bluuebunny/crossref_metadata_2025_split huggingface dataset:
The rough deal protein is a new kinetochore component required for accurate chromosome segregation in Drosophila
Authors: Scaërou Frédéric , Aguilera Isabel , Saunders Robert , Kane Nanci , Blottière Louise , Karess Roger 
Published: April 2021

ABSTRACT
Mutations in the rough deal (rod) gene of Drosophila greatly increase the missegregation of sister chromatids during mitosis, suggesting a role for this gene product in spindle or kinetochore function. The activity provided by rod also appears to be necessary for the recruitment of two known kinetochore components, Zw10 and cytoplasmic dynein. In this paper we describe the cloning of rough deal and an initial cytological characterization of its product. The Rod protein shares no identifiable structural motif with other known proteins, although apparent homologs exist in the genomes of nematode and man. By immunocytochemistry we show that Rod displays a dynamic intracellular staining pattern, localizing first to kinetochores in prometaphase, but moving to kinetochore microtubules at metaphase. Early in anaphase the protein is once again restricted to the kinetochores, where it persists until the end of telophase. This behavior is in all respects similar to that described for Zw10, and suggests that the proteins function together.
Characterization of Dictyostelium discoideum cathepsin D Molecular cloning, gene disruption, endo-lysosomal localization and sugar modifications
Authors: Journet Agnès , Chapel Agnès , Jehan Stéphanie , Adessi Céline , Freeze Hudson , Klein Gérard , Garin Jérôme 
Published: April 2021

ABSTRACT
Previous studies using magnetic purification of Dictyostelium discoideum endocytic vesicles led us to the identification of some major vesicle proteins. Using the same purification procedure, we have now focused our interest on a 44 kDa soluble vesicle protein. Microsequencing of internal peptides and subsequent cloning of the corresponding cDNA identified this protein as the Dictyostelium homolog of mammalian cathepsins D. The only glycosylation detected on Dictyostelium cathepsin D (CatD) is common antigen 1, a cluster of mannose 6- sulfate residues on N-linked oligosaccharide chains. CatD intracellular trafficking has been studied, showing the presence of the protein throughout the entire endocytic pathway. During the differentiation process, the catD gene presents a developmental regulation, which is also observed at the protein level. catD gene disruption does not alter significantly the cell behaviour, either in the vegetative form or the differentiation stage. However, modifications in the SDS-PAGE profiles of proteins bearing common antigen 1 were detected, when comparing parental and catD− cells. These modifications point to a possible role of CatD in the maturation of a few Dictyostelium lysosomal proteins.
The tenascin-C knockout revisited
Authors: Mackie Eleanor J. , Tucker Richard P. 
Published: April 2021

ABSTRACT
In the past seven years, two groups have independently produced tenascin-C-knockout mice. These mice are born alive and, originally, were described as showing no abnormalities. More recent studies, many involving pathological intervention, have shown that tenascin-C-knockout mice have several defects. The mice exhibit abnormal behaviour, as well as abnormalities in brain chemistry. They also show defects in structure and repair of neuromuscular junctions, in the ability to recover from snake-venom-induced glomerulonephritis and in chemically induced dermatitis. Healing of skin wounds is morphologically normal, but the mice exhibit defects in healing after suture injury of corneas. In both skin and corneal wounds, fibronectin expression is abnormally low in tenascin-C-knockout mice. Finally, in vitro studies indicate that haemopoietic activity is defective in bone marrow from these mice. When examined together, these studies provide evidence for precise functions for tenascin-C, as well as an explanation for why the sequence of tenascin-C is so highly phylogenetically conserved.
The sorting determinant guiding Hsp150 to the COPI-independent transport pathway in yeast
Authors: Suntio Taina , Shmelev Anton , Lund Maria , Makarow Marja 
Published: April 2021

ABSTRACT
The COPI coatomer is thought to be required in yeast directly for retrograde transport from the Golgi to the endoplasmic reticulum (ER), and directly or indirectly for ER-to-Golgi transport. Unexpectedly, the secretory glycoproteins Hsp150 and invertase have been found not to require COPI for ER exit. The features according to which cargo proteins are selected for the COPI-independent pathway are not known. The ER form of Hsp150 has three distinct domains: an N-terminal fragment of 54 amino acids (subunit I) is followed by 11 repeats of a 19 amino acid peptide plus a unique C-terminal fragment of 114 amino acids (subunit II). By fusing heterologous proteins to different Hsp150 domains and expressing them in sec21-1 and sec21-3 mutants with temperature-sensitive mutations in the γ-COPI subunit, we show here that the repeats of subunit II function as sorting determinants for COPI-independent ER exit. The C-terminal fragment of Hsp150 could be replaced by E. coli β-lactamase or rat nerve growth factor receptor ectodomain (NGFRe), and subunit I could be deleted, without inhibiting COPI-independent transport. However, when the repetitive region was omitted and β-lactamase was fused directly to the C terminus of subunit I, COPI was required for efficient ER exit. Mass spectroscopic analysis demonstrated that both subunit I and II of Hsp150 were extensively O-glycosylated, suggesting that the O-glycosylation pattern was not decisive for cargo selection.
Differential distribution of mannose-6-phosphate receptors and furin in immature secretory granules
Authors: Dittié Andrea S. , Klumperman Judith , Tooze Sharon A. 
Published: April 2021

ABSTRACT
In neuroendocrine cells sorting of proteins from immature secretory granules (ISGs) occurs during maturation and is achieved by clathrin-coated vesicles containing the adaptor protein (AP)-1. We have investigated the role of the mannose-6-phosphate receptors (M6PRs) in the recruitment of AP-1 to ISGs. M6PRs were detected in ISGs isolated from PC12 cells by subcellular fractionation, and by immuno-EM labelling on cryosections. In light of our previous results, where greater than 80% of the ISGs were found to contain furin, we examined the relationship between furin and M6PR on ISGs. By immunoisolation techniques we find that 50% at most of the ISGs contain the cation-independent (CI)-M6PR. Using sequential immunoisolation we could demonstrate that there are two populations of ISGs: those that have both M6PR and furin, and those which contain only furin. Furthermore, using immobilized GST-fusion proteins containing the cytoplasmic domain of the CI-M6PR we have shown binding of AP-1 requires casein kinase II phosphorylation of the CI-M6PR fusion protein, and in particular phosphorylation of Ser2474. Addition of these phosphorylated GST-CI-M6PR fusion proteins to a cell-free assay reconstituting AP-1 binding to ISGs inhibits AP-1 recruitment to ISGs.
Role of αvβ3 integrin in osteoclast migration and formation of the sealing zone
Authors: Nakamura Ichiro , Pilkington Mary F. , Lakkakorpi Päivi T. , Lipfert Lorraine , Sims Stephen M. , Dixon S. Jeffrey , Rodan Gideon A. , Duong Le T. 
Published: April 2021

ABSTRACT
The αvβ3 integrin is abundantly expressed in osteoclasts and has been implicated in the regulation of osteoclast function, especially in cell attachment. However, in vivo studies have shown that echistatin, an RGD-containing disintegrin which binds to αvβ3, inhibits bone resorption without changing the number of osteoclasts on the bone surface, suggesting inhibition of osteoclast activity. The objective of this study was to examine how occupancy of αvβ3 integrins inhibits osteoclast function, using primary rat osteoclasts and murine pre-fusion osteoclast-like cells formed in a co-culture system. We show that: (1) echistatin inhibits bone resorption in vitro at lower concentrations (IC50 = 0.1 nM) than those required to detach osteoclasts from bone (IC50 ∼1 µM); (2) echistatin (IC50 = 0.1 nM) inhibits M-CSF-induced migration and cell spreading of osteoclasts; (3) αvβ3 integrins are localized in podosomes at the leading edge of migrating osteoclasts, whereas, with echistatin treatment (0.1 nM), αvβ3 disperses randomly throughout the adhesion surface; and (4) when bone resorption is fully inhibited with echistatin, there is visible disruption of the sealing zone (IC50 = 13 nM), and αvβ3 visualized with confocal microscopy re-distributes from the basolateral membranes to intracellular vesicular structures. Taken together, these findings suggest that αvβ3 integrin plays a role in the regulation of two processes required for effective osteoclastic bone resorption: cell migration (IC50 = 0.1 nM) and maintenance of the sealing zone (IC50 ∼10 nM).
The majority of clathrin coated vesicles from lactating rabbit mammary gland arises from the secretory pathway
Authors: Pauloin Alain , Tooze Sharon A. , Michelutti Isabelle , Delpal Serge , Ollivier-Bousquet Michèle 
Published: April 2021

ABSTRACT
Clathrin coated vesicles were isolated from lactating rabbit mammary gland by differential centrifugation, centrifugation on 2OD600-sucrose cushions and Sephacryl S-1000 chromatography. Mammary epithelial cells contain an unexpectedly high quantity of clathrin coated vesicles which appear heterogeneous in size, with a mean diameter of 95.9±10.5 nm and a density of 1.23 g.ml-1.
Analysis of clathrin coated vesicle adaptor composition by SDS-PAGE and western blot showed that only ∼5-10% of total APs consist of AP-2 in isolated mammary gland clathrin coated vesicles whereas it represents ∼70% of the total APs from bovine brain clathrin coated vesicles. Cargo molecules known to be transcytosed such as IgG, IgA, and the pIgR were detected in the clathrin coated vesicles, indicating that part of this vesicle population is involved in transcytotic pathways. However, as the vast majority of the clathrin coated vesicles contained AP-1, it was likely that these clathrin coated vesicles were involved in the secretory pathway.
Relatively high quantities of furin and cation-independent mannose 6-phosphate receptor were detected in mammary clathrin coated vesicles. By immuno electron microscopy, AP-1 and the cation-independent mannose 6-phosphate receptor were localized in Golgi-associated vesicles and on the membrane of secretory vesicles. The presence of AP-1 in the coat patches on the membrane of secretory vesicles containing casein micelles, and the presence of αs1-casein in mammary gland clathrin coated vesicles, support a role for AP-1 in the maturation of secretory vesicles. Our data pinpoint the importance of clathrin coated vesicles in lactating mammary epithelial cells, and suggest these vesicles are involved in the transcytotic pathway, in sorting at the trans-Golgi network and in the biogenesis of casein-containing secretory vesicles.
Gamma irradiation leads to two waves of apoptosis in distinct cell populations of the retina of newborn rats
Authors: Borges Helena L. , Linden Rafael 
Published: April 2021

ABSTRACT
Gamma radiation induces apoptosis in the proliferative zone (neuroblastic layer) of the developing rat retina. We asked whether sensitivity to apoptosis might be related to distinct phases of the cell cycle. Explants of newborn rat retina or newborn pups were gamma-irradiated and apoptosis was detected by chromatin condensation, DNA fragmentation in situ and DNA electrophoresis. After 6 hours, early appearing apoptotic bodies were located mainly towards the outer tier of the neuroblastic layer. In contrast, after 24 hours, late-appearing apoptotic cells were located towards the inner margin of the neuroblastic layer, a region associated with the S phase of the cell cycle. Labeling of a cohort of cells with the nucleotide analog bromo-deoxyuridine (BrdU) at the time of irradiation, showed that these cells die in the late wave of apoptosis. BrdU given 3 hours before fixation labeled a large number of late apoptotic cells, but no early apoptotic cells. After labeling of all cycling cells with BrdU, 40% of the early apoptotic profiles were unlabeled, and thus post-mitotic. The same schedules of cell death were identified after gamma irradiation in vivo. The results show that irradiation leads to two waves of apoptosis in distinct cell populations. An early wave comprises both post-mitotic cells and proliferating cells out of the S phase. The late wave comprises cells in S phase, which pass through this phase again to die. The antioxidant pyrrolidinedithiocarbamate prevented the early but not the late wave of apoptosis following irradiation, and blocked lipid peroxidation at 6 hours after the insult, suggesting that the two waves of apoptosis are indeed mediated by distinct mechanisms.
Removal of calcium ions triggers a novel type of intercadherin interaction
Authors: Troyanovsky Regina B. , Klingelhöfer Jörg , Troyanovsky Sergey 
Published: April 2021

ABSTRACT
Depletion of Ca2+ ions from epithelial cell cultures has been shown to result in the rapid destruction of intercellular junctions. To understand the mechanism of this effect we have examined how removal of calcium ions from the culture medium of A-431 epithelial cells affects complexes incorporating the cell-cell adhesive receptors, E-cadherin, desmoglein or desmocollin. Sedimentation and biochemical analysis demonstrated that calcium removal triggers a rapid formation of a novel type of complex formed via direct lateral E-cadherin-desmoglein, E-cadherin-desmocollin and desmoglein-desmocollin dimerization of the extracellular cadherin regions. Replacement of Trp156 and Val157 of E-cadherin, that has been shown to abolish lateral and adhesive E-cadherin homodimerization in standard cultures, did not influence the formation of these calcium-sensitive complexes. Furthermore, experiments with this mutant revealed that EGTA induced lateral Trp156/Val157-independent homodimerization of E-cadherin. Deletion mutagenesis of E-cadherin showed that these complexes are mediated by at least two extracellular cadherin domains, EC3 and EC4. Notably, protein kinase inhibitor H-7 which confers EGTA-independence of the adhesive E-cadherin complexes does not block this association. We propose that this novel type of intercadherin interaction is involved in the assembly of adherens junctions and their disassembly in low-calcium medium.
5-Methylcytosine distribution and genome organization in Triticale before and after treatment with 5-azacytidine
Authors: Castilho A. , Neves N. , Rufini-Castiglione M. , Viegas W. , Heslop-Harrison J. S. 
Published: April 2021

ABSTRACT
Triticale (2n=6x=42) is a hybrid plant including rye (R) and wheat (A and B) genomes. Using genomic in situ hybridization with rye DNA as a probe, we found the chromosomes of the R genome were not intermixed with the wheat chromosomes in 85% of nuclei. After treatment of seedlings with low doses of the drug 5-azacytidine (5-AC), leading to hypomethylation of the DNA, the chromosomes became intermixed in 60% of nuclei; the next generation showed intermediate organization. These results correlate with previous data showing that expression of R-genome rRNA genes, normally suppressed, is activated by 5-AC treatment and remains partially activated in the next generation. The distribution of 5-methylcytosine (5-mC) was studied using an antibody to 5-mC. Methylation was detected along the lengths of all chromosomes; there were some chromosome regions with enhanced and reduced methylation, but these were not located at consistent positions, nor were there differences between R and wheat genome chromosomes. After 5-AC treatment, lower levels of methylation were detected. After 5-AC treatment, in situ hybridization with rye genomic DNA sometimes showed micronuclei of rye origin and multiple translocations between wheat and rye chromosomes. Genomic DNA was analysed using methylation-sensitive restriction enzymes and, as probes, two rDNA sequences, two tandemly organised DNA sequences from rye (pSc200 and pSc250), and copia and the gypsy group retrotransposon fragments from rye and wheat. DNA extracted immediately after 5-AC treatment was cut more by methylation-sensitive restriction enzymes than DNA from untreated seedlings. Each probe gave a characteristic restriction fragment pattern, but rye- and wheat-origin probes behaved similarly, indicating that hypomethylation was induced in both genomes. In DNA samples from leaves taken 13-41 days after treatment, RFLP (Restriction Fragment Length Polymorphism) patterns were indistinguishable from controls and 5-AC treatments with all probes. Surprising differences in hybridization patterns were seen between DNA from root tips and leaves with the copia-fragment probes.
Functional antagonists of sonic hedgehog reveal the importance of the N terminus for activity
Authors: Williams Kevin P. , Rayhorn Paul , Chi-Rosso Gloria , Garber Ellen A. , Strauch Kathryn L. , Horan Gerald S. B. , Reilly Jennifer O. , Baker Darren P. , Taylor Fred R. , Koteliansky Victor , Pepinsky R. Blake 
Published: April 2021

ABSTRACT
During development, sonic hedgehog functions as a morphogen in both a short-range contact-dependent and in a long-range diffusable mode. Here, we show using a panel of sonic hedgehog variants that regions near the N terminus of the protein play a critical role in modulating these functions. In the hedgehog responsive cell line C3H10T1/2, we discovered that not only were some N-terminally truncated variants inactive at eliciting a hedgehog-dependent response, but they competed with the wild-type protein for function and therefore served as functional antagonists. These variants were indistinguishable from wild-type sonic hedgehog in their ability to bind the receptor patched-1, but failed to induce the hedgehog-responsive markers, Gli-1 and Ptc-1, and failed to promote hedgehog-dependent differentiation of the cell line. They also failed to support the adhesion of C3H10T1/2 cells to hedgehog-coated plates under conditions where wild-type sonic hedgehog supported binding. Structure-activity data indicated that the N-terminal cysteine plays a key regulatory role in modulating hedgehog activity. The ability to dissect patched-1 binding from signaling events in C3H10T1/2 cells suggests the presence of unidentified factors that contribute to hedgehog responses.
Mutations affecting transmembrane segment interactions impair adhesiveness of E-cadherin
Authors: Huber Otmar , Kemler Rolf , Langosch Dieter 
Published: April 2021

ABSTRACT
Lateral clustering of E-cadherin molecules is required for the adhesive properties of this cell-cell adhesion molecule. Both the extracellular domain and the cytoplasmic region of E-cadherin were previously reported to contribute to lateral clustering, but little is known about a role of the transmembrane domain in this respect. Following our previous findings indicating self-assembly of artificial transmembrane segments based on leucine residues, we asked whether the leucine-rich transmembrane segment of E-cadherin participates in lateral clustering. Here, we demonstrate that its transmembrane domain self-assembles as analyzed using the ToxR reporter system. Certain point mutations within the transmembrane domain markedly reduced self-assembly. To study whether the same point mutations also affect E-cadherin-mediated adhesion in vivo, wild-type and mutant E-cadherin cDNAs were transfected into Ltk− cells. Indeed, cell aggregation assays revealed significantly reduced adhesiveness when mutations had been introduced which disrupted transmembrane segment interaction. In control experiments, cell-surface expression, interaction with catenins and the cytoskeleton as well as trypsin-resistance of the protein were unaffected. These data suggest that interactions between the transmembrane segments are important for the lateral association of E-cadherin molecules required for cell-cell adhesion.
P2X4 purinoceptors mediate an ATP-activated, non-selective cation current in rabbit osteoclasts
Authors: Naemsch Lin N. , Weidema A. Frederik , Sims Stephen M. , Underhill T. Michael , Dixon S. Jeffrey 
Published: April 2021

ABSTRACT
Extracellular nucleotides act as signaling molecules in numerous tissues. In bone, nucleotides stimulate osteoclast formation and activity; however, the receptors and signaling mechanisms underlying these effects have yet to be identified. To identify specific P2X purinoceptor subtypes in osteoclasts, degenerate oligonucleotide primers were used to PCR-amplify DNA fragments from a rabbit osteoclast cDNA library. A 372-base-pair fragment was obtained that encoded an amino acid sequence with 88% identity to the rat P2X4 purinoceptor. The presence of P2X4 mRNA in purified osteoclasts was confirmed by reverse transcription-PCR. Endogenous purinoceptors were functionally characterized in isolated rabbit osteoclasts by patch-clamp recording in whole-cell configuration. At negative membrane potentials, application of ATP or ADP rapidly activated an inward current followed by an outward current. In contrast, UTP or ADPβS elicited only an outward current, due to activation of a Ca2+-dependent K+ conductance. The initial inward current was non-selective for cations and inactivated during agonist application. Furthermore, the inward current was insensitive to suramin and Cibacron blue, and was potentiated by Zn2+. These characteristics are consistent with properties of P2X4 purinoceptors. Activation of P2X4 purinoceptors leads to cation influx and depolarization. Nucleotides, released at sites of trauma or inflammation, may act through these receptors on osteoclasts to stimulate bone resorption.
Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat
Authors: Shugrue Christine A. , Kolen E. Robert , Peters Holme , Czernik Andy , Kaiser Chris , Matovcik Lisa , Hubbard Ann L. , Gorelick Fred 
Published: April 2021

ABSTRACT
The regulation of intracellular vesicular trafficking is mediated by specific families of proteins that are involved in vesicular budding, translocation, and fusion with target membranes. We purified a vesicle-associated protein from hepatic microsomes using sequential column chromatography and partially sequenced it. Oliogonucleotides based on these sequences were used to clone the protein from a rat liver cDNA library. The clone encoded a novel protein with a predicted mass of 137 kDa (p137). The protein had an N terminus WD repeat motif with significant homology to Sec31p, a member of the yeast COPII coat that complexes with Sec13p. We found that p137 interacted with mammalian Sec13p using several approaches: co-elution through sequential column chromatography, co-immunoprecipitation from intact cells, and yeast two-hybrid analysis. Morphologically, the p137 protein was localized to small punctate structures in the cytoplasm of multiple cultured cell lines. When Sec13p was transfected into these cells, it demonstrated considerable overlap with p137. This overlap was maintained through several pharmacological manipulations. The p137 compartment also demonstrated partial overlap with ts045-VSVG protein when infected cells were incubated at 15°C. These findings suggest that p137 is the mammalian orthologue of Sec31p.