bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.