bluuebunny/crossref_metadata_2025_split huggingface dataset:
The transition of cells of the fission yeast β-tubulin mutant nda3-311 as seen by freeze-substitution electron microscopy Requirement of functional tubulin for spindle pole body duplication
Authors: Kanbe Tosfflo , Hiraoka Yasushi , Tanaka Kenji , Yanagida Mitsuhiro 
Published: April 2021

ABSTRACT
A previous fluorescence light-microscopic study showed that the fission yeast cold-sensitive β-tubulin mutant nda3-311 was arrested with rod-like condensed chromosomes in a mitotic state at the restrictive temperature. Upon transfer to the permissive temperature, a spindle was formed and the nucleus was divided. In the present study, we employed freeze-substitution electron microscopy to examine the ultrastructure of arrested and released nda3-311 cells. In arrested cells, a single, displaced nucleus was seen with a single spindle pole body. Therefore, spindle pole body duplication seemed to require functional β-tubulin. The nuclear membrane was highly deformed with a leaf-like profile in cross-section, possibly due to an interaction with the rod-like, condensed chromosomes. Upon transfer to the permissive temperature, the spindle pole duplicated and the daughter spindle pole bodies rapidly migrated to the opposite ends of the nucleus, accompanied by the formation of the mitotic spindle. Elongation of the nuclear envelope occurred with concomitant spindle extension, as in a wild-type mitosis. The deformed nuclear membrane became smooth and described a convex curve. The numerous vacuoles that are seen in the arrested cells decreased in number and increased in size. Septation was completed, leaving the two divided nuclei in one half of the cell. Hexagonally arranged microtubules, apparently forming the mitotic spindle, were observed in a cross-section of a cell after return to the permissive conditions.
Variable pathways for developmental changes in composition and organization of microtubules in Physarum polycephalum
Authors: Solnica-Krezel Lilianna , Diggins-Gilicinski Maureen , Burland Timothy G. , Dove William F. 
Published: April 2021

ABSTRACT
The development of uninucleate amoebae into multinucleate plasmodia in myxomycètes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cyto-skeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodiumspecific ββ2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit ββ2-tubulin at earlier or only at later stages of development. Amoeba-specific acetylated α3-tubulin disappears gradually during development. Individual cells differ in the timing of loss of this isotype: α3-tubulin is present in themajority of uninucleate cells, in a fraction of binucleate and quadrinucleate cells, and is absent from larger multinucleate cells. Cytoplasmic microtubules in uninucleate cells are organized by a single microtubule-organizing center (MTOC) juxtaposed to the nucleus. Binucleate cells and quadrinucleate cells exhibit variable numbers of MTOCs. Cytoplasmic microtubules persist during the APT until the stage of plasmodia containing at least 100 nuclei. The lack of a strict correlation between the changes in tubulin composition and changes in organization of microtubular structures indicates that accumulation of β2- tubulin and disappearance of α3tubulin isotypes are not sufficient to bring about reorganization of microtubules during development. Individual cells in a developing population differ not only in the succession of accumulation and loss of developmentally regulated tubulins, but also in the sequences of other cellular changes occurring during the APT.
Alteration in expression of smooth muscle α-Actin associated with transformation of rat 3Y1 cells
Authors: Okamoto-Inoue Mitsuse , Taniguchi Shuntchiro , Ano Hiroyuki Sad , Kawano Toyokazu , Kimura Genki , Gabbiani Giulio , Baba Tsuneo 
Published: April 2021

ABSTRACT
Expression of actin was examined in a cultured rat embryonic cell line 3Y1 and transformed cell lines that originated from 3Y1. An α-actin in addition to cytoplasmic α- and γ-actins was detected in 3Y1 by two-dimensional gel electrophoresis. This α-Actin was hardly detected at all in the transformants induced by Rous sarcoma virus, v-H-ras oncogene or adenovirus type 12, while the α-Actin was retained in the transformed cell lines induced by N-methyl-N′-nitro-N-nitrosoguanidine or in SV40, which are cell lines of relatively low malignancy. Western and Northern blot analyses established that this α-actin was a smooth muscle a-isoform. An immunofluorescence study revealed that smooth muscle α-Actin in 3Y1 cells is present in stress fibers. Thus, smooth muscle α-Actin is also a component of actin stress fibers, as α- and γ-actins are in 3Y1 cells. An alteration in the expression of this actin isoform may be related to phenotypical changes accompanying transformation.
The F1 ATP synthetase β-subunit: A major yeast novobiocin binding protein
Authors: Jenkins John R. , Pocklington Michael J. , Orr Elisha 
Published: April 2021

ABSTRACT
Novobiocin affects DNA metabolism in both prokaryotes and eukaryotes, resulting in cell death. Ln prokaryotes, the drug is a specific inhibitor of DNA gyrase, a type II topoisomerase that can be purified on a novobiocin-Sepharose column. The yeast type II topoisomerase is neither the biochemical, nor the genetic target of the antibiotic. We have purified the major yeast novobiocin binding proteins and identified one of them as the β-subunit of the yeast mitochondrial Fi ATP synthetase, a protein highly conserved throughout evolution. The inactivation of this protein might explain the toxic effects of novobiocin on higher eukaryotic cells.
Differential distribution of xyloglucan glycosyl transferases in pea Golgi dictyosomes and secretory vesicles
Authors: Brummell David A. , Camirand Anne , Maclachlan Gordon 
Published: April 2021

ABSTRACT
Rate-zonal centrifugation of pea (Pisum sativum var. Alaska) stem microsomal membranes on a linear Renografin gradient separated Golgi secretory vesicles from dictyosomes. Secretory vesicles possessed high levels of xyloglucan fucosyl transferase activity, which effects the final decoration of stem xyloglucan side-chains, but lacked substantial xyloglucan xylo-syl transferase activity, which is required for the synthesis of the xyloglucan backbone. In contrast, total dictyosomal membranes possessed both fucosyl and xylosyl transferase activities. Isopycnic centrifugation of a homogenized dictyosome-enriched membrane preparation on a shallower Renografin gradient indicated that lighter dictyosomal membranes possessed xylosyl transferase but relatively little fucosyl transferase activity. The bulk of the dictyosomal membranes formed a denser peak in which xylosyl and fucosyl transferase activities codistributed. Thus a differential localization of function in the Golgi apparatus during biosynthesis of xyloglucan is indicated. A tentative mechanism is suggested in which the elaboration of the glucose-xylose backbone is initiated in lighter dictyosomal membranes, backbone synthesis is concluded and fucosylation begun in denser dictyosomal membranes, and fucosylation completed in Golgi secretory vesicles during transport of xyloglucan to the cell wall.
Measurement of intracellular pH in fungal hyphae using BCECF and digital imaging microscopy: Evidence for a primary proton pump in the plasmalemma of a marine fungus
Authors: Davies Julia M. , Brownlee C. , Jennings D. H. 
Published: April 2021

ABSTRACT
The facultative marine fungus, Dendryphiella salina, has the most negative membrane potential yet recorded for a marine organism. The ionic basis for this is thought to be through the action of a primary proton pump, though there exists the possibility of electrogenic pumping of Na+ or Cl−, given the high ambient concentration of these ions. Fluorescence ratio imaging microscopy with the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein (BCECF) has been used to estimate intracellular pH. Hyphae loaded readily with BCECF after incubation with the acetoxymethyl ester (BCECF/AM). Mean resting intracellular pH (pHi) was 7.3, calculated by comparing 490/450 nm fluorescence ratios with in vivo calibration curvesobtained by pH equilibration using nigericin. Distinct pH compartments could be observed, corresponding to cytoplasmic and smaller vacuolar compartments. Sodium azide reversibly reduced pHi by an average of 0.51 of a pH unit, though the response varied between individual hyphae. Inhibiting the plasmalemma ATPase with orthovanadate also reversibly decreased pHi. The results support the presence of a proton pump in the plasmamembrane. The energetic and evolutionary implications are discussed.
Temporal expression, polar distribution and transition of an epitope domain in the perinuclear theca during mouse spermatogenesis
Authors: Bellvé Anthony R. , Chandrika R. , Barth Alinda 
Published: April 2021

ABSTRACT
A novel domain of epitopes is expressed by a family of high-Mr proteins at the anterior pole of the germ cell nucleus during spermiogenesis, and later by two low-Mr proteins at the anterior and posterior poles of the nucleus during sperm maturation in the epididymis. Initially, monoclonal antibodies (mAbs) PNT-1 (IgG2b) and PNT-2 (IgG2a) bound to antigens present in a cap-like configuration at the apical pole of the spermatid nucleus at step 5 of spermiogenesis. The distribution of epitopes on the nucleus expanded posteriorly until, in testicular sperm they covered the anterior pole down to the distal limits of the subacrosomal perforatorium. By contrast, sperm from the epididymis and vas deferens bound both mAbs in two distinct regions on the nucleus, one on the dorsal margin of the anterior pole, and the other in a ventral zone at the posterior pole. On SDS-PAGE and isoelectric focusing (IEF) immunoblots, both mAbs bound three major proteins with Mr of ∽80 000,77 000 and 75 000 from spermatids and testicular sperm, and proteins of Mr 50 000 and 48 000 in epididymal and vas deferens sperm. Both the high- and low-Mr protein families were recovered in germ cell nuclear/perinuclear matrices. Their mobilities on SDS-PAGE were not altered by exo- or endoglycosidases or by aminoethylation in denaturing conditions. mAb PNT-1 bound to the sperm proteins with a Ka of 3.53 ×1012 M-1 and mAb PNT-2 with a KB of 2.08 ×1012 M-1. From competition binding data, mAbs PNT-1 to -10 appeared to recognize six adjacent or overlapping epitopes on the same proteins. These data suggest the high-Mr proteins, the thecins, present at the anterior pole of haploid germ cells are processed at the onset of sperm maturation to yield two low-Mr proteins that then occupy two distinct domains at the anterior and posterior poles of the nucleus.
Spatial organization of repetitive dna sequences in the bovine sperm nucleus
Authors: Powell Don , Cran David G. , Jennings Carol , Jones Roy 
Published: April 2021

ABSTRACT
During spermatogenesis, DNA in the sperm head becomes more tightly condensed as histones are replaced by protamine-like molecules. In this article, the question is asked whether, during the production of this highly differentiated cell, controls are imposed on the spatial organization of DNA within the nucleus. Heads from bull spermatozoa were isolated by a technique that removed the plasma membrane and acrosomal contents, and the DNA was induced to decondense by addition of 2-mercapto-ethanol and trypsin. Under these conditions, decondensation was induced in all regions of the head. To determine whether there was any spatial restraint on packaging of the genome, three DNA probes were used (pl.709—512, containing an interspersed repetitive sequence; pCSIH, containing a copy of the major bovine centromeric satellite sequence; pl8s and p28s, containing the 18 S and 28 S ribosomal genes) that might be expected to hybridize to different regions. Results showed that the interspersed repetitive probe hybridized to all regions of the head, whereas the ribosomal and centromeric probes hybridized to sequences that were largely confined to the equatorial region of the sperm. We conclude that organization of the genome in the bovine sperm nucleus is not random.
Interzonal microtubules are dynamic during spindle elongation
Authors: Shelden E. , Wadsworth P. 
Published: April 2021

ABSTRACT
The pattern and extent of microtubule assembly during spindle elongation has been examined in PtKi cells by microinjection of biotin–tubulin and immu-nolocalization of biotin–tubulin-containing microtubules using antibodies to biotin. PtK1 cells were microinjected at 30°C, incubated for various intervals to allow incorporation of biotin–tubulin into microtubules, then lysed, fixed and stained for biotin–tubulin and total tubulin. When mid- to late anaphase cells were examined at short times postinjection, using conventional fluorescence light microscopy, rapid incorporation of biotin–tubulin was detected throughout the interzonal region, between the separating chromosomes, and in the spindle asters. Using confocal fluorescence microscopy, the segments of biotin-labeled microtubules in the interzonal region were found to be continuous with the distal, or plus-ends, of unlabeled microtubules. When telophase cells were examined, a marked decline in the extent of incorporation was apparent.
Quantitative analysis of the total length of labeled polymer in the interzonal region of cells from midanaphase through telophase further reveals that the extent of incorporation was maximal during late anaphase, and decreased during telophase. The measured rate of interzonal microtubule growth remained relatively constant during this period. Our results provide direct evidence for plus-end elongation of interzonal microtubules during spindle elongation and further reveal that interzonal microtubules are highly dynamic during late anaphase spindle elongation. The implications of these results for the mechanism of anaphase B are discussed.
Pericytes derived from the retinal microvasculature undergo calcification in vitro
Authors: Schor A. M. , Allen T. D. , Canfield A. E. , Sloan P. , Schor S. L. 
Published: April 2021

ABSTRACT
Pericytes isolated from the bovine retinal microvasculature retain characteristic features of their in vivo counterparts, such as the presence of glycogen deposits, long filamentous processes, prominent microfilament bundles and the ability to display two distinct and reversible phenotypes. Time-lapse video-microscopy demonstrated that pericytes tend to overlap and aggregate, even in sparse cultures. After reaching confluence, they form multilayered areas that retract away from each other, resulting in the formation of multicellular nodules. These nodules increase in size and cellularity by going through repeated 5- to 6-h cycles of anchoring, spreading, cell proliferation and retraction. Alkaline phosphatase was not detected in pericytes at subconfluent or confluent densities, but this enzyme was expressed in areas of high cell density, such as multilayers and nodules. Pericytes synthesise and deposit an extracellular matrix at all stages of their in vitro development, including nodule formation. The matrix within the nodules contains cross-striated collagen fibres and matrix vesicles. Needle-like crystals of hydroxyapatite appear to be deposited within the matrix, thus leading to massive calcification of the nodule. Calcification, as assessed by electron microscopy, histochemical staining and X-ray microprobe analysis, occurred on plastic and collagen substrata in the absence of disodium-βkglycerophosphate. The addition of this compound at 5 or 10 mM or the use of a collagen substratum (rather than plastic), brought forward the process of nodule formation and calcification by 3–6 days. Our results suggest that retinal pericytes may differentiate in vitro along the osteogenic pathway.
Localization of actin, βspectrin, 43×103Mr and 58×103M
r proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes
Authors: Daniels Mathew P. 
Published: April 2021

ABSTRACT
I have examined the possible involvement of specific cytoskeletal and peripheral membrane proteins in the early stages of acetylcholine receptor (AChR) aggregation in rat myotubes in culture by immunofluorescence localization of these proteins on the cytoplasmic face of isolated plasma membranes. A culture procedure utilizing selective replating of myoblasts and subsequent treatment with cytosine arabinoside was devised to obtain large, multipolar myotubes with extensive upper surfaces that are free of fibroblasts. These cultures were exposed for 4–6 h to embryonic pig brain extract (EBX) to induce AChR aggregate formation on the upper cell surface, and the AChRs were labeled with TRITC-copjugated a- bungarotoxin. Large sheets of plasma membranes from the upper cell surface were isolated by adhesion to a coverslip coated with a polypeptide adhesive (Cell-Tak) that was pressed on top of the culture. The membranes were labeled by indirect immunofluorescence with monoclonal antibodies against the 43×103Mr and 58×103Mr proteins, originally identified in the AChR-enriched membranes of Torpedo electroplaques, and with monoclonal anti bodies against isoforms of actin and βspectrin. The labeling patterns showed that all four of these proteins are concentrated in the punctate AChR-enriched domains within the aggregates, suggesting that they may be involved in the early stages of AChR aggregation. Immunofluorescence labeling with monoclonal antibodies against vinculin and clathrin, and with an antiserum to talin, showed that these proteins are also associated with AChR aggregates; however, their labeling patterns did not correspond closely to the AChR-enriched domains. Furthermore, vinculin and talin dissociated from most of the membrane during isolation. The concentration of βspectrin and actin isoforms on the cytoplasmic face of the AChR-enriched domains is consistent with the formation, early in the aggregation process, of a membrane-cytoskeleton association similar to that of erythrocytes.
Immortalization of male genital duct epithelium: an assay system for the cystic fibrosis gene
Authors: Coleman Lindsay , Harris Ann 
Published: April 2021

ABSTRACT
The epithelia lining the vas deferens and epididymis are directly involved in the pathology of the autosomal recessive disease cystic fibrosis (CF). We have established culture systems for these epithelial cells. Long-term cell lines have now been generated from these primary epithelial cells by transformation with a plasmid containing an origin-defective simian virus 40 (SV40). Lines have been established from vas deferens and epididymis and both maintain expression of the CF gene.
Cytokeratins in mesenchymal cells: impact on functional concepts of the diversity of intermediate filament proteins
Authors: Markl Jürgen 
Published: April 2021

ABSTRACT
Shape, motility, viscosity and organelle distribution of animal cells is regulated, or at least influenced, by fibrous proteins constituting a ‘cytoskeleton’ that involves the F-actin-based microfilaments (5–7 nm in diameter) and the microtubules (20–25 nm). In addition, most vertebrate cells contain a third type of highly organized protein bundle, the intermediate filaments (1F; 8–12 nm). Their specific role is less clear, but they certainly contribute to tensile strength of cells and, via their anchorages at desmosomes, to cell-cell interactions (for reviews, see Franke et al. 1982; Traub, 1985; Krohne and Benavente, 1986; Franke, 1987; Steinert and Roop, 1988; Nagle, 1988; Bershadsky and Vasiliev, 1988; Robson, 1989; Carmo-Fonseca and David-Ferreira, 1990). Ultrastructurally, the LFs of different cell types are very similar, but biochemical and immunological data have revealed in mammals at least seven distinct classes comprising, for example in humans, a total of approximately 40 different polypeptides: vimentin (predominantly, but not exclusively in mesenchymally derived cells), desmin (typical of most types of muscle cells), glial filament protein (GFP; predominantly in astrocytes), several different neurofilament proteins (in most neurons), peripherin (in certain neuronal cells), at least four different lamins (structural components of the nuclear lamina), and a whole panopticum of cytokeratins (present in almost all epithelial cells). A given tissue is characterized by a specific pattern of IF polypeptides, which is extensively used for cell typing in tumor diagnosis (see Kartenbeck, 1989), and as a differentiation marker in embryogenesis (e.g. see LaFlamme and Dawid, 1990).
In ultraviolet microbeam irradiations, characteristics of the monochromator and lamp affect the spectral composition of the ultraviolet light and probably the biological results
Authors: Forer Arthur 
Published: April 2021

ABSTRACT
Biological conclusions recently published concerning ultraviolet (u.v.) microbeam irradiation of spindles are different from those we previously published. Several technical differences between the two sets of experiments were investigated. The spectral distributions in the light emitted from mercury-arc, xenon-mercury-arc, and xenon-arc lamps were measured, as were the spectral distributions after the light from these lamps passed through a monochromator that was set to various wavelengths and various half-band-widths. Both the source of the u.v. light and the half-band-width of the monochromator influence the spectral distribution of the light leaving the monochromator: depending on the conditions, the light leaving the monochromator is not necessarily of the same wavelength as that to which the monochromator is set. Differences in these aspects of the experiments could easily give rise to the different biological conclusions reached in the two sets of experiments.
The Golgi sorting domain of coronavirus E1 protein
Authors: Armstrong John , Patel Shaila 
Published: April 2021

ABSTRACT
The coronavirus El membrane protein is confined to the Golgi after it is expressed in cells either by viral infection or via injection of synthetic RNA. We have investigated the features of the protein responsible for intracellular sorting and found that a C-terminal deletion of only 18 amino acids results in its transport to the plasma membrane. However, we have previously shown that this C-terminal region alone is not sufficient for Golgi retention. When El was fused to a cell-surface protein, Thy-1, the resulting molecule was retained in the Golgi. Various mutated forms of El whose destinations were the ER, cell surface orlysosomes were also fused to Thy-1, and in each case the fusion was sorted according to its El component alone. We argue that, in contrast to sorting signals for other membrane compartments, Golgi retention of El is not due to a single short peptide sequence. Instead, the Golgi ‘signal’ of El appears to require for its expression a domain comprising most of the sequence of the protein.