bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ca/Ba/Sr‐induced conformational changes of ciliary axonemes
Authors: Tamm R. Sidney , Tamm Signhild 
Published: February 2005

AbstractMacrocilia of the ctenophore Beroë undergo Ca/Ba/Sr‐dependent activation of ciliary beating and microtubule sliding disintegration [Tamm, J. Comp. Physiol. A163:23–31. 1988a: Tamm, Cell Motil. Cytoskeleton 11:126–138, 1988b; Tamm, Cell Motil. Cytoskeleton 12:104–112, 1989: Tamm and Tamm, Proc. Natl. Acad. Sci. U.S.A. 86:6987–6991, 1989]. Here we report that detergent‐extracted macrocilia show an ATP‐independent conformational change in response to high concentrations of Ca. Ba. or Sr ions. When applied locally by iontophoresis, these ions induce a rapid planar curvature of the distal end of the macrociliary shaft, followed by a slower relaxation to the rest position. Tip curling occurs in a direction opposite to the physiological Ca/Ba/Sr response. When applied uniformly in the bath, a threshold concentration of 10−1 M Sr is required to induce curling of the tip, and the distal ends remain curved. Calmodulin antagonists do not inhibit the tip curling response.Previous workers found that Ca induces changes in the helical shape of isolated doublet microtubules [Miki‐Noumura and Kamiya, Exp. Cell Res. 97:451–453, 1976: Miki‐Noumura and Kamiya. J. Cell Biol. 81:355–360, 1979; Takasaki and Miki‐Noumura. J. Mol. Biol. 158:317–324, 1982] and sperm axonemes [Okuno and Brokaw, Cell Motil. 1:349–362. 1981] and suggested that conformational changes in microtubules may play a role in Ca regulation of ciliary motility. We propose that the Ca/Ba/Sr‐induced curling of the macrociliary tip is due to similar helical changes of doublet microtubules, some of which in macrocilia are prevented from sliding by permanent connections (compartmenting lamellae) between adjacent axonemes within the shaft. Although the tip curling response does not appear to be directly relevant to the physiological Ca response of macrocilia, it provides a novel system for investigating Ca‐induced conformational changes of microtubules independent of dynein‐powered active sliding.
Effect of filamin and controlled linear shear on the microheterogeneity of F‐Actin/Gelsolin gels
Authors: Cortese Jorge Daniel , Frieden Carl 
Published: February 2005

AbstractWe have previously established [Cortese and Frieden, J. Cell Biol. 107:1477–1487, 1988] that actin gels formed under shear are microheterogeneous. In this study, the effect of cross‐linking (by chicken gizzard filamin), severing (by plasma gelsolin), and shear on actin microheterogeneity are investigated using fluorescence photobleaching recovery and video microscopy. We find that filamin and shear form microheterogeneous F‐actin:gelsolin gels by different mechanisms. Bundling of actin:gelsolin filaments by filamin can be explained by an increase in the apparent length of the filaments due to interfilament binding, resulting in a decrease of the polymer number concentration at which filaments organize into anisotropic phases. Some intrafilament binding of filamin to actin filaments may also be present, and those filaments coated with filamin immobilize more slowly than actin under the same polymerization conditions. The length of F‐actin/gelsolin filaments seems to be a major factor in controlling the extent of bundling relative to network formation. In contrast, the effect of shear on the microheterogeneity of actin:gelsolin filaments is consistent with our previous proposal that shear aligns actin filaments, allowing filament‐filament interactions and phase formation to occur. Short filaments are unable to organize into branched actin networks, but they can create large aggregates under low shear. Longer actin filaments will exist as networks with variable levels of branching and are less sensitive to shear. The effect of the intensity of a shear field on the spatial distribution of actin may involve a progressively more random orientation of actin molecules and bundles. A regular pattern develops across the sample at low shear rates (0.04–1.39 s−1), and becomes very irregular at higher shear rates (> 10 s−1). We suggest here that actin‐binding proteins and shear can control the transition between isotropic networks and anisotropic phases by their effect on apparent length and local filament concentration, and also that this transition can have substantial effects on the resistance of cells to mechanical stress.
Plants contain highly divergent actin isovariants
Authors: Gail McLean Barbara , Huang Shurong , McKinney Elizabeth Cohen , Meagher Richard B. 
Published: February 2005

AbstractActin protein isovariants have been identified in animals with distinct cytoplasmic or muscle specific patterns of expression. Analysis of vascular plant actin gene sequences suggests that an even greater diversity should exist within the plant actin protein families, but previous studies on plant proteins have not demonstrated the presence of multiple actin isovariants. Antibodies recognizing a conserved amino‐terminal plant actin peptide, a family of plant actin peptides from a variable region, and two monoclonal antibodies to conserved epitopes within animal actins were used to identify isovariants of soybean actin resolved by two‐dimensional isoelectric focusing (IEF) sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Approximately six to eight actin isovariants with pi values ranging from 5.1 to 5.8 have been identified from soybean hypocotyls. Stems, leaves, and roots with varying amounts of most isovariants present in all four organs. Acidic isovariants were present in much higher levels in leaves and stems. Antisera with λ‐class actin specificity detected a subset of three isovariants in all organs examined. One monoclonal and one antipeptide antisera are shown to react well with a wide variety of plant actin isovariants. Similar patterns of actin isovariants were detected in the distant angiosperms, Arabidopsis petunia, and maize. It is likely that many of these diverse classes of isovariants have been preserved throughout vascular plant evolution and reflect the ancient diversity within plant actin gene families. The extreme difference among isovariants implies the presence of a complex actin‐based cytoskeletal system in plants.
Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope
Authors: Horst Cynthia J. , Johnson Lincoln V. , Besharse Joseph C. 
Published: February 2005

AbstractThe photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent‐extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergentextracted axonemes, was assessed following treatment with the enzymes neuraminidase and O‐glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose‐N‐acetylgalactosamine oligosaccharides which are Olinked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium.Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia. This demonstrates that the photoreceptor connecting cilium and motile cilium transition zone are immunologically related.
Dynamic changes of microtubule and actin structures in CV‐1 cells during electrofusion
Authors: Zheng Qiang , Chang Donald C. 
Published: February 2005

AbstractTo study the involvement of the cytoskeletal system in the fusion of animal cells, we examined the dynamic changes of cytoskeletal proteins during the various stages of cell fusion. CV‐l cells were fused by applying a radio‐frequency electrical pulse. Structural changes of microtubules (MTs) and F‐actin were monitored simultaneously by double‐label fluorescence microscopy. It was observed that in a few minutes after the initiation of cell fusion, MT bundles began to extend into the cytoplasmic bridges which were formed by fusing the membranes of neighboring cells. Later, a network of parallel MT bundles appeared between the adjacent nuclei of the fusing cells; such MT bundles may provide the mechanical links that are responsible for nuclear aggregation. The structural changes of Factin during cell fusion were more complicated. We observed many different patterns of actin distribution in the fusing cells, including some giant, ring‐shaped structures. Reorganization of actin is unlikely to be involved in the nuclear aggregation process. Instead, actin bundles condensed at the cell edges may help to widen the cytoplasmic bridges to allow merging of cellular contents between the fusing cells.
Reorganization of cytoskeletal and junctional proteins during cochlear hair cell degeneration
Authors: Raphael Yehoash , Altschuler Richard A. 
Published: February 2005

AbstractExperiments were carried out to elucidate changes in cytoskeletal elements and intercellular junctions in the organ of Corti, when hair cells degenerate and phalangeal scars form. Hair cell damage was induced by exposing guinea pigs to high intensity noise. The spatial and temporal changes in the organization of micro‐filaments, intermediate filaments, and tight junction‐specific proteins were investigated using scanning and transmission electron microscopy and histochemistry. The results show that microfilaments, cytokeratins, adherens junctions, and tight junctions rearrange their distribution in damaged areas. From the temporal sequence of these changes it appears that phalangeal scars develop simultaneous with hair cell degeneration, and that the integrity of the luminal membranes in the organ of Corti is not interrupted. Each scar is formed by two supporting cells which expand and invade the sub‐apical region of the dying hair cell. This region becomes cytokeratin‐positive. The two supporting cells meet at the mid‐line of the scar, where a new junctional complex is formed. The junctional complex consists of tight junction and adherens‐type junction, but desmosomes are absent.
Reactivation of isolated mitotic apparatus: Metaphase versus anaphase spindles
Authors: Palazzo Robert E. , Lutz Douglas A. , Rebhun Lionel I. 
Published: February 2005

AbstractMitotic spindles isolated from sea urchin eggs can be reactivated to undergo mitotic processes in vitro. Spindles incubated in reactivation media containing sea urchin tubulin and nucleotides undergo pole‐pole elongation similar to that observed in living cells during anaphase‐B. The in vitro behavior of spindles isolated during metaphase and anaphase are compared. Both metaphase and anaphase spindles undergo pole‐pole elongation with similar rates, but only in the presence of added tubulin. In contrast, metaphase but not anaphase spindles increase chromosome‐pole distance in the presence of exogenous tubulin, suggesting that in vitro, tubulin can be incorporated at the kinetochores of metaphase but not anaphase chromosomes. The rate of spindle elongation, ultimate length achieved, and the increase in chromosome‐pole distance for isolated metaphase spindles is related to the concentration of available tubulin. Pole‐pole elongation and chromosome‐pole elongation does not require added adenosine triphosphate (ATP). Guanosine triphosphate (GTP) will support all activities observed. Thus, the force generation mechanism for anaphase‐B in isolated sea urchin spindles is independent of added ATP, but dependent on the availability of tubulin. These results support the hypothesis that the mechanism of force generation for anaphase‐B is linked to the incorporation of tubulin into the mitotic apparatus. (If, in addition, a microtubule‐dependent motor‐protein(s) is acting to generate force, it does not appear to be dependant on ATP as the exclusive energy source).
Alterations in microtubule assembly caused by the microtubule‐active drug LY195448
Authors: Barlow Steven B. , Cabral Fernando 
Published: February 2005

AbstractLY195448 is an experimental drug that blocks cells at metaphase (Boder et al.:Microtubules and Microtubule Inhibitors 1985: 353–361, 1985). A 4 hour exposure of NRK cells to a drug concentration of 46 μM (15 μg/ml) increased the number of mitotic cells in the population from 4.9% to 18.5%. Examination of treated cells by immunofluorescence showed increased numbers of cells blocked at prometaphase, with short microtubules extending from the spindle pole to the kinetochores. The cytoskeleton of interphase cells remained intact at these concentrations. However, the number of microtubules appeared to be reduced, and those that remained appeared kinkier and curled, particularly toward the periphery of the cells. When cytoskeletal microtubules of NRK cells were depolymerized with nocodazole, they reassembled within minutes of transfer to drug‐free media. However, nocodazole‐treated cells transferred to fresh media containing 15 μg/ml of LY 195448 required 2‐3 times longer to reassemble cytoplasmic microtubules. Previously isolated Chinese hamster ovary cell microtubule mutants resistant to either taxol or Colcemid were tested for cross‐resistance to this drug. Cell lines resistant to the depolymerizing drug Colcemid exhibited increased resistance to LY 195448 compared to wild‐type cells, whereas taxol resistant cell lines were more sensitive. Of eleven newly isolated mutant CHO cell lines selected for increased resistance to LY 195448, seven exhibited an altered β‐tubulin protein by two‐dimensional polyacrylamide gel electrophoresis. These 11 cell lines also showed a heterogenous pattern of resistance to several microtubule‐active drugs. These data demonstrate that LY 195448 is cytotoxic to mammalian cells because it inhibits microtubule assembly, most likely through a direct interaction with tubulin.
Effects of interferon‐β on the translocation rate and stationary time in human fibroblasts in culture
Authors: Murphy James S. , Tamm Igor 
Published: February 2005

AbstractThe rate of translocation and the percent of the time that cells are stationary have been measured by computer‐assisted time‐lapse cinemicrography in over 1,000 freshly planted human foreskin fibroblasts (FS‐4 cell strain) for periods of up to a week and the effects of interferon‐β (IFN‐β) on these parameters have been determined. Cells were planted at 2.5 × 103 cells/cm2 in Eagle's minimal essential‐medium (MEM) with 10% fetal bovine serum (FBS). Frames were taken every 2 or 4 minutes and data were collected on both cell location and cell division as a function of time. After planting FS‐4 cells require ∼48 hr to reach maximum motility both with respect to the translocation rate when moving and percent time cells are moving. Recombinant human IFN‐β (800 μ/ml) caused a marked increase in the fraction of time cells were stationary and a decrease of lesser magnitude in the translocation rate, as quantitated during the period during which the stationary fraction for control cells was at a minimum. IFN‐β also decreased the rate of cell proliferation, without any evidence of degeneration or death of cells. Our results contribute new evidence that the fraction of time cells spend moving directionally is an important determinant of their locomotory behavior and that this determinant is responsive to modulation by cytokines.
βVLDL uptake by pigeon monocyte‐derived macrophages: Correlation of binding dynamics with three‐dimensional ultrastructure
Authors: Jones Nancy L. , Lewis Jon C. , Allen Nina S. 
Published: February 2005

AbstractEndocytosis of pigeon beta migrating very‐low‐density lipoprotein (βVLDL) by monocyte‐derived macrophages (monocyte/macrophages), cultured from Random Bred White Carneu (RBWC) pigeons, occurs by both coated and non‐coated regions of the plasma membrane (Henson et al.: Exp. Mol. Pathol. 51:243–263, 1989). Secondary to binding, the βVLDL is translocated to lysosomes for degradation. Ultimately these events lead to foam cell formation in vitro. Utilizing video‐enhanced contrast light microscopy in conjunction with whole mount intermediate‐voltage transmission electron microscopy (IVEM) and high‐resolution scanning EM, the dynamics of βVLDL binding have been correlated with ultrastructure. Beta VLDL conjugated to gold colloids was visualized at the surface of living cells by using Allen video‐enhanced contrast‐differential interference contrast microscopy (AVEC‐DIC). Subsequent to AVEC‐DIC, direct observation of the identical cells by IVEM and SEM was facilitated through the use of gold finder grids, and these EM observations confirmed identification of the videoobserved βVLDL particles.Upon addition of βVLDL, pigeon monocyte/macrophages underwent gross morphological changes. These changes were recorded by video as movements at the cytoplasmic periphery, and the movements involved extension of microvilli, expression of retraction fibers, and elaboration of membrane ruffles. When secondarily observed by stereo (3‐D) IVEM and SEM, the identification of microvilli, retraction fibers, and membrane ruffles was confirmed and the lipoprotein‐gold conjugates were associated with these ligand‐induced membrane structures. Beta VLDL‐gold conjugates were also associated with pit‐like regions at the base of microvilli, while at the base of ruffles, βVLDL‐gold conjugates were located in membrane invaginations and cytoplasmic vesicles.
Pseudopodial activity at the active edge of migrating fibroblast is decreased after drug‐induced microtubule depolymerization
Authors: Bershadsky Alexander D. , Vaisberg Eugeni A. , Vasiliev Juri M. 
Published: February 2005

AbstractIt is known that depolymerization of microtubules by colcemid or other similar drugs abolishes polarization of pseudopodial activity in migrating fibroblasts. In this work the effect of colcemid on the intensity of protrusion and retraction of lamellipodia at the active edges of human fibroblasts migrating into the wound was investigated with video‐enhanced contrast microscopy. To characterize the pseudopodial activity quantitatively the outlines of the active edges in the pairs of frames taken at adjacent 20‐sec intervals were compared and mean areas of protrusions and retractions per unit length of the perimeter of the edge were measured. The mean rates of protrusions and retractions were 4–6 times less in colcemid‐treated cells than in controls. Thus, microtubules depolymerized by colcemid, and/or intermediate filaments undergoing perinuclear collapse in the presence of this drug, are essential not only for the restriction of pseudopodial activity to one particular zone of the cell edge but also for the development of maximal activity in this zone.
Segregation of the nucleolus during mitosis in budding and fission yeast
Authors: Granot David , Snyder Michael 
Published: February 2005

AbstractThe segregation of the nucleolus during mitosis was examined in Saccharomyces cerevisiae and Schizosaccharomyces pombe by indirect immunofluorescence using antibodies directed to highly conserved anti‐nucleolus antigens. In mitotic S. pombe cells, the nucleolus appears to trail the bulk of the DNA. In wild‐type cells of S. cerevisiae, the nucleolus segregates alongside the bulk of the genomic DNA. Based on its distance from the centromere, we would expect the rDNA in both organisms to segregate behind the majority of the genomic DNA, if telomeric regions trail centromeric regions as in other eukaryotes. We therefore suggest that in S. cerevisiae the nucleolus is attached to other parts of the nucleus which enable it to segregate along with the bulk of the DNA. The segregation of the nucleolus in topoisomerase mutants and nuclear division mutants of S. cerevisiae was also investigated. In cdc14 mutants which arrest at late anaphase, the vast majority of the DNA is separated, but the nucleolar antigens remain extended between the mother and daughter cells. Thus, the CDC14 gene of S. cerevisiae appears to be important for the separation of the nucleolus at mitosis.
Vinculin in relation to stress fibers in spread platelets
Authors: Nachmias V. T. , Golla R. 
Published: February 2005

AbstractTo investigate the function of vinculin in blood platelets, we studied its localization in relation to other cytoskeletal proteins as well as its state of phosphorylation in platelets allowed to spread on fibrinogen‐coated surfaces. By 5 minutes after loading the platelets onto the surfaces the 47 and 20 kDa polypeptides became phosphorylated, indicating activation. By 30 minutes, platelets formed small, typical bundles of fibers which stained brilliantly with rhodamine phalloidin. Myosin and tropomyosin, detected with specific antibodies, were localized in periodic arrays along these bundles. By indirect immunofluorescence, a discrete patch of vinculin was observed at each end of every actin‐containing bundle. Vinculin phosphorylation was not detected in immunoprecipitates protected against phosphatases. Interference reflection images showed that regions of close binding to the substratum (adhesion plaques) closely matched the vinculin staining sites. Talin appeared diffusely localized. It could be shown to be present in the plaques when platelets were stabilized with ZnCl2 by the method of Geiger and then sonicated to remove some of the surface membrane. Localizations of vinculin and myosin were unaltered by this treatment. Talin phosphorylation or proteolysis could not account for vinculin translocation.We conclude that platelets, in response to an appropriate physiological surface, form typical actin bundles with vinculin at the termination of each bundle, in close relation to adhesion plaques. The signal for this translocation does not appear to depend on phosphorylation of vinculin or on phosphorylation or proteolysis of talin. Our findings support the conclusion that in platelets, as in nucleated cells, vinculin serves as at least part of the connection between bundled actin fibers and the extracellular matrix. Such a connection seems required for platelets' known ability to exert tension on surfaces.