bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of spindle elongation by taxol
Authors: Amin‐Hanjani S. , Wadsworth P. 
Published: February 2005

AbstractDuring anaphase B spindle elongation, interzonal microtubules lengthen to accomplish pole‐pole separation, while at the same time remaining highly dynamic [Shelden and Wadsworth, J. Cell Sci. 97:273–281, 1990]. To further examine the role of microtubule polymerization and dynamics during spindle elongation, cells have been treated with taxol, which induces microtubule polymerization and stabilizes microtubules. Taxol was added to PtK1 cells 3 minutes after initial chromatid separation, so that the effect on anaphase B could be observed with minimal disruption to anaphase A movement. In 20 μM taxol, the rate and extent of pole‐pole separation, measured from time‐lapse video records, are reduced to 4% and 9.5% of controls, respectively. The organization of microtbules in taxol treated cells was examined using tubulin immunofluorescence and confocal fluorescence microscopy. Taxol induces a dramatic reorganization of interzonal microtubules resulting in a narrow gap, which is nearly completely lacking in MTs, across the center of the interzone. Furthermore, microtubules in taxol treated cells are resistant to nocodazole induced microtubule disassembly. Our results reveal that taxol rapidly inhibits anaphase B spindle elongation; inhibition is accompanied by a depletion of interdigitated interzonal microtubules and a reduction in microtubule dynamic behavior.
Kinky microtubules: Bending and breaking induced by fixation in vitro with glutaraldehyde and formaldehyde
Authors: Cross Alan R. , Williams Jr. Robley C. 
Published: February 2005

AbstractWe have employed video‐enhanced light microscopy to study alterations of the overall shape of microtubules that are produced by the aldehyde fixation methods commonly employed to study them in vitro. Changes brought about by these methods include deformation and breakage. The severity of the effects depends on the fixative employed and increases with its concentration, and with the time of fixation. The changes are observed under a variety of conditions, such as brief exposure to 3.7% formaldehyde, or somewhat longer exposure to glutaraldehyde at concentrations as low as 0.05%. The observed distortion explains why microtubules usually appear curved or sinuous in electron micrographs while appearing relatively rigid and linear in video‐enhanced light microscopy. The observed breakage implies that caution must be used in inferring length distributions from measurements of aldehyde‐fixed microtubules.
Cytoskeletal assembly and vinculin‐cytoskeleton interaction in different phases of the activation of bovine platelets
Authors: Horvath Andrea R. , Asijee Guus M. , Muszbek Laszlo 
Published: February 2005

AbstractVinculin is an Mr 130 kDa protein that has been implicated in membrane‐cytoskeleton interaction in various cell types. It has been demonstrated that vinculin is not a cytoskeletal component in resting platelets, but part of it becomes associated with the cytoskeleton during thrombin‐induced activation. In this study, using a quantitative immunnoblotting technique, the relation of vinculin to the cytoskeleton in different phases of activation of bovine platelets was explored, and the process of incorporation of vinculin into the cytoskeleton was related to that of cytoskeletal assembly. The assembly of cytoskeleton proceeded at a significantly faster rate than the association of vinculin with it, which shows that the latter process is not due to passive trapping of vinculin into the Triton‐insoluble residue, but certain biochemical changes had to occur before such an interaction became possible. When the formation of pseudopodia was prevented by cyto‐chalasin B, but neither aggregation nor the release reaction induced by thrombin were inhibited, the recovery of vinculin in the Triton‐insoluble residue even increased. In both time‐ and thrombin‐concentration‐dependent studies, poor correlation was found between vinculin‐cytoskeleton association and the extent of aggregation. Activation with phorbol‐myristate‐acetate, which is a strong stimulus for aggregation but produces only a slight release in the granular content, resulted in the association of only a negligible amount of vinculin with the cytoskeletal fraction. The incorporation of vinculin into the cytoskeletal fraction of thrombin activated platelets started with the release reaction but still proceeded, and the greatest part of the reaction occurred after secretion had gone to completion. These findings suggest that platelet shape change and pseudopodium extrusion are not prerequisites for, and aggregation is not related to, vinculin‐cytoskeleton interaction. The association of vinculin with the cytoskeleton correlates with the organization of contractile gel, which suggests a role for vinculin in secretion and clot retraction.
Differential sorting of beta tubulin isotypes into colchicine‐stable microtubules during neuronal and muscle differentiation of embryonal carcinoma cells
Authors: Falconer Marcia M. , Echeverri Christophe J. , Brown David L. 
Published: February 2005

AbstractPluripotent P19 embryonal carcinoma (EC) cells were differentiated along the neuronal and muscle pathways. Comparisons of class I, II, III, and IV beta tubulin isotypes in total and colchicine‐stable microtubule (MT) arrays from uncommitted EC, neuronal, and muscle cells were made by immunoblotting and by indirect immunofluorescence microscopy. In undifferentiated EC cells the relative amounts of these four isotypes are the same in both the total and stable MT populations. Subcellular sorting of beta tubulin isotypes was demonstrated in both neuronal and muscle differentiated cells. During neuronal differentiation, class II beta tubulin is preferentially incorporated into the colchicine‐stable MTs while class III beta tubulin is preferentially found in the colchicine‐labile MTs. The subcellular sorting of class II into stable MTs correlates with the increased staining of MAP IB. and with the expression of MAP 2C and tau. Although muscle differentiated cells express class II beta tubulin, stable MTs in these cells do not preferentially incorporate this isotype but instead show increased incorporation of class IV beta tubulin. Muscle cells do not show high levels of MAP IB and do not express MAP 2C or tau. These results are consistent with the hypothesis that a subcellular sorting of tubulin isotypes is the result of a complex interaction between tubulin isotypes and MT‐associated proteins.
Actin based motility on retraction fibers in mitotic PtK2 cells
Authors: Mitchison T. J. 
Published: February 2005

AbstractWhen PtK2 cells round up in mitosis they leave retraction fibers attached between the substrate and the cell body. Retraction fibers and the region where they meet the cell body are rich in actin filaments as judged by phalloidin staining and electron microscopy. Video microscopy was used to study actin dependent motile processes on retraction fibers. Small, phase‐dense nodules form spontaneously on the fibers, and move in to the cell body at a rate of 3 μm/minute. As they move in they increase progressively in phase‐density. This movement appears to be related to actin dependent centripetal movement which has been previously studied in lamellipodia. Despite its generality, the mechanism of such movement is unknown, and retraction fibers present some special advantages for its study. Cytochalasin treatment causes nodules to stop moving and dissolve. Withdrawal of the drug causes them to reform and start moving. Surprisingly, movement after cytochalasin withdrawal was often outward, indicating a local reversal of cortical polarity. After a few minutes correct polarity is reestablished by a global control mechanism. The implications of these observations for the mechanism and polarity of actin dependent motility is discussed. © 1992 Wiley‐Liss, Inc.
Phalloidin binds and stabilizes pea root actin
Authors: Andersland John M. , Parthasarathy M. V. 
Published: February 2005

AbstractPrevious reports about phalloidin binding to plant actins have been indirect. We present here evidence showing that phalloidin does bind and stabilize filaments of actin extracted from pea roots. Criteria for the presence of actin included stabilization as a polymer in the presence of phalloidin, cross‐reaction with antibody against chicken actin, affinity binding to DNase I, and ability to be decorated by the S1 fragment of rabbit muscle myosin.Phalloidin was able to stabilize polymers in pea root extracts against dissociation during SDS gel electrophoresis, and these polymers were shown to be composed exclusively of actin. Pea root actin isolated by affinity chromatography on a DNase I column was incubated with rhodamine phalloidin and electrophoresed on a native gel. The rhodamine fluorescence remained with the stabilized filaments, indicating clearly that phalloidin does bind to actin from a plant source. © 1992 Wiley‐Liss, Inc.
Desmosome assembly in MDCK epithelial cells does not require the presence of functional microtubules
Authors: Pasdar Manijeh , Li Zhi , Krzeminski Kathleen A. 
Published: February 2005

AbstractDesmosomes, complex multisubunit structures that assemble at sites of cell‐cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645–655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin‐Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post‐translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell‐cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstratedan apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell‐cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell‐cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell‐cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules. © 1992 Wiley‐Liss, Inc.
Tubulin dimer formation via the release of α‐ and β‐tubulin monomers from multimolecular complexes
Authors: Zabala Juan C. , Cowan Nicholas J. 
Published: February 2005

AbstractThe functional subunit of microtubules is a heterodimer consisting of α‐ and β‐tubulin. An understanding of tubulin dimerization has been hampered because it has not proved possible to purify native tubulin monomers. To study the process whereby tubulin dimers are formed, we made use of tubulins synthesized by in vitro transcription and translation. We present evidence that the in vitro synthesis of different mouse α‐tubulin isotypes involves a multimolecular complex. The synthesis of mouse β‐tubulin isotypes also involves the formation of multimolecular complexes, though different isotypes behave somewhat differently from one another. The properties of in vitro synthesized α‐ and β‐tubulin multimolecular complexes strongly suggest that they are intermediates in the biosynthesis of tubulin monomers. Upon release, these monomers can exchange with pre‐existing tubulin heterodimers. © 1992 Wiley‐Liss, Inc.
Characterization of smooth muscle caldesmon as a microtubule‐associated protein
Authors: Ishikawa Ryoki , Kagami Osamu , Hayashi Chihiro , Kohama Kazuhiro 
Published: February 2005

AbstractWe have previously shown that nonmuscle caldesmon copurified with brain microtubules binds to microtubules in vitro [Ishikawa et al.: FEBS Lett. 299:54–56, 1992]. To explore the role of caldesmon in the functions of microtubules, further characterization was performed using smooth muscle caldesmon, whose molecular structure and function have been best‐characterized in all caldesmon species.Smooth muscle caldesmon bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with the binding constant of 1.1 × 106M−1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. Partial digestion of the caldesmon with α‐chymotrypsin revealed that the binding site of the caldesmon for microtubules lay in the 34‐kDa C‐terminal domain. When the caldesmon was in the dimeric form in the absence of a reducing agent, the caldesmon cross‐linked microtubules to form bundles. Further, the caldesmon potentiated the polymerization of tubulin, and inhibited the in vitro movement of microtubules on dynein. These results suggest that caldesmon may be involved in the regulation by Ca2+ of the functions of microtubules. © 1992 Wiley‐Liss, Inc.
Thrombin, epidermal growth factor, and phorbol myristate acetate stimulate tubulin polymerization in quiescent cells: A potential link to mitogenesis
Authors: Ball Rebecca L. , Albrecht Thomas , Thompson William C. , James Olapade , Carney Darrell H. 
Published: February 2005

AbstractPrevious studies suggest that alterations in the microtubule (MT)‐tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT‐tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033–1041, 1986). With this assay, 3H‐Ab 1 ‐ 1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified α‐thrombin increases 3H‐Ab 1 ‐ 1.1 binding to cytoskeletons of serum‐arrested mouse embryo (ME) fibroblasts from 1.5‐ to 3‐fold. This stimulation is dose‐dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12‐myristate 13‐acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 μM) eight hours after growth factor addition blocks stimulation of 3H‐thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine‐stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin‐ colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools. © 1992 Wiley‐Liss, Inc.
Native microtubules with a variable number of protofilaments
Authors: Dallai R. , Afzelius B. A. , Lanzavecchia S. , Bellon P. L. 
Published: February 2005

AbstractInsect spermatozoa are characterized by having a set of accessory tubules that surrounds the microtubular doublets of the axoneme and that are formed from the B‐subtubules of the doublets. In trichopteran species, the accessory tubules have an unusually large diameter. Those of one species, Odontocerum albicorne, were seen to have a number of protofilaments that is 19 in the main part of the axoneme, but gradually decreasing to 18, 17, and 16 near the distal tip. The accessory tubule of the trichopteran axoneme has an asymmetrical shape and a skewed orientation, which makes it easy to distinguish a tubule that is viewed from its plus‐end from one viewed from the minus‐end. The shape of a cross‐sectioned protofilament in the trichopteran accessory tubules differs from that of microtubules in general, including accessory tubules of other insects, by being polygonal with the most acute angle pointing centripetally. © 1993 Wiley‐Liss, Inc.
Molecular cloning and expression of the chicken smooth muscle γ‐actin mRNA
Authors: Kovacs Adrienne M. , Zimmer Warren E. 
Published: February 2005

AbstractWe have investigated the expression of chicken smooth muscle γ‐actin mRNA by isolation and characterization of cDNAs representing this actin isoform and utilizing the cDNA to probe RNA from adult and developing cells. Nucleotide sequence elucidated from an apparent full length smooth muscle γ‐actin cDNA revealed that it contained 94 bp of 5′ non‐translated sequence, an open reading frame of 1131 bp, and 97 bp of 3′ non‐translated sequence. Within the 376 amino acid sequence deduced from the chicken cDNA were diagnostic amino acids at the NH2‐ and COOH‐terminal regions which provided unequivocal identification of the γ‐enteric smooth muscle actin isoform. In addition, the chicken γ‐enteric actin deduced from our cDNA clones was found to differ from the sequence reported in earlier protein studies [J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979] by containing a proline rather than a glutamine at position 359 of the protein, indicating that the avian γ‐enteric actin isoform is identical to its mammalian counterpart. Comparison of the 5′ and 3′ non‐translated sequence determined from the chicken cDNA to that elucidated for rat, mouse, and human showed that there is not a high degree of cross‐species sequence conservation outside of the coding regions among these mRNAs. Northern hybridization analyses demonstrated that the γ‐enteric actin mRNA is expressed in adult aorta and oviduct tissues but not in adult skeletal muscle, cardiac muscle, liver, brain, and spleen tissues. The γ‐enteric actin mRNA was first observed in measurable quantities in gizzard tissue from 4–5 day embryos and increased in content in developing smooth muscle cells through 16–17 embryonic days. Following this initial increase during embryonic development, the γ‐enteric actin mRNA exhibits a decline in content until ∼7 days posthatching, after which there is an increase in content to maximal levels found in adult gizzard tissue. In general, the developmental appearance of the γ‐enteric mRNA parallels that observed for this protein in previous studies indicating that the developmental expression of smooth muscle γ‐actin is regulated, in part, by an increased content of mRNA in chicken visceral smooth muscle cells during myogenesis. © 1993 Wiley‐Liss, Inc.
Incorporation of microinjected mutant and wildtype recombinant tropomyosins into stress fibers in fibroblasts
Authors: Ranucci Denise , Yamakita Yoshihiko , Matsumura Fumio , Hitchcock‐DeGregori Sarah E. 
Published: February 2005

AbstractThe structural requirements for assembly of tropomyosin into stress fibers were investigated by microinjecting wildtype and four mutant striated chicken muscle α‐tropomyosins expressed in E. coli as fusion and nonfusion proteins into cultured rat embryo fibroblasts, followed by localization of tropomyosin using indirect immunofluorescence. The results show that the determinants for stress fiber incorporation in living cells correlate with the in vitro actin affinity of these tropomyosins. Wildtype recombinant protein incorporated into stress fibers both when the amino terminus was unacetylated and when it was blocked with an 80‐residue fusion protein [Hitchcock‐DeGregori, S.E., and Heald, R.W. (1987): J. Biol. Chem. 262:9730–9735]. The pattern of incorporation was indistinguishable from that of tropomyosin isolated from chicken pectoral muscle. The striated α‐tropomyosin incorporated into stress fibers, even though this isoform is not found in nonmuscle cells. Three recombinant mutant tropomyosins in which one‐half, two‐thirds, or one actin binding site was deleted were tested [Hitchcock‐DeGregori, S.E., and Varnell, T.A. (1990): J. Mol. Biol. 214:885–896]. Only the fusion protein with a full actin binding site deleted incorporated into stress fibers. However, the unacetylated, nonfusion proteins with one half and one actin binding site deleted incorporated into stress fibers, consistent with the ability of troponin to promote the actin binding in vitro. A fourth mutant, in which the conserved amino‐terminal nine residues were deleted, did not incorporate into stress fibers, consistent with the complete loss of function of this mutant [Cho, Y.J., Liu, J., and Hitchcock‐DeGregori, S.E. (1990): J. Biol. Chem. 265:538–545]. © 1993 Wiley‐Liss, Inc.
Effects of taxol and taxol/hyperthermia treatments on the functional polarization of cytotoxic T lymphocytes
Authors: Knox J. David , Mitchel Ron E. J. , Brown David L. 
Published: February 2005

AbstractImmunofluorescence staining, electron microscopy, and (51Cr) cytolytic release assays are used to investigate the effects of taxol and taxol/hyperthermia treatments on the microtubule organization and cytolytic activity of cytotoxic T lymphocytes (CTLs). A 4 h treatment of CTLs with 1 μM taxol results in an extensive reorganization of the microtubule system to form one to a few large microtubule bundles that extend from the centrosome. The Golgi apparatus is not disrupted by this treatment and remains associated with the microtubule organizing centre (MTOC). This microtubule reorganization has no effect on the ability of CTLs to orient their MTOC towards a bound target cell, nor on their cytolytic activity. In control CTLs, not treated with taxol, a mild hyperthermia treatment (42°C, 30 min) results in an aggregation of the pericentriolar material, a loss of MTOC orientation, an inhibition of cytolytic activity, and a disorganization of the microtubule system [Knox et al.: Exp. Cell Res. 194:275–283, 1991]. In contrast, in taxol‐treated CTLs the stabilized microtubule bundles are unaffected by such hyperthermia treatment; however, the other effects of hyperthermia appear identical in control and taxol‐treated CTLs. These results indicate that a dynamic, radially arranged microtubule array is not required for the functional polarization of CTLs and suggest that a component of the pericentriolar material may play a key role in effecting MTOC orientation. © 1993 Wiley‐Liss, Inc.