bluuebunny/crossref_metadata_2025 huggingface dataset:
Ectopic skeletal muscles derived from myoblasts implanted under the skin
Authors: Irintchev A. , Rosenblatt J. D. , Cullen M. J. , Zweyer M. , Wernig A.
Published: April 2021

We investigated the potential of cultured myoblasts to generate skeletal muscle in an ectopic site. Myoblasts from a clonal cell line or from expanded primary cultures were injected under the skin of the lumbar region of adult syngenic Balb/c mice. One to 7 weeks after injection, distinct muscles, of greater mass in mice injected with clonal myoblasts (6-78 mg, n=37) than in mice injected with primary myoblasts (1-7 mg, n=26), had formed between the subcutaneous panniculus carnosus muscle and the trunk muscles of host animals. These ectopic muscles exhibited spontaneous and/or electrically-evoked contractions after the second week and, when stimulated directly in vitro, isometric contractile properties similar to those of normal muscles. Histological, electron microscopical and tissue culture examination of these muscles revealed their largely mature morphology and phenotype. The fibres, most of which were branched, were contiguous, aligned and capillarised, exhibited normal sarcormeric protein banding patterns, and expressed muscle-specific proteins, including desmin, dystrophin, and isoforms of developmental and adult myosin heavy chain. Enveloping each fibre was a basal lamina, beneath which lay quiescent satellite cells, which could be stimulated to produce new muscle in culture.

Presence of endplates (revealed by α-bungarotoxin and neurofilament staining), and the eventual loss of expression of neural cell adhesion molecule and extrasynaptic acetylcholine receptors, indicated that some fibres were innervated. That these muscle fibres were of implanted-cell origin was supported by the finding of Y-chromosome and a lack of dystrophin in ectopic muscles formed after subcutaneous injection of, respectively, male myoblasts into female mice and dystrophin-deficient (mdx) myoblasts into normal C57Bl/10 muscle. Our results demonstrate that an organised, functional muscle can be generated de novo from a disorganised mass of myoblasts implanted in an extramuscular subcutaneous site, whereby the host contributes significantly in providing support tissues and innervation. Our observations are also consistent with the idea that myogenic cells behave like tissue-specific stem cells, generating new muscle precursor (satellite) cells as well as mature muscle. Subcutaneous implantation of myoblasts may have a range of useful applications, from the study of myogenesis to the delivery of gene products.
Aryl-hydrocarbon receptor is an inhibitory regulator of lipid synthesis and of commitment to adipogenesis
Authors: Alexander David L. , Ganem Leonardo G. , Fernandez-Salguero Pedro , Gonzalez Frank , Jefcoate Colin R.
Published: April 2021

The aryl-hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In mouse embryo fibroblasts, TCDD activates expression of multiple genes, including CYP1B1, the predominant cytochrome P450 expressed in these cells. Here, we analyze constitutive functions of the AhR in primary mouse embryo fibroblasts (MEFs) and spontaneously immortalized MEF cell lines derived from wild-type (WT) C57BL/6 mice and also from congenic mice with a targeted disruption of the AhR gene (AhR−/−). After multiple passages, primary MEFs exhibit spontaneous differentiation, growth cessation and senescence. Eventually, colonies of immortalized MEFs arise to provide clonal lines. The senescent phase occurs much earlier for AhR−/− MEFs, while immortalization is substantially delayed. Comparison of AhR−/− and WT MEFs also indicates that constitutive AhR activity is required for basal expression of CYP1B1 and suppresses lipogenesis in subconfluent cultures. Primary WT and AhR−/− MEFs and the corresponding lines undergo adipogenesis when treated at confluence with the appropriate hormonal inducers. Addition of TCDD before or concurrent with hormonal induction suppressed PPARγ mRNA and adipogenesis, as measured by lipid accumulation, glycerol phosphate dehydrogenase activity and stearoyl CoA desaturase type 1 mRNA expression. This effect of TCDD treatment was absent in AhR−/− MEFs, establishing the role of AhR in hormone-induced adipogenesis. Such hormonal activation of confluent MEFs and preadipocytes results in a limited proliferative expansion followed by irreversible growth arrest. TCDD-treated MEFs undergo the mitotic expansion but fail to exit the cell cycle. In AhR−/− MEFs, there is no such effect of TCDD. These findings implicate the AhR as a constitutive inhibitor of triglyceride synthesis, and as an early regulator of adipocyte differentiation. AhR interference with cell-cycle arrest in differentiation may be linked to the increased rate of senescence.
The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network
Authors: Gavet Olivier , Ozon Sylvie , Manceau Valérie , Lawler Sean , Curmi Patrick , Sobel André
Published: April 2021

Stathmin is a small regulatory phosphoprotein integrating diverse intracellular signaling pathways. It is also the generic element of a protein family including the neural proteins SCG10, SCLIP, RB3 and its two splice variants RB3’ and RB3’’. Stathmin itself was shown to interact in vitro with tubulin in a phosphorylation-dependent manner, sequestering free tubulin and hence promoting microtubule depolymerization. We investigated the intracellular distribution and tubulin depolymerizing activity in vivo of all known members of the stathmin family. Whereas stathmin is not associated with interphase microtubules in HeLa cells, a fraction of it is concentrated at the mitotic spindle. We generated antisera specific for stathmin phosphoforms, which allowed us to visualize the regulation of phosphorylation-dephosphorylation during the successive stages of mitosis, and the partial localization of stathmin phosphorylated on serine 16 at the mitotic spindle. Results from overexpression experiments of wild-type and novel phosphorylation site mutants of stathmin further suggest that it induces depolymerization of interphase and mitotic microtubules in its unphosphorylated state but is inactivated by phosphorylation in mitosis. Phosphorylation of mutants 16A25A and 38A63A on sites 38 and 63 or 16 and 25, respectively, was sufficient for the formation of a functional spindle, whereas mutant 16A25A38A63E retained a microtubule depolymerizing activity. Transient expression of each of the neural phosphoproteins of the stathmin family showed that they are at least partially associated to the Golgi apparatus and not to other major membrane compartments, probably through their different NH2-terminal domains, as described for SCG10. Most importantly, like stathmin and SCG10, overexpressed SCLIP, RB3 and RB3’’ were able to depolymerize interphase microtubules. Altogether, our results demonstrate in vivo the functional conservation of the stathmin domain within each protein of the stathmin family, with a microtubule destabilizing activity most likely essential for their specific biological function(s).
Articulins and epiplasmins: two distinct classes of cytoskeletal proteins of the membrane skeleton in protists
Authors: Huttenlauch Irm , Peck Robert K. , Stick Reimer
Published: April 2021

The cortex of ciliates, dinoflagellates and euglenoids comprises a unique structure called the epiplasm, implicated in pattern-forming processes of the cell cortex and in maintaining cell shape. Despite significant variation in the structural organization of their epiplasm and cortex, a novel type of cytoskeletal protein named articulin is the principal constituent of the epiplasm in the euglenoid Euglena and the ciliate Pseudomicrothorax. For another ciliate, Paramecium, epiplasmins, a group of polypeptides with common biochemical properties, are the major constituents of the epiplasm. Using molecular tools and affinity purification we have selected polyclonal antibodies and identified epitopes of monoclonal antibodies that identify epitopes characteristic of articulins and epiplasmins. With these antibodies we have analysed the occurrence of the two types of cytoskeletal proteins in a dinoflagellate, a euglenoid and several ciliates. Our results indicate that both articulins and epiplasmins are present in these organisms, suggesting that both contribute to the organization of the membrane skeleton in protists. Articulins and epiplasmins represent two distinct classes of cytoskeletal proteins, since different polypeptides were labeled by articulin core domain-specific or epiplasmin epitope-specific antibodies in each organism studied. In one case, a polypeptide in Pseudomicrothorax was identified that reacts with both articulin core domain-specific and with anti-epiplasmin monoclonal antibodies; however, the epiplasmin monoclonal antibody epitope was mapped to the C terminus of the polypeptide, well outside the central VPV-repeat core domain that contains the articulin monoclonal antibody epitope and that is the hallmark of the articulins.
Isolation of the human genes encoding the Pyst1 and Pyst2 phosphatases: characterisation of Pyst2 as a cytosolic dual-specificity MAP kinase phosphatase and its catalytic activation by both MAP and SAP kinases
Authors: Dowd Stephen , Sneddon Alan A. , Keyse Stephen M.
Published: April 2021

We have isolated the human genes encoding the Pyst1 (MKP-3) and Pyst2 (MKP-X) MAP kinase phosphatases. Both genes consist of three exons interrupted by two introns and lack an intron which is conserved in all the other members of this gene family characterised to date. This reinforces the conclusion that Pyst1 and Pyst2 are members of a distinct and structurally homologous subfamily of dual-specificity (Thr/Tyr) MAP kinase phosphatases. We find that Pyst2 mRNA is constitutively expressed in a wide variety of human cell lines including those derived from ovarian, bladder and breast cancers. While there is no evidence for inducible expression of Pyst2 mRNA in human skin fibroblasts in response to cellular stress, Pyst2 mRNA levels are moderately increased in response to serum stimulation. Pyst2 protein is predominantly cytosolic when expressed in COS-1 cells. In common with Pyst1, Pyst2 shows substrate selectivity for the classical p42 (ERK2) isoform of MAP kinase both in vitro and in vivo, displaying much reduced activity towards stress activated MAP kinase isoforms such as JNK-1 and p38/RK. Pyst2 binds p42 MAP kinase in vivo and both MAP kinase binding and substrate selectivity correlate with the ability of different recombinant MAP and SAP kinases to cause catalytic activation of the Pyst2 phosphatase in vitro.
Expression and localization of hepatocyte domain-specific plasma membrane proteins in hepatoma × fibroblast hybrids and in hepatoma dedifferentiated variants
Authors: Bender Virginie , Büschlen Sylvie , Cassio Doris
Published: April 2021

We have studied two aspects of the plasma membrane of hepatocytes, highly differentiated epithelial cells that exhibit a particular and complex polarity. Using a genetic approach, we have distinguished between the expression/regulation of proteins specific for all three hepatocyte membrane domains and their organization into discrete domains. For this analysis we used a panel of previously isolated cell clones, derived from the differentiated rat hepatoma line H4IIEC3, and that present different expression patterns for liver-specific genes. This panel was composed of (1) differentiated clones, (2) chromosomally reduced hepatoma-fibroblast hybrids characterized by a pleiotropic extinction/reexpression of liver-specific genes and (3) dedifferentiated variant and revertant clones. The expression of 16 hepatocyte membrane polarity markers was studied by western blotting and immunolocalization. Even though cells of differentiated clones express all of these polarity markers, they are not polarized, and are therefore suitable for studying the regulation of plasma membrane protein expression, and for identifying gene products implicated in the establishment of membrane polarity. In hepatoma-fibroblast hybrids the expression of four markers, three apical (dipeptidylpeptidase IV, alkaline phosphodiesterase B10 and polymeric IgA receptor) and one lateral (E-cadherin), is down-regulated in extinguished clones and restored in reexpressing subclones, as previously reported for liver-specific functions. The dipeptidylpeptidase IV mRNA was undetectable or strongly reduced in extinguished hybrids, but expressed at a robust level in some of the reexpressing clones. Concerning the dedifferentiated variants, each has its own pattern of membrane marker expression (loss of expression of three to six markers), that differs from that of extinguished hybrids. Revertant cells express all of the membrane markers examined. Among all of these hepatoma derivatives, only cells of reexpressing hybrids are polarized, and form bile canaliculi-like structures, with spherical and even, for one clone, long tubular and branched forms. All apical markers examined are confined in these canalicular structures, whereas the other markers are excluded from them, and present on the rest of the membrane (basolateral markers) or at the cell-cell contacts (lateral markers). Cells of reexpressing hybrids also express simple epithelial polarity. Thus the expression of only a few hepatocyte-domain-specific plasma membrane proteins is subject to down-regulation, as is the case for liver-specific genes so far studied, and the expression of polarity markers and the formation of poles are dissociable events.
Vinculin but not α-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium
Authors: Perez-Moreno M. , Avila A. , Islas S. , Sanchez S. , González-Mariscal L.
Published: April 2021

The establishment of the junctional complex in epithelial cells requires the presence of extracellular calcium, and is controlled by a network of reactions involving G-proteins, phospholipase C and protein kinase C. Since potential candidates for phosphorylation are the tight junction associated proteins ZO1, ZO2 and ZO3, in a previous work we specifically explored these molecules but found no alteration in their phosphorylation pattern. To continue the search for the target of protein kinase C, in the present work we have studied the subcellular distribution and phosphorylation of vinculin and α-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, α-actinin remains unchanged. The increased phosphorylation of vinculin is due to changes in phosphoserine and phosphothreonine content and seems to be regulated by protein kinase C, since: (1) DiC8 (a kinase C stimulator) added to monolayers cultured without calcium significantly increases the vinculin phosphorylation level; (2) H7 and calphostin C (both protein kinase C inhibitors) completely abolish this increase during a calcium switch; (3) inhibition of phosphorylation during a calcium switch blocks the subcellular redistribution of vinculin and α-actinin. These results therefore suggest that vinculin phosphorylation by protein kinase C is a crucial step in the correct assembly of the epithelial junctional complex.
The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle and replication checkpoint functions in fission yeast
Authors: Norbury Randa Craig and Chris
Published: April 2021

Entry into mitosis is normally blocked in eukaryotic cells that have not completed replicative DNA synthesis; this ‘S-M’ checkpoint control is fundamental to the maintenance of genomic integrity. Mutants of the fission yeast Schizosaccharomyces pombe defective in the S-M checkpoint fail to arrest the cell cycle when DNA replication is inhibited and hence attempt mitosis and cell division with unreplicated chromosomes, resulting in the ‘cut’ phenotype. In an attempt to identify conserved molecules involved in the S-M checkpoint we have screened a regulatable murine cDNA library in S. pombe and have identified cDNAs that induce the cut phenotype in cells arrested in S phase by hydroxyurea. One such cDNA encodes a novel protein with multiple calmodulin-binding motifs that, in addition to its effects on the S-M checkpoint, perturbed mitotic spindle functions, although spindle pole duplication was apparently normal. Both aspects of the phenotype induced by this cDNA product, which we term Sha1 (for spindle and hydroxyurea checkpoint abnormal), were suppressed by simultaneous overexpression of calmodulin. Sha1 is structurally related to the product of the Drosophila gene abnormal spindle (asp). These data suggest that calmodulin-binding protein(s) are important in the co-ordination of mitotic spindle functions with mitotic entry in fission yeast, and probably also in multicellular eukaryotes.
Distinct signal transduction pathways are utilized during the tube formation and survival phases of in vitro angiogenesis
Authors: Mahooti Neta Ilan, Sepi , Madri Joseph A.
Published: April 2021

Angiogenesis, the formation of new blood vessels from preexisting ones, occurs during development, wound healing and cancer and involves stages that orchestrate a network of cooperative interactions. Peptide growth factors and extracellular matrix (ECM) components are two major groups of angiogenesis mediators. Among the different ECM proteins, collagens have been well-associated with in vivo angiogenesis. Using human umbilical vein endothelial cells (HUVEC) grown in 3-D collagen gels we show that: (1) HUVEC do not survive well in 3-D collagen gels due to rapid induction of apoptosis. (2) VEGF, a potent in vivo angiogenic factor, fails to induce tube formation. (3) PMA was effective in inducing tube formation and survival in HUVEC dispersed in 3-D collagen gels, activating MAP kinase, phosphoinositide 3-OH kinase (PI-3-kinase) and Akt/PKB (protein kinase B) pathways. (4) VEGF was effective in preventing PMA-induced tube-like structure regression after PMA-withdrawal by (5) activating the mitogen activated protein kinase (MAPK), rather than the Akt/PKB, signaling pathway.
Furrow-associated microtubule arrays are required for the cohesion of zebrafish blastomeres following cytokinesis
Authors: Jesuthasan Suresh
Published: April 2021

During the first few cleavages of the zebrafish embryo, daughter blastomeres are loosely associated immediately after furrow ingression, but then gradually cohere. Cohesion appears to be cadherin-dependent, as cadherin and β-catenin are found at the membrane between cohering blastomeres, and blastomeres fail to cohere in calcium-free medium. Cadherin and β-catenin are not initially found on the blastomere surface, but are deposited specifically at the furrow surface. An array of parallel microtubules is present on either side of the furrow tip during ingression, as seen by confocal microscopy of α-tubulin labelled embryos. Transient incubation of embryos in 1 µg/ml nocodazole at the start of furrowing, which causes a loss of the furrow array, inhibits the localization of β-catenin to the furrow surface but does not prevent furrow ingression. During ingression, intracellular membranes are transported to the furrow, as shown by labelling with DiD or DiOC6(3). Concentration of these membranes near the furrow surface is microtubule-dependent.

These findings suggest that microtubules are required for cohesion of blastomeres because they mediate trafficking of intracellular membranes to the furrow surface, where they are exocytosed and allow cohesion via cadherins.
The kar3p and kip2p motors function antagonistically at the spindle poles to influence cytoplasmic microtubule numbers
Authors: Huyett Abigail , Kahana Jason , Silver Pamela , Zeng Xuemei , Saunders William S.
Published: April 2021

Microtubules provide the substrate for intracellular trafficking by association with molecular motors of the kinesin and dynein superfamilies. Motor proteins are generally thought to function as force generating units for transport of various cargoes along the microtubule polymer. Recent work suggests additional roles for motor proteins in changing the structure of the microtubule network itself. We report here that in the budding yeast Saccharomyces cerevisiae microtubule motors have antagonistic effects on microtubule numbers and lengths. As shown previously, loss of the Kar3p motor stimulates cytoplasmic microtubule growth while loss of Kip2p leads to a sharp reduction in cytoplasmic microtubule numbers. Loss of both the Kip2p and Kar3p motors together in the same cell produces an intermediate phenotype, suggesting that these two motors act in opposition to control cytoplasmic microtubule density. A Kip2p-GFP fusion from single gene expression is most concentrated at the spindle poles, as shown previously for an epitope tagged Kar3p-HA, suggesting both of these motors act from the minus ends of the microtubules to influence microtubule numbers.
The axonemal microtubules of the chlamydomonas flagellum differ in tubulin isoform content
Authors: Johnson Karl A.
Published: April 2021

Little is known of the molecular basis for the diversity of microtubule structure and function found within the eukaryotic flagellum. Antibodies that discriminate between tyrosinated alpha tubulin and post-translationally detyrosinated alpha tubulin were used to localize these complementary tubulin isoforms in flagella of the single-celled green alga Chlamydomonas reinhardtii. Immunofluorescence analysis of intact axonemes detected both isoforms along most of the lengths of flagella; however, each had a short distal zone rich in tyrosinated tubulin. Localizations on splayed axonemes revealed that the microtubules of the central-pair apparatus were rich in tyrosinated tubulin, while outer doublets contained a mixture of both isoforms. Immunoelectron analysis of individual outer doublets revealed that while tyrosinated tubulin was present in both A and B tubules, detyrosinated tubulin was largely confined to the wall of the B hemi-tubules. The absence of detyrosinated tubulin from the A tubules of the outer doublets and the microtubules of the central pair, both of which extend past the B hemi-tubules of the outer doublets in the flagellar tip, explained the appearance of a tyrosinated tubulin-rich distal zone on intact axonemes. Localizations performed on cells regenerating flagella revealed that flagellar assembly used tyrosinated tubulin; detyrosination of the B tubule occurred during later stages of regeneration, well after microtubule polymerization. The developmental timing of detyrosination, which occurs over a period during which the regrowing flagella begin to beat more effectively, suggests that post-translational modification of the B tubule surface may enhance dynein/B tubule interactions that power flagellar beating.
Roles of head and tail domains in α-internexin’s self-assembly and coassembly with the neurofilament triplet proteins
Authors: Ching Gee Y. , Liem Ronald K. H.
Published: April 2021

The roles of the head and tail domains of α-internexin, a type IV neuronal intermediate filament protein, in its self-assembly and coassemblies with neurofilament triplet proteins, were examined by transient transfections with deletion mutants in a non-neuronal cell line lacking an endogenous cytoplasmic intermediate filament network. The results from the self-assembly studies showed that the head domain was essential for α-internexin’s ability to self-assemble into a filament network and the tail domain was important for establishing a proper filament network. The data from the coassembly studies demonstrated that α- internexin interacted differentially with the neurofilament triplet protein subunits. Wild-type NF-L or NF-M, but not NF-H, was able to complement and form a normal filament network with the tailless α-internexin mutant, the α- internexin head-deletion mutant, or the α-internexin mutant missing the entire tail and some amino-terminal portion of the head domain. In contrast, neither the tailless NF-L mutant nor the NF-L head-deletion mutant was able to form a normal filament network with any of these α- internexin deletion mutants. However, coassembly of the tailless NF-M mutant with the α-internexin head-deletion mutant and coassembly of the NF-M head-deletion mutant with the tailless α-internexin mutant resulted in the formation of a normal filament network. Thus, the coassembly between α-internexin and NF-M exhibits some unique characteristics previously not observed with other intermediate filament proteins: only one intact tail and one intact head are required for the formation of a normal filament network, and they can be present within the same partner or separately in two partners.
Strychnine-sensitive stabilization of postsynaptic glycine receptor clusters
Authors: Lévi Sabine , Vannier Christian , Triller Antoine
Published: April 2021

The cellular and molecular mechanisms underlying the postsynaptic aggregation of ionotropic receptors in the central nervous system are not understood. The glycine receptor (GlyR) and its cytoplasmic domain-associated protein, gephyrin, are clustered at the postsynaptic membrane and constitute a good model for addressing these questions. The glycine receptor is inhibited by strychnine. The effects of chronic strychnine treatment on the expression and cellular distribution of gephyrin and glycine receptor were therefore tested using primary cultures of spinal cord neurons. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the glycine receptor α1, α2, β subunits and gephyrin mRNAs were expressed at comparable levels in strychnine-treated and untreated cultures. The number of immunoreactive cells and the subcellular distribution of gephyrin and GlyR subunits was determined with standard and confocal immunofluorescence. The proportion of gephyrin and glycine receptor-immunoreactive (IR) cells was unaffected by strychnine treatment. Confocal microscopy revealed that the glycine receptor was mainly localized intracellularly near the nucleus. This cytoplasmic glycine receptor was not associated with the Golgi apparatus nor with the rough endoplasmic reticulum and therefore is not likely to correspond to neosynthesized proteins. The number of GlyR clusters on the somato-dendritic membrane was dramatically reduced on neurons displaying intracellular staining. In contrast, the subcellular distribution and the number of gephyrin clusters was not modified by the treatment. The fact that gephyrin postsynaptic localization was not modified by strychnine suggests that the aggregation of glycine receptor and gephyrin is governed by different mechanisms. The distribution of other cell surface molecules such as NCAM or GABAA receptor β2/3 subunits was not modified by strychnine treatment. Chronic exposure of the cultures to tetrodotoxin did not affect gephyrin or glycine receptor cluster formation. Taken together, these results indicate that functional glycine receptor, but not electrical synaptic activity, is required for the formation of glycine receptor clusters.
Effects of anti-fibrillarin antibodies on building of functional nucleoli at the end of mitosis
Authors: Fomproix N. , Gébrane-Younès J. , Hernandez-Verdun D.
Published: April 2021

During mitosis some nuclear complexes are relocalized at the chromosome periphery and are then reintegrated into the re-forming nuclei in late telophase. To address questions concerning translocation from the chromosome periphery to nuclei, the dynamics of one nucleolar perichromosomal protein which is involved in the ribosomal RNA processing machinery, fibrillarin, was followed. In the same cells, the onset of the RNA polymerase I (RNA pol I) activity and translocation of fibrillarin were simultaneously investigated. In PtK1 cells, RNA pol I transcription was first detected at anaphase B. At the same mitotic stage, fibrillarin formed foci of increasing size around the chromosomes, these foci then gathered into prenucleolar bodies (PNBs) and later PNBs were targeted into the newly formed nucleoli. Electron microscopy studies enabled the visualization of the PNBs forming the dense fibrillar component (DFC) of new nucleoli. Anti-fibrillarin antibodies microinjected at different periods of mitosis blocked fibrillarin translocation at different steps, i.e. the formation of large foci, foci gathering in PNBs or PNB targeting into nucleoli, and thereby modified the ultrastructural organization of the nucleoli as well as of the PNBs. In addition, antibody-bound fibrillarin seemed localized with blocks of condensed chromatin in early G1 nuclei. It has been found that blocking fibrillarin translocation reduced or inhibited RNA pol I transcription. It is postulated that when translocation of proteins belonging to the processing machinery is inhibited or diminished, a negative feed-back effect is induced on nucleolar reassembly and transcriptional activity.
Map kinase function in amoeboid chemotaxis
Authors: Wang Yiwen , Liu Joey , Segall Jeffrey E.
Published: April 2021

Mutants lacking the MAP kinase DdERK2 show reduced chemotactic responses to folate and cAMP. Analysis of cAMP chemotaxis shows that Dderk2- cells are defective in chemotaxis to high concentrations of cAMP. This defect is due to an inability to repolarize in the continued presence of high concentrations of cAMP. Under these conditions, the speed of movement of mutant cells remains low. Instead of generating a leading pseudopod, mutant cells generate transient crown-like structures over multiple regions of the cell surface. These structures differ from pseudopods in that they contain myosin II as well as F actin and coronin. These studies identify a role for MAP kinases in coordinating the formation of cell projections generated in response to chemoattractants.
dictyostelium γ-tubulin: molecular characterization and ultrastructural localization
Authors: Euteneuer Ursula , Gräf Ralph , Kube-Granderath Eckhard , Schliwa Manfred
Published: April 2021

The centrosome of Dictyostelium discoideum is a nucleus-associated body consisting of an electron-dense, three-layered core surrounded by an amorphous matrix, the corona. To elucidate the molecular and supramolecular architecture of this unique microtubule-organizing center, we have isolated and sequenced the gene encoding γ-tubulin and have studied its localization in the Dictyostelium centrosome using immunofluorescence and postembedding immunoelectron microscopy. D. discoideum possesses a single copy of a γ-tubulin gene that is related to, but more divergent from, other γ-tubulins. The low-abundance gene product is localized to the centrosome in an intriguing pattern: it is highly concentrated in the corona in regularly spaced clusters whose distribution correlates with the patterning of dense nodules that are a prominent feature of the corona. These observations lend support to the notion that the corona is the functional homologue of the pericentriolar matrix of ‘higher’ eukaryotic centrosomes, and that nodules are the functional equivalent of γ-tubulin ring complexes that serve as nucleation sites for microtubules in animal centrosomes.
Cellular activation through the ligation of intercellular adhesion molecule-1
Authors: Clayton Aled , Evans Rachel A. , Pettit Elizabeth , Hallett Maurice , Williams John D. , Steadman Robert
Published: April 2021

In addition to its role in mediating leukocyte adherence to and migration across the endothelium, intercellular adhesion molecule (ICAM)-1 on the surface of interstitial cells has been implicated as a principal adhesion molecule controlling leukocyte infiltration at inflammatory sites. The present study demonstrates that leukocyte binding to fibroblasts isolated from both the human renal cortex and lung and to endothelial cells induced the de novo synthesis of ICAM-1 mRNA and protein through the ICAM-1-dependent activation of the cultured cells. This was mimicked by specifically cross-linking the ICAM-1 receptor with anti-ICAM-1 antibodies. Following cross-linking there was a two- to threefold increase in the steady state levels of ICAM-1 mRNA isolated from the cells. The expression of this mRNA peaked at 1-3 hours and was dose-dependent on the concentration of secondary cross-linking antibody. Peak protein expression was between 18 and 48 hours after cross-linking. Additional data demonstrated a similar increase in the expression of VCAM-1 following ICAM-1 cross-linking. In contrast, there was no reponse as a result of incubation with an isotype-matched control antibody.

Both the binding of leukocytes and the cross-linking of ICAM-1 triggered a rise in cytosolic free calcium as the result, in part, of a calcium influx from the extracellular medium. Using BAPTA-AM to chelate intracellular calcium ions indicated that this increase in cytosolic free calcium was directly involved in the initiation of adhesion molecule upregulation.

The present study demonstrates that both endothelial cells and fibroblasts can be stimulated by the direct cell surface interaction of ICAM-1 with its ligands on inflammatory cells. One phenotypic change resulting from this interaction is the upregulated synthesis and expression of more cellular adhesion molecules. This may have profound implications for the control and persistence of a leukocyte infiltrate and the progression of inflammatory disease.
The synergistic activity of αvβ3 integrin and pdgf receptor increases cell migration
Authors: Woodard Amy S. , García-Cardeña Guillermo , Leong Margaret , Madri Joseph A. , Sessa William C. , Languino Lucia R.
Published: April 2021

Integrins and growth factor receptors act synergistically to modulate cellular functions. The αvβ3 integrin and the platelet-derived growth factor receptor have both been shown to play a positive role in cell migration. We show here that a platelet derived growth factor-BB gradient stimulated migration of rat microvascular endothelial cells on vitronectin (9.2-fold increase compared to resting cells) in a αvβ3 and RGD-dependent manner. In contrast, this response was not observed on a β1 integrin ligand, laminin; background levels of migration, in response to a platelet derived growth factor-BB gradient, were observed on this substrate or on bovine serum albumin (2.4- or 2.0-fold, respectively). Comparable results were obtained using NIH-3T3 cells. Platelet derived growth factor-BB did not change the cells’ ability to adhere to vitronectin, nor did it stimulate a further increase in proliferation on vitronectin versus laminin. In addition, platelet derived growth factor-BB stimulation of NIH-3T3 cells did not alter the ability of αvβ3 to bind RGD immobilized on Sepharose. The αvβ3 integrin and the platelet derived growth factor receptor-β associate in both microvascular endothelial cells and NIH-3T3 cells, since they coprecipitated using two different antibodies to either αvβ3 or to the platelet derived growth factor receptor-β. In contrast, β1 integrins did not coprecipitate with the platelet derived growth factor receptor-β. These results point to a novel pathway, mediated by the synergistic activity of αvβ3 and the platelet derived growth factor receptor-β, that regulates cell migration and, therefore, might play a role during neovessel formation and tissue infiltration.
Biogenesis of tight junctions: The c-terminal domain of occludin mediates basolateral targeting
Authors: Matter Karl , Balda Maria S.
Published: April 2021

Tight junctions form a morphological and physical border between the apical and the basolateral cell surface domains of epithelial cells; hence assembly of tight junctions could occur from both of the two plasma membrane domains. We show here that the C-terminal cytoplasmic domain of occludin, the only known transmembrane protein of tight junctions, was sufficient to mediate basolateral expression of a chimeric protein. Since this chimera was transported directly to the basolateral membrane during biosynthesis, the C-terminal domain of occludin contains a basolateral targeting signal. Additionally, the C-terminal domain of occludin was also able to mediate endocytosis. Thus, the C-terminal cytoplasmic domain appears to govern intracellular transport of occludin. To test whether the basolateral membrane is an obligatory intermediate in transport of occludin to tight junctions, we analyzed the expression of occludin molecules rendered unable to efficiently integrate into tight junctions by the introduction of N-linked glycosylation sites into the two extracellular loops. Indeed, glycosylated occludin accumulated in the basolateral membrane, supporting a model in which the biogenesis of tight junctions occurs from this cell-surface domain.
Nerve growth factor and ceramides modulate cell death in the early developing inner ear
Authors: Frago Laura M. , León Yolanda , de la Rosa Enrique J. , Gómez-Muñoz Antonio , Varela-Nieto Isabel
Published: April 2021

Regulation of normal development involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. We have investigated the signalling mechanisms involved in regulation of the balance between cell proliferation and apoptotic cell death in the otic vesicle. The sphingomyelin pathway signals apoptosis for nerve growth factor upon binding to p75 receptors. It is initiated by sphingomyelin hydrolysis to generate the second messenger ceramide. In the present study, we show that nerve growth factor stimulates sphingomyelin hydrolysis and the concomitant ceramide release in organotypic cultures of otic vesicles. Both nerve growth factor and ceramide induce apoptotic responses to a different extent. Ceramide-induced apoptosis was suppressed by insulin-like growth factor-I which is a strong promoter of cell growth and morphogenesis for the developing inner ear. In contrast, ceramide-1-phosphate protected the explants from apoptosis induced by serum withdrawal but did not antagonise ceramide-induced cell death. This study suggests that sphingomyelin-derived second messengers might be key modulators of programmed cell death during development.
Mechanical compression alters proteoglycan deposition and matrix deformation around individual cells in cartilage explants
Authors: Quinn Thomas M. , Grodzinsky Alan J. , Buschmann Michael D. , Kim Young-Jo , Hunziker Ernst B.
Published: April 2021

We have used new techniques of cell-length scale quantitative autoradiography to assess matrix synthesis, deposition, and deformation around individual chondrocytes in mechanically compressed cartilage explants. Our objectives were to: (1) quantify the effects of static and dynamic compression on the deposition of newly synthesized proteoglycans into cell-associated and further-removed matrices; (2) measure cell-length scale matrix strains and morphological changes of the cell and matrix associated with tissue compression; and (3) relate microscopic physical stimuli to changes in proteoglycan synthesis as functions of compression level and position within mechanically compressed explants. Results indicate a high degree of structural organization in the extracellular matrix, with the pericellular matrix associated with the most rapid rates of proteoglycan deposition, and greatest sensitivity to mechanical compression. Static compression could stimulate directional deposition of secreted proteoglycans around chondrocytes, superimposed on an inhibition of proteoglycan synthesis; these events followed trends for compressive strain in the cell-associated matrix. Conversely, proteoglycan synthesis and pericellular deposition was stimulated by dynamic compression. Results suggest that cell-matrix interactions in the cell-associated matrix may be a particularly important aspect of the chondrocyte response to mechanical compression, possibly involving macromolecular transport limitations and morphological changes associated with fluid flow and local compaction of the matrix around cells.
Two proteins isolated from sea urchin sperm flagella: structural components common to the stable microtubules of axonemes and centrioles
Authors: Hinchcliffe Edward H. , Linck Richard W.
Published: April 2021

Biochemical fractionation of axonemal microtubules yields the protofilament ribbon (pf-ribbon), an insoluble structure of 3-4 longitudinal protofilaments composed primarily of α/β tubulin, tektins A, B and C, and two previously uncharacterized polypeptides of 77 kDa and 83 kDa. We have isolated the 77/83 kDa polypeptides (termed Sp77 and Sp83) from sperm flagella of the sea urchin Stronglyocentrotus purpuratus and raised polyclonal antibodies against them. Sp77 and Sp83 copurify exclusively with the pf-ribbon. Both the anti-Sp77 and anti-Sp83 antibodies detected the nine outer doublets and the basal bodies of sea urchin sperm by immunofluorescence microscopy. In addition, the anti-Sp83 antibody, but not the anti-Sp77 antibody, detected a single 83 kDa polypeptide on immunoblots of unfertilized sea urchin egg cytoplasm, and a single polypeptide of 80 kDa on blots of isolated mitotic spindles from Chinese hamster ovary (CHO) cells. Previous studies have shown that tektins are present in the basal bodies and centrosomes/centrioles of cells ranging from clam to human. We found that anti-Sp83 decorates the spindle poles in sea urchin zygotes, and the interphase centrosome and spindle poles in CHO cells. In CHO cells arrested in S phase with aphidicolin, anti-Sp83 detects multiple centrosomes. The staining of the centrosome was not disrupted by prolonged nocodazole treatment, suggesting that the 80 kDa polypeptide is associated with the centrioles themselves. Our observations demonstrate that, like tektins, Sp77 and Sp83 are structural proteins associated with stable doublet microtubules, and may be components of basal bodies and centrioles of sea urchins and mammalian cells.
PDGF α-receptor mediated cellular responses are not dependent on Src family kinases in endothelial cells
Authors: Hooshmand-Rad R. , Yokote K. , Heldin C.-H. , Claesson-Welsh L.
Published: April 2021

Two novel autophosphorylation sites in the juxtamembrane region of the PDGF α-receptor, Tyr-572 and Tyr-574, were identified. A Y572/574F mutant PDGF α-receptor was generated and stably expressed in porcine aortic endothelial cells. In contrast to the wild-type receptor, the mutant receptor was unable to associate with or activate Src family tyrosine kinases. Tyrosine phosphorylated synthetic peptides representing the juxtamembrane sequence of the receptor dose-dependently inhibited the binding of Src family tyrosine kinases to the autophosphorylated PDGF α-receptor. The mutant receptor showed similar PDGF-induced kinase activity and ability to mediate mitogenicity, actin reorganization and chemotaxis as the wild-type receptor. Thus activation of Src family kinases by the PDGF α-receptor is not essential for PDGF-induced mitogenicity or actin reorganization.
cAMP increasing agents attenuate the generation of apoptosis by etoposide in promonocytic leukemia cells
Authors: García-Bermejo Laura , Pérez Concepción , Vilaboa Nuria E. , Blas Elena de , Aller Patricio
Published: April 2021

Treatment of U-937 promonocytic cells with the DNA topoisomerase II inhibitor etoposide rapidly caused death by apoptosis, as determined by changes in chromatin structure, production of DNA breaks, nucleosome-sized DNA degradation, decrease in mitochondrial membrane potential and phosphatidyl serine translocation in the plasma membrane, and at the same time induced intracellular acidification. Both the execution of the apoptotic process and the intracellular acidification were reduced by the addition of forskolin plus theophylline or other cAMP increasing agents. These agents also attenuated the induction of apoptosis by camptothecin, heat-shock, cadmium chloride and X-radiation. Although etoposide slightly increased the production of reactive oxygen intermediates, this increase was not prevented by forskolin plus theophylline, and the addition of antioxidant agents failed to inhibit apoptosis. Etoposide caused a great increase in NF-κB binding activity, which was not prevented by forskolin plus theophylline, while AP-1 binding was little affected by the topoisomerase inhibitor. The treatments did not significantly alter the levels of Bcl-2 and Bax. By contrast, the expression of c-myc, which was very high in untreated U-937 cells and only partially inhibited by etoposide, was rapidly and almost totally abolished by the cAMP increasing agents. Finally, it was observed that etoposide caused a transient dephosphorylation of retinoblastoma (Rb), which was associated with cleavage of poly(ADP-ribose) polymerase (PARP). Both Rb dephosphorylation and PARP cleavage were inhibited by forskolin plus theophylline. The inhibition of Rb (type I) phosphatase and ICE/CED-3-like protease activities, and the abrogation of c-myc expression, are mechanisms which could explain the anti-apoptotic action of cAMP increasing agents in myeloid cells.
Myogenic conversion of NIH3T3 cells by exogenous MyoD family members: dissociation of terminal differentiation from myotube formation
Authors: Russo Simona , Tomatis Daniela , Collo Ginetta , Tarone Guido , Tatò Franco
Published: April 2021

Myogenic regulatory factors (MRF) of the MyoD family regulate the skeletal muscle differentiation program. Non-muscle cells transfected with exogenous MRF either are converted to the myogenic lineage or fail to express the muscle phenotype, depending on the cell type analysed. We report here that MRF-induced myogenic conversion of NIH3T3 cells results in an incomplete reprogramming of these cells. Transfected cells withdrew from the cell cycle and underwent biochemical differentiation but, surprisingly, terminally differentiated myocytes absolutely failed to fuse into multinucleated myotubes. Analysis of muscle regulatory and structural gene expression failed to provide an explanation for the fusion defectiveness. However, myogenic derivatives of NIH3T3 cells were shown to be unable to accumulate the transcripts encoding muscle-specific isoforms of the integrin subunit β1D and the transcription factor MEF2D1b2, that depend on muscle-specific alternative splicing. Our results suggest that the fusion into myotubes is under a distinct genetic control that might depend, at least partially, on differential splicing.
PUMA1: a novel protein that associates with the centrosomes, spindle and centromeres in the nematode Parascaris
Authors: Esteban M. R. , Giovinazzo G. , Hera A. de la , Goday C.
Published: April 2021

We have identified a 227 kDa spindle- and centromere-associated protein in Parascaris, designated PUMA1 (Parascaris univalens mitotic apparatus), using a monoclonal antibody (mAb403) generated against Parascaris embryonic extracts. PUMA1 distribution was studied by immunofluorescence microscopy in mitotic and meiotic Parascaris cells, where centromere organization differs greatly. In mitosis, PUMA1 associates throughout cell division with the centrosomes and kinetochore-microtubules, and it concentrates at the continuous centromere region of the holocentric chromosomes. PUMA1 also localizes to the spindle mid-zone region during anaphase and at the midbody during telophase. In meiosis, PUMA1 associates with the centrosomes and with the discrete centromeric regions lacking kinetochore structures. The analysis of colchicine-treated embryos indicated that the association of PUMA1 with the centromeric region depends on microtubule integrity. mAb403 also recognizes spindle components in Drosophila. A series of overlapping cDNAs encoding the gene were isolated from a Parascaris embryonic expression library. Analysis of the nucleotide sequence identified an open reading frame capable of encoding a protein of 227 kDa. Analysis of the protein sequence indicated that PUMA1 is predicted to be a coiled-coil protein containing a large central α-helical domain flanked by nonhelical terminal domains. The structural features and cellular distribution of PUMA1 suggest that it may play a role in the organization of the spindle apparatus and in its interaction with the centromere in Parascaris.