bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of motility and cytoskeletal organization of rat bladder carcinoma cells by cyclic AMP
Authors: Morton Diane M. , Tchao Ruy 
Published: February 2005

AbstractCyclic AMP (cAMP) has been implicated in the regulation of movement of certain cultured cell types. We have studied the effects of cAMP on epithelial cell motility using serum‐free NBT‐II cells, derived from a rat bladder carcinoma. The random movement of these cells on type I collagen was reduced upon elevation of intracellular cAMP by several means and this effect was reversible. Alterations in the organization of the cytoskeletal proteins F‐actin and α‐actinin occurred concurrently with the reduction in motility, and the arrangement of these proteins resembled that seen in non‐motile cells on glass. In addition, pretreatment of cells with KT5720, a cAMP‐dependent protein kinase (PKA)‐specific inhibitor, prevented the dibutyryl cAMP‐induced reduction in cell movement as well as the associated cytoskeletal changes. These results suggest that elevation of PKA is responsible for the observed effects on cell motility and cytoskeletal reorganization and demonstrate a role for PKA in the regulation of cell motility in this system. © 1994 Wiley‐Liss, Inc.
Tubulin gene expression during growth and maturation of leaves with different developmental patterns
Authors: Hellmann Andreas , Meyer Claudius U. , Wernicke Wolfgang 
Published: February 2005

AbstractChanges in the tubulin‐protein and ‐poly(A)+RNA contents were monitored by means of Western and Northern blot analyses, respectively, during growth and maturation of leaves of a dicotyledonous (tobacco) and monocotyledonous (barley) plant. It was recently argued from immunofluorescence and preliminary biochemical data that the density of microtubular networks and concomitantly the tubulin content are distinctly reduced after cessation of cell growth in leaves [Jung et al., 1993]. The results presented now confirm and extend this view. There appeared to be clear differences between the monocot and the dicot: (1) the loss of tubulin during leaf development was much slower in the dicot than in the monocot leaves (within months instead of days); (2) the degree of loss was more dramatic in the monocot leaf and only very low threshold levels of tubulin were retained in fully differentiated tissues; and (3) the loss of tubulin in the monocot leaf tissue appeared to be correlated with the decrease in the mRNA content, whereas the high level of tubulin‐RNA in fully differentiated or even almost senescent dicot leaves indicated a gene expression control at the posttranscriptional level.The comparatively rapid and very distinct tubulin‐protein and ‐RNA disappearance during development of the monocot leaf tissues confirm at the molecular level that differentiation proceeds much faster and is much more determinative in these leaves, as was postulated from histological and physiological data. The differences in the behaviour of the microtubular cytoskeleton perhaps even reflect the differences in the ability of the differentiated leaf cells to dedifferentiate, i.e., to establish new sets of microtubules and to reenter the mitotic cell cycle, e.g., during would response, tumour induction or in vitro culture. © 1995 Wiley‐Liss, Inc.
Kinesin does not support the motility of zinc‐macrotubes
Authors: Ray Sanghamitra , Wolf Sharon G. , Howard Jonathon , Downing Kenneth H. 
Published: February 2005

AbstractMoving along a microtubule, kinesin follows a course parallel to the protofilaments; but it is not known whether kinesin binds exclusively on a single protofilament. The presence of zinc during tubulin polymerization induces sheets where neighboring protofilaments are antiparallel. If kinesin could support the motility of these zinc‐sheets, then the binding site for a kinesin molecule would be limited to a single protofilament.Kamimura and Mandelkow [1992: J. Cell Biol. 118:865–75] reported that kinesin moves along zinc‐sheets. We found that zinc‐sheets grown under their conditions often had a microtubule‐like structure along one edge. We confirmed the possibility that the motility observed by Kamimura and Mandelkow [1992: J. Cell Biol. 118:865–75] is attributed to the microtubule‐like structure rather than the zinc‐sheet.To resolve the question of whether kinesin can recognize an antiparallel protofilament lattice, we investigated the kinesin‐mediated motility of zinc‐macrotubes. At higher free zinc concentrations, zinc‐sheets roll up as macrotubes, free of edges. In the presence of 10 m̈M taxol and 100 nM free Zn2+ at pH 6.8, the samples were shown by electron microscopy to contain only macrotubes. Under these buffer conditions, kinesin could bind strongly to axonemal doublets in the presence of AMP‐PNP, and generate motility in the presence of ATP, but kinesin did not bind to nor move the macrotubes. This shows that kinesin cannot bind efficiently to nor move on the anti‐parallel lattice; it is possible (though not necessary) that the groove between two parallel protofilaments is required for kinesin's motility. © 1995 Wiley‐Liss, Inc.
Monoclonal anti‐dipeptide antibodies cross‐react with detyrosinated and glutamylated forms of tubulins
Authors: Kuriyama Ryoko , Levin Andrew , Nelson David , Madl James , Frankfurther Anthony , Kimble Mary 
Published: February 2005

AbstractTwo monoclonal antibodies, GLU‐1 and A1.6, raised against γ‐L‐glutamyl‐L‐glutamic acid dipeptide (Glu‐Glu) and Ca2+ ‐dependent ATPase from Paramecium, respectively, recognized the dipeptide Glu‐Glu sequence. Whereas the antibodies immunofluorescently stained very few, if any, cytoskeletal fibers in cultured mammalian cells, almost all interphase as well as mitotic spindle microtubules became visible after treatment of cells with carboxypeptidase A. Immunoblot analysis demonstrated intense cross‐reaction of the antibodies to the α‐tubulin subunit. α‐Tubulin isotypes produced as fusion proteins in bacteria were labeled by both the antibodies only when the proteins did not contain a tyrosine residue at the C terminus, indicating that GLU‐1 and A1.6 specifically recognize the detyrosinated from of α‐tubulin. When microtubule protein purified from brain was probed, not only α‐but also, to a lesser extent, β‐tubulin were revealed by the dipeptide antibodies. A synthetic tripeptide YED containing one glutamyl group linked to the second residue of the peptide via the γ position was also recognized by the antibodies. Since this peptide sequence corresponds to the amino acid sequence of polyglutamyated class IIIβ isotype at amino acid position 437 to 439, it is suggested that GLU‐1 and A1.6 are able to recognize the glutamylated form of β‐tubulin. These results indicate that the C‐terminal Glu‐Glu sequence displays strong antigenicity, and the antibodies recognize the sequence present in the C terminus of the detyrosinated form of α‐tubulin and the glutamyl side chain of β‐tubulin. Particularly strong immunoreaction was detected with ciliary and flagellar microtubules; thus, stable axonemal microtubules appear to be rich in post‐translationally modified tubulin subunits. © 1995 Wiley‐Liss, Inc.
Chemoreceptor‐mediated polymerization and depolymerization of actin in hair bundles of sea anemones
Authors: Watson Glen M. , Roberts Julia 
Published: February 2005

AbstractHair bundles located on tentacles of sea anemones are morphodynamic mechanoreceptors employed to regulate discharge of nematocysts into swimming prey. Activation of chemoreceptors for N‐acetylated sugars is known to induce anemone hair bundles to elongate while shifting discharge to lower frequencies matching those produced by calmly swimming prey. In the continued presence of N‐acetylated sugars, activation of proline receptors is known to induce hair bundles to shorten while shifting nematocyst discharge to higher frequencies presumed to correspond to movements produced by wounded, struggling prey. In the present study, N‐acetylneuraminic acid (NANA) causes stereocilia to become more intensely fluorescent in confocal optical sections of phalloidin‐stained specimens, suggesting that receptors for N‐acetylated sugars initate processes to increase the density of F‐actin within stereocilia. Computer analysis of electron micrographs is consistent with this interpretation for large diameter stereocilia but not for small diameter stereocilia. In the continued presence of NANA, proline causes flurescence intensity of phalloidin to decrease to or below control levels. DNaseI uniformly stains large diameter stereocilia, suggesting that these stereocilia contain a pool of G‐actin. Fluorescence intensity of DNaseI in stereocilia is significantly less bright in specimens exposed to NANA alone than in specimens exposed to proline in the continued presence of NANA. It appears that whereas activated receptors for NANA induce G‐actin to polymerize in large diameter stereocilia, activated receptors for proline induce F‐actin to depolymerize, restoring G‐actin pools. © 1995 Wiley‐Liss, Inc.
Organization and structure of actin filament bundles in Listeria‐infected cells
Authors: Zhukarev Vladimir , Ashton Francis , Sanger Jean M. , Sanger Joseph W. , Shuman Henry 
Published: February 2005

AbstractDuring its motion inside host cells, Listeria monocytogenes promotes the formation of a column of actin filaments that extends outward from the distal end of the moving bacterium. The column is constructed of short actin filaments that polymerize at the bacteria‐column interface. To get a measure of filament organization in the column, Listeria grown in cultured PtK2 cells were studied with steady state fluorescence polarization, confocal microscopy, and whole cell intermediate voltage electron microscopy. Although actin filament ordering was higher in nearby stress fibers than in the Listeria‐associated actin, four distinct areas of ordering could be observed in fluorescence polarization ratio images of bacteria: (1) the surface of the bacteria, (2) the cytoplasm next to the bacteria, (3) the outer shell of the actin column, and (4) the core of the column. Filaments were preferentially oriented parallel to the long axis of the column with highest ordering along the long axis of the bacterial surface and in the shell of the tail. The lowest ordering was in the core (where filaments are possibly also shorter with respect to the cup and the shell), whereas in the adjacent cytoplasm, filaments were oriented perpendicular to the column. A mutant of Listeria that can polymerize actin around itself but cannot move intracellularly does not have its actin organized along the bacterial surface. Thus the alignment of the actin filaments along the bacterial surfaces may be important for the intracellular movement. These conclusions are also supported by confocal microscopy and whole mount electron microscopic data that also reveal that actin filaments can be deposited asymmetrically around the long axis of the bacteria, a distribution that may affect the direction of motility of Listeria monocytogenes inside infected cells. © 1995 Wiley‐Liss, Inc.
Dynein inner arm heavy chain identification in cAMP‐activated flagella using class‐specific polyclonal antibodies
Authors: Stephens R. E. , Prior G. 
Published: February 2005

AbstractWhile studying cAMP‐dependent dynein α‐heavy chain phosphorylation, we found previously [Stephens and Prior, 1992: J. Cell Sci. 103:999–1012] that high salt extraction of sperm flagella from the mussel Mytilus edulis or the clam Spisula solidissima removed most visible dynein arms, accompanied by an amount of Mg+2‐ATPase that correlated with the mass of dynein α‐and β‐heavy chains removed. However, although almost devoid of ATPase activity, such extracted axonemes retained one third of the heavy chain mass as two sets of electrophoretically‐distinct, vanadate‐cleavable, non‐phosphorylated proteins. To explore the nature of these dynein‐like proteins, antibodies to the α‐ and β‐heavy chains were blot affinity‐purified from a rabbit antiserum raised against gradient‐purified Spisula 18‐20S flagellar outer arm dynein. Although able to recognize common epitopes of the opposite chain type, neither the α‐nor the β‐heavy chain antibody recognized the tightly‐bound proteins in either species, proving that they are immunologically distinct. While the β‐antibody recognized its heavy chain homolog in gill cilia, the α‐antibody did not, demonstrating immunological distinction between flagellar and ciliary dynein α‐heavy chains. Immunization of a mouse with nitrocellulose strips containing one of the two tightly‐bound Spisula flagellar proteins produced an antiserum that cross‐reacted with each tightly‐bound protein in both species and also recognized α‐ and β‐heavy chains. The anti‐molluscan serum cross‐reacted strongly with sea urchin sperm flagellar dynein B‐, C‐, and D‐bands, considered to be inner arm components, but not with sea urchin outer arm α‐ or β‐heavy chains. These data indicate that the electro‐phoretically and immunologically distinct, tightly‐bound proteins of molluscan flagella are inner arm dynein heavy chains. © 1995 Wiley‐Liss, Inc.
β‐Tubulin mutation suppresses microtubule dynamics in vitro and slows mitosis in vivo
Authors: Sage Carleton R. , Davis Ashley S. , Dougherty Cynthia A. , Sullivan Kevin , Farrell Kevin W. 
Published: February 2005

AbstractMicrotubule (MT) dynamics vary both spatially and temporally within cells and are thought to be important for proper MT cellular function. Because MT dynamics appear to be closely tied to the guanosine triphosphatase (GTPase) activity of β‐tubulin subunits, we examined the importance of MT dynamics in the budding yeast S. cerevisiae by introducing a T107K point mutation into a region of the single β‐tubulin gene, TUB2, known to affect the assembly‐dependent GTPase activity of MTs in vitro. Analysis of MT dynamic behavior by video‐enhanced differential interference contrast microscopy, revealed that T107K subunits slowed both the growth rates and catastrophic disassembly rates of individual MTs in vitro. In haploid cells tub2‐T107K is lethal; but in tub2‐T107K/tub2‐590 heterozygotes the mutation is viable, dominant, and slows cell‐cycle progression through mitosis, without causing wholesale disruption of cellular MTs. The correlation between the slower growing and shortening rates of MTs in vitro, and the slower mitosis in vivo suggests that MT dynamics are important in budding yeast and may regulate the rate of nuclear movement and segregation. The slower mitosis in mutant celis did not result in premature cytokinesis and cell death, further suggesting that cell‐cycle control mechanisms “sense” the mitotic slowdown, possibly by monitoring MT dynamics directly. © 1995 Wiley‐Liss, Inc.
Flagellar quiescence response in sea urchin sperm induced by electric stimulation
Authors: Shingyoji Chikako , Takahashi Keiichi 
Published: February 2005

AbstractTo investigate the mechanism of the flagellar quiescence in sperm, we examined the effect of electric stimulation of individual spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Stimulation with a suction electrode attached to the sperm head elicited a flagellar quiescence response, in which the sperm showed a typical cane‐shaped bend in the proximal region of the flagellum when the electrode was used as anode. Cathodic stimulation also induced quiescence, but was much less effective than anodic stimulation. During the quiescence response, which lasted for 1–3 s, no new bend was initiated, and subsequently the flagellum resumed normal beating. The quiescence response required the presence of Ca2+ (>2 mM) in sea water, and was inhibited by Co2+ and La3+. At low Ca2+ concentrations (2–5 mM), the angle of the cane‐shaped bend was smaller than that at 10 mM Ca2+; thus the angle of the cane‐shaped bend, characteristic of the quiescence response is dependent on Ca2+ concentration. These results suggest membrane, followed by an influx of Ca2+ into the flagellum through Ca2+ channels. The increase in Ca2+ concentration within the flagellum affects the amount of sliding and thus produces a cane‐shaped proximal bend of various angles, white inhibiting both the propagation of the proximal bend (principal bend) and the formation of a new reverse bend.
Experimental manipulation of γ‐tubulin distribution in arabidopsis using anti‐microtubule drugs
Authors: Liu Bo , Palevitz Barry A. , Joshi Harish C. 
Published: February 2005

Abstractγ‐Tubulin‐specific antibodies stain the microtubule (Mt) arrays of Arabidopsis suspension cells in a punctate or patchy manner. During division, staining of kinetochore fibers and the phragmoplast is extensive, except in the vicinity of the plus ends at the metaphase plate and cell plate. γ‐Tubulin localization responds to low levels of colchicine, with staining receding farther toward the minus (pole) ends of kinetochore fibers. At higher drug concentrations, γ‐tubulin also associates with abnormal Mt foci as well as with the surface of the daughter nuclei facing the phragmoplast. During UV‐induced recovery from colchicine, γ‐tubulin increases along the presumptive minus ends of mitotic Mts as well as the phragmoplast near the daughter nuclei. With CIPC, immunostaining is concentrated around the centers of focal Mt arrays in multipolar spindles. In the presence of taxol, Mts are more prominent but the mitotic apparatus and phragmoplast are abnormal. As with CIPC, γ‐tubulin is concentrated at focal arrays. Increased punctate staining is also present in interphase arrays, with fluorescent dots often located at the ends of Mts. These results support a preferential association between γ‐tubulin and Mt minus ends, but are also consistent with more general binding along the walls of Mts. Thus, minus ends (and Mt nucleation sites) may be present throughout plant Mt arrays, but γ‐tubulin may also serve another function, such as in structural stabilization.
Exogenous gelsolin binds to sarcomeric thin filaments without severing
Authors: Gonsior Sabine , Hinssen Horst 
Published: February 2005

AbstractWe have investigated the binding of gelsolin to thin myofilaments in situ and their stability against severing. Differentiated myotubes from chicken skeletal muscle containing cross‐striated myofibrils were permeabilized with Triton X‐100 and incubated with gelsolin. Immunoflurorescence microcopy localized both endogenous and exogenous gelsolin in the I‐Z‐I‐regions of the sarcomers. The staining pattern suggested a binding of the exogenous gelsolin along the entire length of the thin filaments. This binding was Ca2+ dependent, but gelsolin was not removed after subsequent addition of EGTA. The fluorescence staining for actin remained unchanged after gelsolin incubation, indicating that thin filaments in cross‐striated myofibrils were resistant to the severing action of gelsolin, in contrast to the microfilaments in stress fibers. After extraction of the permeabilized cells with high ionic strength to remove tropomyosin and myosin, gelsolin stell bound along the entire thin filament and the actin pattern also remained unchanged. After Triton X‐100 permeabilization and high ionic strength extraction, the giant protein nebulin was found to be still present as a myofibrillar component. Gelsolin treatment after high salt extraction affected neither actin nor nebulin in the thin filaments. We therefore conclude that nebulin confers the gelsolin resistance to the sarcomeric actin filaments.
Overexpression of an epitope‐ tagged β‐tubulin in Chinese hamster ovary cells causes an increase in endogenous α‐tubulin synthesis
Authors: Gonzalez‐Garay Manuel L. , Cabral Fernando 
Published: February 2005

AbstractA Chinese hamster β‐tubulin cDNA, engineered to express a 9 amino acid epitope from the influenza hemagglutinin antigen (HA), was transfected into Chinese hamster ovary (CHO) cells. The recombinant protein (HAβ1‐tubulin) appeared to behave normally by the following criteria: immunofluorescence indicated that HAβ1‐tubulin incorporated into all classes of interphase and spindle microtubules as well as microtubule organizing centers. The sensitivity of the cells expressing HAβ1‐tubulin to Colcemid and taxol was unchanged. A 210 kD microtubule associated protein (MAP) remained associated with microtubules that incorporate HAβ1‐tubulin. The synthesis of both endogenous β‐tubulin and HAβ1‐tubulin was repressed by colchicine. The HAβ1‐tubulin incorporated into microtubules to the same extent as the endogenous β‐tubulin, and the overall extent of microtubule assembly in transfected cells was unchanged. Finally, trasfected cells had normal growth rates and morphologies. When effects on endogenous tubulin production were measured, it was found that expression of the HAβ1‐tubulin reduced the synthesis of endogenous wild‐type β‐tubulin but increased the synthesis of α‐tubulin. At steady state, a small increase in total tubulin consistent with the increased synthesis of α‐tubulin was found. The results indicate that expression of excess exogenous β‐tubulin perturbs the synthesis of endogenous α‐tubulin in a manner that is not easily explained by current models of tubulin regulation. The changes in tubulin synthesis along with degradation of excess tubulin subunits may reflect mechanisms that exist to ensure coordinate levels of α‐ and β‐tubulin for assembly. © 1995 Wiley‐Liss, Inc.
Molecular environment of ZO‐1 in epithelial and non‐epithelial cells
Authors: Howarth Andrew G. , Stevenson Bruce R. 
Published: February 2005

AbstractWe previously reported the expression of ZO‐1 in cell types that do not form tight junctions. Here we compare the molecular environments of ZO‐1 in epithelial cells, primary cultures of astrocytes and in the non‐epithelial S180 sarcoma cell line. ZO‐1 co‐localizes with a subset of actin filament in all cell types. In astrocytes, ZO‐1 is found concentrated in discrete bands at points of cell‐cell contact. Indirect immunofluorescent microscopy shows that these bands of ZO‐1 co‐localize with the adherens junction proteins vinculin and α‐actinin, and with the antigen recognized by a pan‐cadherin antibody. In contrast, ZO‐1 in S180 cells, which exhibit limited cell‐cell interactions, is diffusely distributed over the plasma membrane, with concentrations in lamellipodia where actin filaments accumulate. ZO‐1 does not co‐localize with vinculin at focal adhesions in this cell type. Analysis of ZO‐1 immunoprecipitation profiles from different cell types, performed under conditions previously demonstrated to maintain interactions between ZO‐1, ZO‐2 and p130 from the MDCK epithelial cell line, show that the proteins which co‐precipitate with ZO‐1 vary with cell type. Precipitation of polypeptides at 165 kDa, potentially ZO‐2, and 65 kDa occurs in both a mouse kidney tubule epithelial cell line and the non‐epithelial S180 cells. No proteins specifically associate with ZO‐1 immunoprecipitated from astrocytes. Spectrin, α‐actinin, vinculin and cadherin are not detected in immunoblots of ZO‐1 immunoprecipitates from any cell type. © 1995 Wiley‐Liss, Inc.