bluuebunny/crossref_metadata_2025_split huggingface dataset:
Primary cilia cycle in PtK1 cells: Effects of colcemid and taxol on cilia formation and resorption
Authors: Jensen Cynthia G. , Davison Edwin A. , Bowser Samuel S. , Rieder Conly L. 
Published: February 2005

AbstractThe effects of colcemid (0.16–1.0 μM) and taxol (10 μM) on the primary cilia cycle in PtK1 cells were studied by antitubulin immunofluorescence microscopy and by high‐voltage electron microscopy of serial 0.25‐μm sections. Although these dings induce a fully characteristic rearrangement (taxol) or disassembly (colcemid) of cytoplasmic microtubulcs, neither affects the structure of primary cilia formed prior to the treatment or the resorption of primary cilia during the initial stages of mitosis. Cells arrested in mitosis by taxol or colcemid remain in mitosis for 5–7 h at 37°C and then form 4N “micronucleated” restitution nuclei. Formation of primary cilia in these micronucleated cells is blocked by colcemid in a concentration‐dependent fashion: normal cilia with expanded (ie, bulbed) distal ends form at the lower (0.16–0.25 μM) concentrations, while both cilia formation and centriole replication are inhibited at the higher (≥ 1.0 μM) concentrations. However, even in the presence of 1.0 μM colcemid, existing centrioles acquire the appendages characteristically associated with ciliating centrioles and attach to the dorsal cell surface. Continuous treatment with colcemid thus produces a population of cells enriched for the early stages of primary cilia formation. Micronucleated cells formed from a continuous taxol treatment contain two normal centriole pairs, and one or both parenting centrioles possess a primary cilium. Taxol, which has been reported to stabilize microtubules in vitro, does not inhibit the cell‐cycle–dependent assembly and disassembly of axonemal microtubules in vivo.
Effect of inhibition of the catalytic activity of cyclic amp‐dependent protein kinase on mitosis in PtK1 cells
Authors: Browne Carole L. , Bird Mary Lynn , Bower William 
Published: February 2005

AbstractEvidence has suggested that cyclic AMP, acting through activation of the type II cyclic AMP‐dependent protein kinase, may play a role in the regulation of interphase and mitotic microtubules. In order to examine the potential role of the type II cAMP‐dependent kinase during mitosis, dividing PtK1 cells were microinjected with two specific inhibitors of the catalytic activity of the type II kinase. These inhibitors were a specific protein inhibitor of cAMP‐dependent protein kinase (PKI) and an affinity‐purified polyclonal antiserum (anti‐C) directed against the catalytic subunit of the kinase. Both have been shown previously to inhibit kinase activity in vitro. Microinjection of PKI during early‐ to mid‐prophase significantly delayed the progression of the cells through mitosis, with the greatest delay occurring in metaphase. PKI injected during prometaphase also delayed progression through mitosis but to a lesser extent. Microinjection of anti‐C during early‐ to mid‐prophase also caused a significant delay in the completion of mitosis, with many cells becoming “hung up” in prometaphase. Anti‐C injected during prometaphase had little effect on subsequent progression through mitosis. Microinjection of either anti‐C or PKI during metaphase had no discernible effect. No effect on anaphase movement of chromosomes was observed with any treatment. These results provide further evidence that cAMP‐dependent phosphorylation may be involved in the regulation of mitosis, although whether it acts directly through regulation of mitotic spindle microtubules is unclear.
Cytochalasin B‐induced redistribution of cytokeratin filaments in PtK1 cells
Authors: Wolf Kathryn M. , Mullins J. Michael 
Published: February 2005

AbstractIndirect immunofluorescence demonstrated a dramatic reorganization of cytokeratin filaments produced by cytochalasin B (CB) treatment of PtK1 cells. Much of the normal cytokeratin network became arranged into a latticework consisting of bundles of cytokeratin filaments that radiated from, and interconnected, distinct foci, Electron microscopy showed foci to be dense granular regions through which bundles of cytokeratin filaments looped. Composition of the foci included actin, myosin, and alpha‐actinin, as shown by labeling with rhodamine phalloidin or specific antisera. Simultaneous treatment with CB and colchicine was not required for lattice formation, but did produce more extensive development than did CB alone. In cells treated only with CB, the microtubule network remained intact, even in regions of extensive lattice formation. These results contrast sharply with those of Knapp et al (J. Cell Biol. 97:1788 [1983b]), who found lattice formation dependent upon simultaneous CB and colchicine treatment. Time‐course and dose‐response studies of CB treatment showed lattice formation to follow disruption of stress fibers and the concentration of actin into distinct patches that marked the location of lattice foci. Overall results suggest a structural association between microfilaments and cytokeratin filaments that produces the lattice pattern upon CB‐induced disruption of stress fibers. Lattice formation was not limited to a specific cell‐cycle stage, since G1, G2, and M cells displayed the lattice. Treatment of cells with dihydro‐CB and experiments with enucleated cells showed that lattice formation was dependent upon neither the inhibition of sugar transport nor the nuclear extrusion effects of CB.
Amebae of Dictyostelium discoideum respond to an increasing temporal gradient of the chemoattractant cAMP with a reduced frequency of turning: Evidence for a temporal mechanism in ameboid chemotaxis
Authors: Varnum‐Finney Barbara , Edwards Kevin B. , Voss Edward , Soll David R. 
Published: February 2005

AbstractIn an aggregation territory of Dictyostelium discoideum, outwardly moving, nondissipating waves of the chemoattractant cAMP sweep across each ameba. At the front of each wave, an ameba experiences an increasing temporal and a positive spatial gradient of cAMP. At the back of a wave, an ameba experiences a decreasing temporal and a negative spatial gradient of cAMP. Employing a perfusion chamber, we have mimicked the temporal dynamics of these waves in the absence of a spatial gradient and demonstrated that the frequency of lateral pseudopod formation and the frequency of turning are dramatically affected by the direction and dynamics of the temporal gradient. In addition, since an ameba will move in a directed fashion up a shallow, nonpulsatile gradient of cAMP, we also mimicked the increasing temporal gradient generated by an ameba moving up a shallow spatial gradient. The frequency of lateral pseudopod formation and the frequency of turning were depressed. Together, these results demonstrate that amebae can assess the direction of a temporal gradient of chemoattractant in the absence of a spatial gradient and alter both the frequency of pseudopod extension and turning, accordingly. Although these results do not rule out the involvement of a spatial mechanism in assessing a spatial gradient, they strongly suggest that the temporal dynamics of a cAMP wave or the temporal gradient generated by an ameba moving through a spatial gradient may play a major role in chemotaxis.
Endoplasmic microtubules connect the advancing nucleus to the tip of legume root hairs, but F‐actin is involved in basipetal migration
Authors: Lloyd Clive W. , Pearce Karen J. , Rawlins David J. , Ridge Robert W. , Shaw Peter J. 
Published: February 2005

AbstractA prominent feature of tip growth in filamentous plant cells is that the nucleus often migrates in step with the tip as it extends. We have studied this long‐recognized but unexplained relationship in root hairs of the legume Vicia hirsuta by a variety of microscopic techniques. Using rhodaminyl lysine phallotoxin, and antitubulin antibodies, root hairs are shown to contain axial bundles of F‐actin and a complex microtubular system. To the basal side of the nucleus the microtubules are cortical and net axial but in the region between nucleus and tip the arrangement is more complicated. Electron microscopic thin sections demonstrate that internal bundles of microtubles exist in addition to the plasma membrane‐associated kind. Computerized deblurring of through‐focal series of antitubulin stained hairs clarifies the three‐dimensional organization: bundles of endoplasmic microtubules progress from the nuclear region toward the apical dome where they can be seen to fountain out upon the cortex.The relationship between nucleus and tip can be uncoupled with antimicrotubule herbicides. Time lapse video microscopy shows that these agents cause the nucleus to migrate toward the base. This contrary migration can be inhibited by adding cytochalasin D, which fragments the F‐actin bundles.It is concluded that microtubules connect the nucleus to the tip but that F‐actin is involved in basipetal migration as is known to occur when symbiotic bacteria uncouple the nucleus from the tip.
Platelet intermediate filaments: Detection of a vimentinlike protein in human and bovine platelets
Authors: Tablin Fern , Taube David 
Published: February 2005

AbstractHuman and bovine platelets contain a 58,000‐dalton vimentinlike protein that cross‐reacts with antivimentin antibody. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots indicate that this protein is present in whole platelet lysates and triton insoluble cytoskeletons. Transmission electron microscopy of platelets reveals an isotropic network of individual intermediate filaments distributed throughout the platelets. High salt, triton extracted, glutaraldehyde and tannic acid fixed platelets reveal 10‐nm filaments that can be seen to form a peripheral ring, as well as an isotropic network in the body of the cells. Indirect immunofluorescence of resting and spread platelets demonstrates a circumferential staining pattern close to the cell membrane, with additional fibrillar staining throughout the platelets. Our data suggest that the 58,000‐dalton vimentinlike protein may be associated with the microtuble coil and the plasma membrane, and may thus help to maintain the resting platelet's discoid shape.
Reorganization of microtubules during mitosis in Dictyostelium: Dissociation from MTOC and selective assembly/disassembly in situ
Authors: Kitanishi‐Yumura Toshiko , Fukui Yoshio 
Published: February 2005

AbstractWe applied the “agar‐overaly” immunofluorescence techinque (Yumura, S., H. Mori, and Y. Fukui, J. Cell Biol. 99:894–899, 1984) to a semisynchronous culture of Dictyostelium discoideum for studying the organization changes in the microtubule system during mitosis. Using a flurescent DNA dye DAPI (4′,6′ ‐diamidino‐2‐phenylindole), chromatin fibers and individual chromosomes were visible in cells prepared by this method, whereby the mitotic phase could be critically evaluated.We found that a rapid shortening of the cytoplasmic microtubules was preceded by a structural dislocation from their organizing centers (MTOCs) in the midprophase, resulting in the transient occurrence of free microtubules in the cytoplasm. Statistic analyses showed that microtubule disassembly in prophase was diphasic. Initially long, wavy microtubules shortened from their distal ends. Following dissociation of their proximal ends from the MTOC, all microtubules initiated rapid disassembly, probably from both ends. During this process, microtubule assembly from the now duplicated spindle pole body (SPB) resumed.This study also revealed novel information on the dynamics of the Dictyostelium mitotic spindle: 1) Half spindles interdigitate in the spindle center, and the extent of interdigition increases coincidentally with the spindle elongation, and 2) during the anaphase to telophase, a subpopulation of spindle microtubules elongates while the rest of the microtubules disasemble very rapidly.Overall this study indicates the presence of elaborate mechanisms responsible for the selective assembly/disassembly of particular microtubule subpopulations in situ.
Subcortical rotation in Xenopus eggs: A preliminary study of its mechanochemical basis
Authors: Vincent Jean‐Paul , Scharf Stanley R. , Gerhart John C. 
Published: February 2005

AbstractThe amphibian egg undergoes a 30° rotation of its subcortical contents relative to its surface during the first cell cycle, a displacement of 350 μm in 50 min. This is directly visualized by following the movement of an array of Nile blue (a subcortical stain) spots applied to the egg periphery (Vincent, Oster, and Gerhart: Dev Bio 113:484–500, '86). We have investigated the mechanochemical basis of this unusual cell motility. Subcortical rotation depends on microtubule integrity during its entire course and is insensitive to inhibitors of microfilament assembly. It does not depend on newly synthesized proteins for its operation or timing, and it does not involve calcium‐dependent processes. Finally, we show that vegetal fragments of the egg can complete rotation on their own, indicating that mechanochemical components can operate locally in this hemisphere.
A critical role for the polarization of membrane recycling in cell motility
Authors: Kupfer Abraham , Kronebusch Paul J. , Rose John K. , Singer S. J. 
Published: February 2005

AbstractThis paper is concerned with the proposition that the insertion of membrane mass into the leading edge of a motile cell plays a critical role in directed cell migration. We show by immunofluorescence, with cells transfected with a cloned cDNA encoding the G‐protein of a temperature‐sensitive mutant of vesicular stomatitis virus, that the first cell surface appearance of the G‐protein is indeed at the leading edge of the motile cell. Two drugs capable of inhibiting directed cell migration, cytochalasin D and monensin, appear to function independently, the former by affecting the actin cytoskeleton without affecting the polarized insertion of membrane mass into the cell surface and the latter by abrogating membrane mass insertion without affecting the actin cytoskeleton.
Monoclonal antibodies against plant proteins recognise animal intermediate filaments
Authors: Parke J. M. , Miller C. C. J. , Cowell I. , Dodson A. , Dowding A. , Downes M. , Duckett J. G. , Anderton B. J. 
Published: February 2005

AbstractFour monoclonal antibodies were raised against polypetides present in a highsalt detergent‐insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunoflurescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti‐IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti‐IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antiboides labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with varibale intensites. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,0000 Mr (two to three bands) polypetides and a diffuse and around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000–200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypetides putative plant intermediate filament proteins.
Regulation of activation state and flagellar wave form in epididymal rat sperm: Evidence for the involvement of both CA2+ and cAMP
Authors: Lindemann Charles B. , Goltz Jason S. , Kanous Kathleen S. 
Published: February 2005

AbstractRat sperm from the cauda epididymis exhibit increased motility, longevity, and a distinct circular pattern of flagellar curvature in response to 5 mM procaine‐HCI or 0.1 mM 8‐(N, N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate (TMB‐8), reagents that are thought to play a role in the immobilization of free cellular calcium. Triton X‐100‐extracted sperm models will exhibit the same pattern of motility and curvature as procaine‐ or TMB‐8‐activated cells, but only when calcium is removed by a strong chelating agent, and in the pesence of cAMP (3 μM). Demembranated sperm models produced from epididymal rat sperm are quiescent unless cAMP is added. In these sperm models, the presence or absence of free calcium mediates a transition in flagellar curvature. The increased activity of the procaine‐treated intact cells was not accompained by a change in cellular ATP content, nor was ATP availability the limiting factor in the quiescent sperm. Therefore, the increased motility produced by procaine is probably mediated by a fall in free intracellular Ca2+ accompained by a rise in cAMP. Our finding that calcium controls the curvature of sperm flagella may explain altered patterns of flagellar beating, such as the hyperactivated motility that sperm exhibit in the female reproductive tract.
In vitro effects of benzodiazepines on ciliogenesis in the quail oviduct
Authors: Boisvieux‐Ulrich Emmanuelle , Laine Marie‐Christine , Sandoz Daniel 
Published: February 2005

AbstractImmature oviduct implants from quails stimulated by estrogen to induce ciliogenesis were submitted to the in vitro action of benzodiazepines in organotypic culture. Diazepam and medazepam were added to the culture medium for 24 or 48 hours and tissues were examined by transmission and scanning electron microscopy for alterations in ciliary differentiation.Ciliogenesis was inhibited by both diazepam and medazepam, which affected mainly the migration of the basal bodies. Assembly of basal bodies was achieved normally in the cytoplasm, but their separation from generative complexes and migration toward the apical membrane were prevented. They remained in clusters around a deuteosome or eventually anchored to the close lateral plasma membrane.Furthermore, the drugs affected mature beating cilia, which then appeared lying tangentially to the cell surface. Relation between basal bodies and cortical cytoskeleton seemed to be altered by the drugs, which implies that the bearing of cilia and probably the ciliary beating movement were modified. Mocrovillus development was also altered by the action of these drugs.
Identification and characterizaton of a protein associated with the stembody using autoimmune sera from patients with systemic sclerosis
Authors: Kingwell B. , Fritzler M. J. , Decoteau J. , Rattner J. B. 
Published: February 2005

AbstractAn autoantibody that binds an antigen localized to the stembody of dividing cells has been identified in a patient with systemic sclerosis. Initially, this antigen is associated with the surface of the metaphase chromosomes. At the onset of anaphase the antigen becomes preferentially associated with the forming stembodies. This association is maintained as furrowing progresses during telophase and continues after the intercellular bridge is released from the daughter cells during G‐1. Immunoblots indicate that the epitope detected by immunoflurorescence is present on a protein with an apparent molecular weight of 38 kD.
Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A431) cells with nanovid microscopy
Authors: De Brabander M. , Nuydens R. , Geerts H. , Hopkins C. R. 
Published: February 2005

AbstractTransferrin receptors labeled with the B3/25 monoclonal antibody–gold complexes were followed in living A431 cells by using video‐enhanced contrast microscopy. Initially, the antibody‐gold complexes bind to receptors which are freely mobile on the upper cell surface; they then become trapped at the inner margins of the peripheral lamellae and internalize. During endocytosis discrete gold‐loaded vesicular elements first appear, and then, as they fuse, a heterogenous peripheral endosomal compartment forms. The endosomes from this compartment then begin to migrate centripetally through the cytoplasm in a saltatory way so that within 15 min gold label accumulates in a juxtanuclear endosome compartment. This compartment, which consists mainly of multivesicular bodies, is thus formed by the influx and retention of peripheral endosomal elements and their continued fusion in the juxtanuclear area. Although their overall migration is inward, saltating endosomes frequently reverse their direction of movement. As label builds up in the juxtanuclear area, small vesicles containing gold label continuously pinch off from the larger elements and migrate toward the cell periphery.Experiments with nocodazole and sodium azide show that the saltatory movements, the accumulation and retention of endosomes in the juxtanuclear area, and the separation of vesicles from endosomes are driven by a microtubule‐associated, ATP‐dependent, motility‐generating mechanism.Analysis of the movements shows that although each individual vesicle saltation can occur unpredictably toward the centre or the periphery of the cell, a net centripetal flux is observed. Moreover, it is evident that the probability of migration toward and maintenance in the juxtanuclear area is related to the diameter of the vesicles. We propose a mechanism by which bidirectional saltation along microtubules forming a radial network may be instrumental in the selective concentration of large endosomes in the juxtanuclear area while small vesicles are left free to return to the periphery. This process may be responsible for the sorting of receptors and ligands destined either for intracellular degradation in juxtanuclear lysosomes or, alternatively, for recycling to the plasma membrane.