bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structure and function of the cytoskeleton in heliozoa: I. Mechanism of rapid axopodial contraction in Echinosphaerium
Authors: Ando Motonori , Shigenaka Yoshinobu 
Published: February 2005

AbstractIn the heliozoan Echinosphaerium, rapid axopodial contraction often occurs during food capturing. In morphological studies, it has been considered that rapid contraction is caused by conformation is change of the X‐body. We determined that reaction sites or receptors for anion exchange resin, which can induce rapid contraction, were located in every region of the axopodium. However, the probability of locating sites where contraction was induced was lower in the middle region than in the distal region of the axopodium. In cases in which contraction was not induced by resin placed in the middle region of the axopodium, so‐called bead formation was induced instead. At the fine structural level, only granulated forms of the X‐body were observed in this beading region. These results suggest that rapid contraction results from disassembly of axonemal microtubules and the simultaneous contraction of the X‐body and that bead formation represents a stage when only the X‐body contracts. Furthermore, effects of metabolic inhibitors and Ca2+ channel blockers revealed that contraction of the X‐body did not depend on the supply of adenosine triphosphate, but on Ca2+ influx. Ultrastructural observations revealed that the mass of the granulated X‐body was aggregated into the proximal region of the axopodium, suggesting that the X‐body might be associated with the undercoat of axopodial membrane or the axonemal microtubules themselves.
Binding of microtubule‐associated proteins (MAPs) to rat brain mitochondria: A comparative study of the binding of MAP2, Its microtubule‐binding and projection domains, and tau proteins
Authors: Jancsik Veronika , Filliol Dominique , Felter Simone , Rendon Alvaro 
Published: February 2005

AbstractTwo major brain microtubule‐associated proteins (MAPs), MAP2 and tau, were found to be able to bind to purified rat brain mitochondria. The apparent dissociation constants of the binding of thermostable 32P‐labeled MAP2 and tau are 0.9 ± 0.04 × 10−7 and 3.8 ± 0.7 × 10−7 M, respectively. 32P‐labeled MAP2 and tau bound to the mitochondria can be displaced by phosphorylated, nonradioactive MAP2. The binding parameters of MAP2 prepared without heat treatment and those of the thermostable MAP2 were of the same order of magnitude. Microtubule‐binding and projection domains of MAP2 were obtained by chymotryptic digestion of rat brain microtubules (Vallee, Proc. Natl. Acad. Sci. USA, 77:3206‐3210, 1980). Displacement studies with these two domains show that MAP2 bound to mitochondria can be displaced by the microtubule‐binding domain, whereas the projection domain does not displace MAP2. The two domains of MAP2 bind to the mitochondria with similar affinity constants; however, the Bmax for the projection domain was 10 times and 35 times lower than the Bmax of the binding of the intact MAP2 and the microtubule‐binding domain, respectively. Chymotryptic digestion of MAP2 bound to the mitochondria yielded peptide fragments with molecular masses similar to those obtained by the digestion of MAP2 bound to the microtubules. The fragments corresponding to the projection domain were released into the extramitochondrial supernatant, whereas the fragments originating from the microtubule‐binding domain remained bound to the mitochondria. These results suggest that MAP2 binds to mitochondria preferentially via its microtubule‐binding domain.
External mechanical control of the timing of bend initiation in sea urchin sperm flagella
Authors: Eshel Dan , Gibbons I. R. 
Published: February 2005

AbstractThe movement parameters of a sea urchin sperm flagellum can be manipulated mechanically by applying various modes of periodic vibrations to the sperm head held by suction in the tip of a micropipette. The beat frequency of the flagellum readily synchronizes with the frequency of the externally imposed lateral vibration, and the plane of flagellar bending waves adapts itself to the plane of the pipette vibration (Gibbons et al., J. Cell Biol. 101:270a, 1985; Nature 325: 351–352, 1987). In this study, we observed the particular effects of external asymmetric forces on flagellar beating parameters by vibrating the micropipette holding the sperm head in a transverse sawtooth‐like motion composed of a rapid effective stroke and a slower recovery stroke, while keeping the vibration frequency constant. The results demonstrate that the timing of bend initiation within the flagellar beat cycle can be controlled mechanically by changing the time point within the vibration cycle at which the micropipette changes its direction of motion. A switch in the sidedness of the asymmetric movement of the micropipette produces dramatic changes in the profiles of bend growth in the basal 5 μm of the flagellum but has almost no effect on the asymmetry or other parameters of bending in the mid‐ and distal regions of the flagellum. Our results suggest that elastic strain within the basal region of the flagellar structure may play a more significant role in the process of bend initiation than has been realized heretofore.
Rise of internal Ca2+ accompanies the initiation of trout sperm motility
Authors: Cosson Marie Paule , Billard Roland , Letellier Lucienne 
Published: February 2005

AbstractThe initiation of motility of trout spermatozoa is inhibited by the presence of millimolar concentrations of external K+, but external Ca2+ might also be implicated in this control as it has been shown to antagonize the K+ inhibition of motility [S.M. Baynes et al.: J. Fish. Biol., 19:259–267, 1981]. The present work aimed to investigate internal Ca2+ levels during the motility phase of trout spermatozoa. Internal Ca2+ concentrations were monitored by the fluorescent quinoline Ca2+‐indicator, “Quin‐2” [R. Y. Tsien: Nature 290:527–529, 1981]. Trout spermatozoa were loaded with Quin‐/ under conditions that gave efficient intracellular hydrolysis of Quin‐2 and that did not impair the ability of loaded spermatozoa to initiate movement. The beat frequencies, cell velocities, and flagellar asymmetries of sperm movement were not significantly modified by the presence of the internal dye. Upon initiation of flagellar movement, an increase of the internal Quin‐2 fluorescence was observed that reflected a sixfold increase of the free Ca2+ concentration. The free Ca2+ remained elevated after the cessation of movement. The variation of fluorescence was completed within 40 seconds, whereas the initiation of motility was nearly instantaneous, and the total duration of flageliar beating lasted for about 80–100 seconds (measurements at 11°C). The increase in the internal free Ca2+ concentration is completed after the initiation of flagellar beating but its occurrence correlates with that of sperm movement. Fluorescence increase was not observed in the presence of 40 mM K+, a condition in which spermatozoa did not initiate flagellar beating. In the presence of the Ca2+ channel blocker desmethoxyverapamil, neither sperm motility nor fluorescence increases were observed, which suggested that the increase of internal free Ca2+ was produced by a flux of external Ca2+ into the cell rather than by a mobilization of internal Ca2+ stores.
Molecular analysis of the alpha and beta dynein genes of Chlamydomonas reinhardtii
Authors: Mitchell David R. 
Published: February 2005

AbstractWe previously cloned portions of the alpha and beta dynein heavy chain genes of Chlamydomonas reinhardtii by screening a genomic expression library with monoclonal antibodies (Williams et al.: Journal of Cell Biology 103:1–11, 1986). Here we provide further evidence of the identity of these clones and describe the selection of adjacent regions from a large insert genomic library. Southern blots indicate that only a single copy of each gene is present in the Chlamydomonas genome, while Northern blots show that both heavy chains are encoded by 13.5 kilobase mRNAs and that the corresponding transcription units each span approximately 20 kilobase‐pairs of genomic DNA. No similarities were detected between restriction maps of the alpha and beta dynein genes, but extensive regions of sequence similarity were identified by the cross‐hybridization of cloned gene fragments.
Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish xanthophores
Authors: Walker Gary R. , Taylor John D. , Tchen T. T. 
Published: February 2005

AbstractImmunofluorescence and phase‐contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti‐actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal no‐pigment cells and a dedifferentiated cell line (GEM‐81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase‐contrast microscopic images with electron microscopic images of thin sections and of Triton‐insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F‐actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti‐tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti‐actin or with anti‐intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structrues seen with anti‐actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.
Clathrin‐coated membrane: A distinct membrane domain in acetylcholine receptor clusters of rat myotubes
Authors: Pumplin David W. , Bloch Robert J. 
Published: February 2005

AbstractWe have used antibodies to clathrin light chains in immunocytochemical studies of acetylcholine receptor (AChR) clusters of cultured rat myotubes. Immunofluorescence and ultrastructural experiments show that clathrin is present in coated pits and in large plaques of coated membrane. Coated membrane plaques are spatially and structurally distinct from AChR‐rich membrane domains and the bundles of microfilaments that are also present in AChR clusters. Clusters contain a relatively constant amount of clathrin light chain protein, which is not dependent on the amount of AChR. Clathrin plaques remain after AChR domains are disrupted by azide, or after microfilament bundles are destabilized by cytochalasin D. Extraction of myotubes with saponin removes clathrin without disrupting AChR domains. Thus, clathrin plaques, microfilament bundles, and AChR‐rich domains are independently stabilized.
Actin‐dependent carotenoid droplet dispersion in permeabilized cultured goldfish xanthophores
Authors: Yu Fu‐Xin , Taylor John D. , Tchen T. T. 
Published: February 2005

AbstractOrganelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule‐dependent organelle translocations, little is known about actin‐dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actindependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin‐permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti‐actin, but not by anti‐tubulin or anti‐intermediate filament proteins, suggesting a dependence on F‐actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.
Diffuse kinetochores and holokinetic anaphase chromatin movement during mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC‐20
Authors: Rieder Conly L. , Bowser Samuel S. , Cole Richard , Rupp Gerald , Peterson Andrew , Alexander Stephen P. 
Published: February 2005

AbstractMitosis in the hemipteran Agallia constricta (leafhopper) cell line AC‐20 was examined by light microscopy of living and fixed cells. During early prometaphase the numerous small (0.30–3.0‐μm) chromosomes appear as discrete units that lack a primary constriction. However, by late prometaphase the chromosomes are tightly packed at the spindle equator and are no longer clearly resolvable as individuals. When viewed from the side the metaphase chromatin appears as a 2–3‐μm wide band that spans the width of the spindle; when viewed from the pole it appears as a fenestrated disk. The metaphase chromatin splits at anaphase into two sister chromatin plates, each of which exhibits holokinetic poleward movement, i.e., all parts of each plate move as a single unit with the same velocity. In many early‐to‐mid anaphase cells the separating sister plates are connected by chromatin‐containing bridges that break as anaphase progresses. Ultrastructural analyses of serial thick and thin sections from cells fixed by conventional, OsO4/KFeCN, or high pressure rapid freezing methods, reveal that by metaphase all of the chromosomes are interconnected to form a large, irregularly shaped fenestrated disk of chromatin. Similar analyses reveal that adjacent chromatids remain interconnected throughout anaphase. Each disk of metaphase and anaphase chromatin contains numerous kinetochores recessed within its polefacing surface. Kinetochores consist of a fine, faintly staining fibrillar material arranged along the chromatin surface as thin (0.1–0.3 μm dia.) rods varying considerably (0.15–2.3 μm) in length. From these observations we conclude that the polycentric metaphase chromatin of A. constricta, and its holokinetic behavior during anaphase, arises from the aggregation or cohesion of smaller prometaphase chromosomes, each of which contains a single, diffuse kinetochore.
Motion of individual human spermatozoa, both normal and lacking the outer dynein arms, during a continuous temperature rise
Authors: Auger J. , Serres Catherine , Feneux Danielle 
Published: February 2005

AbstractThe effect of increasing temperature from 22–25°C to 37°C on various motion characteristics of individual normal human spermatozoa and spermatozoa lacking the outer dynein arms (LODA) was studied by using a new automatic microscopic tracking method. It was found that: (1) The curvilinear velocity (Vc, measured between 1–3 sec) of both normal and LODA spermatozoia, fluctuated more or less intensely between spermatozoa; this fluctuation was not thermodependent. (2) The average Vc in the two groups of spermatozoa increased with the rise in temperature at a similar rate (1μm/sec/°C), but LODA spermatozoa had an initial Vc lower than that of normal spermatozoa (12.5 ± 5.3 μm/sec and 34.2 ± 8.2 μm/sec, respectively). (3) The profile of the Vc increase associated with the temperature rise was different for the two groups of spermatozoa: for LODA spermatozoa it was linear between 25–37°C, whereas for normal spermatozoa a plateau was reached at about 31°C. (4) Various patterns of trajectory were found for both normal and LODA spermatozoa; these patterns were unrelated to temperature. However, LODA spermatozoa had more linear trajectories than normal spermatozoa. (5) Plots derived from reaction rate theory showed that the activation enthalpy, δH‡ was a function of the increase of Vc for both normal and LODA spermatozoa, but that δH‡ was higher for LODA spermatozoa.
Cytoskeletal dynamics in rabbit synovial fibroblasts: II. Reformation of stress fibers in cells rounded by treatment with collagenase‐lnducing agents
Authors: Aggeler Judith 
Published: February 2005

AbstractModulation of the synthesis and secretion of extracellular matrix proteins and matrix‐degrading metalloproteases by rabbit synovial fibroblasts in an important model system for studying the control of tissue‐specific gene expression. Induction of collagenase expression is correlated with changes in cell shape and actin filament distribution, but the role of the cellular cytoskeleton in the sustained synthesis and secretion of metalloproteases has not been closely examined. When cells were allowed to respread after rounding by trypsin or cytochalasin, two known metalloprotease inducers, reformation of stress fibers was observed within 2 h in the presence of serum. In the absence of serum, trypsin‐treated cells did not respread substantially, even after 24 h in culture. In contrast, cytochalasin‐treated cells recovered almost as rapidly in the absence as in the presence of serum, showing reformation of well‐formed microfilament bundles within 30 min of drug removal, especially at the spreading cell edges. High resolution electron‐microscopic views of detergent‐extracted cytoskeletons confirmed the rapid rebundling of peripheral microfilaments. Acrylamide‐treated cells fell between these two extremes, spreading slowly in the absence of serum, but almost as rapidly as cytochalasin‐treated cells in its presence. Reestablishment of normal intermediate filament distribution generally lagged slightly behind actin for all treatments, and intermediate filaments always appeared to spread back into the cellular cytoplasm within the confines of the reforming peripheral microfilament bundles. No obvious interaction between these two cytoskeletal elements was observed after any treatment, and no specific role for intermediate filaments in modulating gene expression in these cells is suggested by these results. The serum dependence displayed after trypsin or acrylamide treatment may be due to the disturbances in fibronectin synthesis observed in these cells and is consistent with evidence that both induction and sustained expression of matrix‐degrading metalloprotease may involve signals transduced through plasma membrane matrix receptors (integrins).
Differential effects of gelsolins on tissue culture cells
Authors: Huckriede Anke , Füchtbauer Annette , Hinssen Horst , Chaponnier Christine , Weeds Alan , Jockusch Brigitte M. 
Published: February 2005

AbstractGelsolins, prepared from a number of different sources, showed similar severing activity on F‐actin in vitro or on stress fibers of detergent‐extracted cells but differed in their effects on actin in stress fibers of microinjected cells. When human gelsolin isolated from plasma was injected into cells in a Ca++‐containing buffer, stress fibers were degraded, the cellular morphology was changed, and numerous actin patches appeared. These effects were particularly striking when the Ca++‐insensitive N‐terminal proteolytic fragment of this gelsolin was injected. By contrast, Ca++‐sensitive gelsolins isolated from human platelets, pig stomach smooth muscle and pig plasma showed no comparable activity. Furthermore, the Ca++‐independent N‐terminal proteolytic fragments prepared from these gelsolins also had no effect despite their in vitro actin severing activity. Most striking was the finding that human plasma gelsolin expressed in E. coli did not degrade stress fibers, in contrast to the same protein isolated from plasma; nor was there any stress fiber disruption observed with the N‐terminal half of human gelsolin expressed in Escherichia coli.The different behavior of these gelsolins in cells cannot be explained by sequence diversity between plasma and cytoplasmic forms, nor by variability in the Ca++ sensitivity of the preparations. It suggests the presence of factors, as yet unidentified, that may regulate gelsolin activity in the cytoplasm of living cells and discriminate between gelsolins of different origin. Such discrimination could be achieved as a result of post‐translational modification of the gelsolin; only in this way can differences between apparently identical proteins isolated from human plasma and expressed in E. coli be reconciled.
The expression and posttranslational modification of a neuron‐specific β‐tubulin isotype during chick embryogenesis
Authors: Lee Michael K. , Tuttle Jeremy B. , Rebhun Lionel I. , Cleveland Don W. , Frankfurter Anthony 
Published: February 2005

AbstractFive β‐tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene cβ4 and has been assigned to an isotypic family designated as Class III (βIII). In the nervous system of higher vertebrates, βIII is synthesized exclusively by neurons. A βIII‐specific monoclonal antibody was used to determine when during chick embryogenesis cβ4 is expressed, the cellular localization of βIII, and the number of charge variants (isoforms) into which βIII can be resolved by isoelectric focusing. On Western blots, βIII is first detectable at stages 12–13. Thereafter, the relative abundance of βIII in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non‐neural tissue extracts from older embryos (days 10–14) and hatchlings. Western blots of protein separated by two‐dimensional gel electrophoresis (2D–PAGE) reveal that the number of βIII isoforms increases from one to three during neural development. This evidence indicates that βIII is a substrate for developmentally regulated, multiple‐site posttranslational modification. Immunocytochemical studies reveal that while cβ4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non‐proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that cβ4 expression occurs either during or immediately following terminal mitosis, and suggests that βIII may have a unique role during early neuronal differentiation and neurite outgrowth.