bluuebunny/crossref_metadata_2025_split huggingface dataset:
Brain-derived neurotrophic factor-induced phosphorylation of neurofilament-H subunit in primary cultures of embryo rat cortical neurons
Authors: Tokuoka Hirofumi , Saito Taro , Yorifuji Hiroshi , Wei Fan-Yan , Kishimoto Takeo , Hisanaga Shin-ichi 
Published: April 2021

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

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

ABSTRACT
Members of the syntaxin family play a fundamental role in vesicle docking and fusion of diverse transport events. We have molecularly characterized syntaxin 8, a novel member of the syntaxin family. The nucleotide sequence of cloned rat cDNA predicts a polypeptide of 236 residues with a carboxyl-terminal 18-residue hydrophobic domain that may function as a membrane anchor. Characteristic of syntaxins, syntaxin 8 also contain regions that have the potential to form coiled-coil structures. Among the known syntaxins, syntaxin 8 is most homologous to syntaxin 6 which is predominantly associated with the trans-Golgi network (TGN). The syntaxin 8 transcript is detected in all rat tissues examined by northern blot. Antibodies against recombinant syntaxin 8 recognize a 27 kDa protein that is enriched in membrane fractions containing the Golgi apparatus and the endosomal/lysosomal compartments. Syntaxin 8 in membrane extract could be incorporated into a 20S protein complex in a way that is dependent on the soluble N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP), suggesting that syntaxin 8 is indeed a SNAP receptor (SNARE). Indirect immunofluorescence microscopy reveals that the majority of syntaxin 8 is localized to the early endosome marked by Rab5. This is corroborated by immunogold labeling experiments showing enrichment of syntaxin 8 in the early endosome and its co-labeling with Rab5.
Positive and negative regulation of TGF-β signaling
Authors: Miyazono Kohei 
Published: April 2021

ABSTRACT
Cytokines of the transforming growth factor β (TGF-β) superfamily, including TGF-βs, activins and bone morphogenetic proteins (BMPs), bind to specific serine/threonine kinase receptors and transmit intracellular signals through Smad proteins. Upon ligand stimulation, Smads move into the nucleus and function as components of transcription complexes. TGF-β and BMP signaling is regulated positively and negatively through various mechanisms. Positive regulation amplifies signals to a level sufficient for biological activity. Negative regulation occurs at the extracellular, membrane, cytoplasmic and nuclear levels. TGF-β and BMP signaling is often regulated through negative feedback mechanisms, which limit the magnitude of signals and terminate signaling. Negative regulation is also important for formation of gradients of morphogens, which is crucial in developmental processes. In addition, other signaling pathways regulate TGF-β and BMP signaling through cross-talk. Nearly 20 BMP isoforms have been identified, and their activities are regulated by various extracellular antagonists. Regulation of TGF-β signaling might be tightly linked to tumor progression, since TGF-β is a potent growth inhibitor in most cell types.
Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones
Authors: María Frade José 
Published: April 2021

ABSTRACT
During their early postmitotic life, a proportion of the nascent retinal ganglion cells (RGCs) are induced to die as a result of the interaction of nerve growth factor (NGF) with the neurotrophin receptor p75. To analyse the mechanisms by which NGF promotes apoptosis, an in vitro culture system consisting of dissociated E5 retinal cells was established. In this system, NGF-induced apoptosis was only observed in the presence of insulin and neurotrophin-3, conditions that favour the birth of RGCs and other neurones expressing the glycoprotein G4. The pro-apoptotic effect of NGF on the G4-positive neurones was evident after 10 hours in vitro and was preceded by a significant upregulation of cyclin B2, but not cyclin D1, and the presence of mitotic nuclei in these cells. Brain-derived neurotrophic factor prevented both the increase of cyclin B2 expression in the G4-positive neurones and the NGF-induced cell death. Finally, pharmacologically blocking cell-cycle progression using the cyclin-dependent kinase inhibitor roscovitine prevented NGF-induced cell death in a dose-dependent manner. These results strongly suggest that the apoptotic signalling initiated by NGF requires a driving stimulus manifested by the neuronal birth and is preceded by the unscheduled re-entry of postmitotic neurones into the cell cycle.
Chromosome condensation induced by geminivirus infection of mature plant cells
Authors: Bass Hank W. , Nagar Steven , Hanley-Bowdoin Linda , Robertson Dominique 
Published: April 2021

ABSTRACT
Tomato golden mosaic virus (TGMV) is a geminivirus that replicates its single-stranded DNA genome through double-stranded DNA intermediates in nuclei of differentiated plant cells using host replication machinery. We analyzed the distribution of viral and plant DNA in nuclei of infected leaves using fluorescence in situ hybridization (FISH). TGMV-infected nuclei showed up to a sixfold increase in total volume and displayed a variety of viral DNA accumulation patterns. The most striking viral DNA patterns were bright, discrete intranuclear compartments, but diffuse nuclear localization was also observed. Quantitative and spatial measurements of high resolution 3-dimensional image data revealed that these compartments accounted for 1-18% of the total nuclear volume or 2-45% of the total nuclear FISH signals. In contrast, plant DNA was concentrated around the nuclear periphery. In a significant number of nuclei, the peripheral chromatin was organized as condensed prophase-like fibers. A combination of FISH analysis and indirect immunofluorescence with viral coat protein antibodies revealed that TGMV virions are associated with the viral DNA compartments. However, the coat protein antibodies failed to cross react with some large viral DNA inclusions, suggesting that encapsidation may occur after significant viral DNA accumulation. Infection by a TGMV mutant with a defective coat protein open reading frame resulted in fewer and smaller viral DNA-containing compartments. Nevertheless, nuclei infected with the mutant virus increased in size and in some cases showed chromosome condensation. Together, these results established that geminivirus infection alters nuclear architecture and can induce plant chromatin condensation characteristic of cells arrested in early mitosis.
Interaction between the cytodomains of the α3 and β1 integrin subunits regulates remodelling of adhesion complexes on laminin
Authors: Laplantine Emmanuel , Vallar Laurent , Mann Karlheinz , Kieffer Nelly , Aumailley Monique 
Published: April 2021

ABSTRACT
The first step of laminin 1-induced signal transduction is initiated by the formation of α6β1 integrin-specific adhesion complexes. In contrast, on other laminin isoforms the adhesion complexes are α3β1 integrin-specific due to a transdominant regulation of the α6β1 integrin by the α3β1 integrin. To determine the mechanism of this regulation, peptides representing the cytoplasmic domain of the α3 or α6 integrin subunits were microinjected together with recombinant enhanced green fluorescence protein into live fibroblasts. Microinjection of the α3 integrin peptide to laminin 1-adherent cells displaying α6β1 integrin-specific adhesion complexes resulted in the disengagement of the α6β1 integrin, while microinjection of green fluorescence protein alone or in combination with the α6 integrin cytodomain had no effect. Further surface plasmon resonance studies revealed that the cytodomain of the β1 integrin subunit interacts with low affinity with the cytoplasmic tail of the α3 integrin subunit, but not with that of several other α subunits including α6. These results imply that the cytoplasmic tails of the integrin α subunits play a critical role in the regulation of integrin-induced signal transduction. In particular, the intracellular tail of the α3 integrin subunit controls the formation of adhesion complexes in cells adhering to laminins.
The Saccharomyces cerevisiae SDA1gene is required for actin cytoskeleton organization and cell cycle progression
Authors: Buscemi Giacomo , Saracino Francesca , Masnada Debora , Agostoni Carbone Maria Luisa 
Published: April 2021

ABSTRACT
The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiaenovel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G1by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1or in cdc28-13cells released from G1block, prevents recovery of budding and DNA replication. We also show that the block in G1caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G1and that Sda1 is involved in the control of the actin cytoskeleton.
Apical endocytosis of ricin in MDCK cells is regulated by the cyclooxygenase pathway
Authors: Llorente Alicia , Deurs Bo van , Garred Øystein , Eker Per , Sandvig Kirsten 
Published: April 2021

ABSTRACT
Addition of arachidonic acid or stimulation of arachidonic acid production by secretory phospholipase A2 selectively upregulated apical endocytosis of ricin in MDCK cells without affecting basolateral endocytosis. Electron microscopic studies revealed that MDCK cells treated with secretory phospholipase A2 and incubated with horseradish peroxidase had an increased number of normal appearing peroxidase-labeled endosomes and no sign of membrane ruffling. Moreover, inhibition of basal arachidonic acid release, either by decreasing the cytosolic phospholipase A2activity or the diacylglycerol lipase activity, reduced the rate of apical endocytosis. Furthermore, indomethacin, an inhibitor of the cyclooxygenase pathway, counteracted the stimulation of endocytosis seen with both secretory phospholipase A2 and arachidonic acid, suggesting that formation of eicosanoids such as prostaglandins could be essential for the regulation. This idea was supported by the finding that prostaglandin E2, the predominant prostaglandin formed in kidney, also upregulated ricin uptake. The regulatory effect of the cyclooxygenase pathway on apical endocytosis of ricin was found to be independent of protein kinases A and C, which are known to selectively control apical clathrin-independent endocytosis in polarized cells.
Morphology and behaviour of dinoflagellate chromosomes during the cell cycle and mitosis
Authors: Bhaud Yvonne , Guillebault Delphine , Lennon Jean-François , Defacque Hélène , Soyer-Gobillard Marie-Odile , Moreau Hervé 
Published: April 2021

ABSTRACT
The morphology and behaviour of the chromosomes of dinoflagellates during the cell cycle appear to be unique among eukaryotes. We used synchronized and aphidicolin-blocked cultures of the dinoflagellate Crypthecodinium cohniito describe the successive morphological changes that chromosomes undergo during the cell cycle. The chromosomes in early G1phase appeared to be loosely condensed with numerous structures protruding toward the nucleoplasm. They condensed in late G1, before unwinding in S phase. The chromosomes in cells in G2phase were tightly condensed and had a double number of arches, as visualised by electron microscopy. During prophase, chromosomes elongated and split longitudinally, into characteristic V or Y shapes.
We also used confocal microscopy to show a metaphase-like alignment of the chromosomes, which has never been described in dinoflagellates. The metaphase-like nucleus appeared flattened and enlarged, and continued to do so into anaphase. Chromosome segregation occurred via binding to the nuclear envelope surrounding the cytoplasmic channels and microtubule bundles. Our findings are summarized in a model of chromosome behaviour during the cell cycle.
Actin filaments and microtubules play different roles during bristle elongation in Drosophila
Authors: Tilney Lewis G. , Connelly Patricia S. , Vranich Kelly A. , Shaw Michael K. , Guild Gregory M. 
Published: April 2021

ABSTRACT
Developing bristles in Drosophilapupae contain 7-11 bundles of crosslinked actin filaments and a large population of microtubules. During bristle growth the rate of cell elongation increases with bristle length. Thin section EM shows that bundle size is correlated with the amount of cytoplasm at all points along the bristle. Thus, as the bristle elongates and tapers, fewer actin filaments are used. To ensure penetration of inhibitors we isolated thoraces and cultured them in vitro; bristles elongate at rates identical to bristles growing in situ. Interestingly, inhibitors of actin filament assembly (cytochalasin D and latrunculin A) dramatically curtailed bristle elongation while a filament stabilizer (jasplakinolide) accelerated elongation. In contrast, inhibitors of microtubule dynamics (nocodazole, vinblastine, colchicine and taxol) did not affect bristle elongation. Surprisingly, the bristle microtubules are stable and do not turn over. Furthermore, the density of microtubules decreases as the bristle elongates. These two facts coupled with calculations and kinetics of elongation and the fact that the microtubules are short indicate that the microtubules are assembled early in development and then transported distally as the bristle grows. We conclude that actin assembly is crucial for bristle cell elongation and that microtubules must furnish other functions such as to provide bulk to the bristle cytoplasm as well as playing a role in vesicle transport.
Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration
Authors: Eddy Robert J. , Pierini Lynda M. , Matsumura Fumio , Maxfield Frederick R. 
Published: April 2021

ABSTRACT
Buffering of intracellular Ca2+transients in human neutrophils leads to reduced motility due to defective uropod detachment on fibronectin and vitronectin-coated surfaces. Since one potential target of a rise in [Ca2+]iis the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedione monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926) resulted in impaired uropod retraction and a dose-dependent decrease in chemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP). Treatment with ML-9 resulted in a redistribution of F-actin and talin to the non-retracted uropods, mimicking the redistribution observed during [Ca2+]ibuffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin II inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-coated glass. At higher concentrations of ML-9, cell polarization was inhibited and pseudopod extension occurred radially. Using an antibody specific for serine 19-phosphorylated regulatory light chain of myosin II, regions of activated myosin II were found at the leading edge as well as the uropod in motile fMLP-stimulated cells. [Ca2+]idepletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II suggesting that activation of myosin II by intracellular Ca2+transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.
Upregulation of the secretory pathway in cysteine protease inhibitor-resistant Trypanosoma cruzi
Authors: Engel Juan C. , García Cristina Torres , Hsieh Ivy , Doyle Patricia S. , McKerrow James H. 
Published: April 2021

ABSTRACT
A novel chemotherapy in development for Chagas’ disease targets cruzain, the major cysteine protease of Trypanosoma cruzi. Peptidomimetic inhibitors disrupt the intracellular cycle of the parasite and rescue animals from a lethal infection. Inhibitor killing of parasites results from interruption of autocatalytic cruzain processing and transport to lysosomes, and massive accumulation of precursor protein in the Golgi complex. To further understand the mechanisms of protease processing and transport in this primitive eukaryote, and uncover potential mechanisms for resistance to these drugs, we generated cysteine-protease inhibitor (CPI)-resistant epimastigotes in vitro and investigated the mechanisms involved at the biochemical and structural levels. Resistance to 20-fold the lethal CPI concentration, achieved after a year of gradual drug increase, was accompanied by a modest decrease in growth rate. A marked increase in the number of vesicles trafficking from the Golgi complex to the flagellar pocket occurs in resistant cells. No mature protease reaches lysosomes though accumulation of endocytosed gold particles in lysosomes appears to be normal. Higher molecular mass cruzain species, consistentwith complexes of cruzain precursors and inhibitor, are secreted by CPI-resistant parasites into the culture supernatant. Release of these cruzain precursors may be facilitated by an enhanced acidification of trans-Golgi cisternae in resistant parasites. The pH within Golgi cisternae is higher in control epimastigotes and most mature cruzain is lysosomal. Cruzain activity is negligible in CPI-resistant epimastigote extracts compared to the parental clone. Activity is restored following withdrawal of the inhibitor. No cross-resistance to the therapeutic drugs nifurtimox and benznidazole occurred and, conversely, parasites resistant to these drugs were sensitive to CPI. Protease inhibitors are thus potential therapeutical alternatives in cases of nifurtimox/benznidazole resistance. Cumulatively, these results suggest that CPI-resistance induces upregulation of Golgi complex function and post- Golgi secretory pathway, and release of precursors before the enzyme reaches its site of biologic activity.
Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro
Authors: Laoukili Jamila , Perret Eric , Middendorp Sandrine , Houcine Odile , Guennou Christiane , Marano Francelyne , Bornens Michel , Tournier Frédéric 
Published: April 2021

ABSTRACT
Centrin protein is an ubiquitously expressed cytoskeletal component and is a member of the EF-hand superfamily of calcium-binding proteins. It was first discovered in the flagellar apparatus of unicellular green algae where it is involved in contraction of Ca2+-sensitive structures. Centrin protein is associated with centrosome-related structures such as spindle pole body in yeast, and centriole/basal bodies in flagellar and ciliated cells. Three centrin genes have been cloned in human cells. In this work, we have performed a comparative biochemical and functional analysis of centrin isoforms using a primary culture of human nasal epithelial cells which provides an efficient way to obtain a complete ciliated cell differentiation process. RT-PCR experiments show that the expression of the three human centrin genes increases during cell differentiation, and that only centrin 2 and 3 are expressed during cell proliferation. Using polyclonal antibodies raised against recombinant human centrin 2 and 3, we show a specific pattern of protein expression. Ultrastructural immunolocalization suggests that centrin proteins are involved in the early process of centriole assembly, as they are concentrated within the precursor structures of centriole/basal bodies. It also shows a differential localisation of centrin proteins in mature centriole/basal bodies, suggesting different functions for centrins 1/2 and centrin 3. This is also supported by functional analyses showing that centrin 1 and/or centrin 2 are involved in ciliary beating.