bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombospondin-1 differentially induces chemotaxis and DNA synthesis of human venous smooth muscle cells at the receptor-binding level
Authors: Lymn Joanne S. , Patel Mahendra K. , Clunn Gerard F. , Rao Sarafina J. , Gallagher Karen L. , Hughes Alun D.
Published: November 2002

Thrombospondin-1 is a large matricellular protein that acts as a pleiotropic growth factor for human vascular smooth muscle cells, and may play a role in the progression of vascular disease. Although we have previously demonstrated the dependence of both thrombospondin-1-stimulated cell chemotaxis and proliferation on tyrosine kinases, the receptor mechanisms involved remain obscure. This investigation aims to determine the nature of the receptor(s) involved in the cellular responses to thrombospondin-1. Cellular signals were identified by western blotting following cell stimulation, while cellular responses were assessed by measuring DNA synthesis and chemotaxis. These data demonstrate that thrombospondin-1-induced cell chemotaxis can be inhibited by a peptide containing the Arg-Gly-Asp motif, a function-blocking αvβ3 antibody, a function-blocking integrin-associated protein (IAP) antibody and pertussis toxin, while thrombospondin-1-stimulated DNA synthesis is inhibited by a function-blocking α3β1 antibody. Similarly the Arg-Gly-Asp-containing peptide inhibits tyrosine phosphorylation of focal adhesion kinase and the p85 regulatory subunit of phosphatidylinositol 3-kinase, but does not significantly affect tyrosine phosphorylation, or activation, of extracellular-regulated kinase. These data suggest that soluble thrombospondin-1 interacts with human vascular smooth muscle cells via two independent and separable receptor-binding sites, to differentially stimulate cell chemotaxis and DNA synthesis.
Keratin mutations of epidermolysis bullosa simplex alter the kinetics of stress response to osmotic shock
Authors: D'Alessandro Mariella , Russell David , Morley Susan M. , Davies Anthony M. , Lane E. Birgitte
Published: November 2002

The intermediate filament cytoskeleton is thought to confer physical resilience on tissue cells, on the basis of extrapolations from the phenotype of cell fragility that results from mutations in skin keratins. There is a need for functional cell assays in which the impact of stress on intermediate filaments can be induced and analyzed. Using osmotic shock, we have induced cytoskeleton changes that suggest protective functions for actin and intermediate filament systems. Induction of the resulting stress response has been monitored in keratinocyte cells lines carrying K5 or K14 mutations, which are associated with varying severity of epidermolysis bullosa simplex. Cells with severe mutations were more sensitive to osmotic stress and took longer to recover from it. Their stress-activated response pathways were induced faster, as seen by early activation of JNK, ATF-2 and c-Jun. We demonstrate that the speed of a cell's response to hypotonic stress, by activation of the SAPK/JNK pathway, is correlated with the clinical severity of the mutation carried. The response to hypo-osmotic shock constitutes a discriminating stress assay to distinguish between the effects of different keratin mutations and is a potentially valuable tool in developing therapeutic strategies for keratin-based skin fragility disorders.
Conditional ablation of the Mat1 subunit of TFIIH in Schwann cells provides evidence that Mat1 is not required for general transcription
Authors: Korsisaari Nina , Rossi Derrick J. , Paetau Anders , Charnay Patrick , Henkemeyer Mark , Mäkelä Tomi P.
Published: November 2002

The mammalian Mat1 protein has been implicated in cell cycle regulation as part of the Cdk activating kinase (CAK), and in regulation of transcription as a subunit of transcription factor TFIIH. To address the role of Mat1 in vivo, we have used a Cre/loxP system to conditionally ablate Mat1 in adult mitotic and post-mitotic lineages. We found that the mitotic cells of the germ lineage died rapidly upon disruption of Mat1 indicating an absolute requirement of Mat1 in these cells. By contrast, post-mitotic myelinating Schwann cells were able to attain a mature myelinated phenotype in the absence of Mat1. Moreover, mutant animals did not show morphological or physiological signs of Schwann cell dysfunction into early adulthood. Beyond 3 months of age, however, myelinated Schwann cells in the sciatic nerves acquired a severe hypomyelinating morphology with alterations ranging from cells undergoing degeneration to completely denuded axons. This phenotype was coupled to extensive proliferation and remyelination that our evidence suggests was undertaken by the non-myelinated Schwann cell pool. These results indicate that Mat1 is not essential for the transcriptional program underlying the myelination of peripheral axons by Schwann cells and suggest that the function of Mat1 in RNA polymerase II-mediated transcription in these cells is regulatory rather than essential.
A developmentally regulated ARF-like 5 protein (ARL5), localized to nuclei and nucleoli, interacts with heterochromatin protein 1
Authors: Lin Ching-Yi , Li Chun-Chun , Huang Pei-Hsin , Lee Fang-Jen S.
Published: November 2002

ARF-like proteins (ARLs) are distinct group of members of the ARF family of Ras-related GTPases. Although ARLs are very similar in primary structure to ARFs, their functions remain unclear. We cloned mouse (m) and human (h) ARL5 cDNAs to characterize the protein products and their molecular properties. mARL5 mRNA was more abundant in liver than in other adult tissues tested. mARL5, similar to mARL4, was developmentally regulated and localized to nuclei. hARL5 interacted with importin-α through its C-terminal bipartite nuclear localization signal. When expressed in COS-7 cells, mutant hARL5(T35N), which is predicted to be GDP bound, was concentrated in nucleoli. The N-terminus of hARL5, like that of ARF, was myristoylated. Yeast two-hybrid screening and in vitro protein-interaction assays showed that hARL5(Q80L),predicted to be GTP bound, interacted with heterochromatin protein 1α(HP1α), which is known to be associated with telomeres as well as with heterochromatin, and acted as a transcriptional suppressor in mammalian cells. The interaction was reproduced in COS cells, where hARL5(Q80L) was co-immunoprecipitated with HP1α. hARL5 interaction with HP1α was dependent on the nucleotide bound, and required the MIR-like motif. Moreover,hARL5(Q80L), but not hARL5 lacking the MIR-like motif, was partly co-localized with overexpressed HP1α. Our findings suggest that developmentally regulated ARL5, with its distinctive nuclear/nucleolar localization and interaction with HP1α, may play a role(s) in nuclear dynamics and/or signaling cascades during embryonic development.
The postsynaptic density and dendritic raft localization of PSD-Zip70,which contains an N-myristoylation sequence and leucine-zipper motifs
Authors: Konno Daijiro , Ko Ji-Ae , Usui Shinichi , Hori Kei , Maruoka Hisato , Inui Makoto , Fujikado Takashi , Tano Yasuo , Suzuki Tatsuo , Tohyama Koujiro , Sobue Kenji
Published: November 2002

The postsynaptic site of the excitatory synapse, which is composed of the postsynaptic density (PSD) attached to the postsynaptic membrane, is a center for synaptic plasticity. To reveal the molecular organization and functional regulation of the postsynaptic site, we cloned a 70 kDa protein that is concentrated in PSDs using a monoclonal antibody against the PSD. This protein, named PSD-Zip70, is highly homologous to the human FEZ1/LZTS1 gene product. PSD-Zip70 contains an N-myristoylation consensus sequence, a polybasic cluster in the N-terminal region and four leucine-zipper motifs in the C-terminal region. Light and electron microscopy showed that this protein was localized to the dendritic spines, especially in the PSD and the postsynaptic membrane. Fractionation of the synaptic plasma membrane demonstrated that PSD-Zip70 was localized to the PSD and the dendritic raft. In Madin-Darby canine kidney (MDCK) cells, exogenous PSD-Zip70 was targeted to the apical plasma membrane of microvilli, and its N-myristoylation was necessary for this targeting. In hippocampal neurons,N-myristoylation was also required for the membrane localization and the C-terminal region was critically involved in the synaptic targeting. These results suggest that PSD-Zip70 may be involved in the dynamic properties of the structure and function of the postsynaptic site.
Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse
Authors: Stepp Mary Ann , Gibson Heather E. , Gala Purvi H. , Iglesia Drina D. Sta. , Pajoohesh-Ganji Ahdeah , Pal-Ghosh Sonali , Brown Marcus , Aquino Christopher , Schwartz Arnold M. , Goldberger Olga , Hinkes Michael T. , Bernfield Merton
Published: November 2002

Mice lacking syndecan-1 are viable, fertile and have morphologically normal skin, hair and ocular surface epithelia. While studying the response of these mice to corneal epithelial and skin wounding, we identified defects in epithelial cell proliferation and regulation of integrin expression. mRNA profiling of corneal epithelial tissues obtained from wild-type and syndecan-1-/- mice suggest that these defects result from differences in overall gene transcription. In the cornea,syndecan-1-/- epithelial cells migrate more slowly, show reduced localization of α9 integrin during closure of wounds and fail to increase their proliferation rate 24 hours after wounding. In the skin, we did not document a migration defect after full thickness wounds but did observe cell proliferation delays and reduced localization of α9 integrin in the syndecan-1-/- epidermis after dermabrasion. Despite increased cell proliferation rates in the uninjured syndecan-1-/- epidermis and the corneal epithelium, morphologically normal epithelial thickness is maintained prior to injury; however, wounding is accompanied by prolonged hypoplasia in both tissues. Analyses of integrin protein levels in extracts from full thickness skin, revealed increased levels of α3 and α9 integrins both prior to injury and after hair removal in syndecan-1-/- mice but no increase 2 days after dermabrasion. These data for the first time show involvement of α9 integrin in skin wound healing and demonstrate essential roles for syndecan-1 in mediating cell proliferation and regulation of integrin expression in normal and wounded epithelial tissues.
The scaffolding domain of caveolin 2 is responsible for its Golgi localization in Caco-2 cells
Authors: Breuza Lionel , Corby Séverine , Arsanto Jean-Pierre , Delgrossi Marie-Hélène , Scheiffele Peter , Le Bivic André
Published: November 2002

In this work, we showed that in Caco-2 cells, a polarized cell line derived from human colon cancer that does not express caveolin 1 (Cav-1), there was no detectable expression of caveolin 2 (Cav-2). When Cav-2 was reintroduced in these cells, it accumulated in the Golgi complex. A chimera, in which the scaffolding domain of Cav-1 was replaced by the one from Cav-2, induced a prominent Golgi staining of Cav-1, strongly indicating that this domain was responsible for the accumulation of Cav-2 in the Golgi complex. Cav-2 was able to interact with Cav-1 in the Golgi complex but this interaction was not sufficient to export it from this compartment. Several chimeras between Cav-1 and 2 were used to show that surface expression of caveolin was necessary but not sufficient to promote caveolae formation. Interestingly, levels of incorporation of the chimeras into Triton insoluble rafts correlated with their ability to trigger caveolae formation raising the possibility that a critical concentration of caveolins to discrete domains of the plasma membrane might be necessary for caveolae formation.
Transient association of titin and myosin with microtubules in nascent myofibrils directed by the MURF2 RING-finger protein
Authors: Pizon Véronique , Iakovenko Andrei , van der Ven Peter F. M. , Kelly Raymond , Fatu Cristina , Fürst Dieter O. , Karsenti Eric , Gautel Mathias
Published: November 2002

Assembly of muscle sarcomeres is a complex dynamic process and involves a large number of proteins. A growing number of these have regulatory functions and are transiently present in the myofibril. We show here that the novel tubulin-associated RING/B-box protein MURF2 associates transiently with microtubules, myosin and titin during sarcomere assembly. During sarcomere assembly, MURF2 first associates with microtubules at the exclusion of tyrosinated tubulin. Then, MURF2-labelled microtubules associate transiently with sarcomeric myosin and later with A-band titin when non-striated myofibrils differentiate into mature sarcomeres. Finally, MURF2 labelled microtubules disappear from the sarcomere after the incorporation of myosin filaments and the elongation of titin. This suggests that the incorporation of myosin into nascent sarcomeres and the elongation of titin require an active,microtubule-dependent transport process and that MURF2-associated microtubules play a role in the alignment and extension of nascent sarcomeres. MURF2 is expressed in at least four isoforms, of which a 27 kDa isoform is cardiac specific. A C-terminal isoform is generated by alternative reading frame use,a novelty in muscle proteins. In mature cardiac sarcomeres, endogenous MURF2 can associate with the M-band, and is translocated to the nucleus. MURF2 can therefore act as a transient adaptor between microtubules, titin and nascent myosin filaments, as well as being involved in signalling from the sarcomere to the nucleus.
Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo
Authors: Allard Patrick , Champigny Marc J. , Skoggard Sarah , Erkmann Judith A. , Whitfield Michael L. , Marzluff William F. , Clarke Hugh J.
Published: November 2002

The stem-loop binding protein (SLBP) binds to the 3′ end of histone mRNA and participates in 3′-processing of the newly synthesized transcripts, which protects them from degradation, and probably also promotes their translation. In proliferating cells, translation of SLBP mRNA begins at G1/S and the protein is degraded following DNA replication. These post-transcriptional mechanisms closely couple SLBP expression to S-phase of the cell cycle, and play a key role in restricting synthesis of replication-dependent histones to S-phase. In contrast to somatic cells,replication-dependent histone mRNAs accumulate and are translated independently of DNA replication in oocytes and early embryos. We report here that SLBP expression and activity also differ in mouse oocytes and early embryos compared with somatic cells. SLBP is present in oocytes that are arrested at prophase of G2/M, where it is concentrated in the nucleus. Upon entry into M-phase of meiotic maturation, SLBP begins to accumulate rapidly,reaching a very high level in mature oocytes arrested at metaphase II. Following fertilization, SLBP remains abundant in the nucleus and the cytoplasm throughout the first cell cycle, including both G1 and G2 phases. It declines during the second and third cell cycles, reaching a relatively low level by the late 4-cell stage. SLBP can bind the histone mRNA-stem-loop at all stages of the cell cycle in oocytes and early embryos, and it is the only stem-loop binding activity detectable in these cells. We also report that SLBP becomes phosphorylated rapidly following entry into M-phase of meiotic maturation through a mechanism that is sensitive to roscovitine, an inhibitor of cyclin-dependent kinases. SLBP is rapidly dephosphorylated following fertilization or parthenogenetic activation, and becomes newly phosphorylated at M-phase of mitosis. Phosphorylation does not affect its stem-loop binding activity. These results establish that, in contrast to Xenopus, mouse oocytes and embryos contain a single SLBP. Expression of SLBP is uncoupled from S-phase in oocytes and early embryos, which indicates that the mechanisms that impose cell-cycle-regulated expression of SLBP in somatic cells do not operate in oocytes or during the first embryonic cell cycle. This distinctive pattern of SLBP expression may be required for accumulation of histone proteins required for sperm chromatin remodelling and assembly of newly synthesized embryonic DNA into chromatin.
The centrosome is a dynamic structure that ejects PCM flares
Authors: Megraw Timothy L. , Kilaru Sandhya , Turner F. Rudolf , Kaufman Thomas C.
Published: November 2002

The Drosophila Centrosomin (Cnn) protein is an essential core component of centrosomes in the early embryo. We have expressed a Cnn-GFP fusion construct in cleavage stage embryos, which rescues the maternal effect lethality of cnn mutant animals. The localization patterns seen with GFP-Cnn are identical to the patterns we see by immunofluorescent staining with anti-Cnn antibodies. Live imaging of centrosomes with Cnn-GFP reveals surprisingly dynamic features of the centrosome. Extracentrosomal particles of Cnn move radially from the centrosome and frequently change their direction. D-TACC colocalized with Cnn at these particles. We have named these extrusions`flares'. Flares are dependent on microtubules, since disruption of the microtubule array severs the movement of these particles. Movement of flare particles is cleavage-cycle-dependent and appears to be attributed mostly to their association with dynamic astral microtubules. Flare activity decreases at metaphase, then increases at telophase and remains at this higher level of activity until the next metaphase. Flares appear to be similar to vertebrate PCM-1-containing `centriolar satellites' in their behavior. By injecting rhodamine-actin, we observed that flares extend no farther than the actin cage. Additionally, disruption of the microfilament array increased the extent of flare movement. These observations indicate that centrosomes eject particles of Cnn-containing pericentriolar material that move on dynamic astral microtubules at a rate that varies with the cell cycle. We propose that flare particles play a role in organizing the actin cytoskeleton during syncytial cleavage.
An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin
Authors: Tange Yoshie , Hirata Aiko , Niwa Osami
Published: November 2002

We identified a novel fission yeast gene, ned1+, with pleiotropic mutations that have a high incidence of chromosome missegregation, aberrantly shaped nuclei, overdeveloped endoplasmic reticulum-like membranes, and increased sensitivity to a microtubule destabilizing agent. Ned1 protein, which was phosphorylated in a growth-related manner, interacted in a yeast two-hybrid system with Dis3 as well as with Pim1/RCC1 (nucleotide exchange factor for Ran). Ned1 also interacted with an essential nucleoporin, a probable homologue of mammalian Nup98/96. The ned1 gene displayed a variety of genetic interactions with factors involved in nuclear transport and chromosome segregation, including the crm1 (exportin), spi1 (small GTPase Ran), pim1, and dis genes. A substitution mutation that affected the two-hybrid interaction with Dis3 increased chromosome instability, suggesting the functional importance of the interaction. Overproduction of Ned1 protein induced formation of an abnormal microtubule bundle within the nucleus, apparently independently of the spindle pole body, but dependent on pim1+ activity. The ned1+ gene belongs to an evolutionarily conserved gene family, which includes the mouse Lpin genes, one of whose mutations is responsible for lipodystrophy.
Chromosomal association of Ran during meiotic and mitotic divisions
Authors: Hinkle Beth , Slepchenko Boris , Rolls Melissa M. , Walther Tobias C. , Stein Pascal A. , Mehlmann Lisa M. , Ellenberg Jan , Terasaki Mark
Published: November 2002

Recent studies in Xenopus egg extracts indicate that the small G protein Ran has a central role in spindle assembly and nuclear envelope reformation. We determined Ran localization and dynamics in cells during M phase. By immunofluorescence, Ran is accumulated on the chromosomes of meiosis-II-arrested Xenopus eggs. In living cells, fluorescently labeled Ran associated with the chromosomes in Xenopus and remained associated during anaphase when eggs were artificially activated. Fluorescent Ran associated with chromosomes in mouse eggs, during meiotic maturation and early embryonic divisions in starfish, and to a lesser degree during mitosis of a cultured mammalian cell line. Chromosomal Ran undergoes constant flux. From photobleach experiments in immature starfish oocytes, chromosomal Ran has a koff of ∼0.06 second-1, and binding analysis suggests that there is a single major site. The chromosomal interactions may serve to keep Ran-GTP in the vicinity of the chromosomes for spindle assembly and nuclear envelope reformation.
RNA trafficking and stabilization elements associate with multiple brain proteins
Authors: Snee Mark , Kidd Grahame J. , Munro Trent P. , Smith Ross
Published: November 2002

Two of the best understood somatic cell mRNA cytoplasmic trafficking elements are those governing localization of β-actin and myelin basic protein mRNAs. These cis-acting elements bind the trans-acting factors fibroblast ZBP-1 and hnRNP A2, respectively. It is not known whether these elements fulfil other roles in mRNA metabolism. To address this question we have used Edman sequencing and western blotting to identify six rat brain proteins that bind the β-actin element (zipcode). All are known RNA-binding proteins and differ from ZBP-1. Comparison with proteins that bind the hnRNP A2 and AU-rich response elements, A2RE/A2RE11 and AURE, showed that AURE and zipcode bind a similar set of proteins that does not overlap with those that bind A2RE11. The zipcode-binding protein, KSRP, and hnRNP A2 were selected for further study and were shown by confocal immunofluorescence microscopy to have similar distributions in the central nervous system, but they were found in largely separate locations in cell nuclei. In the cytoplasm of cultured oligodendrocytes they were segregated into separate populations of cytoplasmic granules. We conclude that not only may there be families of trans-acting factors for the same cis-acting element, which are presumably required at different stages of mRNA processing and metabolism, but independent factors may also target different and multiple RNAs in the same cell.
The CBEL glycoprotein ofPhytophthora parasiticavar-nicotianaeis involved in cell wall deposition and adhesion to cellulosic substrates
Authors: Gaulin Elodie , Jauneau Alain , Villalba François , Rickauer Martina , Esquerré-Tugayé Marie-Thérèse , Bottin Arnaud
Published: November 2002

The cell wall of the oomycete plant pathogen Phytophthora parasitica var. nicotianae contains a protein called CBEL that shows cellulose-binding (CB), elicitor (E) of defense in plants and lectin-like (L) activities. The biological role of this molecule in Phytophthora was investigated by generating transgenic strains suppressed in CBEL expression. Phenotypic characterization of these strains showed that they were severely impaired in adhesion to a cellophane membrane, differentiation of lobed structures in contact with cellophane, and formation of branched aggregating hyphae on cellophane and on flax cellulose fibres. Infection assays revealed that the strains suppressed in CBELexpression were not greatly affected in pathogenicity and formed branched aggregating hyphae in contact with the roots of the host plant, thereby indicating that CBEL is involved in the perception of cellulose rather than in the morphogenesis of hyphal aggregates. Interestingly, the absence of CBEL was correlated with abnormal formation of papillae-like cell wall thickenings in vitro, suggesting that CBEL is involved in cell wall deposition in Phytophthora. Reverse genetics in oomycetes has long been hampered by their diploid nature and difficulties in transformation and regeneration. The gene inactivation approach reported in this work provides the first direct evidence for intrinsic functions of an elicitor and cell wall protein in oomycetes.
Integrin α8β1 mediates adhesion to LAP-TGFβ1
Authors: Lu Min , Munger John S. , Steadele Melissa , Busald Christina , Tellier Marinka , Schnapp Lynn M.
Published: November 2002

The development of fibrosis is a common response to a variety of injuries and results in the net accumulation of matrix proteins and impairment of normal organ function. We previously reported that the integrinα8β1 is expressed by alveolar interstitial cells in normal lung and is upregulated during the development of fibrosis. TGFβ1 is an important mediator of the inflammatory response in pulmonary fibrosis. TGFβ1 is secreted as a latent protein that is non-covalently associated with latency-associated peptide (LAP) and requires activation to exert its effects. LAP-TGFβ1 and LAP-TGFβ3 contain the tripeptide sequence,arginine-glycine-aspartic acid (RGD), a known integrin recognition motif. The integrin α8β1 binds to several ligands such as fibronectin and vitronectin through the RGD sequence. Recent reports demonstrate that the integrins αvβ1, αvβ6 and αvβ8 adhere to LAP-TGFβ1 through the RGD site. Therefore, we asked whether LAP-TGFβ1 might be a ligand for α8β1 and whether this may be important in the development of fibrosis. We found that cell lines transfected with α8 subunit were able to spread on and adhere to recombinant LAP-TGFβ1 significantly better than mock transfected cell lines.α8-transfected cells were also able to adhere to LAP-TGFβ3 significantly better than mock transfected cells. Adhesion to LAP-TGFβ1 was enhanced by activation of α8β1 by Mn2+, or 8A2, an integrin β1 activating antibody. Furthermore, cell adhesion was abolished when we used a recombinant LAP-TGFβ1 protein in which the RGD site was mutated to RGE. α8β1 binding to LAP-TGFβ1 increased cell proliferation and phosphorylation of FAK and ERK, but did not activate of TGFβ1. These data strongly suggest that LAP-TGFβ1 is a ligand ofα8β1 and interaction of α8β1 with LAP-TGFβ1 may influence cell behavior.
Effects of Charcot-Marie-Tooth-linked mutations of the neurofilament light subunit on intermediate filament formation
Authors: Perez-Olle Raul , Leung Conrad L. , Liem Ronald K. H.
Published: November 2002

Neurofilaments (NFs) are the major intermediate filaments (IFs) of mature neurons. They play important roles in the structure and function of axons. Recently, two mutations in the neurofilament light (NFL) subunit have been identified in families affected by Charcot-Marie-Tooth (CMT) neuropathy type 2. We have characterized the effects of these NFL mutations on the formation of IF networks using a transient transfection system. Both mutations disrupted the self-assembly of human NFL. The Q333P mutant in the rod domain of NFL also disrupted the formation of rat and human NFL/NFM heteropolymers. The phenotypes produced by the P8R mutation in the head domain of NFL were less severe. The P8R mutant NFL co-polymerized with NFM to form bundled filaments and, less often, aggregates. Our results suggest that alterations in the formation of a normal IF network in neurons elicited by these NFL mutations may contribute to the development of Charcot-Marie-Tooth neuropathy.
A role for myosin VI in actin dynamics at sites of membrane remodeling duringDrosophilaspermatogenesis
Authors: Rogat Aaron D. , Miller Kathryn G.
Published: November 2002

Myosin VI has been implicated in membrane dynamics in several organisms. The mechanism of its participation in membrane events is not clear. We have used spermatogenesis in Drosophila to investigate myosin VI's in vivo role. We demonstrate that myosin VI colocalizes with and is required for the accumulation of the actin polymerization regulatory proteins, cortactin and arp2/3 complex, on actin structures that mediate membrane remodeling during spermatogenesis. In addition, we show that dynamin localizes to these actin structures and that when dynamin and myosin VI function are both impaired,major defects in actin structures are observed. We conclude that during spermatogenesis myosin VI and dynamin function in parallel pathways that regulate actin dynamics and that cortactin and arp2/3 complex may be important for these functions. Regions of myosin VI accumulation are proposed as sites where actin assembly is coupled to membrane dynamics.
The small GTPase Rho3 and the diaphanous/formin For3 function in polarized cell growth in fission yeast
Authors: Nakano Kentaro , Imai Jun , Arai Ritsuko , Toh-e Akio , Matsui Yasushi , Mabuchi Issei
Published: November 2002

We identified a novel Rho gene rho3+ and studied its interaction with diaphanous/formin for3+ in the fission yeast Schizosaccharomyces pombe. Both rho3 null cells and for3 null cells showed defects in organization of not only actin cytoskeleton but also cytoplasmic microtubules (MTs). rho3 for3double null cells had defects that were more severe than each single null cell: polarized growth was deficient in the double null cells. Function of For3 needed the highly conserved FH1 and FH2 domains, an N-terminal region containing a Rho-binding domain, and the C-terminal region. For3 bound to active forms of both Rho3 and Cdc42 but not to that of Rho1. For3 was localized as dots to the ends of interphase cells and to the mid-region in dividing cells. This localization was probably dependent on its interaction with Rho proteins. Overexpression of For3 produced huge swollen cells containing depolarized F-actin patches and thick cytoplasmic MT bundles. In addition, overexpression of a constitutively active Rho3Q71L induced a strong defect in cytokinesis. In conclusion, we propose that the Rho3-For3 signaling system functions in the polarized cell growth of fission yeast by controlling both actin cytoskeleton and MTs.
Temporal pattern of NFκB activation influences apoptotic cell fate in a stimuli-dependent fashion
Authors: Fan Chenguang , Yang Jusan , Engelhardt John F.
Published: November 2002

The transcription factor NFκB is a critical immediate early response gene involved in modulating cellular responses and apoptosis following diverse environmental injuries. The activation of NFκB is widely accepted to play an anti-apoptotic role in cellular responses to injury. Hence, enhancing NFκB activation in the setting of injury has been proposed as one potential therapeutic approach to environmental injuries. To this end, we constructed a recombinant adenoviral vector (Ad.IκBαAS) expressing antisense IκBα mRNA that is capable of augmenting NFκB activation prior to and following four types of cellular injury [TNF-α,UV, hypoxia/reoxygenation (H/R) or pervanadate treatment]. Biochemical and functional analyses of NFκB activation pathways for these injuries demonstrated two categories involving either serine (S32/36) phosphorylation(TNF-α, UV) or tyrosine (Y42) phosphorylation (H/R or PV) of IκBα. We hypothesized that activation of NFκB prior to injury using antisense IκBα mRNA would reduce apoptosis. As anticipated, recombinant adenoviral IκBα phosphorylation mutants(Ad.IκBαS32/36A or Ad.IκBαY42F) preferentially reduced NFκB activation and enhanced apoptosis following injuries associated with either serine or tyrosine phosphorylation of IκBα,respectively. These studies demonstrate for the first time that an IκBαY42F mutant can effectively modulate NFκB-mediated apoptosis in an injury-context-dependent manner. Interestingly, constitutive activation of NFκB following Ad.IκBαAS infection reduced apoptosis only following injuries associated with IκBα Y42, but not S32/36, phosphorylation. These findings demonstrate that the temporal regulation of NFκB and the apoptotic consequences of this activation are differentially influenced by the pathway mediating NFκB activation. They also provide new insight into the therapeutic potential and limitations of modulating NFκB for environmental injuries such as ischemia/reperfusion and pro-inflammatory diseases.
Wnt regulation of chondrocyte differentiation
Authors: Church Vicki , Nohno Tsutomu , Linker Claudia , Marcelle Christophe , Francis-West Philippa
Published: November 2002

The Wnt family of growth factors are important regulators of several developmental processes including skeletogenesis. To further investigate the role of Wnts we analysed their expression in the developing chick limb and performed functional analyses in vivo and in vitro. We found that Wnt5b and Wnt11 are restricted within the prehypertrophic chondrocytes of the cartilage elements, Wnt5a is found in the joints and perichondrium, while Wnt4 is expressed in the developing joints and, in some bones, a subset of the hypertrophic chondrocytes. These Wnts mediate distinct effects on the initiation of chondrogenesis and differentiation of chondrocytes in vitro and in vivo. Wnt4 blocks the initiation of chondrogenesis and accelerates terminal chondrocyte differentiation in vitro. In contrast, Wnt5a and Wnt5b promote early chondrogenesis in vitro while inhibiting terminal differentiation in vivo. As Wnt5b and Wnt11 expression overlaps with and appears after Indian hedgehog (Ihh), we also compared their effects with Ihh to see if they mediate aspects of Ihh signalling. This showed that Ihh and Wnt5b and Wnt11 control chondrogenesis in parallel pathways.
Protein kinase CKII regulates the interaction of β-catenin withα-catenin and its protein stability
Authors: Bek Stephan , Kemler Rolf
Published: November 2002

β-Catenin is a multi-functional cellular component and a substrate for several protein kinases. Here we investigated the interaction of protein kinase CKII (casein kinase II) and β-catenin. We show that CKII phosphorylates the N-terminal region of β-catenin and we identified Ser29, Thr102, and Thr112 as substrates for the enzyme. We provide evidence that CKII regulates the cytoplasmic stability of β-catenin and acts synergistically with GSK-3β in the multi-protein complex that controls the degradation of β-catenin. In comparing wild-type and Ser/Thr-mutantβ-catenin, a decreased affinity of the mutant protein to α-catenin was observed. Moreover, kinase assays in vitro demonstrate a CKII-dependent increase in the binding of wild-type β-catenin with α-catenin. In line with that, cells expressing Ser/Thr-mutant β-catenin exhibit an increased migratory potential, which correlates with an enhanced cytosolic localization and a reduced association with the cytoskeleton of the mutant protein. From these results we conclude that CKII regulates the function ofβ-catenin in the cadherin adhesion complex as well as its cytoplasmic stability.
Intracellular localization and dynamics of myosin-II and myosin-IC in live Acanthamoeba by transient transfection of EGFP fusion proteins
Authors: Kong Hyun-Hee , Pollard Thomas D.
Published: November 2002

We developed a reliable method for transient transfection of Acanthamoeba using Superfect (Qiagen) and a vector with the Acanthamoeba ubiquitin promoter and enhanced green fluorescent protein (EGFP) as the reporter gene. The transfection efficiency was 3% for profilin-I-EGFP and EGFP-myosin-II tail, and less than 0.5% for larger constructs such as full length myosin-II or myosin-IC. Profilin-I-EGFP was distributed throughout the cytoplasm as observed previously with rhodamine-labeled profilin, while EGFP alone accumulated in the nucleus. EGFP fused to full length myosin-II or to the C-terminal 256 residues of the myosin-II tail concentrated in fluorescent spots similar to thick filaments and minifilaments identified previously in fixed cells with fluorescent antibodies. Thick filaments were located in the dorsal cytoplasm and along the lateral margins of the back half of the cell. Thick filaments formed behind the leading edge and moved continuously towards the rear of the cell, where they disassembled. If phosphorylation of the myosin-II heavy chain was prevented by mutation of all three phosphorylated serines to alanine, thick filaments of unphosphorylated myosin-II accumulated around vesicles of various sizes. EGFP-myosin-IC was spread throughout the cytoplasm but concentrated transiently around contractile vacuoles and macropinocytosis cups providing that the construct included both the head and a tail with the SH3 domain.
The architecture of interphase chromosomes and gene positioning are altered by changes in DNA methylation and histone acetylation
Authors: Santos Ana Paula , Abranches Rita , Stoger Eva , Beven Alison , Viegas Wanda , Shaw Peter J.
Published: November 2002

Wheat nuclei have a remarkably well defined interphase organisation, and we have made use of this to determine the relationship between interphase chromosome organisation, the positioning of specific transgenes and induced changes in DNA methylation and histone acetylation, using in situ hybridisation and confocal 3D imaging. After germinating seeds either in the presence of 5-Azacytidine (5-AC), which leads to DNA hypomethylation, or trichostatin A (TSA), which results in histone hyperacetylation, the architecture of the interphase chromosome arms changes significantly even though the overall Rabl configuration is maintained. This suggests that specific chromosome segments are remodelled by these treatments but that there is a strong link of both centromeres and telomeres to the nuclear envelope. In lines carrying multiple transgene integrations at widely separated sites, we show that the multiple transgenes, which are usually colocalised during interphase, are dispersed after 5-AC or TSA treatment and that there is an increase in transgene activity. This suggests that the colocalisation/dispersion of the transgenes may be a function of specific interphase chromosome organisation and that these lines containing multiple transgene copies may all be partially transcriptionally repressed.
Loss of Rb overrides the requirement for ERK activity for cell proliferation
Authors: D'Abaco Giovanna M. , Hooper Steven , Paterson Hugh , Marshall Christopher J.
Published: November 2002

The Ras GTPase is a critical transducer of mitogenic signals ultimately leading to inactivation of the retinoblastoma (Rb) protein, but the molecular basis underlying Ras-dependent control of cell cycle kinetics remains to a great extent unknown. In an effort to further elucidate the role of Ras activation in cell cycle control, we have studied the role of the downstream Mek-ERK pathway in facilitating exit from the quiescent G0 state and passage through the G1/S transition. We have adopted a genetic approach in combination with U0126, an inhibitor of Mek activation to study the role of Mek in cell cycle progression. Here we report that whereas wild-type (Wt) mouse embryo fibroblasts (MEFs) depend on ERK activation to enter the cell cycle,Rb-deficient (Rb-/-) MEFs have a reduced requirement for ERK signalling. Indeed in the presence of U0126 we found that Rb-null MEFs can exit G0, make the G1/S transition and proliferate. Analysis of Rb-deficient tumour cell lines also revealed a reduced requirement for ERK signalling in asynchronous growth. We discuss the molecular mechanism that may underlie this escape from MAP kinase signalling.
Localisation of human DNA polymerase κ to replication foci
Authors: Bergoglio Valérie , Bavoux Clarisse , Verbiest Vincent , Hoffmann Jean-Sebastien , Cazaux Christophe
Published: November 2002

The replication of the undamaged genomic DNA requires error-free DNA polymerases δ and ϵ as part of a protein complex that acts continuously along the double helix. In contrast, when the genomic structure is perturbed, DNA replication needs to function more flexibly to bypass DNA distortions. It has been proposed that the newly discovered error prone DNA polymerases play a role in the replication of irregular structure. Here we report that one of them, the human Polκ, is mostly localised uniformly in the nucleus of undamaged cells, but could be also concentrated in PCNA-containing replication foci. Following treatment with anti-replicative agents, the proportion of foci-containing cells was increased. These data suggest that Polκ may function as part of the replication machinery itself and could be recruited when replicative complexes are stalled. Mutagenesis experiments also indicated that Polκ involvement may affect the accuracy of DNA replication. The results are discussed within the context of the oncogenic process since Polκ has been found as overexpressed in some cancers.
Essential role of citron kinase in cytokinesis of spermatogenic precursors
Authors: Cunto Ferdinando Di , Imarisio Sara , Camera Paola , Boitani Carla , Altruda Fiorella , Silengo Lorenzo
Published: November 2002

During spermatogenesis, the first morphological indication of spermatogonia differentiation is incomplete cytokinesis, followed by the assembly of stable intercellular cytoplasmic communications. This distinctive feature of differentiating male germ cells has been highly conserved during evolution,suggesting that regulation of the cytokinesis endgame is a crucial aspect of spermatogenesis. However, the molecular mechanisms underlying testis-specific regulation of cytokinesis are still largely unknown. Citron kinase is a myotonin-related protein acting downstream of the GTPase Rho in cytokinesis control. We previously reported that Citron kinase knockout mice are affected by a complex neurological syndrome caused by cytokinesis block and apoptosis of specific neuronal precursors. In this report we show that, in addition,these mice display a dramatic testicular impairment, with embryonic and postnatal loss of undifferentiated germ cells and complete absence of mature spermatocytes. By contrast, the ovaries of mutant females appear essentially normal. Developmental analysis revealed that the cellular depletion observed in mutant testes is caused by increased apoptosis of undifferentiated and differentiating precursors. The same cells display a severe cytokinesis defect, resulting in the production of multinucleated cells and apoptosis. Our data indicate that Citron kinase is specifically required for cytokinesis of the male germ line.
Retinoids induce lumen morphogenesis in mammary epithelial cells
Authors: Montesano Roberto , Soulié Priscilla
Published: November 2002

Lumen formation is a fundamental step in the development of the structural and functional units of glandular organs, such as alveoli and ducts. In an attempt to elucidate the molecular signals that govern this morphogenetic event, we set up an in vitro system in which cloned mammary epithelial cells grown in collagen gels under serum-free conditions form solid, lumen-less colonies. Addition of as little as 0.1% donor calf serum (DCS) was sufficient to induce the formation of a central cavity. Among a number of serum constituents analyzed, retinol was found to mimic the effect of DCS in inducing lumen morphogenesis. Since the biological activities of retinol are largely dependent on its conversion to all-trans-retinoic acid (RA), we examined in more detail the effect of RA on lumen formation. RA induced the formation of lumen-containing colonies (cysts) in a concentration- and time-dependent manner, a half-maximal effect after 9 days of culture being observed with 100 pM RA. The pleiotropic effects of retinoids are mediated by nuclear retinoic acid receptors (RARs; α, β and γ) and retinoid X receptors (RXRs; α, β and γ). To identify the signaling pathway involved in RA-induced lumen formation, we used receptor-specific synthetic retinoids. TTNPB, a selective RAR agonist,promoted lumen morphogenesis, whereas RXR-selective ligands lacked this activity. Lumen formation was also induced at picomolar concentrations by Am-580, a synthetic retinoid that selectively binds the RARα receptor subtype. Moreover, co-addition of Ro 41-5253, an antagonist of RARα,abrogated the lumen-inducing activity of both RA and DCS, indicating that this biological response is mediated through an RARα-dependent signaling pathway. To gain insight into the mechanisms underlying RA-induced lumen formation, we assessed the potential role of matrix metalloproteinases (MMP). Using gelatin zymography, we observed a dose-dependent increase in latent and active forms of gelatinase B (MMP-9) upon RA treatment. In addition, lumen formation was abrogated by addition of the synthetic MMP inhibitor BB94,indicating that this morphogenetic process is likely to require MMP activity. Collectively, our results provide evidence that RA promotes lumen formation by mammary epithelial cells in vitro and suggest that it plays a similar role during mammary gland development in vivo.
Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells
Authors: Amasheh Salah , Meiri Noga , Gitter Alfred H. , Schöneberg Torsten , Mankertz Joachim , Schulzke Jörg D. , Fromm Michael
Published: November 2002

Tight junctions seal the paracellular pathway of epithelia but, in leaky tissues, also exhibit specific permeability. In order to characterize the contribution of claudin-2 to barrier and permeability properties of the tight junction in detail, we studied two strains of Madin-Darby canine kidney cells(MDCK-C7 and MDCK-C11) with different tight junctional permeabilities.

Monolayers of C7 cells exhibited a high transepithelial resistance (>1 kΩ cm2), compared with C11 cells (<100 Ωcm2). Genuine expression of claudin-1 and claudin-2, but not of occludin or claudin-3, was reciprocal to transepithelial resistance. However,confocal microscopy revealed a marked subjunctional localization of claudin-1 in C11 cells, indicating that claudin-1 is not functionally related to the low tight junctional resistance of C11 cells.

Strain MDCK-C7, which endogenously does not express junctional claudin-2,was transfected with claudin-2 cDNA. In transfected cells, but not in vector controls, the protein was detected in colocalization with junctional occludin by means of immunohistochemical analyses. Overexpression of claudin-2 in the originally tight epithelium with claudin-2 cDNA resulted in a 5.6-fold higher paracellular conductivity and relative ion permeabilities of Na+≡1, K+=1.02, NMDG+=0.79,choline+=0.71, Cl-=0.12, Br-=0.10 (vector control, 1:1.04:0.95:0.94:0.85:0.83). By contrast, fluxes of (radioactively labeled) mannitol and lactulose and (fluorescence labeled) 4 kDa dextran were not changed. Hence, with regular Ringer's, Na+ conductivity was 0.2 mS cm-2 in vector controls and 1.7 mS cm-2 in claudin-2-transfected cells, while Cl- conductivity was 0.2 mS cm-2 in both cells. Thus, presence of junctional claudin-2 causes the formation of cation-selective channels sufficient to transform a `tight'tight junction into a leaky one.
Absence of retinoids can induce motoneuron disease in the adult rat and a retinoid defect is present in motoneuron disease patients
Authors: Corcoran Jonathan , So Po Lin , Maden Malcolm
Published: November 2002

We generated retinoid-deficient adult rats by the removal of retinoids from their diet. We show that their motoneurons undergo neurodegeneration and that there is an accumulation of neurofilaments and an increase in astrocytosis,which is associated with motoneuron disease. These effects are mediated through the retinoic acid receptor α. The same receptor deficit is found in motoneurons from patients suffering from spontaneous amyotrophic lateral sclerosis. Furthermore, we show that there is a loss of expression of the retinaldehyde dehydrogenase enzyme II in motoneurons. Therefore, we propose that a defect in the retinoid signalling pathway is in part be responsible for some types of motoneuron disease.