bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of an amino acid substitution in the benA, β‐tubulin gene of Aspergillus nidulans that confers thiabendazole resistance and benomyl supersensitivity
Authors: Jung M. Katherine , Oakley Berl R. 
Published: February 2005

AbstractWe are using molecular genetic techniques to identify sites of interaction β‐tubulin with benzimidizole anti‐microtubule agents. We have developed a marker‐rescue technique for cloning mutant alleles of the benA, β‐tubulin gene of Aspergillus nidulans and have used the technique to clone two mutant benA alleles, benA16 and benA19. These are the only A. nidulans alleles known to confer resistance to the benzimidazole antimicrotubule agent thiabendazole and supersensitivity to other benzimidazole antimicrotubule agents including benomyl and its active breakdown product, carbendazim. benA16 has been shown, moreover, to reduce thiabendazole binding to β‐tubulin. We have sequenced the two mutant alleles and have found that they carry different nucleotide changes that cause the same single amino acid substitution, valine for alanine at amino acid 165. Since thiabendazole and carbendazim differ at only one side chain, the R2 group, we conclude that the region around amino acid 165 is involved in the binding of the R2 group of benzimidazole antimicrotubule agents to β‐tubulin.
Microtubule‐associated proteins from antarctic fishes
Authors: Detrich H. William , Neighbors Bonnie W. , Sloboda Roger D. , Williams Robley C. 
Published: February 2005

AbstractMicrotubules and presumptive microtubule‐associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol‐dependent, microtubule‐affinity procedure (cf. Vallee: Journal of Cell Biology 92:435–442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000–430,000, 220,000–280,000, 140,000–155,000, 85,000–95,000, 40,000–45,000, and 32,000–34,000. The surfaces of MAP‐rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0–5°C, induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2–5°C, and proteins corresponding to MAPs 1 and 2 co‐sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33°C, but the microtubules depolymerized at 0°C, We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold‐induced microtubule depolymerization.
The iris diaphragm model of centriole and basal body formation
Authors: Albrecht‐Buehler Guenter 
Published: February 2005

AbstractThis paper suggests that the formation and structure of the microtubular skeleton of centrioles and basal bodies can be derived from the following simple geometric principle. A closed ring of nine microtubular initiation sites defines (1) a template for the packing of 18 additional microtubular initiation sites, and (2) the shape of nine rigid arms. Upon swivelling of each arm around a point located four initiation sites away on the initial ring, the array unfolds in a manner similar to the opening of an iris diaphragm.As a consequence, the curved shape of the microtubular triplet blades arises together with the clockwise rotational sense of the slanted blades of the centriole or basal body. The final diameter of the centriole (basal body) self‐adjusts. Furthermore, the pitch of the triplet blades, the taper of centrioles and basal bodies, and the change of slant of the blades towards the distal end can be derived. In addition, the model points to a method of replication of pro‐centrioles (pro‐basal bodies). The hypothesis was tested by the fitting of electron microscopical cross sections of centrioles of 3T3 cells to the geometric shapes predicted by the model.
p34cdc2 Kinase is localized to distinct domains within the mitotic apparatus
Authors: Rattner J. B. , Lew John , Wang Jerry H. 
Published: February 2005

AbstractAntibodies to both the C‐terminal and the N‐terminal regions of the 34 kd serinethreonine specific protein kinase, p34cdc2, were used to study the distribution of this protein in dividing cells and isolated chromosomes of the Indian muntjac. p34cdc2 was found to be present throughout the cytoplasm of dividing cells. In addition, a portion of cellular p34cdc2 was localized to the centrosome, kinetochore, and intercellular bridge and along kinetochore‐to‐pole microtubules during cell division. Tubulin‐denuded metaphase kinetochores retained their association with p34cdc2. The detection of p34cdc2 within a variety of domains of the mitotic apparatus, in addition to the previous reported association with the centrosome [Bailly et al., EMBO J. 8:3985–3995, 1989; Raibowol et al., Cell 57:393–401, 1989] suggests that p34cdc2 may play a role in events associated with anaphases A and B as well as with the transition between interphase and mitosis.
Polypeptides from the myxomycete Physarum polycephalum interacting in vitro with microtubules
Authors: Albertini Catherine , Akhavan‐Niaki Haleh , Wright Michel 
Published: February 2005

AbstractMicrotubule‐interacting proteins have been studied in the lower eukaryote Physarum polycephalum. We show for the first time 1) the presence in Physarum amoebal crude extracts of at least six polypeptides that bind specifically to amoebal microtubules, 2) the binding between these proteins and mammalian microtubules, 3) the heat stability of two of these polypeptides (125 and 235 kDa), 4) the functional properties of a fraction containing a heat‐soluble 125 kDa polypeptide, and 5) the phosphorylation of the 125 kDa polypeptide during two distinct periods of the cell cycle in Physarum synchronous plasmodia, first at late S/early G2 phase and second at late G2/prophase.
Yeast myosin heavy chain mutant: Maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene
Authors: Rodriguez José R. , Paterson Bruce M. 
Published: February 2005

AbstractRecent studies with myosin heavy chain mutants in the slime mold Dictyostelium discoideum and the yeast Saccharoymyces cerevisiae indicate that the myosin heavy chain gene is not essential for cell survival under laboratory growth conditions. However, cells lacking a normal myosin heavy chain gene demonstrate substantial alterations in growth and cell division. In this study, we report that a disruption mutant in the rod portion of the yeast myosin heavy chain gene, MYOl, produces abnormal chitin distribution and cell wall organization at the mother‐bud neck in a high proportion of dividing cells. It is suggested that this phenotype is the cause of the cell division defect and the osmotic sensitivity of yeast MYOl mutants. In the absence of a normal MYOl polypeptide, yeast cells alter their cell type specific budding pattern. It is concluded that an intact myosin heavy chain gene is required to maintain the cell type specific budding pattern and the correct localization and deposition of chitin and cell wall components during cell growth and division.
Calyculin‐A increases the level of protein phosphorylation and changes the shape of 3T3 fibroblasts
Authors: Chartier Lynn , Rankin Lucinda L. , Allen Ronald E. , Kato Yuko , Fusetani Nobuhiro , Karaki Hideaki , Watabe Shugo , Hartshorne David J. 
Published: February 2005

AbstractCalyculin‐A, an inhibitor of type 1 and 2A phosphatases, was applied extracel‐lularly to 3T3 fibroblasts. At 0.1 μM, calyculin‐A caused a marked increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca2+. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phos‐phorylation following treatment with calyculin‐A was observed in vimentin, the 20‐kD myosin light chain, and an unidentified 440‐kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells.Calyculin‐A also caused marked shape changes of 3T3 cells. Within minutes after addition of calyculin‐A (0.1 μM) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments, and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes were reversible and after removal of calyculin‐A the rounded cells attached to the substratum, resumed a flattened shape, and were active mitotically. In the cells treated with calyculin‐A an unusual “ball‐like” structure was observed with transmission electron microscopy. This unique structure was 2–3 μM in diameter and was located close to the nucleus.The use of calyculin‐A adds further support to the idea that cell shape is controlled, at least in part, by concerted actions of a kinase‐phosphatase couple.
Dilution‐induced disassembly of microtubules: Relation to dynamic instability and the GTP cap
Authors: Voter William A. , O'Brien E. Timothy , Erickson Harold P. 
Published: February 2005

AbstractMicrotubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15‐fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubules of 60 μm/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.: Journal of Cell Biology 107:1437–1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP‐tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respectively.
Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of α‐tubulin
Authors: Alfa Caroline E. , Hyams Jeremy S. 
Published: February 2005

AbstractThe state of tubulin tyrosination in the fission yeast Schizosaccharomyces pombe was investigated using a combination of indirect immunofluorescence microscopy and Western blotting. Antibodies specific for the tyrosinated form of α‐tubulin stained all microtubule arrays in wild type cells and recognised the two α‐tubulin polypeptides in Western blots of cell extracts enriched for tubulin by DEAE‐Sephadex chromatography. Antisera that specifically recognised the detyrosinated, glu, form, on the other hand, gave consistently negative results, both in cells undergoing rapid exponential growth and in those allowed to accumulate in stationary phase. Neither the “ageing” of microtubules, by arresting cells at different points (late G1 or G2/M) in the cell division cycle, nor stabilising them, using D2O, lead to any detectable tubulin detyrosination. These results suggest that S. pombe lacks the carboxypeptidase that carries out the tubulin detyrosination reaction. This is the first report of an organism that possesses the correct C‐terminal α‐tubulin sequence yet fails to carry out this post‐translational modification. The implication of this novel finding for the biological role of these events is discussed.
Cytoskeleton of the mouse egg and embryo: Reorganization of planar elements
Authors: Ian Gallicano G. , McGaughey Robert W. , Capco David G. 
Published: February 2005

AbstractExamination of detergent‐extracted mouse eggs and embryos reveals the existence of two cytoskeletal networks. One network is the typical thin filament network observed in somatic cells while the other is composed of large planar elements. These latter cytoskeletal structures, with individual widths of 60.0±6.8 nm, alter their spatial organization in a developmental stage‐specific manner. The planar elements are composed of filaments with a diameter of 10 nm aligned side‐by‐side with these filaments exhibiting a linear periodicity of 20.0±1.6 nm. A biochemical fraction containing components of the planar elements has been prepared from different stages of development and disappearance of prominent polypep‐tides from this fraction correlates with the altered spatial organization of the planar elements. Ultrastructure and biochemistry of cytoskeletal planar elements in eggs and embryos of the mouse are comparable with cytoskeletal sheets of Syrian hamster eggs and embryos, suggesting these cytoskeletal components may have a functional role in mammalian embryogenesis. Because such structures have not been identified in eggs or embryos of species other than mammals, their function may be unique to mammalian embryogenesis.
Direct binding of F‐actin to ponticulin, an Integral plasma membrane glycoprotein
Authors: Chia Catherine P. , Hitt Anne L. , Luna Elizabeth J. 
Published: February 2005

AbstractWe have developed an 125I‐labeled F‐actin blot overlay assay for the identification of F‐actin‐binding proteins after transfer to nitrocellulose from SDS‐polyacryl‐amide gels. Two major F‐actin‐binding proteins from Dictyostelium discoideum, a cytoplasmic 30 kDa protein and a 17 kDa integral membrane protein, and two minor membrane polypeptides of 19 kDa and 15 kDa were detected by this method. Using F‐actin affinity and immunoaffinity chromatography, the 17 kDa polypeptide was identified as ponticulin, a previously described actin‐binding glycoprotein from D. discoideum plasma membranes (Wuestehube, L.J., and Luna, E.J. [1987]: J. Cell Biol. 105:1741–1751). The binding of F‐actin to ponticulin on blots is specific because unlabeled F‐actin competes with 125I‐labeled F‐actin and because G‐actin does not bind. Nitrocellulose‐bound ponticulin displays binding characteristics similar to those of purified plasma membranes in solution, e.g., F‐actin binding is sensitive to high salt and to elevated temperatures. Under optimal conditions, 125I‐labeled F‐actin blot overlays are at least as sensitive as are immunoblots with an antibody specific for ponticulin. When blotted onto nitrocellulose after 2‐D gel electrophoresis, all isoforms of ponticulin and of the 19 kDa and 15 kDa polypeptides appear to bind F‐actin in proportion to their abundance. Thus the actin‐binding activities of these proteins do not appear to be regulated by modifications that affect isoelectric point. However, the actin‐binding activity of nitrocellulose‐bound ponticulin is diminished when the protein is exposed to reducing agents, suggesting an involvement of disulfide bond(s) in ponticulin function. The 125I‐labeled F‐actin blot overlay assay also may enable us to identify F‐actin‐binding proteins in other cell types and should provide a convenient method for monitoring the purification of these proteins.
Spatial distribution of posttranslationally modified tubulins in polarized cells of developing Artemia
Authors: Macrae Thomas H. , Langdon Carrie M. , Freeman John A. 
Published: February 2005

AbstractIn many differentiated cells, posttranslationally modified tubulins exhibit restricted subcellular distribution, leading to the proposal that they are required for the production and maintenance of polarity. To study this possibility, we used immunological approaches to examine tubulin isoforms in developing Artemia larvae and to determine their location in several types of cells within the organism. The amount of tubulin in relation to total protein remained relatively constant during early larval development while detyrosinated tubulin increased, an event correlated with the differentiation of larval gut muscle cells. Except for epidermal cells of the developing thorax, each type of cell within the Artemia larvae exhibited characteristic staining patterns which were very similar for each antitubulin antibody. Within epidermal cells, microtubules containing acetylated tubulin appeared patchy or punctate in their distribution, an image not seen with the other antibodies. In most polarized cells, staining for tubulin and actin colocalized in discrete areas, demonstrating enrichment of both proteins within the same cellular compartment and suggesting functional interactions. Mitotic figures were stained with qualitatively equal intensity by all of the antitubulin antibodies, but asters were not observed. Midbodies were intensely stained with phalloidin as well as the antibodies to tubulin. It was clear that microtubules exhibited a preferential localization in cells of Artemia but in no case was a tubulin isoform found exclusively in one area of a cell. The results support the contention that microtubules influence the organization of polarized cell structure and function but they do not permit the conclusion that this capability is dependent on the localization of posttranslationally modified tubulins to restricted subcellular positions.
Cell locomotion: New research tests old ldeas on membrane and cytoskeletal flow
Authors: Heath Julian P. , Holifield Bruce F. 
Published: February 2005

AbstractRecent studies on the mobility of membrane markers on crawling cells indicate that there is no long‐range centripetal flow of membrane proteins or lipids during cell locomotion. In this article we reflect on the history of ideas about membrane flow in cells, and we discuss how these new findings will shift the focus of research in cell locomotion away from the cell surface to the molecular interactions and dynamics of the actin cytoskeleton.
Microtubule binding and translocation by inner dynein arm subtype i1
Authors: Smith Elizabeth F. , Sale Winfield S. 
Published: February 2005

AbstractStructural, biochemical, and genetic evidence has demonstrated there are three inner dynein arm subforms, I1, I2, and I3, which differ in organization and composition (see Piperno et al.: J. Cell Biol. 110:379–389, 1990). Using dynein extracted from Chlamydomonas outer dynein armless mutant pf28, we have begun to define the structural and functional properties of isolated inner arm subforms. Inner dynein arm I1 was purified either by sucrose density gradient centrifugation or microtubule binding affinity. I1, composed of heavy chains 1α and 1β, sedimented at 21S and selectively bound to and cross‐linked purified microtubules in and ATP‐sensitive manner. Deep etch electron microscopy revealed that the 21S sedimenting fraction contained two‐headed structures in which large globular heads are connected by long, flexible‐stem domains. In contrast, components derived from I2 and I3 sedimented as a mixture of 11S particles with single globular heads which did not bind to purified microtubules. Both the 21S and 11S sedimenting fractions supported microtubule translocation in in vitro motility assays. In 1 mM MgATP the I1‐containing fraction produced very slow microtubulegliding velocities (0.76 μm/sec) compared to the I2, I3‐containing fraction (4.1 μm/sec).
Flagellar photoresponses of Chlamydomonas cells held on micropipettes: II. Change in flagellar beat pattern
Authors: Rüffer Ursula , Nultsch Wilhelm 
Published: February 2005

AbstractIn response to step‐up as well as step‐down blue or white light stimuli, changes of beat pattern were observed in the two flagella of Chlamydomonas. The front amplitude was either increased or decreased, always in reverse in the two flagella. Again, two opposite combinations of step‐up and step‐down responses were found roughly in parallel to the two types of beat frequency changes. It is shown that positive phototaxis is probably achieved by the first type [called type (+)] and negative phototaxis by the second one [called type (−)]. Comparative measurements have revealed that frequency is not only related to the rate of flagellar movement, but also to the beat pattern. The rate of movement may change in different ways in the recovery and in the effective stroke. Though beat frequency and pattern changes are opposite in the two types, the rates of movement of the two flagella during the effective stroke are not always. In type (−) divergent changes were found in the rates of effective stroke movement, perhaps indicating the involvement of an additional mechanism.
Post‐mitotic cellular reorganisation in the diatom Cymatopleura solea: The role of microtubules and the microtubule centre
Authors: Pickett‐Heaps Jeremy D. 
Published: February 2005

AbstractCell division in Cymatopleura requires precise and major relocations of the nucleus and chioroplast which have been followed by time‐lapse cinematography and with the electron microscope. These movements are (1) the premitotic nucleus migrating to one end of the cell; (2) after cytokinesis, the daughter nuclei moving back to the cell centre, often oscillating several times while establishing their final location; (3) the single chloroplast folding over and sandwiching the central nucleus; and (4) the folded end of the chloroplast stretching back to fill the empty half of the cell. In all cases, straight, actively moving, transient strands of cytoplasm are associated with the movement of the nucleus and chloroplast, and these often appear to be pulling on the surface or the fold of the chloroplast which undergoes transient distortion.These movements are rapid and colchicine‐sensitive. Ultrastructurally, they appear to be mediated by the prominent microtubule centre (MC) and its associated cytoskeleton of microtubules (MTs) although MTs do not attach directly to either nucleus or chloroplast. The MC is located close to the moving nucleus. Later, it moves ahead of the moving chloroplast and its MTs ensheath the tip. Later still, it is seen embedded in the fold of the chloroplast. In all three situations, MTs from it are seen in the strands of cytoplasm radiating from this area across the vacuole. After these events, the MC resumes its usual interphase situation on the nuclear surface.