bluuebunny/crossref_metadata_2025_split huggingface dataset:
Disorganization of microfilaments and intermediate filaments interferes with the assembly and stability of desmosomes in MDCK epithelial cells
Authors: Pasdar Manijeh , Li Zhi 
Published: February 2005

AbstractTo investigate the possible role(s) of cytoskeletal elements in desmosome assembly we have studied the effects of cytostatic drugs on the assembly of desmosomes in MDCK epithelial cells. We showed previously [Pasdar et al.: Cell Motil. Cytoskeleton 23:201–213, 1992] that selective disruption of microtubules has no effect on desmosome assembly. Here, we have treated MDCK cells with cytochalasin B and a combination of cytochalasin B and nocodazole and analysed the effects on desmosome assembly. Immunofluorescence analysis of MDCK cultures following drug treatment indicated complete disruption of actin microfilaments and disorganization of cytokeratin intermediate filaments. Biochemical analysis of newly synthesized desmosomal membrane core glycoproteins as well as the cell adhesion proteir. E‐cadherin revealed no effect of these drugs on the kinetics of synthesis, intracellular processing, or transport to the plasma membrane either in the presence or absence of cell‐cell contact. However, morphological analyses revealed a significant disruption in the spatial organization of desmosomal proteins and E‐cadherin. Drug treatment in the absence of cell‐cell contact resulted in the disruption of the normally observed homogenous punctate staining pattern and appearance of aggregate staining. Induction of cell‐cell contact in these cultures resulted in redistribution of some of the aggregate staining to the plasma membrane. In contrast to control cultures, significant amount of intracellular staining was retained for all desmosomal proteins. Biochemical analyses of turnover rates of newly synthesized desmosomal proteins indicated a significant decrease in metabolic stability of these proteins while the turnover rate of E‐cadherin was not significantly different among control and drug‐treated cultures. Taken together, these results suggest that intact actin and cytokeratin filaments are necessary for the stability, efficient assembly, and spatial organization of the junctional components at the membrane. The regulatory role of cytokeratins and actin filaments in assembly and stability of desmosomes on the plasma membrane is discussed. © 1993 Wiley‐Liss, Inc.
CENP‐F is a ca 400 kDa kinetochore protein that exhibits a cell‐cycle dependent localization
Authors: Rattner J. B. , Rao A. , Fritzler M. J. , Valencia D. W. , Yen T. J. 
Published: February 2005

AbstractWe have identified a novel .ca 400 kDa cell‐cycle dependent kinetochore associated protein in human cells, designated CENP‐F, using human autoimmune serum. Immunofluorescence staining using the native serum, affinity purified antibodies, or antibodies raised against a cloned portion of CENP‐F first reveals CENP‐F homogeneously distributed throughout the nucleus of HeLa cells in the G2 stage of the cell cycle. Progression into prophase is accompanied by the localization of CENP‐F to all the kinetochore regions of the karyotype. Kinetochore association is maintained throughout metaphase, but at the onset of anaphase CENP‐F is no longer detected in association with the kinetochore but is found at the spindle mid‐zone. By telophase, it is concentrated into a narrow band on either side of the midbody. Studies of the interaction of CENP‐F with the kinetochore indicate that this protein associates with the kinetochore independent of tubulin and dissociation is dependent on events connected with the onset of anaphase. Nuclease digestion studies and immunoelectron‐microscopy indicate that CENP‐F is localized to the kinetochore plates and specifically to the outer surface of the outer kinetochore plate. The distribution of CENP‐F closely parallels that of another high molecular weight kinetochore associated protein, CENP‐E. Comparative studies indicate that there are antibodies in the CENP‐F reactive autoimmune serum that recognize determinants present in the central helical rod domain of CENP‐E. Immune depletion experiments confirm that CENP‐F exhibits the distribution pattern in cells that was seen with the native autoimmune serum. © 1993 Wiley‐Liss, Inc.
Centrosome repositioning immediately following karyokinesis and prior to cytokinesis
Authors: Mack G. , Rattner J. B. 
Published: February 2005

AbstractThe behaviour of the centrosome immediately following cell division in tissue culture cells has been investigated. We find that following karyokinesis, but preceding cytokinesis, sister centrosomes relocate from the spindle poles to a position adjacent to the intercellular bridge. This repositioning is accompanied by the appearance of a microtubule bundle that extends from the poleward region of the cell to the centrosome and increases in length as the centrosome approaches the intercellular bridge. Disruption of this bundle with colcemid interrupts centrosome repositioning. In contrast, centrosome repositioning persists in late mitotic cells grown in the presence of cytochalasin D. However, the position of the microtubule‐centrosome complex within the cell is randomized suggesting that the path, but not the process, of centrosome repositioning is dependent on an intact actin filament network. This study points out, for the first time, that the complex migration of the centrosome preceding mitosis is paralleled by an equally complex set of events following cell division. We suggest that post‐mitotic centrosome repositioning may play a role in ensuring that daughter cells have equal but opposite polarity and may reflect an interrelationship between the establishment of the interphase cytoskeleton and the completion of cytokinesis. © 1993 Wiley‐Liss, Inc.
β‐actin specific monoclonal antibody
Authors: Gimona Mario , Vandekerckhove Joel , Goethals Marc , Herzog Monika , Lando Zeev , Small J. Victor 
Published: February 2005

AbstractUsing a synthetic peptide mimicking the NH2‐terminus of β‐actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the β isoform, by its exclusive recognition of the synthetic β‐actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the β‐actin spot in two‐dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function. © 1994 Wiley‐Liss, Inc.
Effects of silver ions (Ag+) on contractile ring function and microtubule dynamics during first cleavage in Ilyanassa obsoleta
Authors: Conrad Abigail H. , Stephens Andy P. , Paulsen Avelina Q. , Schwarting Scott S. , Conrad Gary W. 
Published: February 2005

AbstractThe terminal phase of cell division involves tight constriction of the cleavage furrow contractile ring, stabilization/elongation of the intercellular bridge, and final separation of the daughter cells. At first cleavage, the fertilized eggs of the mollusk, Ilyanassa obsoleta, form two contractile rings at right angles to each other in the same cytoplasm that constrict to tight necks and partition the egg into a trefoil shape. The cleavage furrow contractile ring (CF) normally constricts around many midbody microtubules (MTs) and results in cleavage; the polar lobe constriction contractile ring (PLC) normally constricts around very few MTs and subsequently relaxes without cleavage. In the presence of Ag+ ions, the PLC 1) begins MT‐dependent rapid constriction sooner than controls, 2) encircles more MTs than control egg PLCs, 3) elongates much more than control PLCs, and 4) remains tightly constricted and effectively cleaves the polar lobe from the egg. If Ag+‐incubated eggs are returned to normal seawater at trefoil, tubulin fluorescence disappears from the PLC neck and the neck relaxes. If nocodazole, a drug that depolymerizes MTs, is added to Ag+‐incubated eggs during early PLC constriction, the PLC is not stabilized and eventually relaxes. However, if nocodazole is added to Ag+‐incubated eggs at trefoil, tubulin fluorescence disappears from the PLC neck but the neck remains constricted. These results suggest that Ag+ accelerates and gradually stabilizes the PLC constriction by a mechanism that is initially MT‐dependent, but that progressively becomes MT‐independent. © 1994 Wiley‐Liss, Inc.
Microtubule sliding in reduced‐amplitude bending waves of Ciona sperm flagella: Bending waves attenuated by lithium
Authors: Brokaw Charles J. 
Published: February 2005

AbstractThe distinct damped, or attenuated, bending pattern observed when demembranated sperm flagella of the tunicate, Ciona, are reactivated in the presence of 2 mM Li+ has been analysed in detail. In these patterns, bends are initiated at the base of the flagellum, but die out after they start to propagate along the flagellum, so that little or no bending is seen in the distal half of the flagellum. A quantitative descriptive analysis shows that the distinctive feature of this attenuation of bending wave amplitude is an asymmetric interbend decay, or slippage, occuring, on average, only at the transitions between a reverse bend and the preceding principal bend. This attenuation is combined with a significant amount of synchronous sliding in the distal half of the flagellum and a decrease in propagation velocity of transitions between bends in the mid‐region of the flagellum.Computer simulations demonstrate that the synchronous sliding in the distal half of these flagella can be an entirely passive consequence of the mechanical interaction between active sliding and bending in the basal third of the flagellum and viscous resistances to movement of the distal region of the flagellum through the fluid environment. The current computer models do not contain a mechanism for asymmetric interbend decay that can reproduce these attenuated bending patterns. © 1994 Wiley‐Liss, Inc.
Role of cAMP in the reactivation of demembranated ram spermatozoa
Authors: San Agustin Jovenal T. , Witman George B. 
Published: February 2005

AbstractEjaculated ram sperm were demembranated with Triton X‐100, separated from the detergent‐soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264–273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was ∼100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP‐dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5–24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5–24) prior to reactivation. The cytosol‐free models retained >90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X‐100 alone, but only by cAMP and Triton X‐100 in combination. We conclude that cAMP‐dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg‐lys‐arg‐ala‐arg‐lys‐glu, which has been reported to be a selective inhibitor of cGMP‐dependent protein kinase. © 1994 Wiley‐Liss, Inc.
Microtubule converging centers and reorganization of the interphase cytoskeleton and the mitotic spindle in higher plant Haemanthus
Authors: Smirnova Elena A. , Bajer Andrew S. 
Published: February 2005

AbstractWe analyzed the distribution and orientation of transitory microtubule structures, microtubule converging centers, during interphase and mitosis in endosperm of the higher plant Haemanthus. In interphase the pointed tips of microtubule converging centers are associated with the nuclear envelope. Their orientation gradually reverses during prophase, and the tips tend to point away from the nucleus. From prometaphase through early telophase, microtubule converging centers are present predominantly in the cytoplasm at the polar region. They are either “free” or associated with chromosomes or microtubule bundles. In late telophase, pointed tips of microtubule converging centers are again associated with the reconstructed nuclear envelope and, additionally, they often appear in the phragmoplast area. The orientation of microtubule converging centers seems to be directly correlated to the previously determined microtubule polarity, with the converging tip being minus and the diverging one, plus.Elevated temperature (35°–37°) enhances the number of microtubule converging centers in the cytoplasm and at the nuclear envelope. This is especially pronounced during the telophase‐interphase transition and in some interphase cells, indicating temperature and stage dependence.Our data imply that microtubule converging centers bind together MT minus ends and, thus, control the predominant direction of elongation and shortening of microtubule arrays. We argue that these configurations are instrumental during the reorganization of interphase cytoskeleton and mitotic spindle in Haemanthus endosperm. © 1994 Wiley‐Liss, Inc.
Antisense MAP‐2 oligonucleotides induce changes in microtubule assembly and neuritic elongation in pre‐existing neurites of rat cortical neurons
Authors: Sharma Nishi , Kress Yvonne , Shafit‐Zagardo Bridget 
Published: February 2005

AbstractMicrotubule‐associated protein 2 (MAP‐2) is an abundant component of the cytoskeleton present in dendrites and cell bodies of neurons of the CNS. To examine the biological function of MAP‐2, two MAP‐2 antisense (AS) oligonucleotides complementary to the 5′ region of the rat MAP‐2 cDNA were added to rat primary embryonic day 17–18 (E17‐18) cultured cortical neurons 24 h after plating and neurite outgrowth and morphology studied. The treatment of primary cortical cultures with either of the two MAP‐2 AS oligonucleotides resulted in decreased MAP‐2 and reduction in the number of neuritic processes relative to the control or MAP‐2 sense‐treated cultures. By immunostaining and light microscopy the AS‐treated neurons appeared smaller, more rounded, and less intensely stained for MAP‐2 than the untreated or the MAP‐2 sense‐treated cultures. By electron microscopy disorganized microtubules and a reduction in the number of microtubules within neurites of the AS‐treated cultures were observed. We conclude that MAP‐2 continues to be required for microtubule spacing and stability within neurites once they have formed. © 1994 Wiley‐Liss, Inc.
Regulation and evolution of the single alpha‐tubulin gene of the ciliate Tetrahymena thermophila
Authors: McGrath Kathleen E. , Yu Su May , Heruth Daniel P. , Kelly Anne A. , Gorovsky Martin A. 
Published: February 2005

AbstractThe single alpha‐tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha‐tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha‐tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha‐tubulins. The single alpha‐tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha‐tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha‐tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, alpha‐tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha‐tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts alpha‐tubulin message amounts via message decay. However the complex control of alpha‐tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates. © 1994 Wiley‐Liss, Inc.
The premyofibril: Evidence for its role in myofibrillogenesis
Authors: Rhee Dukhee , Sanger Jean M. , Sanger Joseph W. 
Published: February 2005

AbstractWhen cardiac muscle cells are isolated from embryonic chicks and grow in culture they attach to the substrate as spherical cells with disrupted myofibrils, and over several days in culture, they spread and extend lamellae. Based on antibody localizations of various cytoskeletal proteins within the spreading cardiomyocyte, three types of myofibrils have been identified: 1) fully formed mature myofibrils that are centrally positioned in the cell, 2) premyofibrils that are closest to the cell periphery, and 3) nascent myofibrils located between the premyofibrils and the mature myofibrils. Muscle‐specific myosin is localized in the A‐bands in the mature, contractile myofibrils, and along the nascent myofibrils in a continuous pattern, but it is absent from the premyofibrils. Antibodies to non‐muscle isoforms of myosin IIB react with the premyofibrils at the cell periphery and with the nascent myofibrils, revealing short bands of myosin between closely spaced bands of α‐actinin. In the areas where the nascent myofibrils border on the mature myofibrils, the bands of non‐muscle myosin II reach lengths matching the lengths of the mature A‐bands. With the exception of a small transition zone consisting of one myofibril, or sometimes several sarcomeres, bordering the nascent myofibrils, there is no reaction of these non‐muscle myosin IIB antibodies with the mature myofibrils in spreading myocytes. C‐protein is found only in the mature myofibrils, and its presence there may prevent co‐polymerization of non‐muscle and muscle myosins. Antibodies directed against the non‐muscle myosin isoforms, IIA, do not stain the cardiomyocytes. In contrast to the cardiomyocytes, the fibroblasts in these cultures stain with antibodies to both non‐muscle myosin IIA and IIB. The premyofibrils near the leading edge of the lamellae show no reaction with antibodies to either titin or zeugmatin, whereas the nascent myofibrils and mature myofibrils do. The spacings of the banded α‐actinin staining range from 0.3 to 1.4 μm in the pre‐ and nascent myofibrils and reach full spacings (1.8‐2.5 μm) in the mature myofibrils. Based on these observations, we propose a premyofibril model in which non‐muscle myosin IIB, titin, and zeugmatin play key roles in myofibrillogenesis. This model proposes that pre‐ and nascent myofibrils are composed of minisarcomeres that increase in length, presumably by the concurrent elongation of actin filaments, the loss of the non‐muscle myosin II filaments, the fusion of dense bodies or Z‐bodies to form wide Z‐bands, and the capture and alignment of muscle myosin II filaments to form the full spacings of mature myofibrils. © 1994 Wiley‐Liss, Inc.
Differences in the effect of Ca2+ on isolated microtubules from cod and cow brain
Authors: Strömberg Elisabeth , Wallin Margareta 
Published: February 2005

AbstractIsolated microtubules from cod and cow brains were compared with respect to their response to calcium ions. The effect of Ca2+ on cod microtubules was found to be temperature dependent. In contrast to cow microtubules, cod microtubules assembled at 18°C. At this temperature the assembly was inhibited by Ca2+ concentrations of 2 mM and higher. This was also found for cow microtubules at 37°C. However, at 30°C there was no effect of 2 mM Ca2+ of the amount of assembly or disassembly of cod microtubules consisting of only tubulin or of tubulin and microtubule‐associated proteins (MAPs). The morphology was affected though, since some coiled ribbons formed from tubulin and MAPs. The calcium‐binding calmodulin did not alter the effect of calcium on cod microtubules markedly. At higher Ca2+ concentrations (>4 mM), coiled ribbons were formed from cod tubulin and MAPs, but mainly amorphous aggregates and very few coiled ribbons were formed from cod tubulin alone, indicating that the Ca2+ effect is modulated by cod MAPs. The modulatory effect of cod MAPs was however not species specific, since both cod and cow MAPs had the same effect on cod microtubules, in spite of a different protein composition. A MAP‐dependent effect of Ca2+ was also found for cow microtubule proteins. The assembly of pure cow tubulin, as well as that of cow tubulin and MAPs, was inhibited by 2 mM Ca2+. In the presence of 10 and 20 mM Ca2+, pure cow tubulin formed amorphous aggregates, rings, and even paracrystals, while the assembly of cow tubulin and MAPs was inhibited. Our results suggest therefore that the effect of Ca2+ can be moderated by MAPs, but depends on intrinsic properties of the different tubulins. © 1994 Wiley‐Liss, Inc.
Intact alpha‐actinin molecules are needed for both the assembly of actin into the tails and the locomotion of Listeria monocytogenes inside infected cells
Authors: Dold Frederick G. , Sanger Jean M. , Sanger Joseph W. 
Published: February 2005

AbstractAfter the infectious bacterium, Listeria monocytogenes, is phagocytosed by a host cell, it leaves the lysosome and recruits the host cell's cytoskeletal proteins to assemble a stationary tail composed primarily of actin filaments cross‐linked with alpha‐actinin. The continual recruitment of contractile proteins to the interface between the bacterium and the tail accompanies the propulsion of the bacterium ahead of the elongating tail. When a bacterium contacts the host cell membrane, it pushes out the membrane into an undulating tubular structure or filopodium that envelops the bacterium at the tip with the tail of cytoskeletal proteins behind it. Previous work has demonstrated that alpha‐actinin can be cleaved into two proteolytic fragments whose microinjection into cells interferes with stress fiber integrity. Microinjection of the 53 kD alpha‐actinin fragment into cells infected with Listeria monocytogenes, induces the loss of tails from bacteria and causes the bacteria to become stationary. Infected cells that possess filopodia when injected with the 53 kD fragment lose their filopodia. These results indicate that intact alpha‐actinin molecules play an important role in the intracellular motility of Listeria, presumably by stabilizing the actin fibers in the stationary tails that are required for the bacteria to move forward. Fluorescently labeled vinculin associated with the tails when it was injected into infected cells. Talin antibody staining indicated that this protein, also, is present in the tails. These observations suggest that the tails share properties of attachment plaques normally present in the host cells. This model would explain the ability of the bacterium (1) to move within the cytoplasm and (2) to push out the surface of the cell to form a filopodium. The attachment plaque proteins, alpha‐actinin, talin, and vinculin, may bind and stabilize the actin filaments as they polymerize behind the bacteria and additionally could also enable the tails to bind to the cell membrane in the filopodia. © 1994 Wiley‐Liss, Inc.