bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of an Spc110p-related protein in vertebrates
Authors: Tassin A. M. , Celati C. , Paintrand M. , Bornens M. 
Published: April 2021

ABSTRACT
Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components. In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly. Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner. Western blot experiments identified a 100 kDa protein specifically enriched in cen-trosome preparations from human cells. This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it. Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulosetubulin or Xenopus egg extract tubulin. This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex. To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract. We performed a partial purification of the γ-tubulin-ring complex from egg extract. Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and γ-tubulin were found in the same complex. Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation.
Scaffold attachments within the human genome
Authors: Craig Jeff M. , Boyle Shelagh , Perry Paul , Bickmore Wendy A. 
Published: April 2021

ABSTRACT
It is generally agreed that, above the level of the 30 nm fibre, eukaryotic chromatin is constrained into loops, but there is disagreement about the nature of the substructure that serves to anchor loops and the DNA sequences that act as the attachment sites. This problem may stem from the very different methods that all purport to separate loop and attached DNAs. We have tested ideas about how the genome is arranged into loops by analysing the average loop size over different cytologically resolvable regions of human chromosomes using fluorescence in situ hybridisation with loop and attached DNA fractions. Variations in average loop size, along and between chromosomes, measurable at this level of resolution were small but sig-nificant and were dependent on the extraction method. This emphasises the fundamental differences between the nuclear substructure probed by different protocols. DNA attached to the nuclear ‘scaffold’ or ‘matrix’ hybridises preferentially to gene-poor regions of the genome (G-bands). Conversely, fractions attached to the nuclear ‘skeleton’ hybridise preferentially to gene-rich R-bands and sites of high levels of transcription. The inactive X chromosome has a deficit of associations with the nuclear skeleton but not with the matrix or scaffold. A large excess of attached sequences is found at some sites of constitutive heterochromatin, but not at centromeres.
A novel protein cross-reacting with antibodies against spectrin is localised in the nucleoli of amphibian oocytes
Authors: Carotenuto Rosa , Maturi Giovanna , Infante Vincenzo , Capriglione Teresa , Petrucci Tamara C. , Campanella Chiara 
Published: April 2021

ABSTRACT
Cytoskeletal proteins such as actin and myosin are important constituents of the nucleoplasm. Spectrin is an actin binding protein typically related to plasma membrane; recently, it has been found that it is widespread and forms distinct membrane protein domains in such organelles as the Golgi. In this paper, the large germinal vesicle of amphibian oocytes was chosen as a particularly suitable system to investigate the presence and location of spectrin in the nucleus. We manually isolated the germinal vesicles of both Discoglossus pictus and Xenopus laevis oocytes, and processed them for SDS-PAGE, immunoblotting and immunoprecipitation. By the use of an antibody against the general form of brain β spectrin (βIIΣ1) and of an anti-α brain spectrin (αIIΣ*), a band of 230 kDa was identified as a nuclear spectrin-like molecule. Moreover the 230 kDa protein was extracted from the nuclei by 1 M KCl, similarly to spectrin in other systems. In oocyte sections and nuclear spreads incubated with anti-αIIΣ* and/or anti-βIIΣ1 antibodies, the immunostain was localised in the nucleoplasm and in the outer shell of the round bodies abundantly present in the germinal vesicle. Sections of the same oocytes, stained with a monoclonal antibody against nucleolar fibrillarin and anti-αIIΣ*, showed colocalisation of the two antibodies. It was concluded that, in the germinal vesicle of amphibian oocytes, a spectrin-like molecule is a part of the outer shell of nucleoli. It is hypothesised that spectrin, together with actin, might be instrumental in keeping nucleoli attached to the inner nuclear membrane, as nucleoli migrate during oogenesis to the inner aspect of the nuclear envelope, where they are stably kept until the end of their growth. Furthermore, these results strongly suggest that the 230 kDa band might comprise both an α and a β chain of the same apparent molecular mass, thus constituting a novel form of a spectrin-like molecule.
Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery
Authors: Broughton Jon , Swennen Dominique , Wilkinson Barrie M. , Joyet Philippe , Gaillardin Claude , Stirling Colin J. 
Published: April 2021

ABSTRACT
The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit. In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved. In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum. Sequence comparisons reveal that S. pombe Sec61p is 58.6% identical to that of S. cerevisiae. The deduced amino acid sequence of the Y. lipolytica protein shares 68.8% sequence identity with S. cerevisiae Sec61p.
Gene disruption studies have shown that the SEC61 is required for viability in both S. pombe and Y. lipolytica demonstrating that the essential nature of this protein is not unique to S. cerevisiae. Moreover, heterologous complementation studies indicate that the Y. lipolytica SEC61 gene can complement a null mutation in S. cerevisiae. Sequence comparisons between the various eukaryotic Sec61p homologues reveal a number of highly conserved domains, including several transmembrane sequences and the majority of cytosolic loops. These comparisons will provide an important framework for the detailed analysis of interactions between Sec61p and other components of the translocation machinery and between Sec61p and translocating polypeptide chains.
The glucocorticoid receptor and AP-1 are involved in a positive regulation of the muscle regulatory gene myf5 in cultured myoblasts
Authors: Auradé Frédéric , Pfarr Curt M. , Lindon Catherine , Garcia Alphonse , Primig Michael , Montarras Didier , Pinset Christian 
Published: April 2021

ABSTRACT
The muscle regulatory factor, myf5, is involved in the establishment of skeletal muscle precursor cells. Little is known, however, about the control of the expression of the gene encoding this basic helix-loop-helix (bHLH) factor. We have addressed this question in the mouse myogenic cell line, C2, and in a derivative of this cell line where the myf5 gene is the only muscle-specific bHLH factor to be expressed at the myoblast stage. We present evidence that the synthetic glucocorticoid dexamethasone, and the pharmacological agent anisomycin, act synergistically to rapidly upregulate the levels of myf5 transcript and protein. The glucocorticoid antagonist RU 486 abolishes this synergy, demonstrating the involvement of the glucocorticoid receptor. The expression of a dominant negative mutant of c-jun which interferes with the transactivating properties of all AP-1 family members also blocks the induction of myf5 by anisomycin and dexamethasone. An activator of protein kinase C (PKCs), 12-O-tetradecanoyl phorbol 13-acetate (TPA), abolishes the up-regulation of myf5 gene expression by dexamethasone and anisomycin, and its effect is counteracted by an inhibitor of PKCs, GF 109203X. These results point to the possible involvement of PKCs in the negative control of myf5. Evidence that both positive and negative regulation of myf5 transcripts, described here, does not require the fresh synthesis of transcription factors suggests that myf5 may behave like an immediate early gene.
Morphological effects of wortmannin on the endosomal system and GLUT4-containing compartments in rat adipose cells
Authors: Malide Daniela , Cushman Samuel W. 
Published: April 2021

ABSTRACT
Studies using functional and pharmacological approaches have implicated PI 3-kinase as a key intermediate in the glucose transport and GLUT4 translocation responses to insulin. Confocal microscopy was used to investigate the effects of the PI 3-kinase inhibitor wortmannin in isolated rat adipose cells. Independent of insulin, wortmannin induces the appearance of phase-lucent vacuoles containing the endosomal markers TfR, Rab4, M6PR, and cellubrevin. When added before or with insulin, wortmannin blocks insulin-stimulated GLUT4 translocation, but does not influence the basal VAMP2-containing GLUT4 compartment. These results substantiate the concept of a specialized basal GLUT4 compartment mostly distinct from that of the recycling receptors. However, when added after insulin, wortmannin induces a rapid redistribution of GLUT4 from the cell surface into those endosomal-derived vacuoles where the GLUT4 co-localize with TfR, Rab4, cellubrevin, and VAMP2, but not with clathrin, M6PR, Golgi complex markers TGN38-mannosidase II and γ-adaptin, and lysosomal marker lgp-120. Therefore, wortmannin also disrupts insulin-stimulated GLUT4 traffic in the recycling endosomal pathway, at a step distal to the sorting of recycling proteins from late endosomal and TGN markers; wortmannin does not appear to affect internalization from the plasma membrane, and delivery from early to late endosomes or from late endosomes to the TGN. In combination with previous kinetic biochemical studies, these results suggest that: (i) insulin stimulates the exocytosis of GLUT4 through a direct pathway from a specialized basal compartment to the plasma membrane, (ii) during endocytosis in the presence of insulin, GLUT4 is sorted out of the TfR compartment into a separate recycling pathway back to the plasma membrane, and (iii) both of these pathways involve wortmannin sensitive enzymes.
Intracellular Ca2+ signals in Dictyostelium chemotaxis are mediated exclusively by Ca2+ influx
Authors: Nebl T. , Fisher P. R. 
Published: April 2021

ABSTRACT
We measured folate- and cAMP-induced changes in cytoplasmic free calcium concentration ([Ca2+]i) using recombinant aequorin reconstituted in living Dictyostelium cells with coelenterazine-h. The resulting semi-synthetic protein displayed increased sensitivity to Ca2+ allowing accurate measurement of chemoattractant-induced transients at low resting levels. Both folate- and cAMP-induced Ca2+ responses were developmentally regulated, exhibited remarkably similar kinetics and were dependent on the relative rather than the absolute magnitude of increases in attractant concentration. They began after a short delay of 5-10 seconds, leading to a maximum increase in cytosolic calcium concentration after ∼25 seconds and a return to basal level within ∼60 seconds after stimulation. Responses elicited by the two chemoattractants were dose-dependent and saturated between 4 and 20 μM. They depended on the presence of free extracellular calcium ions and were inhibited in a concentration-dependent manner between 10−4 and 10−5 M. In accordance with 45Ca2+-uptake measurements by Milne and Coukell (J. Cell Biol. (1991) 112, 103-110), both responses were also completely inhibited by 15 μM Ruthenium Red, 15 μM carbonylcyanide m-chlorophenylhydrazone (CCCP) and 500 μM gadolinium ions. Under conditions that prohibited influx of Ca2+ from the extracellular medium there were no detectable changes in [Ca2+]i that could be related to a separate release of the ion from intracellular stores. Together, these results show that the Ca2+ signals involved in chemotaxis correlate temporally with actin depolymerization (not polymerization) and are mediated by Ca2+ influx, not IP3-mediated intracellular release.
Altered expression of the α7β1 integrin in human and murine muscular dystrophies
Authors: Hodges B. L. , Hayashi Y. K. , Nonaka I. , Wang W. , Arahata K. , Kaufman S. J. 
Published: April 2021

ABSTRACT
The α7β1 integrin is the primary laminin receptor on skeletal myoblasts and adult myofibers. It has distinct functions during muscle development and contributes to muscle structural integrity. We have studied this integrin in cases where expression of dystrophin or laminin are compromised. Immunofluorescence demonstrates an increase in α7β1 in patients with Duchenne muscular dystrophy and in mdx mice that lack dystrophin. Analysis of RNA from mdx mice and from patients with Duchenne and Becker muscular dystrophies indicates that the increase in the α7β1 integrin is regulated at the level of α7 gene transcription. In contrast, the levels of α7β1 integrin are severely diminished in patients with laminin α2 chain congenital dystrophy muscular dystrophy and in dy/dy mice that also do not make the α2 laminin chain. Analysis of RNA from the hindlimbs of dy/dy mice demonstrated that in the absence of laminin α7 gene transcription is inhibited and limited to specific alternatively spliced isoforms. We suggest that the increased expression of α7β1 integrin in the absence of dystrophin compensates for the reduced dystrophin-mediated linkage of fibers with the basal lamina and modulates the development of pathology associated with these diseases. The decrease in α7β1 integrin and its transcripts in the absence of laminin likely contributes to the severe myopathy that results from laminin α2 chain deficiency and suggests that laminin-2 regulates expression of the α7 integrin gene. The role of the α7β1 integrin in muscle integrity also suggests that compromised expression of this receptor may underlie as yet undefined myopathies.
HSF1 transcription factor concentrates in nuclear foci during heat shock: relationship with transcription sites
Authors: Jolly Caroline , Morimoto Richard I. , Robert-Nicoud Michel , Vourc’h Claire 
Published: April 2021

ABSTRACT
In this paper, we show that upon heat shock, HSF1 concentrates in the nucleus of diploid human fibroblasts in two large foci. The relative distribution of HSF1 nuclear foci and active heat shock protein (hsp) genes was investigated by combining fluorescence in situ hybridization (FISH) for the detection of hsp nuclear transcripts and immunofluorescence for the detection of HSF1. We show that the HSF1 foci are distinct from the sites of hsp70 and hsp90 genes transcription. This is the second report of ploidy-dependent foci of transcription factors that are independent of their specific transcription sites. However, the correlation between the number of HSF1 foci and the ploidy of the cells strongly supports the existence of a specific chromosomal target for HSF1 foci.
A structure/function analysis of Rat7p/Nup159p, an essential nucleoporin of Saccharomyces cerevisiae
Authors: Priore Veronica Del , Heath Catherine V. , Snay Christine A. , MacMillan Amanda , Gorsch Lisa C. , Dagher Suzanne , Cole Charles N. 
Published: April 2021

ABSTRACT
Rat7p/Nup159p is an essential nucleoporin of Saccharomyces cerevisiae originally isolated in a genetic screen designed to identify yeast temperature-sensitive mutants defective in mRNA export. Here we describe a detailed structural-functional analysis of Rat7p/Nup159p. The mutation in the rat7-1 ts allele, isolated in the original genetic screen, was found to be a single base pair change that created a stop codon approximately 100 amino acids upstream of the actual stop codon of this 1,460 amino acid polypeptide, thus eliminating one of the two predicted coiled-coil regions located near the carboxyl terminus of the protein. These coiled-coil regions are essential since an allele lacking both coiled-coil regions was unable to support growth under any conditions. In contrast, no other region of the protein was absolutely required. The SAFG/PSFG repeat region in the central third of the protein was completely dispensable for growth at temperatures between 16°C and 37°C and cells expressing this mutant allele were indistinguishable from wild type. Deletion of the aminoterminal third of the protein, upstream from the repeat region, or the portion between the repeat region and the coiled-coils resulted in temperature-sensitivity, but the two alleles showed distinct phenotypes with respect to the behavior of nuclear pore complexes (NPCs). Taken together, our data suggest that Rat7p/Nup159p is anchored within the NPC through its coiled-coil region and adjacent sequences. In addition, we postulate that the N-terminal third of Rat7p/Nup159p plays an important role in mRNA export.
Post-Golgi biosynthetic trafficking
Authors: Keller Patrick , Simons Kai 
Published: April 2021

ABSTRACT
Eukaryotic cells have developed complex machineries to distribute proteins and lipids from the Golgi complex. Contrary to what has originally been postulated, delivery of proteins to the cell surface is not a simple bulk flow process but involves sorting into distinct pathways from the trans-Golgi network. Here we describe the various routes emerging from the trans-Golgi network in different cell types, and we discuss the mechanisms that mediate sorting into these pathways. While much remains to be learned about these sorting mechanisms, it is apparent that a number of pathways previously believed to be restricted to certain cell types might be used more commonly.
A nuclear matrix-associated high molecular mass nuclear antigen, HMNA, of chicken and marked decrease of its immunoreactivity during the progression of S phase
Authors: Shimada Kenji , Harata Masahiko , Mizuno Shigeki 
Published: April 2021

ABSTRACT
A hnRNP-free nuclear matrix prepared from chicken MSB-1 cells was used to raise monoclonal antibodies. The monoclonal antibodies 2H3 and 3B7 showed identical non-homogeneous immunofluorescence staining patterns of nuclei in MSB-1 cells and chicken embryonic fibroblasts. In a synchronized culture of MSB-1 cells, the immunore-activity of nuclei with 2H3, but not with 3B7, antibody decreased markedly during the progression of S phase, but returned to the normal level at the next G1 phase. When cells were treated with Triton X-100 prior to fixation with paraformaldehyde or cells were fixed in methanol, nuclei were reactive with 2H3 antibody throughout the S phase. Both 2H3 and 3B7 antibodies recognized a high molecular mass nuclear antigen (HMNA) of approximately 550 kDa, which was associated with the nuclear matrix. HMNA was resistant to extraction with 0.5 M NaCl from the nuclei at the G1/S boundary but became extractable by the end of S phase. A cDNA clone, pBHB36, containing a partial sequence for HMNA was isolated by immunoscreening as a double positive clone with 2H3 and 3B7 antibodies. The deduced 1,150 residue-long sequence of pBHB36 shows no homology with any molecules in the nucleotide and protein sequence databases, and contains different epitope regions for 2H3 and 3B7 antibodies. A possibility of hydrophobic association of HMNA with nuclear protein(s) during the progression of S phase is discussed.
Overexpression of MAP4 inhibits organelle motility and trafficking in vivo
Authors: Chloë Bulinski J. , McGraw Timothy E. , Gruber Dorota , Nguyen H. Lan , Sheetz Michael P. 
Published: April 2021

ABSTRACT
We previously prepared cell lines that inducibly overexpress MAP4, a microtubule (MT)-associated protein widely expressed in non-neuronal cells. Overexpression of either the full-length MAP4 molecule or its MT-binding domain, MTB, stabilized MTs and retarded cell growth, suggesting that overexpressed MAP4 impacts on MT-dependent functions in vivo. To test this hypothesis, we examined MT-based vesicle movements in living cells, using high resolution DIC microscopy. Overexpression of either MAP4 or MTB yielded a dose-dependent reduction in the frequency of MT-dependent organelle movements, relative to control cells. At steady state, both MAP4- and MTB-overexpressing cells showed unusual distributions of transferrin, LDL, dextran, and Golgi elements, as compared to control cells. MAP4 preferentially inhibited receptor-dependent uptake and degradation of LDL, and repositioning of Golgi elements after disruption by the drug, brefeldin A. L-MOCK cells treated with Taxol to stabilize the MTs to an extent equiva-lent to MAP4 overexpression did not show similar inhibition of vesicle motility or organellar trafficking, suggesting that deficits in organelle movements in vivo represent a direct effect of the presence of MAP4 or MTB, rather than an indirect effect of the stabilization of MTs by overexpressed MAP constructs. Our results show that MAP4 has the capacity to affect transport along MTs in vivo; these findings suggest a potential mechanism by which MAP4 could con-tribute to polarization or morphogenesis of cells.