bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteolytic fragmentation of Dictyostelium myosin and localization of the in vivo heavy chain phosphorylation site
Authors: Kuczmarski Edward R. , Routsolias Lisa , Parysek Linda M. 
Published: February 2005

AbstractDictyostelium myosin was associated into dimers and small oligomers at very low ionic strength, filamentous at intermediate ionic strength, and monomeric in solution conditions of high ionic strength. These different associations were probed by fragmenting myosin with chymotrypsin, trypsin, or V‐8 protease. All three proteases digested monomeric myosin giving rise to multiple fragments with a wide range of molecular weights. Filamentous myosin was not digested by the V‐8 protease, was preferentially cleaved at a single site in the middle of the heavy chain by chymotrypsin, and was cleaved at several sites by trypsin. If the reaction was carried out in very low ionic strength, however, two of these proteases generated stable fragments of high molecular weight. Electron microscopic analysis of these stable fragments showed that tails were shorter than in intact myosin, indicating that the cleavage sites were in the rod portion of the molecule. Under the same conditions of enzymatic digestion, myosin that had been radio labeled in vivo with 32P was analyzed by SDS‐PAGE and autoradiography. By comparing the state of phosphorylation and the size of the stable fragments, it was determined that the heavy chain phosphorylation site was located between 55 and 70 kD from the tip of the myosin tail, near a region where the tail displayed sharp bends.
Characterization of gliding motility inFlexibacter polymorphus
Authors: Ridgway H. F. , Lewin R. A. 
Published: February 2005

AbstractMotility of the marine gliding bacteriumFlexibacter polymorphuswas studied by using microcinematographic techniques. Following adhesion to a glass surface, multicellular filaments and individual cells usually began to glide within a few seconds at a speed of approximately 12 μm per second (at 23°C). Adhesion to the glass surface was evidently mediated by multitudes of extremely fine extracellular fibrils. Gliding velocity was independent of filament length but directly related to electron‐transport activity and substratum temperature in the range 3–35°C. The rate of gliding was inversely related to medium viscosity, suggesting that the locomotor apparatus functions at constant torque. Forward motion was occasionally interrupted by direction reversals, somersaults (observed primarily in single cells of short filaments), or spinning of filaments tethered by one pole. The frequency of direction reversal was found to be an inverse function of filament length. Translational motility was invariably accompanied by sinistral revolution about the longitudinal axis of a filament. The sense and pitch of revolution were constant among filaments of different length. Polystyrene microspheres or India ink particles adsorbed to gliding cells were actively displaced in either direction, their movement tracing either a regular zigzag or helical path along the filament surface. Because microspheres were also observed to move on nonmotile filaments, particle translocation was evidently not obligatorily linked to gliding locomotion. Multiple particles adsorbed to a single filament often moved independently. The data are consistent with a motility mechanism involving limited motion in numerous mechanically independent (yet functionally coordinated) domains on the cell surface.
The molecular mobility of α‐actinin and actin in a reconstituted model of gelation
Authors: Simon John R. , Furukawa Ruth H. , Ware Bennie R. , Taylor D. Lansing 
Published: February 2005

AbstractDictyostelium discoideum α‐actinin (D.d.α‐actinin) is a calcium and pH‐regulated actin‐binding protein that can cross‐link F‐actin into a gel at a submicromolar free calcium concentration and a pH <7 [Fechheimer, et al., 1982]. We examined mixtures of actin and D.d. α‐actinin at four pH and calcium concentrations that exhibited various degrees of gelation or solation. The macroscopic viscosities of these mixtures were measured by falling ball viscometry (FBV) and compared to the translational diffusion coefficients measured by gaussian spot and periodic‐pattern fluorescence photobleaching recovery (FPR) of both the actin filaments and D.d. α‐actinin. A homogeneous, macroscopic gel was not composed of a static actin network. Instead, the filament diffusion coefficient decreased to ∼65% of the control value. If the D.d. α‐actinin concentration was increased, the solution became inhomogeneous, consisting of domains of higher actin concentration. These domains were often composed of a static actin network. The mobility of D.d. α‐actinin consisted of a major fraction that freely diffused and a minor fraction that appeared immobile under the conditions employed. This suggested that D.d. α‐actinin binding to the actin filaments was static over the time course of measurement (∼5 sec). Under solation conditions, there was no apparent interaction of actin with D.d. α‐actinin. These results demonstrate that (1) actin filaments need not be cross‐linked into an immobile, static array in order to have macroscopic properties of a gel, (2) interpretation of the rheological properties of actin:α‐actinin gels are complicated by spatial heterogeneity of the filament concentration and mobility; and (3) a fraction of D.d. α‐actinin binds statically to actin in undisturbed gels. The implications of these results are discussed in relation to cytoplasmic structure and contractility.
Arrangement of desmin intermediate filaments in smooth muscle cells as shown by high‐resolution immunocytochemistry
Authors: Stromer Marvin H. , Bendayan Moïse 
Published: February 2005

AbstractTo gain additional information about the arrangement of intermediate filaments (IF) in normal smooth muscle, fresh avian gizzard was processed for immunoelectron microscopy. The protein A‐gold immunocytochemical technique was applied for the localization of desmin antigenic sites. Desmin‐containing IFs were located in an axial bundle that partially surrounds the nucleus and were associated with numerous mitochondria near the poles of the nucleus. The bundle probably extends the length of the cell. Antibody labeling also showed concentrations of IF around and between cytoplasmic dense bodies (CDB) and also between CDB and membrane‐associated dense bodies (MADB). The relationship between the axial bundle and the nucleus and associated mitochondria suggests that the bundle may support and define the position of these organelles in the cell. A fraying or branching of the bundle may integrate the bundle into the remaining cytoskeletal network of the cell.
Cytokinesis occurs at boundaries of domains delimited by nuclear‐based microtubules in sporocytes of Conocephalum conicum (Bryophyta)
Authors: Brown Roy C. , Lemmon Betty E. 
Published: February 2005

AbstractStudies of the microtubular cytoskeleton during sporogenesis in the hepatic Conocephalum conicum (Bryophyta) have revealed several unusual phenomena that contribute to understanding the cytokinetic apparatus in plant cell division. Although a typical phragmoplast forms in the interzonal microtubules of the first division spindle and expands to the cell periphery, no cell plate develops. There is no evidence of predetermined division sites and the orientation of both first and second meiotic spindles is imprecise. Simultaneous division of the cytoplasm follows second nuclear division. Equal apportionment of the cytoplasm appears to be a function of the establishment of cytoplasmic domains in the coenocyte, the boundaries of which are delimited by interaction of postmeiotic microtubule systems radiating from the four nuclei. Primary phragmoplasts are initiated in phragmoplasts that are initiated between nonsister nuclei. Depending upon the arrangement of nuclei in the nonpolar sporocyte, from one to three secondary phragmoplasts develop in the zones of contact between opposing sets of microtubules. Except for the site and time of initiation, the two types of phragmoplasts are identical. Eventually the phragmoplasts become confluent and cell plates form in all second division phragmoplasts. It is clear that typical functional phragmoplasts can form in sites determined by interaction of postmeiotic microtubule systems as well as in interzonal spindles as is common in plant cell division.
Effects of taxol on slow and fast axonal transport
Authors: Komiya Yoshiaki , Tashiro Tomoko 
Published: February 2005

AbstractAxonal transport of tubulin in the rat sciatic nerve is almost completely inhibited by a single subepineural injection of taxol, without affecting that of neurofilament proteins. Actin and a large number of polypeptides cotransported with actin as minor components are also blocked by taxol, although to a lesser extent. Fast axonal transport is essentially free from the inhibitory effect of this drug. Although previous models have suggested that slow axonal transport involves the bulk movement of cytoskeletal structures, these results suggest that such transport may involve an equilibrium between polymerised and depolymerised forms of the axonal cytoskeleton.
Structure and mass of mammalian respiratory ciliary outer arm 19S dynein
Authors: Hastie Annette T. , Marchese‐Ragona Silvio P. , Johnson Kenneth A. , Wall Joseph S. 
Published: February 2005

AbstractMammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 ± 1×10 ± 2 nm dimensions. The mass of this structure averaged 1.22 ± 0.34 million daltons with the individual globular heads averaging 310 ± 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 ± 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 ± 1×10 ± 2 nm often with 2 ± 1 nm centrally accumulated stain and with or without an appendage. This one‐headed particle thus resembled one‐half of the two‐headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 ± 111 kD. A third type of particle observed was a ring‐like structure with 4 ± 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 ± 1 nm, and had a greater peripheral accumulation of negative stain than either of the one‐ or two‐headed particles, suggesting that it was not derived therefrom. Structures appearing as three globular heads were the least frequently observed. Mammalian respiratory ciliary outer arm 19S dynein is therefore a two‐headed structure and conforms to descriptions of outer arm dyneins from mammalian spermatozoa flagella and from invertebrate cilia or flagella.
The ciliary cycle during hyperpolarization‐induced activity: An analysis of axonemal functional parameters
Authors: Sugino Kazuyuki , Machemer Hans 
Published: February 2005

AbstractMotor responses of the frontal cirri of the ciliate Stylonychia were recorded at the axial view of the ciliary base with high‐speed cinematography. Voltage‐clamp applying sustained hyperpolarizing voltage steps was used to explore the properties of the ciliary cycle modulated by the membrane potential. Upon hyperpolarization between – 1 and – 13 mV, a previously inactive frontal cirrus reoriented from a neutral posture and started beating so that the axis of the beating cone of a proximal cirral segment assumed an orientation near 100° (proceeding counterclockwise from posterior = 0°) and inclination near 60° (0° = perpendicular to the cell surface). The major beating amplitude was limited to about 150°. Increasing hyperpolarization increased the spatial polarity of the cycle (ratio of major over minor amplitude, from 2 to 2.4). Rates of the power stroke increased with hyperpolarizations up to – 4 mV but were consistently smaller than those of the return stroke during the ciliary cycle (ratio: 0.4 to 0.6; = temporal polarity). Comparison of different hypothetical beat forms (0‐shape, D‐shape, and egg‐shape) showed that the orientation‐time data are the major determinants of the angular velocity and rate of reorientation of the cilium during the cycle. Geometric transformation of these data led to descriptions of the cycle of a proximal ciliary segment in terms of active sliding velocities and rates of unidirectional sliding translocation between identified doublets. Three voltage‐sensitive functional parameters of the cilium—the inclination (which is noncyclic) and the rates of active sliding and sliding translocation (both of which are cyclic in nature)—are discussed as generating the spatial and temporal properties of the ciliary beat.
Effect of metabolic inhibitors on sucrose‐induced metaphase spindle elongation and spindle recovery
Authors: Snyder Judith Armstrong 
Published: February 2005

AbstractHyperosmotic sucrose treatment of metaphase PtK‐1 cells has been shown to produce a reversible concentration‐dependent effect on spindle elongation linked to a functional alteration in the connection of the chromosome to the spindle (Pover et al.: European Journal of Cell Biology 39:366–372, 1985). Spindle elongation, similar to that which occurs at anaphase B, is thought to be driven by the compression stored in the form of microtubule curvature in the nonkinetochore (nkMT) population of microtubules at metaphase (Snyder et al.: European Journal of Cell Biology 35:62–69, 1984 and 39:373–379, 1985). Addition of metabolic inhibitors to Ham's F‐12 salts with deoxyglucose (D/F‐12 medium) containing 0.4 M sucrose and 1 mM DNP does not within statistical error affect the rate and extent of sucrose‐induced spindle elongation; rates and extents are 60–75% of normal anaphase B motions. Electron microscopic analysis of metaphase cells treated with D/F‐12 medium and 0.4 M sucrose with 1 mM DNP demonstrates that spindle microtubules lose curvature and become straight in appearance, typical of microtubule organization in untreated anaphase cells. Sucrose‐treated cells released into D/F‐12 medium show a rapid reduction in spindle length; however, cells treated with either 0.4 M sucrose or 0.4 M sucrose and 1 mM DNP‐containing D/F‐12 medium and released into DNP‐containing D/F‐12 medium do not exhibit a significant reduction in spindle length. Electron microscopic analysis links changes in spindle length with microtubule/kinetochore associations. These data suggest that energy required for the initial phases of spindle elongation during anaphase is preloaded into the mitotic spindle by metaphase and does not require additional energy to be expressed as examined by sucrose‐induced spindle elongation in the presence of metabolic inhibitors. Second, energy is required to make or maintain (or both) functional chromosome associations with the spindle as measured by reduction in spindle length following sucrose removal.
Unique isoactins in the brush border of rat intestinal epithelial cells
Authors: Sawtell N. M. , Hartman A. L. , Lessard J. L. 
Published: February 2005

AbstractThe mammalian genome contains 20–30 genes encoding a family of actins. To date, however, only six proteins (four muscle and two nonmuscle isoforms) encoded by this multigene complex have been identified. We have isolated two actins from the brush border of rat intestinal epithelial cells that have isoelectric points and N‐terminal peptides characteristic of the cytoplasmic β‐ and γ‐actins. However, using a panel of actin‐specific monoclonal antibodies, we show that these actins contain a set of epitopes that distinguishes them from any of the known cytoplasmic or muscle isoforms. These unique actins share features of both the nonmuscle and muscle isoforms, suggesting that they represent an intermediate in the evolution of the specialized muscle actins.
Calcium‐dependent protein kinase is localized with F‐actin in plant cells
Authors: Putnam‐Evans Cindy , Harmon Alice C. , Palevitz Barry A. , Fechheimer Marcus , Cormier Milton J. 
Published: February 2005

AbstractWe recently purified a calcium‐dependent but calmodulin‐ and phospholipid‐independent protein kinase (CDPK) from cultured plant cells (Harmon et al.: Plant Physiology 83:830–837, 1987). A monoclonal antibody (mAb 3B9) directed against CDPK was used to localize this protein in Allium root cells and Tradescantia pollen tubes using immunofluorescence techniques. The mAb 3B9 staining pattern showed that CDPK is localized within a fibrous network in the cytoplasm resembling the normal interphase network of F‐actin. Treatment of tissue with 10 μM cytochalasin D (CD) prior to fixation abolished the staining pattern. Double‐localization experiments in which pollen tubes were first stained with mAb 3B9 and then with rhodamine‐phalloidin (RP) demonstrated that CDPK and F‐actin were colocalized. Monoclonal antibody 3B9 did not react with purified actin from rabbit muscle or Dictyostelium and did not bind to proteins corresponding to the Mr of actin in crude extracts of Allium root tips and Tradescantia pollen tubes.CDPK did not phosphorylate purified rabbit muscle or Dictyostelium actin in vitro. Binding studies showed that CDPK (1) does not cosediment with actin filaments and (2) does not form a complex with G‐actin. The data indicate that although CDPK does not interact directly with actin, it may be associated with an actin‐binding protein and therefore could play a role in the regulation of the plant cytoskeleton.
A model for slow axonal transport and its application to neurofilamentous neuropathies
Authors: Blum J. J. , Reed M. C. 
Published: February 2005

AbstractA model for slow axonal transport is developed in which the essential features are reversible binding of cytoskeletal elements and of soluble cytosolic proteins to each other and to motile elements such as actin microfilaments. Computer simulation of the equations of the model demonstrate that the model can account for many of the features of the SCa and SCb waves observed in pulse experiments. The model also provides a unified explanation for the increase and decrease of neurofilament transport rates observed in various toxicant‐induced neuropathies.
225‐Kilodalton phosphoprotein associated with mitotic centrosomes in sea urchin eggs
Authors: Kuriyama Ryoko 
Published: February 2005

AbstractProtein phosphorylation during development of sea urchin eggs from fertilization to first cleavage was examined by labeling cells with specific antiphosphoprotein antibodies. Indirect immunofluorescence staining with monoclonal antithiophos‐phoprotein antibody (Gerhart et al.: Cytobios 43:335–347, 1985) has revealed that nuclei as well as centrosomes, kinetochores, and midbodies were specifically thiophosphorylated in developing eggs incubated with adenosine 5′‐O (3‐thiotriphosphate) (ATP‐γ‐S). The phosphorylation reaction required Mg2+ but was not dependent on cAMP or calmodulin in detergent‐extracted models. Centrosomes were purified by fractionation of isolated mitotic spindles with 0.5 M KCl extraction. The thiophosphoproteins were retained in the purified centrosomes and the antibody recognized a major 225‐Kd polypeptide on immunoblots. In an independent preparation, a monoclonal antiphosphoprotein antibody (CHO3) was found also to react with mitotic poles and stained a 225‐Kd polypeptide, confirming the centrosome specificity of this protein. Immunoelectron microscopy showed that the 225‐Kd thiophosphoprotein was found at mitotic poles associated with granules to which mitotic microtubules were directly attached. Unlike centrosomes in permeabilized eggs, those in isolated spindles could not be thiophosphorylated, possibly due to inactivation or loss of either phosphorylation enzymes or cofactors, or both, during isolation. The immunofluorescence labeling of thiophosphate could be inhibited by ATP and AMP‐PNP in a concentration‐dependent manner. Exogenous ATP could abolish thiophosphate‐staining more effectively when added with phosphatase inhibitors, suggesting a dynamic state in which centrosomal proteins are being phosphorylated and dephosphorylated in rapid succession by the action of protein kinase(s) and phosphatase(s).
Visualization of intermediate filaments in living cells using fluorescently labeled desmin
Authors: Mittal B. , Sanger J. M. , Sanger J. W. 
Published: February 2005

AbstractFluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10‐nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells.