bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glutamylated tubulin probed in ciliates with the monoclonal antibody GT335
Authors: Bré Marie Hélène , de Néchaud Béatrice , Wolff Annie , Fleury Anne 
Published: February 2005

AbstractMicrotubular networks are extensively developped in many ciliate species. In several of them, we investigate the occurrence of the post‐translational glutamylation of tubulin [Eddé et al., 1990: Science 247:82–85; Eddé et al., 1991: J. Cell. Biochem. 46:134–142] using as a probe for such modified tubulin, the monoclonal antibody GT335 [Wolff et al., 1992: Eur. J. Cell Biol. 59:425–432]. Results obtained in Paramecium strongly suggest that both axonemal and cytoplasmic tubulin are glutamylated. As in the vertebrate brain tubulin so far tested, the GT335 epitope is located at the carboxy‐terminal fragment of cytoplasmic tubulin removed by subtilisin treatment. Immunoblotting and immunofluorescence experiments reveal that, unlike tubulin acetylation, glutamylation is not restricted to cold‐resistant microtubules. In addition, immunofluorescence studies performed on dividing cells show that glutamylation takes place soon after the polymerization of microtubules.Finally, glutamylated tubulin is also detected in the ciliate species Euplotes, Tetrahymena, and Paraurostyla. Together with results obtained on flagellate species, this suggests that tubulin glutamylation came out early in the course of eukaryotic evolution and has been widely exploited in various cellular strategies. © 1994 Wiley‐Liss, Inc.
Structural and biochemical properties of kinesin heavy chain associated with rat brain mitochondria
Authors: Jellali Abdeljelil , Metz‐Boutigue Marie‐Hélène , Surgucheva Irina , Jancsik Veronika , Schwartz Christian , Filliol Dominique , Gelfand Vladimir I. , Rendon Alvaro 
Published: February 2005

AbstractKinesin, a mechanochemical enzyme that translocates membranous organelles, was initially identified and purified from soluble extracts from vertebrate brains. However, immunocytochemical and morphological approaches have demonstrated that kinesin could be associated to intracellular membranous organelles. We used an antibody raised against the head portion of the Drosophila kinesin heavy chain to reveal the presence of this protein in membranous organelles from rat brain. By using differential centrifugation and immunoblotting we observed a 116 kDa protein that crossreacts with this antibody in microsomes, synaptic vesicles, and mitochondria. This protein could be extracted from mitochondria with low salt concentrations or ATP. The 116 kDa solubilized protein has been identified as conventional kinesin based on limited sequence analysis. We also show that a polyclonal antibody raised against mitochondria‐associated kinesin recognizes soluble bovine brain kinesin. The soluble and mitochondrial membrane‐associated kinesins show a different isoform pattern. These results are consistent with the idea that kinesin exists as multiple isoforms that might be differentially distributed within the cell. In addition digitonin fractionation of mitochondria combined with KI extraction revealed that kinesin is a peripheral protein, preferentially located in a cholesterol‐free outer membrane domain; this domain has the features of contact points between the mitochondrial outer and inner membranes. The significance of these observations on the functional regulation of the mitochondria‐associated kinesin is discussed. © 1994 Wiley‐Liss, Inc.
Purification of microtubule proteins from Xenopus egg extracts: Identification of a 230K MAP4‐like protein
Authors: Faruki Shamsa , Karsenti Eric 
Published: February 2005

AbstractWe describe the purification of microtubule proteins from Xenopus egg extracts by temperature‐dependent assembly and disassembly in the presence of dimethyl sulfoxide and identify a number of presumptive microtubule‐associated proteins (MAPs). One of these proteins has a molecular weight of 230 kDa and is immunologically related to HeLa MAP4. We show that this MAP is heat stable and phosphorylated, and that it promotes elongation of microtubules from axonemes. © 1994 Wiley‐Liss, Inc.
Cytoskeletal alterations in cultured cardiomyocytes following exposure to the lipid peroxidation product, 4‐hydroxynonenal
Authors: VanWinkle W. Barry , Snuggs Mark , Miller Joseph C. , Buja L. Maximilian 
Published: February 2005

AbstractDamage to the cardiac myocyte sarcolemma following any of several pathological insults such as ischemia (anoxia) alone or followed by reperfusion (reoxygenation), is most apparent as progressive sarcolemmal blebbing, an event attributed by many investigators to a disruption in the underlying cytoskeletal scaffolding. Scanning electron microscopic observation of tissue cultured rat neonatal cardiomyocytes indicates that exposure of these cells to the toxic aldehyde 4‐hydroxynonenal (4‐HNE), a free radical‐‐induced, lipid peroxidation product, results in the appearance of sarcolemmal blebs, whose ultimate rupture leads to cell death. Indirect immunofluorescent localization of a number of cytoskeletal components following exposure to 4‐HNE reveals damage to several, but not all, key cytoskeletal elements, most notably microtubules, vinculin‐containing costameres, and intermediate filaments. The exact mechanism underlying the selective disruption of these proteins cannot be ascertained at this time. Colocalization of actin indicated that whereas elements of the cytoskeleton were disrupted by increasing length of exposure to 4‐HNE, neither the striated appearance of the myofibrils nor the lateral register of neighboring myofibrils was altered. Monitoring systolic and diastolic levels of intracellular calcium ([Ca2+]i) indicated that increases in [Ca2+]i occurred after considerable cytoskeletal changes had already taken place, suggesting that damage to the cytoskeleton, at least in early phases of exposure to 4‐HNE, does not involve Ca2+ ‐dependent proteases. However, 4‐HNE–induced cytoskeletal alterations coincide with the appearance of, and therefore suggest linkage to, sarcolemmal blebs in cardiac myocytes.Although free radicals produced by reperfusion or reoxygenation of ischemic tissue have been implicated in cellular damage, these studies represent the first evidence linking cardiomyocyte sarcolemmal damage to cytoskeletal disruption produced by a free radical product. © 1994 Wiley‐Liss, Inc.
Characterization of keratin densities in mitotic wish cells
Authors: Meek William D. , Henderson David A. 
Published: February 2005

AbstractThree dimensional (3‐D) reconstruction of four mitotic WISH cells from ultrathin sections gave an informative representation of the spatial distribution of keratin densities in these cells. The correspondence between the densities as studied by transmission electron microscopy (TEM) and the Keratin bodies initially revealed by immunoflourescent colabeling of cultures, was confirmed by immunoelectron‐microscopy. The smaller, and sometimes more elongated densities, were relatively abundant just beneath the subplasmalemmal microfilament band; and at certain levels of the mitotic cell they were observed to be connected to neighboring densities by intact intermediate filaments (IFs). The larger and more spherical densities appeared to be somewhat more discrete and randomly distributed. Other observed associations of the keratin densities included the telophase contractile ring of microfilaments, chromosomes, the reformed telophase nucleus, and desmosomal junctions with neighboring interphase cells. Cytochalasin D (CD) treatment of cells displaced the peripheral keratin densities toward the cell membrane. The density volume constituted 0.52% to 1.57% of the total cell volume, and the proportional density size was decreased in the cells that had progressed into anaphase and telophase. The observed formation and subsequent dissolution of keratin densities during mitosis may represent a dynamic mechanism of restructuring the keratin cytoskeleton in an unpolymerized form in order to allow for rapid reformation of interphase cell junctions. The physical associations observed between intact IFs and the keratin densities may provide support at certain depths of the mitotic cell, and the juxtaposition of densities with nuclear components suggests a possible source of and role for keratin IFs during nuclear events. © 1994 Wiley‐Liss, Inc.
Complex intermolecular interactions maintain a stable linkage between the photoreceptor connecting cilium axoneme and plasma membrane
Authors: Muresan Virgil , Besharse Joseph C. 
Published: February 2005

AbstractMicrotubule‐membrane cross‐linkers in motile and nonmotile cilia are supramolecular structures, held together by strong interactions between the constituent molecules. We have characterized these interactions in the photoreceptor connecting cilium, where cross‐linkers co‐fractionate and maintain their in situ location after Triton X‐100 extraction of axonemes. In bovine photoreceptor cells, the transmembrane assemblage that is cross‐linked to the connecting cilium axoneme contains three high molecular mass glycoconjugates of 425, 600, and 700 kDa (Horst et al., 1987). The relative amounts of the three glycoconjugates, as judged from band intensity in electrophoretograms, depend strongly on sample treatment prior to electrophoresis. The electrophoretic pattern was reproducible after several weeks of storage of the axoneme fraction in extraction buffer containing 50% sucrose. Removal of sucrose from the buffer by dialysis eliminated the 600 kDa and 700 kDa, and decreased the detected amount of the 425 kDa glycoconjugate. When samples were incubated in Laemmli sample buffer at increasing temperatures (23°, 60°, 95°C), a gradual reduction in the intensity of the three bands was observed. The quantitative reduction of high molecular mass glycoconjugates was accompanied by the appearance of novel protein species of lower molecular mass, as detected by lectin and antibody overlays of axonemal transblots. These results suggest that the previously characterized cross‐linker glycoconjugates are complex, SDS‐resistant multi‐molecular conglomerates. We have further used fluorescent lectins to monitor the presence of glycoconjugates on whole‐mounted axonemes, in conditions aimed to selectively solubilize the cross‐linkers. The cross‐linker complexes could not be dissociated from the axoneme by incubation with buffers containing 1 M of either Na2SO4 or NaI. The results indicate that the connecting cilium‐specific cross‐linker complexes are bound via high‐affinity interactions to both axoneme and overlying plasma membrane. © 1994 Wiley‐Liss, Inc.
Movement of axoplasmic organelles on actin filaments from skeletal muscle
Authors: Kuznetsov Sergei A. , Rivera Domingo T. , Severin Fedor F. , Weiss Dieter G. , Langford George M. 
Published: February 2005

AbstractIt was recently shown that, in addition to the well‐established microtubule‐dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992, Nature 356:722‐725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video‐enhanced contrast differential interference contrast (AVEC‐DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine‐phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC‐DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments. © 1994 Wiley‐Liss, Inc.
Cloning and sequencing of cDNAs encoding the actin cross‐liking protein transgelin defines a new family of actin‐associated proteins
Authors: Prinjha R. K. , Shapland C. E. , Hsuan J. J. , Totty N. F. , Mason I. J. , Lawson D. 
Published: February 2005

AbstractWe have used degenerate oligonucleotides, derived from the amino acid sequence of transgelin peptides [Shapland et al., 1993: J. Cell Biol. 121:1065‐1073], to isolate and sequence overlapping cDNA clones encoding this actin gelling protein. Primers with 5′ restriction enzyme sites directed against the N and C terminal amino acids present in these clones were then used to amplify and clone the entire transgelin coding region from reverse transcribed rat small intestine cDNA (RT‐PCR). These studies have shown that transgelin is the product of a single gene which is conserved between yeast, Drosophila, molluscs, and humans. Transgelin is expressed as a single message that is regulated at the level of transcription in SV40 transformed 3T3 cells. Our data have shown that transgelin and several other proteins of unknown function, SM22α [Pearlstone et al., 1987: J. Biol. Chem. 262:5985‐5991], mouse p27 [Almendral et al., 1989: Exp. Cell Res. 181:518‐530], and human WS3‐10 [Thweatt et al., 1992: Biochem. Biophys. Res. Commun. 187:1‐7], share extensive homology. More limited regions of homology shared between transgelin and other proteins such as rat NP25 (unpublished), chicken calponins α and β [Takahashi and Nadal‐Ginard, 1991: J. Biol. Chem. 266:13284‐13288], and Drosophila mp20 [Ayme‐Southgate et al., 1989: J. Cell Biol. 108:521‐531] suggest that all of these proteins may be classified as members of a new transgelin multigene family. © 1994 Wiley‐Liss, Inc.
Human neutrophil motility: Time‐dependent three‐dimensional shape and granule diffusion
Authors: Felder Stephen , Kam Zvi 
Published: February 2005

AbstractThe locomotion of human polymorphonuclear leukocytes (PMNs) was studied with two complementary methods: Three‐dimensional shapes were reconstructed from time series of optical sectioning microscopy using differential interference contrast (DIC) optics, and the diffusion of cytoplasm granules within individual cells was measured using quasielastic laser light scattering (QELS). The three‐dimensional cell edges outlined in the optical sections were analyzed qualitatively in time‐lapse film strips and quantitatively from morphometry. The fastest locomotion occurred in chemotactic gradient with cell velocity that oscillated between 10 and 30 μm/min with a period of 50–55 seconds. Within the periodic bursts of speed, a fibroblast‐like locomotory cycle was observed, with leading lamella extended and contacts formed with the substrate surface, followed by rapid motion of the cell body and nucleus over the immobile contacts. Consistent with this apparent staged motion, correlation analysis revealed a phase lag of 2‐3 seconds in velocities between the bottom (ventral) and the top layers of the cell. In addition there was a tendency to a lower cell profile at times of higher velocity. The diffusion of natural cytoplasmic granules within resting PMNs was not affected by cytoskeleton disrupting drugs. During the stage of most rapid motion, when cytoplasmic streaming could be seen, diffusion of the granules decreased two‐ to 2.5‐fold, and then returned to resting levels. These observations suggest that PMN locomotion consists of extensions near the surface to form forward contacts and then stiffening or possibly contraction of the cytoskeleton when the body of the cell is moved forward.Three‐dimensional movies of PMN cells are included in the video supplement. © 1994 Wiley‐Liss, Inc.
Differential regulation of skeletal muscle myosin‐II and brush border myosin‐I enzymology and mechanochemistry by bacterially produced tropomyosin isoforms
Authors: Fanning A. S. , Wolenski J. S. , Mooseker M. S. , Izant J. G. 
Published: February 2005

AbstractIn this report, we have compared the physical properties and actin‐binding characteristics of several bacterially produced nonmuscle and striated muscle tropomyosins, and we have examined the effects of these isoforms on the interactions of actin with two structurally distinct classes of myosin: striated muscle myosin‐II and brush border (BB) myosin‐I. All of the bacterially produced nonmuscle tropomyosins bind to F‐actin with the expected stoichiometry and with affinities comparable to that of a tissue produced α‐tropomyosin, although the striated muscle tropomyosin CTm7 has a lower affinity of F‐actin than a tissue‐purified striated muscle α tropomyosin. The bacterially produced isoforms also protect F‐actin from severing by villin as effectively as tissue‐purified striated muscle α‐tropomyosin. The bacterially produced 284 amino acid striated muscle tropomyosin isoform CTm7, the 284 amino acid nonmuscle tropomyosin isoform CTm4, and two chimeric tropomyosins (CTm47 and CTm74) all inhibit the actin‐activated MgATPase activity of muscle myosin S1 by ∼ 70–85%, comparable to the inhibition seen with tissue‐purified striated muscle α tropomyosin. The 248 amino acid tropomyosin XTm4 stimulated the actin‐activated MgATPase activity of muscle myosin S1 approximately two‐ to threefold. The in vitro sliding of actin filaments translocated by muscle myosin‐II (2.4 μm/sec at 19°C, 5.0 μm/s at 24°C) increased 25–65% in the presence of XTm4. Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin‐II motility. The actin‐activated MgATPase activity of BB myosin‐I was inhibited 75–90% by all of the tropomyosin isoforms tested, including the 248 amino acid tropomyosin XTm4. BB myosin‐I motility (50 nm/s) was completely inhibited by both the 248 and 284 amino acid tropomyosins. These results demonstrate that bacterially produced tropomyosins can differentially regulate myosin enzymology and mechanochemistry, and suggest a role for tropomyosin in the coordinated regulation of myosin isoforms in vivo. © 1994 Wiley‐Liss, Inc.
Differential localization of α‐actinin and the 30 kD actin‐bundling protein in the cleavage furrow, phagocytic cup, and contractile vacuole of Dictyostelium discoideum
Authors: Furukawa Ruth , Fechheimer Marcus 
Published: February 2005

AbstractDictyostelium discoideum amoebae possess eight different actin crosslinking proteins. Immunofluorescence microscopy has been employed in this study to investigate the intracellular localization of two of these proteins, α‐actinin and the 30 kD actin‐bundling protein, to investigate whether they are redundant, or alternatively, make distinct contributions to cell structure and movement. The 30 kD protein is concentrated in the cleavage furrow of dividing cells, while enhanced staining for α‐actinin is not apparent in this region. By contrast, α‐actinin is concentrated around the contractile vacuole, while the 30 kD protein is not preferentially localized in the area of this organelle. Association of α‐actinin with the contractile vacuole was confirmed by colocalization with calmodulin, a marker of this organelle. There are temporal differences in the localization of the 30 kD protein and α‐actinin during phagocytosis. The 30 kD protein is localized in the phagocytic cup, but disassociates from phagosomes soon after internalization [Furukawa et al., 1992: Protoplasma 169: 18–27]. α‐actinin enters the phagocytic cup after the 30 kD protein, and remains associated with the phagosome after the 30 kD protein has disassociated. These results support the hypothesis that α‐actinin and the 30 kD protein play distinct roles in cell structure and movement in Dictyostelium. © 1994 Wiley‐Liss, Inc.
Fluorescence studies of spectrin and its subunits
Authors: Subbarao Nanda K. , MacDonald Robert C. 
Published: February 2005

AbstractTo better understand the solution structure of spectrin, the environments of its tryptophan residues have been examined by fluorescence spectroscopy. The spectra and the extent of quenching by several quenching agents have been determined for intact spectrin and its α and β subunits. The arsenal of quenchers used in the study represented both hydrophilic and hydrophobic species including anionic, cationic and neutral compounds. Effects on spectrin fluorescence of ethanol and ionic strength, which extend and/or rigidify spectrin, and of glycerol, which is commonly used in electron microscopy of the protein, have also been assessed in the presence and absence of quenchers. Most of the tryptophans of spectrin are either internally quenched or are sequestered, hindering the approach of hydrophilic quenching agents. Both the spectral shape and the extent of quenching by acrylamide indicate that some tryptophans of the β subunit are slightly more exposed in the isolated chain than in the dimer. Similar effects on spectra and on quenching of the intact dimer and of the isolated β chain are seen when the ionic strength is reduced. Ethanol and glycerol reduce spectrin tryptophan accessibility to 2‐p‐toluidinyl napthalene‐6‐sulfonic acid (TNS). It therefore appears that low ionic strength, α‐β association and neutral solute (or lowered dielectric constant) all induce a similar, but modest conformational change in the domain structure. The extent of TNS binding is not increased by lowering the ionic strength, suggesting that the expansion and/or stiffening of the molecule in low electrolyte solutions does not involve exposure of significant numbers of hydrophobic sites. © 1994 Wiley‐Liss, Inc.
Novel touch‐induced, Ca2+‐dependent phobic response in a flagellate green alga
Authors: Kreimer Georg , Witman George B. 
Published: February 2005

AbstractThe biflagellate green algaSpermatozopsis similisexhibits a remarkable avoidance reaction in addition to the photophobic or stop response characteristic of such algae.S. similisnormally swims forward with its anteriorly attached flagella directed posteriorly and propagating sine‐like waves from base to tip. Upon contact with surfaces or other cells,S. similisresponds with rapid backward swimming, covering distances of up to 50 μm in 140 to 220 msec. This reaction, which we term the mechanoshock response, also can be triggered by vigorous mechanical stimulation, but not by physiological light intensities. It consists of 3 phases: (1) a rapid acceleration phase with average duration of 31 msec; (2) a phase of about 66 msec with constant high speed (maximal velocities of > 600 μm·sec−1) or slow deceleration; and (3) a deceleration phase of ∼ 83 msec, followed by a stop or short period of circling. The cells then resume forward swimming in a random direction. Prior to the mechanoshock response the flagella rapidly are brought together into a close parallel configuration extending anteriorly of the cell body. They then appear to propel the cell by undulatory beating, while the cell describes a pronounced helical path. Small decreases in the extracellular Ca2+concentration, as well as low concentrations of Ba2+, strongly suppress the probability of this phobic reaction. We conclude that this mechanoshock response involves large Ca2+influxes, probably mediated by mechanosensitive and/or stretch‐activated ion‐channel(s). © 1994 Wiley‐Liss, Inc.