-end cap that includes an ATPase with properties similar to kinesins.
Calsequestrin, a calcium sequestering protein localized at the sarcoplasmic reticulum, is not essential for body-wall muscle function in Caenorhabditis elegans
Authors: Cho Jeong Hoon , Oh Young Soo , Park Kye Won , Yu Jae-Ran , Choi Kyu Yeong , Shin Ji-Yeon , Kim Do Han , Park Woo Jin , Hamada Tomoyo , Kagawa Hiroaki , Maryon Edward B. , Bandyopadhyay Jaya , Ahnn Joohong
Published: April 2021

Calsequestrin is the major calcium-binding protein of cardiac and skeletal muscles whose function is to sequester Ca2+ in the lumen of the sarcoplasmic reticulum (SR). Here we describe the identification and functional characterization of a C. elegans calsequestrin gene (csq-1). CSQ-1 shows moderate similarity (50% similarity, 30% identity) to rabbit skeletal calsequestrin. Unlike mammals, which have two different genes encoding cardiac and fast-twitch skeletal muscle isoforms, csq-1 is the only calsequestrin gene in the C. elegans genome. We show that csq-1 is highly expressed in the body-wall muscles, beginning in mid-embryogenesis and maintained through the adult stage. In body-wall muscle cells, CSQ-1 is localized to sarcoplasmic membranes surrounding sarcomeric structures, in the regions where ryanodine receptors (UNC-68) are located. Mutation in UNC-68 affects CSQ-1 localization, suggesting that the two possibly interact in vivo. Genetic analyses of chromosomal deficiency mutants deleting csq-1 show that CSQ-1 is not essential for initiation of embryonic muscle formation and contraction. Furthermore, double-stranded RNA injection resulted in animals completely lacking CSQ-1 in body-wall muscles with no observable defects in locomotion. These findings suggest that although CSQ-1 is one of the major calcium-binding proteins in the body-wall muscles of C. elegans, it is not essential for body-wall muscle formation and contraction.
Processing and trafficking of cysteine proteases in Leishmania mexicana
Authors: Brooks Darren R. , Tetley Laurence , Coombs Graham H. , Mottram Jeremy C.
Published: April 2021

Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPBC25G). When CPBC25G was expressed in a L. mexicana mutant lacking all CPB genes, the inactive pro-enzyme was processed to the mature protein and trafficked to the lysosome. These results show that auto-activation is not required for correct processing of CPB in vivo. When CPBC25G was expressed in a L. mexicana mutant lacking both CPA and CPB genes, the majority of the pro-enzyme remained unprocessed and accumulated in the flagellar pocket. These data reveal that CPA can directly or indirectly process CPBC25G and suggest that cysteine proteases are targeted to lysosomes via the flagellar pocket. Moreover, they show that another protease can process CPB in the absence of either CPA or CPB, albeit less efficiently. Abolition of the glycosylation site in the mature domain of CPB did not affect enzyme processing, targeting or in vitro activity towards gelatin. This indicates that glycosylation is not required for trafficking. Together these findings provide evidence that the major route of trafficking of Leishmania cysteine proteases to lysosomes is via the flagellar pocket and therefore differs significantly from cysteine protease trafficking in mammalian cells.
Structure and dynamics of hnRNP-labelled nuclear bodies induced by stress treatments
Authors: Chiodi Ilaria , Biggiogera Marco , Denegri Marco , Corioni Margherita , Weighardt Florian , Cobianchi Fabio , Riva Silvano , Biamonti Giuseppe
Published: April 2021

We have previously described HAP, a novel hnRNP protein that is identical both to SAF-B, a component of the nuclear scaffold, and to HET, a transcriptional regulator of the gene for heat shock protein 27. After heat shock, HAP is recruited to a few nuclear bodies. Here we report the characterisation of these bodies, which are distinct from other nuclear components such as coiled bodies and speckles. The formation of HAP bodies is part of a general cell response to stress agents, such as heat shock and cadmium sulfate, which also affect the distribution of hnRNP protein M. Electron microscopy demonstrates that in untreated cells, similar to other hnRNP proteins, HAP is associated to perichromatin fibrils. Instead, in heat shocked cells the protein is preferentially associated to clusters of perichromatin granules, which correspond to the HAP bodies observed in confocal microscopy. Inside such clusters, perichromatin granules eventually merge into a highly packaged ‘core’. HAP and hnRNP M mark different districts of these structures. HAP is associated to perichromatin granules surrounding the core, while hnRNP M is mostly detected within the core. BrU incorporation experiments demonstrate that no transcription occurs within the stress-induced clusters of perichromatin granules, which are depots for RNAs synthesised both before and after heat shock.
Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts
Authors: Lampson Michael A. , Racz Attila , Cushman Samuel W. , McGraw Timothy E.
Published: April 2021

Insulin-responsive trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) in adipose and muscle cells is well established. Insulin regulation of GLUT4 trafficking in these cells underlies the role that adipose tissue and muscle play in the maintenance of whole body glucose homeostasis. GLUT4 is expressed in a very limited number of tissues, most highly in adipose and muscle, while IRAP is expressed in many tissues. IRAP’s physiological role in any of the tissues in which it is expressed, however, is unknown. The fact that IRAP, which traffics by the same insulin-regulated pathway as GLUT4, is expressed in ‘non-insulin responsive’ tissues raises the question of whether these other cell types also have a specialized insulin-regulated trafficking pathway. The existence of an insulin-responsive pathway in other cell types would allow regulation of IRAP activity at the plasma membrane as a potentially important physiological function of insulin. To address this question we use reporter molecules for both GLUT4 and IRAP trafficking to measure insulin-stimulated translocation in undifferentiated cells by quantitative fluorescence microscopy. One reporter (vpTR), a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor, has been previously characterized. The other is a GLUT4 construct with an exofacial HA epitope and a C-terminal GFP. By comparing these reporters to the transferrin receptor, a marker for general endocytic trafficking, we demonstrate the existence of a specialized, insulin-regulated trafficking pathway in two undifferentiated cell types, neither of which normally express GLUT4. The magnitude of translocation in these undifferentiated cells (approximately threefold) is similar to that reported for the translocation of GLUT4 in muscle cells. Thus, undifferentiated cells have the necessary retention and translocation machinery for an insulin response that is large enough to be physiologically important.
The armadillo repeat region targets ARVCF to cadherin-based cellular junctions
Authors: Kaufmann Ulrike , Zuppinger Christian , Waibler Zoe , Rudiger Manfred , Urbich Carmen , Martin Bernd , Jockusch Brigitte M. , Eppenberger Hans , Starzinski-Powitz Anna
Published: April 2021

The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites. Several members of the armadillo repeat protein family mediate this linkage. We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail. Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay. Immunoprecipitation and in vitro binding assays confirmed this interaction. Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, β-catenin and p120 (ctn). Hence, the N terminus of ARVCF is not required for junctional localization. In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes. Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.
The S. pombe rlc1 gene encodes a putative myosin regulatory light chain that binds the type II myosins myo3p and myo2p
Authors: Goff Xavier Le , Motegi Fumio , Salimova Ekaterina , Mabuchi Issei , Simanis Viesturs
Published: April 2021

In order to identify additional components important for cell division in the fission yeast Schizosaccharomyces pombe we have screened a bank of conditional cold-sensitive mutants for cytokinesis defects. One of these mutants showed a delay in cell cleavage, and strong genetic interactions with other genes implicated in medial ring formation. Cloning of the corresponding gene indicates that it encodes a protein with significant homology to the regulatory light chain of non-muscle myosins. We have named the gene rlc1 (regulatory light chain 1). The gene is not essential for division, but null mutants display a cell cleavage defect and form an aberrant F-actin ring. Two myosin-II heavy chains have been identified in fission yeast: Co-immunoprecipitation experiments indicate that rlc1p associates more strongly with myo3p than myo2p.
Immunoelectron microscopic evidence that GLUT4 translocation explains the stimulation of glucose transport in isolated rat white adipose cells
Authors: Malide Daniela , Ramm Georg , Cushman Samuel W. , Slot Jan W.
Published: April 2021

We used an improved cryosectioning technique in combination with quantitative immunoelectron microscopy to study GLUT4 compartments in isolated rat white adipose cells. We provide clear evidence that in unstimulated cells most of the GLUT4 localizes intracellularly to tubulovesicular structures clustered near small stacks of Golgi and endosomes, or scattered throughout the cytoplasm. This localization is entirely consistent with that originally described in brown adipose tissue, strongly suggesting that the GLUT4 compartments in white and brown adipose cells are morphologically similar. Furthermore, insulin induces parallel increases (with similar magnitudes) in glucose transport activity, approximately 16-fold, and cell-surface GLUT4, approximately 12-fold. Concomitantly, insulin decreases GLUT4 equally from all intracellular locations, in agreement with the concept that the entire cellular GLUT4 pool contributes to insulin-stimulated exocytosis. In the insulin-stimulated state, GLUT4 molecules are not randomly distributed on the plasma membrane, but neither are they enriched in caveolae. Importantly, the total number of GLUT4 C-terminal epitopes detected by the immuno-gold method is not significantly different between basal and insulin-stimulated cells, thus arguing directly against a reported insulin-induced unmasking effect. These results provide strong morphological evidence (1) that GLUT4 compartments are similar in all insulin-sensitive cells and (2) for the concept that GLUT4 translocation almost fully accounts for the increase in glucose transport in response to insulin.
Activated Raf kinase inhibits muscle cell differentiation through a MEF2-dependent mechanism
Authors: Winter Barbara , Arnold Hans-Henning
Published: April 2021

Muscle cell development is dependent on the activity of cell type-specific basic-helix-loop-helix transcription factors, MyoD, Myf-5, myogenin, and MRF4 which collaborate with myocyte enhancer factor 2 proteins to activate muscle-specific gene expression. Growth factors and activated Ras prevent differentiation of myoblasts in culture but the downstream signalling pathways are not well understood. Here, we demonstrate that active Raf kinase (Raf-BxB) completely inhibits myogenic conversion of 10T1/2 cells mediated by Myf-5 and differentiation of L6 myoblasts as indicated by the absence of myotubes, lack of myogenin expression, and markedly reduced expression of myosin heavy chain. However, activated Raf inhibits transcriptional activation by Myf-5 only partially suggesting that other potential targets of Ras/Raf signalling may be involved. Significantly, we observed that elevated Raf kinase activity in L6 muscle cells suppresses the accumulation of MEF2 protein in nuclei, while MEF2 transcription appears unaffected. Moreover, forced expression of MEF2A in 10T1/2 cells rescues MyoD dependent myogenic conversion in the presence of constitutively active Raf kinase and partially restores transactivation of a myogenin promoter-dependent reporter gene in L6 muscle cells containing activated Raf kinase. From these observations we conclude that persistent activation of Raf signalling affects nuclear MEF2 functions which may explain why myogenin expression and myoblast differentiation are inhibited.
The genomic silencing of position-effect variegation in Drosophila melanogaster: interaction between the heterochromatin-associated proteins Su(var)3-7 and HP1
Authors: Delattre Marion , Spierer Anne , Tonka Chia-Hwa , Spierer Pierre
Published: April 2021

Position-effect variegation results from mosaic silencing by chromosomal rearrangements juxtaposing euchromatin genes next to pericentric heterochromatin. An increase in the amounts of the heterochromatin-associated Su(var)3-7 and HP1 proteins augments silencing. Using the yeast two-hybrid protein interaction trap system, we have isolated HP1 using Su(var)3-7 as a bait. We have then delimited three binding sites on Su(var)3-7 for HP1. On HP1, the C-terminal moiety, including the chromo shadow domain, is required for interaction. In vivo, both proteins co-localise not only in heterochromatin, but also in a limited set of sites in euchromatin and at telomeres. When delocalised to the sites bound by the protein Polycomb in euchromatin, HP1 recruits Su(var)3-7. Finally, and in contrast with euchromatin genes, a decrease in the amounts of both proteins enhances variegation of the light gene, one of the few genetic loci mapped within pericentric heterochromatin. This body of data supports a direct link between Su(var)3-7 and HP1 in the genomic silencing of position-effect variegation.
Immobilization of Notch ligand, Delta-1, is required for induction of Notch signaling
Authors: Varnum-Finney Barbara , Wu Lizi , Yu Monica , Brashem-Stein Carolyn , Staats Steven , Flowers David , Griffin James D. , Bernstein Irwin D.
Published: April 2021

Cell-cell interactions mediated by Notch and its ligands are known to effect many cell fate decisions in both invertebrates and vertebrates. However, the mechanisms involved in ligand induced Notch activation are unknown. Recently it was shown that, in at least some cases, endocytosis of the extracellular domain of Notch and ligand by the signaling cell is required for signal induction in the receptive cell. These results imply that soluble ligands (ligand extracellular domains) although capable of binding Notch would be unlikely to activate it. To test the potential activity of soluble Notch ligands, we generated monomeric and dimeric forms of the Notch ligand Delta-1 by fusing the extracellular domain to either a series of myc epitopes (Delta-1ext-myc) or to the Fc portion of human IgG-1 (Delta-1ext-IgG), respectively. Notch activation, assayed by inhibition of differentiation in C2 myoblasts and by HES1 transactivation in U20S cells, occurred when either Delta-1ext-myc or Delta-1ext-IgG were first immobilized on the plastic surface. However, Notch was not activated by either monomeric or dimeric ligand in solution (non-immobilized). Furthermore, both non-immobilized Delta-1ext-myc and Delta-1ext-IgG blocked the effect of immobilized Delta. These results indicate that Delta-1 extracellular domain must be immobilized to induce Notch activation in C2 or U20S cells and that non-immobilized Delta-1 extracellular domain is inhibitory to Notch function. These results imply that ligand stabilization may be essential for Notch activation.
The Spd1p S phase inhibitor can activate the DNA replication checkpoint pathway in fission yeast
Authors: Borgne Annie , Nurse Paul
Published: April 2021

Spd1p (for S phase delayed) is a cell cycle inhibitor in Schizosaccharomyces pombe. Spd1p overexpression blocks the onset of both S phase and mitosis. In this study, we have investigated the mechanisms by which Spd1p overexpression blocks cell cycle progression, focussing on the block over mitotic onset. High levels of Spd1p lead to an increase in Y15 phosphorylation of Cdc2p and we show that the block over G2 requires the Wee1p kinase and is dependent on the rad and chk1/cds1 checkpoint genes. We propose that high levels of Spd1p in G2 cells activate the DNA replication checkpoint control, which leads to a Wee1p-dependent increase of Cdc2p Y15 phosphorylation blocking onset of mitosis. The Spd1p block at S phase onset may act by interfering directly with DNA replication, and also activates the G2rad/hus checkpoint pathway to block mitosis.
Recent structural insights into transcription preinitiation complexes
Authors: Nogales Eva
Published: April 2021

Our understanding of the elaborate mechanism of gene transcription initiation in eukaryotes has been widened by recent structural information on some of the key components of the complex preinitiation transcriptional machinery. The high-resolution structures of both bacterial and eukaryotic polymerases are technical landmarks of great biological significance that have given us the first molecular insight into the mechanism of this large enzyme. While new atomic structures of different domains of general transcription factors, such as the double bromodomain of TAF250, have become available by means of X-ray crystallography and NMR studies, more global pictures of multisubunit transcription complexes, such as TFIID, TFIIH or the yeast mediator, have now been obtained by electron microscopy and image-reconstruction techniques. A combination of methodologies may prove essential for a complete structural description of the initial steps in the expression of eukaryotic genes.
Protease-activated receptor-2 mediates proliferative responses in skeletal myoblasts
Authors: Chinni Carla , Niese Michael R. de , Jenkins Alison L. , Pike Robert N. , Bottomley Stephen P. , Mackie Eleanor J.
Published: April 2021

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by proteases within the N terminus, exposing a new tethered ligand that binds and activates the receptor. Activators of PAR-2 include trypsin and mast cell tryptase. Skeletal myoblasts are known to express PAR-1, a thrombin receptor. The current study was undertaken to determine whether myoblasts express PAR-2. Primary neonatal rat and mouse skeletal myoblast cultures were shown to express PAR-2 in polymerase chain reaction and immunocytochemical studies. Expression of PAR-2 was also demonstrated by immunohistochemistry in developing mouse skeletal muscle in vivo. Trypsin or a synthetic peptide corresponding to the rat PAR-2 tethered ligand caused a dose-dependent elevation in intracellular calcium in cultured rat myoblasts, with an EC50 of 13 nM or 56 μM, respectively. Studies aimed at identifying the function of PAR-2 in myoblasts demonstrated no effect of the receptor-activating peptide on survival or fusion in serum-deprived myoblasts. The PAR-2-activating peptide did, however, stimulate proliferation of serum-deprived myoblasts. These results demonstrate that skeletal muscle cells express PAR-2, activation of which leads to stimulation of myoblast proliferation.
Characterisation of transcriptionally active and inactive chromatin domains in neurons
Authors: Akhmanova Anna , Verkerk Ton , Langeveld An , Grosveld Frank , Galjart Niels
Published: April 2021

The tandemly organised ribosomal DNA (rDNA) repeats are transcribed by a dedicated RNA polymerase in a specialised nuclear compartment, the nucleolus. There appears to be an intimate link between the maintenance of nucleolar structure and the presence of heterochromatic chromatin domains. This is particularly evident in many large neurons, where a single nucleolus is present, which is separated from the remainder of the nucleus by a characteristic shell of heterochromatin. Using a combined fluorescence in situ hybridisation and immunocytochemistry approach, we have analysed the molecular composition of this highly organised neuronal chromatin, to investigate its functional significance. We find that clusters of inactive, methylated rDNA repeats are present inside large neuronal nucleoli, which are often attached to the shell of heterochromatic DNA. Surprisingly, the methylated DNA-binding protein MeCP2, which is abundantly present in the centromeric and perinucleolar heterochromatin, does not associate significantly with the methylated rDNA repeats, whereas histone H1 does overlap partially with these clusters. Histone H1 also defines other, centromere-associated chromatin subdomains, together with the mammalian Polycomb group factor Eed. These data indicate that neuronal, perinucleolar heterochromatin consists of several classes of inactive DNA, that are linked to a fraction of the inactive rDNA repeats. These distinct chromatin domains may serve to regulate RNA transcription and processing efficiently and to protect rDNA repeats against unwanted silencing and/or homologous recombination events.
ALT-associated PML bodies are present in viable cells and are enriched in cells in the G2/M phase of the cell cycle
Authors: Grobelny Jennifer V. , Godwin Andrew K. , Broccoli Dominique
Published: April 2021

Telomere maintenance is essential for the unlimited proliferative potential of human cells, and hence immortalization. However, a number of tumors, tumorderived cell lines and in vitro immortalized cell lines have been described that do not express detectable telomerase activity. These lines utilize a mechanism, termed Alternative Lengthening of Telomeres (ALT), to provide telomere maintenance. A subset of the cells in each ALT cell line contain a novel form of the promyelocytic leukemia nuclear body (PML NB) in which telomeric DNA and the telomere binding proteins TRF1 and TRF2 co-localize with the PML protein, termed ALT-associated PML bodies (AA-PBs). In contrast, in non-ALT, telomerase-positive cell lines these telomeric proteins and the PML NB occupy distinct and separate subnuclear domains. PML NBs have been implicated in terminal differentiation, growth suppression and apoptosis. The role, if any, of AA-PBs in telomere maintenance or culture viability in telomerase negative cell lines is unclear, but it has been suggested that cells containing these structures are no longer viable and are marked for eventual death. We utilized a series of human ovarian surface epithelium (HOSE) cell lines that use ALT for telomere maintenance to determine if AA-PBs are indeed markers of cells in these cultures that are no longer cycling. We show that AA-PB positive cells incorporate BrdU and thus are able to carry out DNA replication. In addition, AA-PBs are present in mitotic cells and the frequency of cells containing these structures is increased when cultures are enriched for cells in the G2/M phase of the cell cycle suggesting that the formation of AA-PBs is coordinately regulated with the cell cycle. Finally, we demonstrate that the majority of the AA-PB positive cells in the culture are not destined for immediate apoptosis. Taken together the data argue against AA-PBs marking cells destined for death and, instead, raise the possibility that these structures may be actively involved in telomere maintenance via the ALT pathway.
Evidence for a novel pathway for the targeting of a Saccharomyces cerevisiae peroxisomal protein belonging to the isomerase/hydratase family
Authors: Karpichev Igor V. , Small Gillian M.
Published: April 2021

We, and others, have identified a novel Saccharomyces cerevisiae peroxisomal protein that belongs to the isomerase/hydratase family. The protein, named Dci1p, shares 50% identity with Eci1p, a Δ3-cis-Δ2-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the β-oxidation of unsaturated fatty acids. Both of these proteins are localized to peroxisomes, and both contain motifs at their amino- and carboxyl termini that resemble peroxisome targeting signals (PTS) 1 and 2. However, we demonstrate that the putative type 1 signaling motif is not required for the peroxisomal localization of either of these proteins. Furthermore, the correct targeting of Eci1p and Dci1p occurs in the absence of the receptors for the type 1 or type 2 peroxisome targeting pathway. Together, these data suggest a novel mechanism for the intracellular targeting of these peroxisomal proteins.
Expression and nuclear localization of BLM, a chromosome stability protein mutated in Bloom’s syndrome, suggest a role in recombination during meiotic prophase
Authors: Moens Peter B. , Freire Raimundo , Tarsounas Madalina , Spyropoulos Barbara , Jackson Stephen P.
Published: April 2021

Bloom’s syndrome (BS) is a recessive human genetic disorder characterized by short stature, immunodeficiency and elevated risk of malignancy. BS cells have genomic instability and an increased frequency of sister chromatid exchange. The gene mutated in BS, BLM, encodes a 3′-5′ helicase (BLM) with homology to bacterial recombination factor, RecQ. Human males homozygous for BLMmutations are infertile and heterozygous individuals display increased frequencies of structural chromosome abnormalities in their spermatozoa. Also, mutations in the Saccharomyces cerevisiaehomolog of BLM, Sgs1, cause a delay in meiotic nuclear division and a reduction in spore viability. These observations suggest that BLM may play a role during meiosis. Our antibodies raised against the C terminus of the human protein specifically recognize both mouse and human BLM in western blots of cell lines and in successive developmental stages of spermatocytes, but fail to detect BLM protein in a cell line with a C-terminally truncated protein. BLM protein expression and location are detected by immunofluorescence and immunoelectron microscopy as discrete foci that are sparsely present on early meiotic prophase chromosome cores, later found abundantly on synapsed cores, frequently in combination with the recombinases RAD51 and DMC1, and eventually as pure BLM foci. The colocalization of RAD51/DMC1 with BLM and the statistically significant excess of BLM signals in the synapsed pseudoautosomal region of the X-Y chromosomes, which is a recombinational hot spot, provide indications that BLM protein may function in the meiotic recombination process.
Comparison of the glycosyl-phosphatidylinositol cleavage/attachment site between mammalian cells and parasitic protozoa
Authors: White Ian J. , Souabni Abdallah , Hooper Nigel M.
Published: April 2021

It was previously hypothesised that the requirements for glycosyl-phosphatidylinositol (GPI) anchoring in mammalian cells and parasitic protozoa are similar but not identical. We have investigated this by converting the GPI cleavage/attachment site in porcine membrane dipeptidase to that found in the trypanosomal variant surface glycoprotein 117 and expressing the resulting mutants in COS-1 cells. Changing the entire ω, ω+1 and ω+2 triplet in membrane dipeptidase from Ser-Ala-Ala to Asp-Ser-Ser resulted in efficient GPI anchoring of the mutant proteins, as assessed by cell-surface activity assays and susceptibility to release by phosphatidylinositol-specific phospholipase C. Immunoelectrophoretic blot analysis with antibodies recognising epitopes either side of the native ω residue in porcine membrane dipeptidase, and expression of a mutant in which potential alternative cleavage/attachment sites were disrupted, indicated that alternative GPI cleavage/ attachment sites had not been used. These results indicate that the requirements for GPI anchoring between mammalian and protozoal cells are not as different as previously suggested, and that rules for predicting the probability of a sequence acting as a GPI cleavage/ attachment site need to be applied with caution.
Endo180, an endocytic recycling glycoprotein related to the macrophage mannose receptor is expressed on fibroblasts, endothelial cells and macrophages and functions as a lectin receptor
Authors: Sheikh Humma , Yarwood Helen , Ashworth Alan , Isacke Clare M.
Published: April 2021

Endo180 was previously characterized as a novel, cell type specific, recycling transmembrane glycoprotein. This manuscript describes the isolation of a full length human Endo180 cDNA clone which was shown to encode a fourth member of a family of proteins comprising the macrophage mannose receptor, the phospholipase A2receptor and the DEC-205/MR6 receptor. This receptor family is unusual in that they contain 8-10 C-type lectin carbohydrate recognition domains in a single polypeptide backbone, however, only the macrophage mannose receptor had been shown to function as a lectin. Sequence analysis of Endo180 reveals that the second carbohydrate recognition domain has retained key conserved amino acids found in other functional C-type lectins.

Furthermore, it is demonstrated that this protein displays Ca2+-dependent binding to N-acetylglucosamine but not mannose affinity columns. In order to characterize the physiological function of Endo180, a series of biochemical and morphological studies were undertaken. Endo180 is found to be predominantly expressed in vivo and in vitro on fibroblasts, endothelial cells and macrophages, and the distribution and post-translational processing in these cells is consistent with Endo180 functioning to internalize glycosylated ligands from the extracellular milieu for release in an endosomal compartment.
The transmembrane protein p23 contributes to the organization of the Golgi apparatus
Authors: Rojo Manuel , Emery Gregory , Marjomäki Varpu , McDowall Alasdair W. , Parton Robert G. , Gruenberg Jean
Published: April 2021

In previous studies we have shown that p23, a member of the p24-family of small transmembrane proteins, is highly abundant in membranes of the cis-Golgi network (CGN), and is involved in sorting/trafficking in the early secretory pathway. In the present study, we have further investigated the role of p23 after ectopic expression. We found that ectopically expressed p23 folded and oligomerized properly, even after overexpression. However, in contrast to endogenous p23, exogenous p23 molecules did not localize to the CGN, but induced a significant expansion of characteristic smooth ER membranes, where they accumulated in high amounts. This ER-derived, p23-rich subdomain displayed a highly regular morphology, consisting of tubules and/or cisternae of constant diameter, which were reminiscent of the CGN membranes containing p23 in control cells. The expression of exogenous p23 also led to the specific relocalization of endogenous p23, but not of other proteins, to these specialized ER-derived membranes. Relocalization of p23 modified the ultrastructure of the CGN and Golgi membranes, but did not affect anterograde and retrograde transport reactions to any significant extent. We conclude (i) that p23 has a morphogenic activity that contributes to the morphology of CGN-membranes; and (ii) that the presence of p23 in the CGN is necessary for the proper organization of the Golgi apparatus.
Brain-derived neurotrophic factor-induced phosphorylation of neurofilament-H subunit in primary cultures of embryo rat cortical neurons
Authors: Tokuoka Hirofumi , Saito Taro , Yorifuji Hiroshi , Wei Fan-Yan , Kishimoto Takeo , Hisanaga Shin-ichi
Published: April 2021

Phosphorylation of the neurofilament-H subunit (NF-H) was investigated in rat embryonic brain neurons in culture. A portion of the NF-H was phosphorylated in vivo at embryonic day 17 when brain neurons were prepared. When the neurons were isolated and cultured, the NF proteins disappeared once and then reappeared over the next several days in the following order: (1) NF-L/NF-M, (2) dephosphorylated NF-H and (3) phosphorylated NF-H. Phosphorylation of NF-H began around 4 days after cell plating, at about the time of synapse formation. Treatments that appeared to modulate the timing of synapse formation also affected the timing of NF-H phosphorylation: (1) earlier phosphorylation was observed at higher neuronal cell density, (2) earlier phosphorylation was observed in neurons cultured on a coating substrate that promotes rapid neurite extension and (3) phosphorylation was suppressed when neurite extension was inhibited by brefeldin A. Three possible synapse formation-induced events, excitation, cell-cell contact through adhesion proteins and elevated concentrations of neurotrophic factors, were examined for their possible involvement in generating the signal for NF-H phosphorylation. Neither excitation nor cell contact enhanced NF-H phosphorylation. Neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) stimulated phosphorylation of NF-H. The BDNF-stimulated phosphorylation was inhibited by an anti-BDNF antibody and K252a, an inhibitor of BDNF receptor TrkB tyrosine kinase. Among known NF-H kinases of cyclin-dependent kinase 5 (CDK5), external signal-regulated protein kinase (ERK) and stress-activated protein kinase (SAPK), CDK5 and SAPK showed an increase in kinase activity or an active form with a time course similar to NF-H phosphorylation in control culture. On the other hand, BDNF stimulated the kinase activity of CDK5 and induced appearance of an active form of ERK transiently. These results suggest a possibility that synapse formation induces NF-H phosphorylation, at least in part, through activation of CDK5 by BDNF.
The parting of the endothelium: miracle, or simply a junctional affair?
Authors: Johnson-Léger Caroline , Aurrand-Lions Michel , Imhof Beat A.
Published: April 2021

Leukocyte extravasation from the blood across the endothelium is vital for the functioning of the immune system. Our understanding of the early steps of this process has developed rapidly. However, it is still unclear how leukocytes undergo the final step, migrating through the junctions that mediate adhesion between adjacent endothelial cells, while preserving the barrier function of the endothelium. The first stage of transmigration – tethering and rolling – is mediated by interactions between selectins on the surface of leukocytes and glycosylated proteins such as GlyCAM-1 on the surface of endothelial cells. Stimulation of the leukocyte by chemokines then induces tight adhesion, which involves binding of activated leukocyte integrins to endothelial ICAM-1/VCAM-1 molecules. Passage of the leukocyte across the endothelium appears to require delocalization of certain endothelial cell molecules and proteolytic degradation of junctional complexes.
Preferential association of syntaxin 8 with the early endosome
Authors: Subramaniam V. Nathan , Loh Eva , Horstmann Heinz , Habermann Anja , Xu Yue , Coe John , Griffiths Gareth , Hong Wanjin
Published: April 2021

Members of the syntaxin family play a fundamental role in vesicle docking and fusion of diverse transport events. We have molecularly characterized syntaxin 8, a novel member of the syntaxin family. The nucleotide sequence of cloned rat cDNA predicts a polypeptide of 236 residues with a carboxyl-terminal 18-residue hydrophobic domain that may function as a membrane anchor. Characteristic of syntaxins, syntaxin 8 also contain regions that have the potential to form coiled-coil structures. Among the known syntaxins, syntaxin 8 is most homologous to syntaxin 6 which is predominantly associated with the trans-Golgi network (TGN). The syntaxin 8 transcript is detected in all rat tissues examined by northern blot. Antibodies against recombinant syntaxin 8 recognize a 27 kDa protein that is enriched in membrane fractions containing the Golgi apparatus and the endosomal/lysosomal compartments. Syntaxin 8 in membrane extract could be incorporated into a 20S protein complex in a way that is dependent on the soluble N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP), suggesting that syntaxin 8 is indeed a SNAP receptor (SNARE). Indirect immunofluorescence microscopy reveals that the majority of syntaxin 8 is localized to the early endosome marked by Rab5. This is corroborated by immunogold labeling experiments showing enrichment of syntaxin 8 in the early endosome and its co-labeling with Rab5.