bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ultrastructure and motion analysis of permeabilized paramecium capable of motility and regulation of motility
Authors: Lieberman Steven J. , Hamasaki Toshikazu , Satir Peter 
Published: February 2005

AbstractStructural and behavioral features of intact and permeabilized Paramecium tetraurelia have been defined as a basis for study of Ca2+ control of ciliary reversal. Motion analysis of living paramecia shows that all the cells in a population swim forward with gently curving spirals at speeds averaging 369 ± 19 μm/second. Ciliary reversal occurs in 10% of the cell population per second. Living paramecia, quick‐fixed for scanning electron microscopy (SEM), show metachronal waves and an effective stroke obliquely toward the posterior end of the cell. Upon treatment with Triton X‐100, swimming ceases and both scanning and transmission electron microscopy reveal cilia that uniformly project perpendicularly from the cell surface. Thin sections of these cells indicate that the ciliary, cell, and outer alveolar membranes are greatly disrupted or entirely missing and that the cytoplasm is also disrupted. These permeabilized paramecia can be reactivated and are capable of motility and regulation of motility. Motion analysis of cells reactivated with Mg2+ and ATP in low Ca2+ buffer (pCa7) shows that 71% swim forward in straight or curved paths at speeds averaging 221 ± 20 μm/second. When these cells are quick‐fixed for SEM the metachronal wave patterns of living, forward swimming cells reappear. Motion analysis of permeabilized cells reactivated in high Ca2+ buffers (pCa 5.5) shows that 94% swim backward in tight spirals at a velocity averaging 156 ± 7 μm/second. SEM reveals a metachronal wave pattern with an effective stroke toward the anterior region. Although the permeabilized cells do not reverse spontaneously, the pCa response is preserved and the Ca2+ switch remains intact. The ciliary axonemes are largely exposed to the external environment. Therefore, the behavioral responses of these permeabilized cells depend on interaction of Ca2+ with molecules that remain bound to the axonemes throughout the extraction and reactivation procedures.
Membrane IgM: Interactions with the cortical cytoskeleton in the human lymphoblastoid cell line WiL2
Authors: Salisbury J. L. , Baron A. T. , Keller G. A. , Skiest D. 
Published: February 2005

AbstractCell‐surface IgM (antigen receptor) sediments with the membrane fraction following osmotic lysis and homogenization of cells of the human lymphoblastoid cell line WiL2. In nonreducing buffers, SDS PAGE analysis of membrane pellets demonstrates that “native” membrane IgM exists as a dimer. In contrast to osmotic lysis, lysis of cells with the nonionic detergent Triton X‐100 releases approximately 90% of the membrane‐bound IgM into the supernatant; approximately 10% of the IgM pellets with the cytoskeletal fraction on centrifugation. Ligand challenge with either m̈‐chain‐specific antibodies or concanavalin A induces a change in the state of membrane IgM making it refractory to detergent extraction, such that 43% of the IgM pellets during centrifugation. This ligand‐induced retention of IgM is significantly diminished by the microfilament‐disrupting agent cytochalasin D, whereas pretreatment of cells with sodium azide or colchicine results in no significant change in the percentage of membrane IgM retained by Triton X‐100 residues. These results indicate that retention of IgM involves an association with the cortical actin‐based cytoskeleton. Investigation of the structural basis for ligand‐induced Triton X‐100 retention of membrane IgM by using ferritin‐conjugated antibodies, myosin subfragment S1, and stereo‐imaging electron microscopy has revealed linkages between ligand‐receptor (antigen‐IgM) complexes and elements of the cortical actin‐based cytoskeleton.
Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization
Authors: Hamaguchi Yukishisa , Mabuchi Issei 
Published: February 2005

AbstractActin from sea urchin eggs was fluorescently labeled with fluorescein isothiocyanate (FITC), N‐(7‐dimethylamino‐4‐methylcoumarinyl)‐maleimide (DACM), or 5‐iodoacetamidofluorescein (IAF) and microinjected into sea urchin eggs and oocytes. It distributed evenly in the cytoplasm of unfertilized eggs. Upon fertilization, actin accumulated first around the sperm binding site and, soon afterwards, in the fertilization cone. The accumulation propagated all over the cortex after a latent period of 10–20 sec. In the case of Clypeaster japonicus eggs, propagation of the accumulation coincided with a shape change in the egg, suggesting that the accumulated actin in the cortex generates forces. FITC‐actin was incorporated into microvilli and retained in the cortex after cleavage. On the other hand, DACM‐ or IAF‐actin was not incorporated into microvilli and was dispersed from the cortex by cleavage. These differences may be attributable to differences in the properties of the actins labeled at different sites. After photobleaching by laser light irradiation, FITC‐ or IAF‐actin redistributed in the cortex of fertilized egg as quickly as it did before fertilization. When an unfertilized egg was injected with both actin and a calcium buffer (intracellular free Ca2+ concentration 9 μM), the actin accumulation was similar to that during fertilization but without the latent period. This suggests that the accumulation depended on the increase in the intracellular free Ca2+ concentration. When the unfertilized egg was injected with 0.2 M EGTA after injection of labeled actin and then inseminated, it accumulated only in the protrusion of cytoplasm where the sperm had entered, and fertilization was not completed. In immature oocytes, the accumulation was observed in the cortical region, including the huge protrusion of the cytoplasm where the sperm had entered. These results suggest that actin accumulation in the sperm binding site plays an important role in the sperm reception mechanism of the egg.
Stepwise sliding disintegration of tetrahymena ciliary axonemes at higher concentrations of ATP
Authors: Tanaka Mikako , Miki‐Noumura Taiko 
Published: February 2005

AbstractSeveral characteristics of the sliding disintegration of Tetrahymena ciliary axonemes were found by turbidimetric assay, the ATP‐regenerating system, and quantitative determination of the ATP concentration. At ATP concentrations exceeding 40 μM, the response in terms of turbidity was biphasic and could be divided into three phases. The dependence of each phase on ATP concentration was examined. The time duration of phase 2 increased with ATP concentration. When the ATP concentration was kept constant by the ATP‐regenerating system, consisting of pyruvate kinase and phosphoenol pyruvate, the time duration of phase 2 increased with the concentration of phosphoenol pyruvate. On examining the change in turbidity with decreasing ATP concentration, the transition from phase 2 to phase 3 was found to occur at an ATP concentration of 40 μM.Dark‐field and electron microscopy indicated the sliding disintegration to be closely correlated with the degree of tubidity. At phase 1, one or two doublets extruded from most of the axonemes, and disintegration failed to progress during phase 2. At the transition point from phase 2 to 3, at about 40 μM, ATP, other doublets were noted to extrude from the axonemes one after the other, causing turbidity to be minimal by the end of phase 3.The ATP concentration dependence of stepwise sliding disintegration suggests that each axoneme may possess the ability to regulate doublet microtubule sliding at lower or higher concentrations of ATP. In response to local differences or gradients of ATP concentration along the axoneme, the axonemes may cause localized sliding of doublets, thus subsequently generating active bending movement.
Microtubules in ascidian eggs during meiosis, fertilization, and mitosis
Authors: Sawada Tomo‐o , Schatten Gerald 
Published: February 2005

AbstractThe sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic rescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.
Distribution of acetylated α‐tubulin in retina and in in vitro—assembled microtubules
Authors: Sale Winfield S. , Besharse Joseph C. , Piperno Gianni 
Published: February 2005

AbstractWe have used the mouse monoclonal antibody 6‐11B‐1, specific for acetylated α‐tubulin, to determine the distribution of acetylated α‐tubulin in in vitro–assembled microtubules and retinal tissue. Analysis by immunoblots revealed that microtubules assembled from bovine brain extracts contain both acetylated and nonacetylated α‐tubulin. Immunofluorescence, using 6‐11B‐1 and antitubulin B‐5‐1‐2, a monoclonal antibody specific for α‐tubulin, demonstrated the colocalization of both α‐tubulin species in neurons of the retina and that acetylated microtubules are relatively abundant in neurons. However, analysis at higher resolution revealed that rod photoreceptors contain spatially distinct microtubule arrays which differ in content of acetylated α‐tubulin and differ in stability. Acetylated microtubules which composed those of the rod outer segment and connecting cilium were resistant to depolymerization in nocodazole or colchicine. In contrast, the nonacetylated microtubules which composed those of the rod‐inner segment were depolymerized in nocodazole or colchicine. Therefore, these acetylated microtubules are more resistant to depolymerization than non‐acetylated microtubules.
Microtubule rearrangements during mitosis in multinucleate cells
Authors: Armas‐Portela R. , Paweletz N. , Zimmermann H.‐P. , Ghosh S. 
Published: February 2005

AbstractThe peroxidase‐antiperoxidase (PAP) method for the detection of polymerized tubulin has been used to study the microtubule rearrangements during mitosis in PtK1 and HeLa multinucleate cells obtained by polyethyleneglycol (PEG)‐mediated fusion. We demonstrate here that the transition of the microtubular cytoskeleton from interphase to mitosis is an inducible event and independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. However, for the induction of the microtubule rearrangements nuclear envelope breakdown is required. At midprophase, cytoskeletal microtubule rearrangements start for multinucleate PtK1 cells, whereas in HeLa cells such changes are delayed, and a more abrupt transition is observed here. After complete nuclear envelope breakdown (prometaphase) mitotic asters and spindles but no cytoplasmic (interphase) microtubuli can be observed in both systems. Metaphase is characterized by an interaction between the different mitotic poles which show the form of bipolar spindles, but individual separated mitotic poles far removed from the chromatin can also be seen.
Comparative study of the beat patterns of american and asian horseshoe crab sperm: Evidence for a role of the central pair complex in forming planar waveforms in flagella
Authors: Ishijima Sumio , Sekiguchi Koichi , Hiramoto Yukio 
Published: February 2005

AbstractAmerican horseshoe crab sperm flagella have the typical 9+2 structure whereas Asian horseshoe crab sperm flagella have a 9+0 axoneme lacking central pair and central sheaths. Beat patterns of the American and the Asian horseshoe crab sperm were recorded by means of a high‐speed video system (200 fields/second) and were compared in order to study the role of the central pair of the axoneme in ciliary and flagellar movement.The American horseshoe crab sperm beat with relatively planar waves, whereas the Asian horseshoe crab sperm beat with right‐handed helical waves. These results suggest that the central complex plays an important role in forming planar waves, whereas it is not essential for the conversion of microtubule sliding into axonemal bends.
Cell model contraction in the ciliate spirostomum
Authors: Ishida Hideki , Shigenaka Yoshinobu 
Published: February 2005

AbstractAs the species name indicates, the large heterotrichous ciliate Spirostomum ambiguum is characterized by a twisting contraction of the cell body that is easily triggered by various kinds of external stimuli. On the basis of morphological studies, contraction and extension of this organism have been considered to result from antagonistic actions of myoneme and microtubular ribbons. After many trials, we have succeeded in preparing cell models to examine induced contractions and extensions of the cell body. The contraction of this model was induced by increasing the free Ca2+ concentrations even in the absence of Mg‐ATP and was reversed by adding Mg‐ATP without Ca2+. Using dynein ATPase inhibitors such as vanadate and ATP analogs, furthermore, the experiments revealed that the ATPase that generated the force between the two neighboring microtubular ribbons might be a dynein‐like ATPase.
Cytochalasin‐Induced reorganization of actin in allium root cells
Authors: Palevitz Barry A. 
Published: February 2005

AbstractThe effect of cytochalasins on F‐actin was investigated in Allium root cells stained with rhodamine‐phalloidin. With cytochalasin D (CD), the normal interphase network of actin fibers is replaced by dispersed rods and specks similar to those seen in animal cells. However, during division, the specks accumulate at the poles in the form of one to a few large aggregates. The effects intensify with increasing concentration (0.5–5 m̈g/ml) and exposure time (0.5–3 hr). Further, similar behavior is observed with cytochalasin B, but dihydroCB has little effect. Double localizations show that during preprophase, aggregates cluster in association with microtubule foci at the new poles located near the nuclear envelope. From metaphase through anaphase, the aggregates are often found near the tips of kinetochore fibers, while in telophase they are often appressed to the pole side of the daughter nuclei. No association is seen between actin and the other microtubule arrays. The reorganization of F‐actin into small specks is unaffected by sodium azide, but aggregation at the poles is very sensitive to this agent. Polar clustering is also blocked by oryzalin, colchicine, and isopropyl n‐(3‐chlorophenyl) carbamate, but taxol has no effect. Experiments with scleroderma serum 5051 show that CD‐induced aggregates are embedded in centrosomal material at the poles. The results reveal that the reorganization of actin in response to cytochalasins differs during the cell cycle. Furthermore, the aggregation of actin during division is probably governed by an energy‐dependent interaction with microtubules.
Developmental organization of the intestinal brush‐border cytoskeleton
Authors: Takemura Reiko , Masaki Tomoh , Hirokawa Nobutaka 
Published: February 2005

AbstractAt the terminal web of the chicken intestinal epithelial cell, the actin bundles are cross‐linked by a fine filamentous network of actin‐associated cross‐linkers. Myosin, fodrin, and TW 260/240 have been identified as major components of the cross‐linkers. We studied the development of the cross‐linkers by quick‐freeze, deep‐etch electron microscopy, and the expression of cross‐linker proteins (myosin, fodrin 240, and TW 260) by immunofluorescence and immunoblotting analysis during the embryogenesis. Microvilli start to form at 5–7 days, and the rootlets begin to elongate at 10 days. At an early stage of the development of the terminal web (13 days), fodrin 240 and a small amount of myosin are expressed, and a few actin‐associated cross‐linkers are present between the rootlets. However, TW 260 is not expressed at this stage. At an intermediate stage (19 days), the amount of myosin increases, and TW 260 begins to be expressed. The number of cross‐linkers associated with the unit length of the rootlets is 24/μm. At the final stage of the terminal web formation (2 days after hatching), the amount of fodrin 240, myosin, and TW 260 is similar to the adult level, and the number of the actin‐associated cross‐linkers per unit length of the rootlet is 27/μm (∼︁85% of the adult). These results suggest that the synthesis of cross‐linker proteins may be intricately regulated to achieve the desired density of cross‐linkages at each developmental stage: at early and intermediate stages, sufficient and not an excess of cross‐linkages are formed; and at a final stage, a higher complexity of cross‐linkages is achieved. In addition, there is a differential expression of the components of the actin‐associated cross‐linkers: myosin and fodrin could be early components of the cross‐linkers involved in the basic stabilization of the terminal web structure, whereas TW 260/240 becomes incorporated later, possibly involved in the stabilization preparatory to the rapid elongation of microvilli, which occurs after the formation of the terminal web.
Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle
Authors: Otey C. A. , Kalnoski M. H. , Bulinski J. C. 
Published: February 2005

AbstractIn vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle‐specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal α isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle γ actin to stain cultured myotubes and adult skeletal myofibrils by double‐indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double‐stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin‐containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.
Three‐dimensional light microscopy of diploid Drosophila chromosomes
Authors: Agard David A. , Hiraoka Yasushi , Sedat John W. 
Published: February 2005

AbstractFluorescence microscopy, uniquely, provides the ability to examine specific components within intact, even living, cells. Unfortunately, high‐resolution conventional fluorescence microscopy is intrinsically a two‐dimensional technique and performs poorly with specimens thicker than about 0.5 μm. Probing the spatial organization of components within cells has required the development of new methods optimized for three‐dimensional data collection, processing, display, and interpretation. Our interest in understanding the relationship between chromosome structure and function has led us to develop the necessary methodology for exploring cell structures in three dimensions. It is now possible to determine directly the three‐dimensional spatial organization of diploid chromosomes within intact nuclei throughout most of the mitotic the cell cycle.
Subcellular compartmentalization by local differentiation of cytoplasmic structure
Authors: Luby‐Phelps Katherine , Taylor D. Lansing 
Published: February 2005

AbstractThe compartmentalization of eukaryotic cells by internal membranes and the subcellular localization of endogenous macromolecules by specific binding mechanisms are familiar concepts. In this report we present evidence that the cytoplasmic ground substance, which surrounds and contains the membranebound compartments, may also be compartmentalized by local differentiations of its submicroscopic structure that sort subcellular particles on the basis of size. The subcellular distribution of size‐fractionated, fluorescent tracer particles was studied in living cells by ratio imaging and fluorescence recovery after photobleaching (FRAP). Large and small particles showed different distributions within the cytoplasmic volume, suggesting that the large particles were relatively excluded from some domains. While the structural basis for this phenomenon is not yet understood in detail, ratio imaging of large and small particles can be used as an empirical tool to identify cytoplasmic compartments for further study. The cytoplasmic diffusion coefficient (Dcyto) and % mobile fraction of the large particles showed considerable spatial variation over the projected area of the cell, while Dcyto and % mobile fraction of the small particles did not. A model is presented to account for this difference. Based on this model, a method is proposed by which FRAP can be used to detect sol‐gel transitions in the cytoplasmic ground substance of living cells.
Results obtained with a sensitive confocal scanning system designed for epifluorescence
Authors: Amos W. B. 
Published: February 2005

AbstractA wide variety of specimens has been examined with our apparatus, a commercial version of which is being manufactured by Bio‐Rad/Lasersharp. The advantages expected of a confocal system have been realised in practice, the most striking advantage being the exclusion of glare from out‐of‐focus structures. This has made it possible to image cytological details in unflattened cells and intact tissues that were previously inaccessible to the light microscope.