bluuebunny/crossref_metadata_2025_split huggingface dataset:
Divergent neural β tubulin from the antarctic fish notothenia coriiceps neglecta: Potential sequence contributions to cold adaptation of microtubule assembly
Authors: Detrich H. William , Parker Sandra K. 
Published: February 2005

AbstractThe cytoplasmic microtubules of the cold‐adapted Antarctic fishes, unlike those of homeotherms and temperate poikilotherms, assemble and function at body temperatures in the range −1.8 to +2°C. To determine whether alterations to the primary sequence of β tubulin may contribute to enhancement of microtubule assembly at cold temperatures, we have cloned and sequenced a 1.8‐kilobase neural β‐chain cDNA, Ncnβ1, from an Antarctic rockcod, Notothenia coriiceps neglecta. Based on nucleotide sequence homology, Ncnβ1 probably corresponds to a class‐II β‐tubulin gene. The 446‐residue β chain encoded by Ncnβ1 is closely related (sequence homology ∼95%) both to the neural class‐I/II isotypes and to the neural/testicular class‐IV variants of higher vertebrates, but the sequence of its carboxy‐terminal isotype‐defining region (residues 431–446) has diverged markedly (≥ 25% change relative to the I/II/IV referents). Furthermore, the NcnβsZ1 polypeptide contains six unique amino‐acid substitutions (five conservative, one nonconservative) not found in other vertebrate brain isotypes, and the carboxyterminal region possesses a unique tyrosine inserted at position 442. We conclude that Ncnβ1 encodes a class‐II β tubulin that contains sequence modifications, located largely in its interdimer contact domain, that may contribute to cold adaptation of microtubule assembly. © 1993 Wiley‐Liss, Inc.
Centripetal movement of homologs occurs at the onset of anaphase A in primary oocytes of Ephestia kuehniella Z. (Pyralidae, Lepidoptera)
Authors: Wolf Klaus Werner 
Published: February 2005

AbstractThe transition from metaphase to anaphase was examined in primary oocytes of the Mediterranean mealmoth, Ephestia kuehniella. Isolated spindles were analysed using phase‐contrast microscopy and antitubulin immunofluorescence. Metaphase I bivalents in female Lepidoptera contain material derived from the synaptonemal complexes, the elimination chromatin, which is located between the homologs. Upon the onset of anaphase I, the homologs detach from the elimination chromatin. The present observations revealed that prior to the start of anaphase A, the homologs move centripetally, i.e., toward the central spindle axis, while the elimination chromatin remains stationary. This type of movement is not directly comparable with conventional chromosome movements such as prometaphase congression, anaphase A, and anaphase B, since it occurs at right angles to the preferential orientation of the spindle MTs. A MT‐based mechanism may nevertheless account for the centripetal shift of homologs in meiosis I of the moth: bundling of kinetochore MTs of different chromosomes could transfer the chromosomes toward the spindle axis. Since the homologs move along the poleward surfaces of the plate of elimination chromatin, a MT‐independent force producer could exist as well at the interface between the chromosomes and the plate of elimination chromatin. © 1993 Wiley‐Liss, Inc.
Reactivation of demembranated, cytosol‐free ram spermatozoa
Authors: San Agustin Jovenal T. , Witman George B. 
Published: February 2005

AbstractA procedure for preparing cytosol‐free ram sperm models was developed. Sperm are introduced to a Triton X‐100‐containing demembranation medium layered on top of a discontinuous Percoll gradient. After brief exposure to the demembranating solution, the sperm are separated from the detergent‐soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they are recovered. Optimum conditions consisted of Triton X‐100 at 0.20% and a demembranation time of 35 sec. Cross‐sections of midpieces and principal pieces of the demembranated sperm were examined by electron microscopy. With 0.20% Triton X‐100 in the demembranation medium, 86% of the cross‐sections showed no plasma membranes and the rest had broken plasma membranes. The remaining tail structures appeared to be morphologically intact. Assay of phosphoglucose isomerase as a marker enzyme confirmed that at least 98% of the cytosolic protein was removed. Ram sperm models obtained by this procedure could be reactivated, and the percent motility and beat parameters were similar to those of the intact sperm. Reconstitution with the detergent‐soluble components was neither required for, nor enhanced, reactivation. Therefore, demembranated ram sperm do not require a detergent‐soluble protein factor for reactivation. © 1993 Wiley‐Liss, Inc.
Colchicine‐induced stimulation of PMN motility related to cytoskeletal changes in actin, α‐actinin, and myosin
Authors: Keller H. U. , Niggli V. 
Published: February 2005

AbstractColchicine‐induced stimulation of polymorphonuclear leukocyte (PMN) locomotion is an interesting model because extension of blebs at the front occurs at a rate (about 2.4 μm/s) which is far above that reported for growth of actin filaments. The following cytoskeletal changes were observed in colchicine‐treated PMNs: (1)a small increase in cytoskeleton‐associated actin was noted, as well as a somewhate more pronounced increase in cytoskeleton‐associated α‐actinin, as compared with untreated or DMSO‐treated controls. There was, however, no measurable increase in F‐actin as determined by NBD‐phallacidin blinding; (2)the values for the ratio (α‐actinin/actin) are lower in PMNs treated with colchicine for 30 min, as compared with PMNs stimulated with fNLPNTL for 1 minute (non‐polar ruffling cells) or 30 min (polarized locomoting cells); thus, this ratio may depend on the type of PMN motility; (3) in polarized PMNs F‐actin was mainly located linearly all along the cell membrane; there was more intense staining at the front of the cells; (4) α‐actining appeared to colocalize with F‐actin at the leading front, but not with F‐actin at the tail of polarized cells; (5) myosin was preferentially found at the rear part of polarized cells but not or only to a small extent at the front. Our data indicate a close functional correlation between microtubules and microfilaments. We speculate that F‐actin in combination with α‐actinin promotes expansion of pseudopods, whereas myosin combined with F‐actin promotes contraction. In more general terms we suggest that different forms of PMN motility are generated by differential selective interaction of cytoskeletal compnents and variations in the composition of the cytoskeleton in different sites of the same cells. © 1993 Wiley‐Liss, Inc.
βIV is the major β‐tubulin isotype in bovine cilia
Authors: Renthal Robert , Schneider Barbara G. , Miller Margaret M. , Ludueña Richard F. 
Published: February 2005

AbstractFour different isotypes of β‐tubulin are known to be expressed in mammalian brain. Monoclonal antibodies against βII, βIII, and βIV were used to characterize the β‐tubulin isotypes in two ciliated bovine tissues: non‐motile sensory cilia of retinal rod cells and motile cilia of tracheal epithelium. Retinal rod outer segment (ROS) connecting cilia and cytoskeletons were purified by density gradient centrifugation. This preparation contained more than 20 major protein protein components, as shown by dodecyl sulfate polyacrylamide gel electrophoresis. Electroblots were used to quantitate the relative amounts of βII, βIII, and βIV. The connecting cilium and cytoskeleton of the rod outer segment has less type III β‐tubulin than brain and more type IV. The ratio of βIV to βII in the ROS is nearly a factor of 8 larger than in brain. Electron microscopic immunocytochemistry showed extensive labeling of cilia by anti‐type IV in thin sections of retinas and trachea, and also in purified ROS cilia and cyoskeletons. Labeling of cilia by anti‐βII was also observed, although in the purified ROS cilia and cytoskeleton, the anti‐βII labeling was primarily on amorphous non‐ciliary material. The results suggest that both motile and non‐motile cilia are enriched in the type IV β‐tubulin subunit. © 1993 Wiley‐Liss, Inc.
Analysis of the elongation of the coiled filamentous structure, the truncated cone, during the acrosome reaction of abalone sperm by computer simulation and quick‐freeze, deep‐etch electron microscopy
Authors: Sakai Yoshi T. , Shinagawa Yoshihisa , Shiroya Yoko , Kunii Tosiyasu L. 
Published: February 2005

AbstractA novel structure named the truncated cone was located in the apex of the acrosomal vesicle right beneath the outer acrosomal membrane of abalone sperm head. This truncated cone structure was composed of about 12 helically coiled filaments, each 3.5–3.6 μm long with a diameter of 8–12 nm, forming a tightly compressed helicoid. During the acrosome reaction, the truncated cone elongated more than three times the original height and transformed into a thin cylinder by further coiling up of the filaments from the initial 2.5 to final 5 turns. The diameter and the lenght of each filament did not change during the elongation of the truncated cone into thin cylinder.Calculation from the equation of helical movement (spiral motion) applying the actual values of the truncated cone structure measured by electron microscopy gave the theoretical values nearly coincident with the actual measurements. The computer animation simulated the process of the movement of the coiled filaments composing the truncated cone and suggested that the elongation of the truncated cone into thin cylinder can be elucidated as a helical movement of the coiled filaments keeping their length constant.Quick‐freeze, deep‐etch electron microscopy further revealed that each of the coiled filaments was characterized by its beaded configuration, closely resembling that of the intermediate filametns of our previous results by immunoelectron microscopy and immunoblot analysis. The movement of the helically coiled filaments of the truncated cone may provide first example of the intermediate filaments to participate in motility and fertilization. © 1993 Wiley‐Liss, Inc.
Multiple sites for subtilisin cleavage of tubulin: Effects of divalent cations
Authors: Lobert Sharon , Hennington Bettye Sue , Correia John J. 
Published: February 2005

AbstractLimited digestion of pig brain GDP‐tubulin by subtilisin was carried out in the presence of Mg2+, Mn2+, Ca2+, Zn2+, or Be2+. Isoelectric focusing, followed by SDS‐PAGE, revealed characteristic divalent cation‐dependent changes in the α‐ and β‐tubulin cleavage patterns. Previous studies revealed that the β‐cleavage pattern is different for heterodimers and microtubules [Lobert and Correia, 1992: Arch. Biochem. Biophys. 296:152–160]. Divalent cation effects on subtilisin digestion of tubulin indicate different classes of divalent cation binding sites. Western blot analysis locates the proteolytic zone at residue 430 or higher in both subunits for all conditions. Turbidity and electron microscopy reveal that GDP‐tubulin cleaved by subtilisin in the presence of Mg2+, Ca2+, or Mn2+ forms sheets of rings. Mn2+ induces ring formation in uncleaved GDP‐tubulin. Isotype‐depleted tubulin was generated by the removal of class III β‐tubulin using immunoaffinity chromatography. Subtilisin digestion of the depleted fraction and the purified class III β‐tubulin demonstrates that cleavage occurs at three to four distinct sites. Thus, subtilisin‐digested tubulin is more heterogeneous than was previously reported and the cleavage sites depend on solution conditions, divalent cations, and the state of assembly. This has important implications for experiments that utilize subtilisin‐digested tubulin for studying microtubule‐associated protein binding. © 1993 Wiley‐Liss, Inc.
Recent quantitative studies of actin filament turnover during cell locomotion
Authors: Zigmond Sally H. 
Published: February 2005

AbstractCell locomotion depends on polymerization and depolymerization of filamentous actin. Net polymerization at the cell front occurs fast enough to fill the extending lamellipod, and since total F‐actin is essentially constant over time, depolymerization must equal polymerization. Indeed, the fastest moving cell types have the highest rates of depolymerization. Accounting for the high rate of depolymerization raises several problems. One is that net depolymerization requires the concentration of G‐actin to be low (below the critical concentration), but rapid polymerization (occurring <1 μm away) requires the concentration of G‐actin to be high (well above the critical concentration). This may be accomplished by spatial compartmentalization of factors that favor polymerization or depolymerization, and/or by proteins that bind G‐actin and prevent spontaneous polymerization while allowing barbed‐end elongation. A second problem is that depolymerization proceeds faster than would seem possible from studies of F‐actin in vitro (as calculated from number and lenghts of filaments present and in vitro rate constants). Rapid depolymerization may be accomplished by filament cutters or by cytoplasmic components (as yet undiscovered) that increase the rate of depolymerization. © 1993 Wiley‐Liss, Inc.
Preferential dendritic localization of pericentriolar material in hippocampal pyramidal neurons in culture
Authors: Ferreira Adriana , Palazzo Robert E. , Rebhun Lionel I. 
Published: February 2005

AbstractCentrosomes are unique cytoplasmic structures which serve as microtubule organizing centers (MTOC). In most animal cells centrosomes consist of one or more pair of centrioles surrounded by electron dense amorphous pericentriolar material (PCM) responsible for nucleation of microtubules. In the present study we analyzed the pattern of induction and localization of proteins of the PCM at different stages of neuronal development in cell cultures prepared from the embryonic hippocampus. For this purpose we used a human polyclonal antibody that recognizes two proteins of the PCM (100 kd and 60 kd, respectively). The results indicate that in mature neurons, pericentriolar immunoreactive material is preferentially localized in dendritic processes, and that throughout the course of neurite development and differentiation it is systematically excluded from the neuron's axon. Western blot analysis showed that during neuronal development in situ, there is an increase in he immunoreactivity for both proteins recognized by this antibody. In contrast, in hippocampal pyramidal neurons that develop in culture, there is an increase in the 60 kd polypeptide, while the 100 kd one is not detected after 7 days in vitro. © 1993 Wiley‐Liss, Inc.
Dynamics of organelles in the mitotic spindles of living cells: Membrane and microtubule interactions
Authors: Waterman‐Storer Clare M. , Sanger Joseph W. , Sanger Jean M. 
Published: February 2005

AbstractThe distribution and dynamics of the membranous organelles in two cell types were investigated during cell division. Live cells (either PtK2 or LLC‐PK1) labeled with the vital dye 3,3′‐dihexyloxacarbocyanine iodide [DiOC6(3)] were observed via serial optical sectioning with the laser‐scanning confocal microscope. Z‐series of labeled, dividing cells were collected every 1–2 minutes throughout mitosis, beginning at prophase and extending to the spreading of the daughter cells. Membrane distribution began to change from the onset of prophase in both cell types. When the mitotic spindle formed in prometaphase, fine tubular membranes, similar to those extending out to the edges of interphase cells aligned along the kinetochore spindle fibers. The lacy polygonal network typical of interphase cells persisted beneath the spindle, and a membrane network was also associated with the dorsal layer of the cell. As PtK2 cells reached metaphse, their spindles were nearly devoid of membrane staining, whereas the spindles of LLC‐PK1 cells contained many tubular and small vesicular membranous structures. X–Z series of the LLC‐PK1 metaphase spindle revealed a small cone of membranes that was separated from the rest of the cytoplasm by kinetochore MTs. In both cell types, as chromosome separation proceeded, the interzone remained nearly devoid of membranes until the onset of anaphase B. At this time the elongating interzonal microtubules were closely associated with the polygonal network of endoplasmic reticulum. Cytokinesis caused a compression, and then an exclusion of organelles from the midbody. Immunofluorescence staining with anti‐tubulin antibodies suggested that spindle membranes were associated with microtubules throughout mitosis. In addition, taxol induced a dense and extensive collection of small vesicles to collect at the spindle poles of both cell types. Nocodazole treatment induced a distinct loss of organization of the membranous components of the spindles. Together these results suggest that microtubules organize the membrane distribution in mitotic cells, and that this organization may vary in different cell types depending on the quantity of microtubules within the spindle. © 1993 Wiley‐Liss, Inc.
Probing actin incorporation into myofibrils using Asp11 and His73 actin mutants
Authors: Xia Dong , Peng Bonnie , Sesok Deborah A. , Peng Isaac 
Published: February 2005

AbstractWe used a cell free system [Bouché et al.: J. Cell Biol. 107:587–596, 1988] to study the incorporation of actin into myofibrils. We used α‐skeletal muscle actin and actins with substitutions of either His73 [Solomon and Rubenstein: J. Biol. Chem. 262:11382, 1987], or Asp11 [Solomon et al.: J. Biol. Chem. 263:19662, 1988]. Actins were translated in reticulocyte lysate and incubated with myofibrils. The incorporated wild type actin could be cross‐linked into dimers using N,N′‐1,4‐phenylenebismaleimide (PBM), indicating that the incorporated actin is actually inserted into the thin filaments of the myofibril. The His73 mutants incorporated to the same extent as wild type actin and was also cross‐linked with PBM. Although some of the Asp11 mutants co‐assembled with carrier actin, only 1–3% of the Asp11 mutant actins incorporated after 2 min and did not increase after 2 hr. Roughly 17% of wild type actin incorporated after 2 min and 31% after 2 hr. ATP increased the release of wild type actin from myofibrils, but did not increase the release of Asp11 mutants. We suggest that (1) the incorporation of wild type and His73 mutant actins was due to a physiological process whereas association of Asp11 mutants with myofibrils was non‐specific. (2) the incorporation of wild type actin involved a rapid initial phase, followed by a slower phase, and (3) since some of the Asp11 mutants can co‐assemble with wild type actin, the ability to self‐assemble was not sufficient for incorporation into myofibrils. Thus, incorporation probably includes interaction between actin and a thin filament associated protein. We also showed that incorporation occurred at actin concentrations which would cause disassembly of F‐actin. Since the myofibrils did not show large scale disassembly but incorporated actin, filament stability and monomer incorporation are likely to be mediated by actin associated proteins of the myofibril. © 1993 Wiley‐Liss, Inc.
Coactosin, a 17 kDA F‐actin binding protein from Dictyostelium discoideum
Authors: de Hostos E. L. , Bradtke B. , Lottspeich F. , Gerisch G. 
Published: February 2005

AbstractA 17 kDa protein, designated as coactosin, has been purified from an actinmyosin complex reconstituted in vitro from a soluble fraction of Dictyostelium discoideum cells. The protein binds to F‐actin in vitro without significantly altering its viscosity. Immunoblots labeled with monoclonal antibodies indicate that part of the protein is associated with the detergent‐insoluble cytoskeleton. cDNA clones comprising the entire coding region of coactosin have been isolated from an expression library. The cDNA‐derived amino‐acid sequence reveals similarities of coactosin to the drebrins identified in neurons and to actin‐binding proteins from other organisms, including yeast ABP1p, and yeast and vertebrate cofilins. © 1993 Wiley‐Liss, Inc.
Sarcomeric myosin heavy chain expressed in nonmuscle cells forms thick filaments in the presence of substoichiometric amounts of light chains
Authors: Vikstrom Karen L. , Rovner Art S. , Saez Claudia G. , Bravo‐Zehnder Marcela , Straceski Anthony J. , Leinwand Leslie A. 
Published: February 2005

AbstractCentral to the function of myosin is its ability to assemble into thick filaments which interact precisely and specifically with other myofibrillar proteins. We have established a novel experimental system for studying myofibrillogenesis using transient transfections of COS cells, a monkey kidney cell line. We have expressed both full‐length rat α cardiac myosin heavy chain (MHC) and a truncated heavy meromyosin‐like α MHC (sHMM) and shown that immunoreactive MHC proteins of the expected sizes were detected in lysates of transfected cells. Surprisingly, the full‐length MHC formed large spindle‐shaped structures throughout the cytoplasm of transfected cells as determined by immunofluorescence microscopy. The structures were not found in cells expressing the sHMM construct, indicating that their formation required an MHC rod. The spindle‐shaped structures ranged in length from approximately 1 μm to over 20 μm in length and were birefringent suggesting that they are ordered arrays of thick filaments. This was confirmed by electron microscopic analysis of the transfected cells which revealed arrays of filamentous structures approximately 12 nm in diameter at their widest point. In addition, the vast majority of transfected MHC did not associate with the endogenous nonmuscle myosin light chains, demonstrating that myosin thick filaments can form in the absence of stoichiometric amounts of myosin light chains. © 1993 Wiley‐Liss, Inc.
In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants
Authors: Novy Robert E. , Sellers James R. , Liu Li‐Fei , Lin Jim Jung‐Ching 
Published: February 2005

AbstractAt least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsmα) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full‐length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5. PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F‐actin was stronger than the PEThTM3 isoform. However, analysis of actin‐binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N‐terminal fragment of hTM5 fused to the C‐terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N‐terminal fragment of hTM3 fused to the C‐terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F‐actin than their parental tropomyosins. A bacterially made C‐terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin‐binding of these bacterially expressed tropomyosins. However, PETCaD39′s enhancement of binding to F‐actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low Mr isoform PEThTM5 appeared to be able to amplify the actin‐activated HMM ATPase activity by 4.7 fold, while the high Mr isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low Mr isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell. © 1993 Wiley‐Liss, Inc.