bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro nucleation of microtubules from microtubule‐organizing center prepared from cellular slime mold
Authors: Kuriyama Ryoko , Sato Chikako , Fukui Yoshio , Nishibayashi Soryu 
Published: February 2005

AbstractNucleus associated bodies (NABs) were isolated from Dictyostelium discoideum or Dictyostelium mucoroides and their ability to nucleate microtubules in vitro was examined.NABs were localized at the tapered ends of the nuclei and released from lysed cells in complex with the nuclei. Microtubules radiating from the NAB could also be isolated with the complex under microtubule stabilizing conditions. The ultrastructure of the isolated NAB showed it to be composed of a core structure surrounded by an amorphous matrix.The ability of isolated NABs to nucleate microtubules in vitro was demonstrated by incubation with exogenous brain microtubule protein. Microtubule assembly was easily visualized by dark‐field or immunofluorescence microscopy. Polymerization of microtubules seemed to be initiated not from the core structure but from the surrounding matrix.The number of microtubules polymerized from the NAB was directly counted in whole‐mount preparations by electron microscopy, which provided a quantitative assay for the NAB activity. The nucleating activity of NAB was quite unstable and its half‐life was calculated as about 5 hours. The activity was sensitive to protease digestion and was also temperature sensitive but could be stabilized by addition of glycerol or storage at – 80°C or in liquid nitrogen. These characteristics are analogous to those of the centrosomes in cultured mammalian cells and a possible explanation of their similarity is discussed.
Isolation of a new actin‐binding protein from dictyostelium discoideum
Authors: Condeelis John , Geosits Stephen , Vahey Maryanne 
Published: February 2005

AbstractA new actin binding protein has been purified to homogeneity from amoebae of Dictyostelium discoideum. This protein is a single polypeptide with a molecular weight of 120,000 upon sodium dodecyl sulfate gel electrophoresis. It is soluble and trypsin‐sensitive, contains no carbohydrate, increases the viscosity and sedimentation rate of F actin, and inhibits the actin‐stimulated Mg ATPase of rabbit muscle heavy meromyosin. The interaction of 120,000‐dalton protein with F actin is not inhibited by millimolar ATP, pyrophosphate, or micromolar calcium. The 120,000‐dalton actin binding protein increases the initial rate of actin polymerization and decreases the critical concentration of actin at steady state.These properties demonstrate that 120,000‐dalton protein from Dictyostelium discoidum is not a myosinlike protein. Rather, this protein is probably involved in regulating the assembly of the actin cytoskeletion.
Binding stoichiometry of 21S dynein to A and B subfiber microtubules
Authors: Warner F. D. , McIlvain Jane H. 
Published: February 2005

AbstractThe binding properties of Tetrahymena 21S dynein to doublet A and B subfiber microtubules were analyzed by both a turbidimetric assay (Δ A350 nm) and electron microscopy. KCl‐extracted, sucrose‐gradient, purified 21S dynein binds to each of the two kinds of axonemal microtubules in both ATP‐insensitive and ATP‐sensitive modes, even though only a single type of binding occurs to each of the subfibers in situ. Total dynein bound to axonemal microtubules is a composite of binding that is sensitive to dissociation by ATP and binding that is insensitive to ATP. Each exhibits a different binding profile. Total binding exhibits a sigmoid profile (h = 1.93) and saturates at 1.49 mg D/mg T. ATP‐sensitive binding likewise exhibits a sigmoid profile (h = 2.66) but saturates at 1.06 mg D/mg T. Binding occurs with a similar affinity for both A and B subfibers. The Hill coefficient (h) for ATP‐sensitive binding implies positive cooperativity between binding events. ATP‐insensitive binding was studied independently in 20 μM ATP, 10 μM vanadate, which blocks ATP‐sensitive binding. ATP‐insensitive binding exhibits a hyperbolic profile (h = 1.0) and likewise occurs along each of the two kinds of axonemal tubules. Binding saturates at 0.87 mg D/mg T. The binding data suggest that the tubulin dimer has conserved both ATP‐sensitive and ATP‐insensitive binding sites for 21S dynein, even though the sites may not be expressed in vivo.
Dictyostelium calmodulin: Production of a specific antiserum and localization in amoebae
Authors: Bazari Wendy L. , Clarke Margaret 
Published: February 2005

AbstractA rabbit antiserum was raised against calmodulin from the eukaryotic microorganism Dictyostelium discoideum. In double immunodiffusion experiments, the antiserum formed an immunoprecipitation line with Dictyostelium calmodulin but not bovine brain calmodulin; competition radioimmunoassays showed no cross reactivity between the antiserum and calmodulins from bovine brain and spinach. The calmodulin content of vegetative Dictyostelium amoebae, determined by competition radioimmunoassay, was 0.5 μg/mg protein; similar levels were found in developing cells. The antiserum was used to visualize the distribution of calmodulin in Dictyostelium amoebae by indirect immunofluorescence. Cells were examined under various conditions: in suspension, attached to a substrate, and while phagocytosing yeast cells. In all cases, anticalmodulin staining was concentrated in the cell cortex. Parallel experiments using a monoclonal antibody against Dictyostelium myosin showed that this protein is also enriched in the cortical region of the cell.
Purification and polypeptide composition of dynein ATPases from chlamydomonas flagella
Authors: Pfister K. Kevin , Fay Rose B. , Witman George B. 
Published: February 2005

AbstractExtraction of isolated, demembranated flagellar axonemes of Chlamydomonas reinhardii with 0.6 M KCl solubilized 77–92% of the total axonemal Mg++ or Ca++‐ATPase activity, which sedimented as 18S and 12S peaks in sucrose density gradients. The ATPases of these two peaks were further purified by hydroxyapatite (HAP) column chromatography. The ATPase activity of the 18S peak eluted from the HAP column as a single peak coinciding with the protein peak. The HAP purified 18S ATPase had a specific activity of ∼2.0 ± 0.5 μmoles Pi hydrolyzed min/mg and was associated with four high molecular weight (HMW) polypeptides of ∼ 310,000‐340,000 daltons, two intermediate molecular weight (IMW) polypeptides of 78,000 and 69,000 daltons, and eight low molecular weight (LMW) polypeptides of 7,800–19,600 daltons. When the 12S sucrose gradient peak together with a trailing shoulder were chromatographed on HAP, the ATPase activity was eluted in two peaks designated 12S and 10.5S on the basis of the sedimentation properties of their associated polypeptides. The 12S peak contained a single dynein ATPase having a specific activity of ∼ 0.6 ± 0.3 μmoles Pi hydrolyzed min/mg and associated with ∼ 330,000‐, 21,700‐, and 18, 100‐dalton polypeptides. The 10.5S peak contained several high, intermediate, and low molecular weight polypeptides; of these, on HMW polypeptide and one 28,700‐dalton polypeptide correlated well with the ATPase activity. The purified ATPases had no polypeptides in common; each therefore represents a discrete dynein. Based on protein recovered in the purified fractions, 18S dynein represents ∼ 9.2% of the total axonemal protein; 12S dynein represents ∼ 4.7% of the axonemal protein.
Motility, cell shape, and locomotion of neutrophil granulocytes
Authors: Keller H. U. 
Published: February 2005

AbstractActivation of the motile apparatus by chemokinetic factors cannot be reliably assessed in cells that are attached to a solid substratum because motility can be totally abolished by excessive adhesion. It is however, necesary to quantify the activation of the motile apparatus in order to analyze and understand chemokinetic responses.It was the purpose of the present work to establish morphological criteria that can be used to quantify motility in nonadherent (floating) neutrophils and to predict the locomotor response under conditions of limited adhesion. The proportion of neutrophils performing crawling‐like movements (polarized cells) in suspension correlates very closely with stimulated locomotion at low to optimal concentration of f‐Met‐Leu‐Phe, ie, under conditions of limited adhesion. Reduced locomotion at supraoptimal concentrations of f‐Met‐Leu‐Phe has also morphological correlates. The major feature is the decrease in the proportion of neutrophils performing crawling‐like movements and the corresponding appearance of cells that are motile but not polarized in suspension and that do not locomote on the substratum. Concentration‐dependent changes in neutrophil length and in the proportion of polarized neutrophils with and without tail were also observed. The locomotor potential of neutrophils under conditions of limited contact with the substratum can be predicted on the basis of their motile behavior, in particular the proportion of cells showing crawling‐like movements, in suspension. In combination with measurements of adhesion the procedure should permit a more complete analysis of the regulation of chemokinetic responses.
Coelomocyte motility
Authors: Edds K. T. , Chambers C. , Allen R. D. 
Published: February 2005

AbstractWe have utilized a video‐enhanced contrast system coupled to a DIC‐equipped microscope to examine the motility of both whole coelomocytes and individual filopodia. When the cells are left in diluted coelomic fluid, they exhibit a fibroblast‐like mode of translocation across the substrate. These cells extend lamellipodia at their advancing margin and develop retraction fibers at the trailing edge. Filopodia are actively extended from the lamellipodia of the advancing margin. Cells that are washed free of the coelomic fluid and placed in an isotonic buffer lose their ability to translocate. Filopodia on these stationary cells are seen to undergo a series of waving and bending motions. These motions are rapid and result in a filopodium folding back upon itself only to reextend later. Both forms of motility are discussed in light of the existing structural and biochemical knowledge of this and other cell types.
Ciliary but not saltatory movements are inhibited by vanadate microinjected into living cultured cells
Authors: Buckley Ian , Stewart Murray 
Published: February 2005

AbstractTo test the idea that saltatory organelle movements of nonmuscle cells might be driven by microtubule‐dynein interactions, we microinjected vanadate into several different types of cultured cell. Solutions of sodium metavanadate made up in a simple buffered salt solution were pressure microinjected into fully spread cells in an open‐topped culture chamber placed on the stage of an inverted microscope. The cells were observed by oil‐immersion phase‐contrast optics and results were recorded on movie film. Vanadate, at 10−5‐10−2 M, microinjected into cultured chick embryo fibroblasts, failed to inhibit organelle movements. To test the effectiveness of vanadate's inhibitory action under living cell conditions, ciliated epithelial cells were micro‐injected. In these cells even the smallest microinjection of 5 × 10−5 M vanadate caused an immediate cessation of ciliary beating. Moreover, in cells that were well spread it was found that whereas vanadate, at 5 × 10−5 × 10−3M, inhibited ciliary motion, it failed to inhibit organelle saltations in the same cell. To determine whether vanadate would inhibit a living actin‐myosin system, myocardial cells were also microinjected. Following microinjection of 5 × 10−5 and 5 × 10−4M vanadate a temporary tonic contraction (which also occurred following microinjection of buffer alone) was followed by regular beating. Taken together these results demonstrate that in living cell systems microtubule‐dynein interactions are as sensitive to vanadate inhibition as they are in demembranated model systems, and that a working actin‐myosin system in a living muscle cell does not share this great sensitivity. In light of the pronounced differential inhibitory effects of vanadate on the movements of cilia and organelles, our results suggest that saltatory organelle movements in chick embryo fibroblasts and rabbit oviduct epithelial cells are unlikely to be brought about by microtubule‐dynein interactions.
The interaction of cAMP with modeled bull sperm
Authors: Lindemann Charles B. , Lipton Melissa , Shlafer Roman 
Published: February 2005

AbstractDemembranated and membrane disrupted bull sperm models exhibit an increase in motility when exposed to cAMP. Tritium‐labeled cAMP was used to locate the initial site of action of cAMP in the modeled sperm preparations. cAMP did not bind selectively to the modeled cells, and the presence or absence of plasma membrane fragments on the models did not significantly alter this result. When suspension medium taken from modeled sperm preparations was subjected to gel filtration on Sephadex G25‐150 columns, cAMP bound to a high molecular weight component that eluted with the void volume. The responsible binding factor is a soluble component that is released when the plasma membranes of the sperm are disrupted during the modeling procedure. To test the importance of the cAMP binding factor, modeled sperm were centrifuged, the super‐natant solution was decanted, and the cells were resuspended in fresh medium. After this treat‐ment the cells could be restored to motility with Mg‐ATP but no longer exhibited a response to cAMP. Furthermore, addition of cAMP binding factor isolated by gel filtration partially restored the response of these sperm to cAMP. Investigation of the properties of the cAMP‐binding factor have confirmed that it is specific for cAMP, with a much lower affinity for AMP and cGMP. In the pre‐sence of a large excess of unlabeled cAMP the labeled complex has a half‐life of approximately 1 hour. Our results indicate that the action of cAMP on the motility of modeled sperm is mediated by its attachment to a high molecular weight, soluble component of the cell cytoplasm.
Cell movement and its coordination in swarms of myxococcus xanthus
Authors: Kaiser Dale , Crosby Cathy 
Published: February 2005

AbstractThe coordinated movement of many cells—a process called swarming—permits myxobacteria to spread rapidly over a surface. We have investigated the mechanism of swarming in Myxococcus xanthus by making time‐lapse motion pictures and by measuring the dependence of cell movement and spreading rate on the concentration of cells. Motion pictures of spreading zones showed that spreading resulted from motility, not growth, and that a swarm spread outward by establishing a loose reticulum of cells, then later filling it in. The spreading rate of wildtype strains was found to be highly dependent on cell density, increasing about 8‐fold as the cell density was increased from 2.5 to 200 units. Mutants swarmed if they possessed only the A‐motile component (A+S−) or only the S‐motile component (A−S+) of wild type (A+S+); their spreading rate increased with cell density but was always less than A+S+. Individual A+S+, A+S−, and A−S+ cells executed typical gliding movements and (when moving) progressed at approximately the same speed, as if A and S motility were different ways of engaging the same gliding machine. Photographic studies of an A−S+ strain showed that cells moved only if they were separated by less than approximately one cell length from each other. This provided further evidence that pili, which are present on A+S+ and A−S+ cells and which extend about one cell length, could be responsible for switching on movement in S‐motile cells, and presumably in wild type as well.
The effects of taxol on cytoskeletal components in cultured fibroblasts and epithelial cells
Authors: Green Kathleen J. , Goldman Robert D. 
Published: February 2005

AbstractTaxol promotes microtubule (MT) assembly in vitro and induces the reorganization of the cytoskeleton into unusual MT arrays in cultured cells. The possibility that taxol also has an indirect effect on intermediate filaments (IF) was investigated. In baby hamster kidney (BHK‐21) and human skin (ENSON) fibroblasts treated with 1–10 μM taxol for 1–24 h, the drug induces changes which are similar to those produced by colchicine. These include a loss of major cellular extensions, a redistribution of organelles to a perinuclear location, and an inhibition of locomotion. Saltatory particle movements are not inhibited, however. Ruffling and filopod formation continue, indicating that cells are viable up to 24 h.Polarized light microscopy of living fibroblasts treated with taxol reveals the presence of perinuclear birefringent material which has been examined by immunofluorescence. In control cells, IF and MT radiate from a juxtanuclear region and extend to the cell periphery. In taxol‐treated cells, MT and IF are excluded from cell margins, forming large central bundles.In the epithelial cell lines PtK2 and PAM, the keratin system of IF does not become redistributed; in PtK2, however, a second fibroblastlike system of IF does become redistributed to a perinuclear position during taxol treatment.Ultrastructural analyses show that taxol‐treated fibroblasts contain parallel arrays of cross‐bridged MT‐IF as well as bundles of MT exclusive of IF. Epithelial cells contain a predominance of IF‐free MT bundles which are organized into hexagonally packed arrays. In these bundles MT frequently exhibit hooks or other incomplete MT profiles and are linked by filamentous material.
Differential response to contact during embryonic nerve‐nonnerve cell interactions
Authors: Nuttall Robert P. , Zinsmeister Philip P. 
Published: February 2005

AbstractThe outcome of contact interactions involving neurons and nonneurons varies depending on the cell types involved. When neuronal growth cones from either ciliary (motor) or dorsal root (sensory) ganglia directly contact the lamellipodium of an embryonic heart fibroblast, both neurite elongation and fibroblast locomotion are inhibited. This occurs in spite of the fact that cell‐surface activity in both cells continues unabated. Such contact inhibition is not observed when homologous ganglionic nonneurons are involved in the interaction. In fact, these cells become intimately associated with growth cones and/or neuritic shafts as a result of the contact. The detailed nature of the respose to contact exhibited by nerves and nonnerves varies not only with cell type but also with the portion of the cell involved in the contact. Growth cone filopodia tend to actively palpate the fibroblast surface, whereas spread regions, termed “veils,” form areas of apposition with fibroblast lamellipodia. This latter situation resembles the “typical” contact inhibition of locomotion that occurs following embryonic heart fibroblast‐fibroblast interactions. Growth cones also frequently exhibit contact guidance when interacting with nonruffling lateral surfaces of heart fibroblasts.
A quantitative comparison of cellular motile systems
Authors: Nicklas R. Bruce 
Published: February 2005

AbstractCellular motile systems as diverse as muscle and the mitotic spindle have been compared by their specific power output: the maximum power they develop per unit of engine volume. Striated muscles and flagella have high specific output; their performance is comparable to that of typical automobile engines. The cytokinetic furrow and the mitotic spindle have very much lower specific power output. The furrow's output is 7,000 times lower than muscle and the spindle's is 300,000 times lower. Different macromolecules have been used to generate power in systems with similar output (muscles and flagella) and, conversely, the same macromolecular motor has been used in systems with very different output (muscles and cytokinetic furrows). The common feature amid this diversity is adaptation to a particular biological role, which specific power output reflects very well. High values are found where a powerful, compact engine should be advantageous, while low values are found where precision, not power, matters most.
A flagellar surface glycoprotein mediating cell‐substrate interaction in Chlamydomonas
Authors: Bloodgood Robert A. , Workman Lisa J. 
Published: February 2005

AbstractThe Chlamydomonas flagellar surface exhibits interesting adhesive properties that are associated with flagellar surface motility. This dynamic surface property can be exhibited as the binding and movement of small polystyrene microspheres or as the interaction of the flagellar surface with a solid substrate followed by whole cell locomotion, termed “gliding.” In order to identify flagellar surface proteins that mediate substrate interaction during flagellar surface motility, two immobilized iodination systems were employed that mimic the conditions for flagellar surface motility: small polystyrene microspheres derivatized with lactoperoxidase, and large glass beads derivatized with Iodogen. Use of these iodination conditions resulted in preferential iodination of a high‐molecular‐weight glycoprotein with apparent molecular weight of 300,000–350,000. These results suggest this glycoprotein as a major candidate for the surface‐exposed adhesive component that directly interacts with the substrate and couples the substrate to a system of force transduction presumed to be located within the flagellum.
Selective immunocytochemical detection of fluorescent analogs with antibodies specific for the fluorophore
Authors: Luby‐Phelps Katherine , Amato Philip A. , Taylor D. Lansing 
Published: February 2005

AbstractFluorescent analogs of cellular components are finding increasing use in the field of cell biology. The power of this technique can be augmented by the use of antibodies specific for the fluorophore to visualize selectively the fluorescent analog at the electron microscope level. Rabbit antibodies specific for fluorescein were elicited and purified according to published methods (Lopatin and Voss [1971]: Biochemistry 10:208). Immune sera and IgG formed precipitin lines with fluorescein‐labeled proteins in Ouchterlony immunodiffusion assays, and significantly quenched the fluorescence of fluorescein‐labeled proteins. Immune IgG and Fab fragments decorated fluorescein‐labeled actin, but not unlabeled actin, in negative‐stained preparations. Anti‐fluorescein IgG was used for immunofluorescent localization of fluorescein‐labeled actin following microinjection of the fluorescent analog into living cells. This approach was extended to the immunoelectron microscopic localization of the injected analog at the subcellular level by the use of an electron‐dense marker coupled to goat anti‐rabbit IgG. Many other fluorescent probes also can be used as haptens for production of antibodies. Therefore, a general method for localizing fluorescently labeled molecules at the electron microscopic level is now available. Several other applications of anti‐fluorescein antibody in studies involving fluorescent analogs are also suggested.
Three‐dimensional bend propagation in hamster sperm models and the direction of roll in free‐swimming cells
Authors: Yeung Ching‐Hei , Woolley David M. 
Published: February 2005

AbstractIontophoretic application of ATP to the flagellum of the demembranated hamster spermatozoon produced a planar pair of bends at the two ends of the stimulated site. During bend propagation, torsion appeared in the vicinity of the interbend in some responses such that the distal bend was twisted clockwise when viewed from the base of the flagellum. This pattern of propagation is consistent with the instantaneous configurations of free‐swimming cells previously described. The technique used here establishes that the three dimensionality arises from propagation per se, and does not depend on forces developed during swimming. The rolling of both free‐swimming intact and demembranated spermatozoa was examined by two‐color darkground videomicroscopy and the direction of rotation was, as predicted, always anticlockwise. A hypothetical mechanism, involving differential speeds of propagation of active sliding within the active microtubule subset, is proposed to account for the observed waveforms.