bluuebunny/crossref_metadata_2025_split huggingface dataset:
Examination of the calcium‐modulated protein S100α and its target proteins in adult and developing skeletal muscle
Authors: Zimmer Danna B. 
Published: February 2005

AbstractIn this study radioimmunoassay, immunohistochemistry, Northern blot analysis, and a gel overlay technique have been used to examine the level, subcellular distribution, and potential target proteins of the S100 family of calcium‐modulated proteins in adult and developing rat skeletal muscles. Adult rat muscles contained high levels of S100 proteins but the particular form present was dependent on the muscle type: cardiac muscle contained exclusively S100α, slow‐twitch skeletal muscle fibers contained predominantly S100α, vascular smooth muscle contained both S100α and S100β, and fast‐twitch skeletal muscle fibers contained low but detectable levels of S100α and S100β. While the distribution of S100 mRNAs paralled the protein distribution in all muscles there was no direct correlation between the mRNA and protein levels in different muscle types, suggesting that S100 protein expression is differentially regulated in different muscle types. Immunohistochemical analysis of the cellular distribution of S100 proteins in adult skeletal muscles revealed that S100α staining was associated with muscle cells, while S100β staining was associated with nonmuscle cells. Radioimmunoassays of developing rat skeletal muscles demonstrated that all developing muscles contained low levels of S100α at postnatal day 1 and that as development proceeded the S100α levels increased. In contrast to adult muscle, S100α expression as confined to fast‐twitch fibers in developing skeletal muscle until postnatal day 21. At postnatal day 1, developing contractile elements were S100α positive, but no staining periodicity was detectable. At postnatal day 21, S100α exhibited the same subcellular localization as seen in the adult: colocalization with the A‐band and/or longitudinal sarcoplasmic reticulum. Comparison of the S100α‐binding protein profiles in fast‐ and slow‐twitch fibers of various species revealed few, if any, species‐ or fiber type‐specific S100 binding proteins. Isolated sarcoplasmic reticulum fractions and myo fibrils contained multiple S100α‐hinding proteins. The colocalization of S100α and S100α‐binding proteins with the contractile apparatus and sarcoplasmic reticulum suggest that S100α may regulate excitation and/or contraction in slow‐twitch fibers.
Buffer conditions and non‐tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulin
Authors: Simon John R. , Parsons Stephen F. , Salmon E. D. 
Published: February 2005

AbstractThe dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video‐enhanced, differential‐interference contrast (VE‐DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 μM) displayed only plus‐ended growth. The elongation velocity was 7.8 μm/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 μm/ min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 μM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 μM) were assayed in ultrafiltiates (MW cut‐off <30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 μm/min for 26 min before a catastrophe and rapid shortening at 11.8 μm/min. Rescue was less frequent than unfiltered extracts, minus‐ended growth was observed, and self‐assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non‐buffer factors which stimulate elongation velocity by 6.5‐fold without self‐assembly, increase catastrophe frequency by 20‐fold, and block minus‐ended growth.
Functional coupling to brush border creatine kinase imparts a selective energetic advantage to contractile ring myosin in intestinal epithelial cells
Authors: Gordon Phillip V. , Keller Thomas C. S. 
Published: February 2005

AbstractThe B‐CK isozyme of cytoplasme creatine kinase is localized distinctly in the terminal web region of the intestinal epithelial cell brush border (Keller and Gordon: Cell Motil. Cytoskeletoa 19:169–179, 1991). Experiments were performed to determine whether this CK is energetically coupled to the myosin II that is present in the circumferential ring and interrootlet structural domains of the brush border terminal web. In isolated brush borders, ATP‐dependent circumferential ring contraction and interrootlet myosin solubilization were supported either by an exogenous PEP‐pyruvate kinase‐based ATP‐regeneration system (PEP‐PK) or by the addition of phosphocreatine to the endogenous B‐CK‐based ATP‐regeneration system (PCr‐B‐CK). Addition of an exogenous hexokinase‐glucose ATP‐hydrolysis system (HK‐G) effectively blocked both contraction and myosin solubilization in the PEP‐PK assay. In contrast, HK‐G had no significant effect on PCr‐B‐CK‐supported brush border contraction, although it did inhibit interrootlet myosin solubilization. Thus, when high‐energy phosphate is supplied as phos‐phocreatine, brush border B‐CK imparts to the circumferential ring myosin a selective energetic advantage over other ATPases. These results suggest that myosin and B‐CK are functionally coupled in the brush border circumferential ring, where they might comprise one end of an energy circuit that supplies energy for contraction, but that colocalizaton of CK with myosin in the brush border interrootlet domain is insufficient to establish functional coupling.
Cytochalasins induce actin polymerization in human leukocytes
Authors: Rao K. Murali Krishna , Padmanabhan Jaya , Cohen Harvey J. 
Published: February 2005

AbstractWe studied the effect of cytochalasins (B, D, and E) on the F‐actin content in human neutrophils and lymphocytes using NBD‐phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration‐ and time‐dependent increase in the F‐actin content in both cell types. The order of potency was cytochalasin D > E > B. The increase in F‐actin content was accompanied by a decrease in the G‐actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin‐binding or sequestering proteins. 2‐deoxyglucose (20 mM) caused a decrease in the basal F‐actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than current models suggest.
An immunoreactive homolog of mammalian kinesin in Nicotiana tabacum pollen tubes
Authors: Tiezzi Antonio , Moscatelli Alessandra , Cai Giampiero , Bartalesi Alessandro , Cresti Mauro 
Published: February 2005

AbstractA cytoskeletal apparatus is involved in the movement of vesicles, organelles, and gametes in the pollen tube. The function of microfilaments has been defined quite precisely, but the role of microtubules needs to be further clarified. On the basis of immunological and biochemical investigations, we have identified a polypep‐tide showing common properties with kinesin, a microtubule‐based motor mainly described in nonplant tissues, in the pollen tube of Nicotiana tabacum. Like mammalian kinesin, the kinesin‐immunoreactive homolog from Nicotiana tabacum pollen tubes binds to mammalian microtubules in an AMP‐PNP dependent manner. The kinesin‐like component is likely to be involved in the movement of vesicular material in the growing pollen tube.
Isolation and characterization of a unique 15 kilodalton trypanosome subpellicular microtubule‐associated protein
Authors: Balaban N. , Goldman R. 
Published: February 2005

AbstractA protein of 15 kDa (p15) was isolated from Trypanosoma brucei subpellicular microtubules by tubulin affinity chromatography. The protein bound tubulin specifically both in its native form and after SDS‐PAGE in tubulin overlay experiments. p15 promoted both the in vitro polymerization of purified calf brain tubulin and the bundling of preformed mammalian microtubules. Immunolabeling identified p15 at multiple sites along microtubule polymers comprising calf brain tubulin and p15 as well as on the subpellicular microtubules of cryosectioned trypanosomes. Antibodies directed against p15 did not cross react with mammalian microtubules. It is suggested that p15 is a trypanosome‐specific microtubule‐associated protein (MAP) that contributes to the unique organization of the sub‐pellicular microtubules.
Characterization of tetramethylrhodaminyl‐phalloidin binding to cellular F‐actin
Authors: Cano Manuel L. , Cassimeris Lynne , Joyce Michael , Zigmond Sally H. 
Published: February 2005

AbstractFluorescent derivatives of phallcidin are widely used to measure filamentous actin (F‐actin) levels and to stabilize F‐actin. We have characterized the kinetics and affinity of binding of tetramethylrhodaminy (TRITC)‐phalloidin to rabbit skeletal muscle F‐actin and to F‐actin in lysates of rabbit polymorphonuclear leukocytes (PMNs). We have defined conditions where TRITC‐phalloidin can be used to inhibit F‐actin depolymerization and to quantify F‐actin without prior fixation. By equi librium measurements, the affinity of TRITC‐phalloidin binding to rabbit skeletal muscle F‐actin (pyrene labeled) or to PMN lysate F‐actin was 1–4 × 10−7 M. In both cases, the stoichiometry of binding was approximately 1:1. Kinetic measurements of TRITC‐phalloidin binding to PMN lysate F‐actin resulted in an association rate constant of 420 ± 120 M−1 sec−1 and a dissociation rate constant of 8.3 ± 0.9 ± 10−5 sec−1. The affinity calculated from the kinetic measurements. (2 ± 1 × 10−7 M) agreed well with that obtained by equilibrium measurements. The rate with which 0.6 μM TRITC‐phalloidin inhibited 0.1 μM pyrenyl F‐actin depolymerization (90% inhibition in 10 sec) was much faster than the rate of binding to pyrenyl F‐actin (<1% bound in 10 sec), suggesting that phalloidin binds to filament ends more rapidly than to the rest of the filament. We show that TRITC‐phalloidin can be used to measure F‐actin levels in cell lysates when G‐actin is also present (i.e., in cell lysates at high concentrations) if DNase I is included to prevent phalloidin‐induced polymerization.
Reactivation of outer‐arm‐depleted lung axonemes: Evidence for functional differences between inner and outer dynein arms in situ
Authors: Hard Robert , Blaustein Kathy , Scarcello Lisa 
Published: February 2005

AbstractDemembranated axonemes isolated from newt lung ciliated cells show a complex beat frequency response to varying [MgATP] and temperature [Hard and Cypher, 1992, Cell Motil. Cytoskeleton 21:187–198]. The present study was undertaken to ascertain whether the beat frequency of outer‐arm‐depleted newt lung axonemes is controlled in a manner similar to that of intact axonemes. Populations of demembranated ciliary axonemes were isolated by Triton X‐100 extraction of lungs from the newt, Taricha granulosa. Aliquots of the demembranated axonemes were further treated with solutions containing high salt (0.375 M KCl) and 1.25 mM MgATP. This treatment resulted in the selective removal of outer dynein arms and a concomitant decrease in beat frequency to a stable level, 33–35% of control values. The effects of pH, salt concentration, nucleotides, and temperature on the beat frequency of reactivated outer‐arm‐depleted axonemes were ascertained and compared with those of intact axonemes. Some reactivation properties, such as nucleotide specificity, the effect of pH on beat frequency and the threshold [MgATP] required for reactivation (approximately 5 μM) were similar to those observed for intact axonemes. Other properties, such as the relationship between beat frequency and varying [MgATP] or salt concentration, differed both qualitatively and quantitatively from those of control axonemes, as did their response to temperature over the range, 5°–32°C. The nature of the results obtained with temperature and MgATP suggests that inner and outer dynein arms are not functionally equivalent in situ.
Actin in the ciliated protozoan Climacostomum virens: Purification by DNAse I affinity chromatography, electrophoretic characterization, and immunological analysis
Authors: Fahrni José F. 
Published: February 2005

AbstractThe anti‐actin monoclonal antibody (mab) JLA20 (Lin: Proc. Natl. Acad. Sci. U.S.A. 78:2335–2339, 1981) labels a 43 kD protein on Western blots of Climacostomum cell extracts; this protein does not react with an anti‐α‐smooth muscle actin mab (Skalli et al.: J. Cell Biol. 103:2787–2796, 1986) nor with an anti‐α‐sarcomeric actin mab (Skalli et al.: Am. J. Pathol. 130:515–531, 1988). This protein binds to DNAse I and can be purified by DNAse I affinity chromatography. The affinity‐purified actin also reacts with mab JLA20. Two‐dimensional gel analysis reveals that Climacostomum actin focuses as three spots which are more basic than the mammalian actin isoforms. After addition of KC1, the affinity‐purified actin polymerizes into filaments as shown by electron microscopy after negative staining. © 1992 Wiley‐Liss, Inc.
Polar organizers in monoplastidic mitosis of hepatics (Bryophyta)
Authors: Brown Roy C. , Lemmon Betty E. 
Published: February 2005

AbstractArchesporial cells of the hepaticMonoclea gottscheiLindb. undergo a series of monoplastidic mitotic divisions prior to enlarging into sporocytes. Interphase cells have a nuclear‐based endoplasmic microtubule system that is predominantly aligned in the long axis of the cell and lack a hoop‐like cortical system. No preprophase bands (PPBs) are formed. Prior to mitosis, the single plastid divides and daughter plastids move to the incipient spindle poles as is typical of monoplastidic cell division. However, the plastids do not serve as microtubule organizing centers (MTOCs) as they do in both mitosis and meiosis of hornworts and lycopsids, and in meiosis of mosses. Rather, microtubules of the developing spindle emanate from distinct polar organizers (POs) arising just outside the nuclear envelope as in polyplastidic mitosis in other hepatics. The POs, which appear to arise de novo on opposite ends of the nucleus during preprophase, consist of vesicles, endoplasmic reticulum, and radiating microtubules. The developmental and evolutionary significance of distinct POs, plastid MTOCs, and the diffuse MTOCs of higher plants is discussed. © 1992 Wiley‐Liss, Inc.
Actin dynamics during the cell cycle in Chlamydomonas reinhardtii
Authors: Harper John D. I. , McCurdy David W. , Sanders Mark A. , Salisbury Jeffrey L. , John Peter C. L. 
Published: February 2005

AbstractWe have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349–362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335–2339, 1981) identify Chlamydomonasactin as a ∼43,000‐Mr protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415–430, 1985) first identified Chlamydomonas actin using NBD‐phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F‐actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the ante‐ rior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrange‐ ment forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F‐actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis. © 1992 Wiley‐Liss, Inc.
Association of the β isoform of protein kinase C with vimentin filaments
Authors: Spudich Annamma , Meyer Tobias , Stryer Lubert 
Published: February 2005

AbstractProtein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305–312, 1986; Nature 334:661–665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621–623, 1983). The β isoform of PKC is translocated and degraded much more rapidly than the β isoform in phorbolester‐stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238–4243, 1989). We report here immunofluorescence evidence that the distributions of PKC α and β are strikingly different in antigen‐activated RBL cells. PKC β associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC β concentrates in regions of the cell periphery. This distribution of PKC β is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti‐tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC β and to the intermediate filament protein vimentin are almost identical, indicating that PKC β associates with vimentin filaments. These bundles of 100 Å filaments may provide docking sites for interactions of PKC β with its substrates and thus confer specificity to the actions of this isoform. © 1992 Wiley‐Liss, Inc.
Three microtubule‐organizing centers are required for ascus growth and sporulation in the fungus Sordaria macrospora
Authors: Thompson‐Coffe Catherine , Zickler Denise 
Published: February 2005

AbstractThe microtubule system of the Sordaria macrospora ascus was examined by antitubulin immunofluorescence, without the removal of the cell wall. The complex cytoskeleton revealed three possible microtubule‐organizing centers (MTOCs): the spindle pole body (SPB), the nuclear envelope, and an apical organizing center. MPM‐2, a mitotic phosphoprotein antibody which reacts with MTOCs, stained the apical center in a developmentally specific manner, and the nuclear envelope and SPB in a cell cycle‐dependent fashion. Nocodazole was used in both high (10–15 μg/ml) and low (0.5 μg/ml) concentrations to depolymerize the networks and reveal their points of origin and recovery. The apical center was active from prophase I to the end of first meiosis. The nuclear envelope was the site of microtubule nucleation in early prophase and at the telophase/interphase transition, while SPBs were active in both nuclear division and sporulation.Mutant strains deficient in sporulation and with aberrant morphology were analyzed by antitubulin and MPM‐2 immunofluorescence. Shape mutants showed abnormal or absent apical organizing centers and abnormal cortical microtubule patterns, indicating a possible role for the cortical network in the establishment and maintenance of ascus shape. © 1992 Wiley‐Liss, Inc.
Distribution of detyrosinated microtubules in motile NRK fibroblasts is rapidly altered upon cell‐cell contact: Implications for contact inhibition of locomotion
Authors: Nagasaki T. , Chapin C. J. , Gundersen G. G. 
Published: February 2005

AbstractFibroblasts migrating into an experimental wound contain an extensive array of detyrosinated microtubules (Glu MTs) oriented in the direction of migration, whereas nonmotile cells in the interior of a monolayer contain Glu MTs that are primarily coiled around the nucleus. To determine the role of cell‐cell contact in the formation of these distinct arrays of Glu MTs, we studied the distribution of Glu MTs by immunofluorescence in NRK fibroblasts that had been fixed at different intervals after they had established contact with other cells. Time‐lapse video recordings were made of the contacting cells to provide a record of cellular behavior. In motile cells that became completely surrounded by virtue of contact with other cells, Glu MTs were found mostly coiled around the nucleus. The proportion of cells whose Glu MTs extended to the original leading edge decreased dramatically after the cells had been surrounded for 10 min or more. At earlier times, when the contact was confined to a portion of the cell margin, Glu MTs were absent from the area behind the contact site, yet were still oriented toward the noncontacting and ruffling margins. The contact‐induced alteration of Glu MTs was not due to the cessation of forward locomotion of cells per se, since immobilization of cells with cytochalasin D did not cause a dramatic change in Glu MTs. That cell‐cell contact also specifies the type of Glu MTs formed in cells was shown by experiments in which MTs were regrown following complete depolymerization with nocodazole. The remodeling of Glu MTs during cell‐cell contact may be involved in cellular repolarization during contact inhibition of locomotion and will be a useful marker for further dissecting the molecular events of contact inhibition of motility. © 1992 Wiley‐Liss, Inc.
A high molecular weight centrosomal protein of mammalian cells is antigenically related to myosin II
Authors: Bailly Eric , Bordes Nicoles , Bornens Michel , Klotz Catherine 
Published: February 2005

AbstractAvailable data on the molecular composition of the centrosome, the typical microtubule‐organizing center of animal cells, are still fragmentary. To address this important issue we have taken advantage of centrosome isolation from a human lymphoblastic cell line (KE37) to generate a monoclonal antibody (mAb) library. Here we present the characterization of one of these mAbs (CTR56). On the basis of both its immunofluorescence staining pattern and its reactivity with a major 200 kD antigen on immunoblots, CTR56 has been tentatively classified as an anticellular myosin heavy chain. In light of cytological and biochemical data obtained in parallel with two other well‐characterized myosin antibodies, it appears that myosin cannot be considered as a genuine centrosomal protein. We have resolved the paradoxical results with CTR56 by showing that in addition to the cellular myosin heavy chain, this antibody also recognizes a high molecular weight protein specifically enriched in centrosomal fractions. The possible biological significance of this finding is discussed in structural and functional terms. © 1992 Wiley‐Liss, Inc.