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1
Vollrath, Michael, and Michael Altmannsberger. "Chemically Induced Esthesioneuroepithelioma: Ultrastructural Findings." Annals of Otology, Rhinology & Laryngology 98, no. 4 (April 1989): 256–66. http://dx.doi.org/10.1177/000348948909800404.
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Tumors of the olfactory epithelium of rats were induced with two different nitrosamines: 2,6-dimethylnitrosomorpholine and N-nitrosopiperidine. Both carcinogens yielded identical tumors consisting of small, undifferentiated, neuroblastic cell elements without specialized cell contact. Cell processes contained microtubuli, centrioles, and neurosecretory granules. Two kinds of rosettes were encountered frequently: Neuroblastic Homer Wright rosettes consisted of undifferentiated cells, surrounding a minute lumen filled with amorphous material; and Flexner rosettes showed a higher degree of maturation. Inside their central lumen, cell processes with characteristic features of olfactory sensory cells (basal bodies, cilia, centrioles, microtubuli) could be demonstrated. The stem cell of this tumor is most likely the undifferentiated light basal cell inside the olfactory epithelium, since its ultrastructural appearance and its cytoskeleton are alike. At least under neoplastic conditions, this stem cell may likewise differentiate into epithelial cells, since transition to squamous cell carcinomas has been observed. In view of their overwhelming similarity to their human counterpart, the induced tumors are most likely to represent esthesioneuroepitheliomas.
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Teng, Miao, Yong-Ming Dang, Jia-ping Zhang, Qiong Zhang, Ya-dong Fang, Jun Ren, and Yue-sheng Huang. "Microtubular stability affects cardiomyocyte glycolysis by HIF-1α expression and endonuclear aggregation during early stages of hypoxia." American Journal of Physiology-Heart and Circulatory Physiology 298, no. 6 (June 2010): H1919—H1931. http://dx.doi.org/10.1152/ajpheart.01039.2009.
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Hypoxia-inducible factor (HIF)-1α is a key regulator of anaerobic energy metabolism. We asked the following question: Does the breakdown of microtubular structures influence glycolysis in hypoxic cardiomyocytes by regulating HIF-1α? Neonatal rat cardiomyocytes were cultured under hypoxic conditions, while microtubule-stabilizing (paclitaxel) and -depolymerizing (colchicine) agents were used to change microtubular structure. Models of high microtubule-associated protein 4 (MAP4) expression and RNA interference of microtubulin expression were established. Microtubular structural changes and intracellular HIF-1α protein distribution were observed with laser confocal scanning microscopy. Content of key glycolytic enzymes, viability, and energy content of cardiomyocytes were determined by colorimetry and high-performance liquid chromatography. HIF-1α protein content and mRNA expression were determined by Western blotting and real-time PCR, respectively. Low doses of microtubule-stabilizing agent (10 μmol/l paclitaxel) and enhanced expression of MAP4 stabilized the reticular microtubular structures in hypoxic cardiomyocytes, increased the content of key glycolytic enzymes, ameliorated energy supply and enhanced cell viability, and upregulated HIF-1α protein expression and endonuclear aggregation. In contrast, the microtubule-depolymerizing agent (10 μmol/l colchicine) or reduced microtubulin expression had adverse affects on the same parameters, in particular, HIF-1α protein content and endonuclear aggregation. We conclude that microtubular structural changes influence glycolysis in the early stages of hypoxia in cardiomyocytes by regulating HIF-1α content. Stabilizing microtubular structures increases endonuclear and total HIF-1α expression, content of key glycolytic enzymes, and energy supply. These findings provide potential therapeutic targets for ameliorating cell energy metabolism during early myocardial hypoxia.
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Theron, J. J., H. Bosman, R. De Winter, and C. N. Henning. "Sekresiemeganisme van atriale natriuretiese peptied (ANP)." Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie 12, no. 2 (July 9, 1993): 37–39. http://dx.doi.org/10.4102/satnt.v12i2.557.
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Mechanical stretch of atrial muscle in vitro induces movement of ANP granules from central perinuclear areas to the periphery of the sarcoplasm at the sarcolemma. This centrifugal movement is associated with maximal secretion of ANP into the suspension medium, without morphological evidence of exocytosis. Since the addition of vinblastine suppresses the movement of granules and ANP secretion, microtubuli are probably involved in the intracellular movement of ANP granules.
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Ookata, K., S. Hisanaga, E. Okumura, and T. Kishimoto. "Association of p34cdc2/cyclin B complex with microtubules in starfish oocytes." Journal of Cell Science 105, no. 4 (August 1, 1993): 873–81. http://dx.doi.org/10.1242/jcs.105.4.873.
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The microtubular cytoskeleton exhibits a dramatic reorganization, progressing from interphase radial arrays to a mitotic spindle at the G2/M transition. Although this reorganization has been suspected to be caused by maturation promoting factor (MPF: p34cdc2/cyclin B complex), little is known about how p34cdc2 kinase controls microtubule networks. We provide evidence of the direct association of the p34cdc2/cyclin B complex with microtubules in starfish oocytes. Anti-cyclin B staining of detergent-treated oocytes, isolated asters and meiotic spindles revealed fluorescence associated with microtubule fibers, chromosomes and centrosomes. Microtubules prepared from starfish oocytes were associated with cyclin B and p34cdc2 proteins. Microtubule-bound p34cdc2 and cyclin B were released from microtubules by a high-salt solution and possessed a complex form as shown by the adsorption to suc1-beads and by immunoprecipitation with the anti-cyclin B antibody. The p34cdc2/cyclin B complex associated to microtubules had high histone H1 kinase activity at meiotic metaphase. However, it was not necessary for the p34cdc2/cyclin B complex to be active for microtubule binding, as an inactive form in immature oocytes was also observed to bind to microtubules. The coprecipitation of suc1-column purified p34cdc2/cyclin B with purified porcine brain microtubules in the presence of starfish oocyte microtubule-associated proteins (MAPs) indicates that the association of p34cdc2/cyclin B with microtubules in vitro is mediated by MAPs.
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Bajer, A. S., and J. Molè-Bajer. "Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo." Journal of Cell Biology 102, no. 1 (January 1, 1986): 263–81. http://dx.doi.org/10.1083/jcb.102.1.263.
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The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizating organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self-reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubule-related processes.
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Buljan, Vlado A., Manuel B. Graeber, R. M. Damian Holsinger, Daniel Brown, Brett D. Hambly, Edward J. Delikatny, Vladimira R. Vuletic, et al. "Calcium–axonemal microtubuli interactions underlie mechanism(s) of primary cilia morphological changes." Journal of Biological Physics 44, no. 1 (October 31, 2017): 53–80. http://dx.doi.org/10.1007/s10867-017-9475-2.
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Roychoudhury, Sonali, and Martha J. Powell. "Ultrastructure of mitosis in the algal parasitic fungus Polyphagus euglenae." Canadian Journal of Botany 69, no. 10 (October 1, 1991): 2201–14. http://dx.doi.org/10.1139/b91-277.
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Ultrastructure of mitosis in the parasitic fungus, Polyphagus euglenae, was investigated with emphasis on centrosome structure and prophase events. The interphase centrosome included a diplosome, scattered electron-dense satellites, and multiple ring-shaped microtubule foci. As centrosomes separated during prophase, microtubular arrays extended between the replicated centrosomes and radiated out along the outer surface of the nuclear envelope. The asymmetric configuration of these microtubular arrays suggests that intersecting microtubules provide tension forces on elongating centrosome to centrosome microtubules during centrosome separation. After centrosome migration, multiple microtubule foci appeared to fuse into crescent-shaped microtubule organizing centers. Condensing chromatin was concentrated in the region of the future equatorial plane of the mitotic spindle prior to the appearance of discontinuities in the nuclear envelope and incursion of the spindle. The nucleolus fragmented during prometaphase, and fragments were discarded with the interzonal region during telophase. Nucleoli appeared in daughter nuclei before chromatin became diffuse. Similarities in the mitotic apparatus of P. euglenae with that previously reported for Monoblepharella sp. support a phylogenetic affinity between members of the orders Chytridiales and Monoblepharidales. Key words: mitosis, Polyphagus euglenae, Chytridiales, centrosomes, phylogeny, ultrastructure.
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Lloyd, C. W., and B. Wells. "Microtubules are at the tips of root hairs and form helical patterns corresponding to inner wall fibrils." Journal of Cell Science 75, no. 1 (April 1, 1985): 225–38. http://dx.doi.org/10.1242/jcs.75.1.225.
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Root hairs have sometimes provided contradictory evidence for microtubule/microfibril parallelism. This tissue was re-examined using optimized conditions for the fixation, before immunofluorescence, of root hairs. In phosphate buffer, microtubules did not enter the apical tip of radish root hairs and were clearly fragmented. However, in an osmotically adjusted microtubule-stabilizing buffer, microtubules were observed within the apical dome and appeared unfragmented. Microtubules are not, therefore, absent from the region where new cell wall is presumed to be generated during tip growth. A spiralling of microtubules was seen at the apices of onion root hairs. Using shadow-cast preparations of macerated radish root hairs, it was confirmed that steeply helical microtubules matched the texture of the inner wall. In onion, the 45 degrees microtubular helices are accompanied by similarly wound inner wall fibrils. Results do not support the view that microtubules are not involved in the oriented deposition of fibrils in root hairs. Instead, they are interpreted in terms of a flexible helical cytoskeleton, which is capable of changing its pitch but is sensitive to fixation conditions.
9
Schroeder, C. C., A. K. Fok, and R. D. Allen. "Vesicle transport along microtubular ribbons and isolation of cytoplasmic dynein from Paramecium." Journal of Cell Biology 111, no. 6 (December 1, 1990): 2553–62. http://dx.doi.org/10.1083/jcb.111.6.2553.
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Cytoplasmic microtubule-based motility in Paramecium was investigated using video-enhanced contrast microscopy, the quick-freeze, deep-etch technique, and biochemical isolations. Three distinct vesicle populations were found to be transported unidirectionally along the cytopharyngeal microtubular ribbons. This minus-end-directed movement exhibited unique in vivo features in that the vesicle transport was nonsaltatory, rapid, and predominantly along one side of the microtubular ribbons. To identify candidate motor proteins which may participate in vesicle transport, we prepared cytosolic extracts of Paramecium and used bovine brain microtubules as an affinity matrix. These preparations were found to contain a microtubule-stimulated ATPase which supported microtubule gliding in vitro. This protein was verified as a cytoplasmic dynein based upon its relative molecular mass, sedimentation coefficient of 16S, susceptibility to vanadate photocleavage, elevated CTPase/ATPase ratio, and its typical two-headed dynein morphology. This dynein was directly compared with the axonemal dyneins from Paramecium and found to differ by five criteria: morphology, sedimentation coefficient, CTPase/ATPase ratio, vanadate cleavage patterns, and polypeptide composition. The cytoplasmic dynein is therefore not an axonemal dynein precursor, but rather it represents a candidate for supporting the microtubule-based vesicle transport which proceeds along the microtubular ribbons.
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Caplow, Michael, John Shanks, and Bruna Pegoraro Brylawski. "Concerning the location of the GTP hydrolysis site on microtubules." Canadian Journal of Biochemistry and Cell Biology 63, no. 6 (June 1, 1985): 422–29. http://dx.doi.org/10.1139/o85-061.
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The kinetics for GTP hydrolysis associated with microtubule assembly with microtubular protein has been analyzed under reaction conditions where tubulin–GDP does not readily assemble into microtubules. The GTPase rate is only slightly faster during the time when net microtubule assembly occurs, as compared with steady state. The slightly slower steady-state GTPase rate apparently results from GDP product inhibition, since the progressive decrease in the rate can be quantitatively accounted for using the previously determined GTP dissociation constant and the Ki value for GDP. Since the GTPase rate is not a function of the rate for net microtubule assembly, it is concluded that GTP hydrolysis is not required for tubulin subunit incorporation into microtubules. The constancy of the rate indicates that the GTPase reaction occurs at a site, the concentration of which does not change during the assembly process. This result is consistent with a reaction scheme in which GTP hydrolysis occurs primarily at microtubule ends. We propose that hydrolysis occurs at microtubule ends, at the interface between a long core of tubulin–GDP subunits and a short cap of tubulin–GTP subunits.
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Afzelius, B. A., P. L. Bellon, and S. Lanzavecchia. "Microtubules and their protofilaments in the flagellum of an insect spermatozoon." Journal of Cell Science 95, no. 2 (February 1, 1990): 207–17. http://dx.doi.org/10.1242/jcs.95.2.207.
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Spermatozoa of stick insects have nine accessory tubules, which surround the nine outer microtubular doublets and the two inner microtubular singlets. When fixed in a fixative that was designed to minimize protein denaturation (glutaraldehyde and tannic acid, no osmium post-fixation but block staining with uranyl acetate in water) the accessory tubules were seen to contain 17 protofilaments of the same type as those in the 9 + 2 microtubular doublets and singlets. The protofilaments in accessory tubules and other microtubules were roughly triangular. When studied by Markham's photographic method a somewhat different tilt of the two longer sides was seen; this makes it possible to distinguish a polarity in the microtubules, i.e. to differentiate between a microtubule that is viewed from its (-)end to its (+)end from one that is viewed in the opposite direction. The dynein arms of the doublets can be used as an independent type of marker for the polarity. In a computer-aided analysis of the fine structure of the tail axoneme, the A-subtubules of the outer doublets were seen to be not quite equidistant; rather, there were somewhat widened electron-dense interspaces in the ring of protofilaments in four places. The locations of these widened interspaces coincide with the attachment sites for the spoke, the inner dynein arm, the outer dynein arm, and the intertubular material. It is tentatively concluded that proteins of these structures, and perhaps also other microtubule-associated proteins, may be anchored deep within the wall of a microtubule rather than just superficially along it.
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Coquelle, Frédéric M., Benjamin Vitre, and Isabelle Arnal. "Structural basis of EB1 effects on microtubule dynamics." Biochemical Society Transactions 37, no. 5 (September 21, 2009): 997–1001. http://dx.doi.org/10.1042/bst0370997.
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+TIPs (plus-end tracking proteins) are an increasing group of molecules that localize preferentially to the end of growing microtubules. +TIPs regulate microtubule dynamics and contribute to the organization of the microtubular network within the cell. Thus they participate in a wide range of cellular processes including cell division, motility and morphogenesis. EB1 (end-binding 1) is a highly conserved key member of the +TIP group that has been shown to modulate microtubule dynamics both in vitro and in cells. EB1 is involved in accurate chromosome segregation during mitosis and in the polarization of the microtubule cytoskeleton in migrating cells. Here, we review recent in vitro studies that have started to reveal a regulating activity of EB1, and its yeast orthologue Mal3p, on microtubule structure. In particular, we examine how EB1-mediated changes in the microtubule architecture may explain its effects on microtubule dynamics.
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Dumontet, Charles, and Branimir I. Sikic. "Mechanisms of Action of and Resistance to Antitubulin Agents: Microtubule Dynamics, Drug Transport, and Cell Death." Journal of Clinical Oncology 17, no. 3 (March 1999): 1061. http://dx.doi.org/10.1200/jco.1999.17.3.1061.
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PURPOSE: To analyze the available data concerning mechanisms of action of and mechanisms of resistance to the antitubulin agents, vinca alkaloids and taxanes, and more recently described compounds. DESIGN: We conducted a review of the literature on classic and recent antitubulin agents, focusing particularly on the relationships between antitubulin agents and their intracellular target, the soluble tubulin/microtubule complex. RESULTS AND CONCLUSION: Although it is widely accepted that antitubulin agents block cell division by inhibition of the mitotic spindle, the mechanism of action of antitubulin agents on microtubules remains to be determined. The classic approach is that vinca alkaloids depolymerize microtubules, thereby increasing the soluble tubulin pool, whereas taxanes stabilize microtubules and increase the microtubular mass. More recent data suggest that both classes of agents have a similar mechanism of action, involving the inhibition of microtubule dynamics. These data suggest that vinca alkaloids and taxanes may act synergistically as antitumor agents and may be administered as combination chemotherapy in the clinic. However, enhanced myeloid and neurologic toxicity, as well as a strong dependence on the sequence of administration, presently exclude these combinations outside the context of clinical trials. Although the multidrug resistance phenotype mediated by Pgp appears to be an important mechanism of resistance to these agents, alterations of microtubule structure resulting in altered microtubule dynamics and/or altered binding of antitubulin agents may constitute a significant mechanism of drug resistance.
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Dang, Yongming, Xiaodong Lan, Qiong Zhang, Lingfei Li, and Yuesheng Huang. "Analysis of Grayscale Characteristics in Images of Labeled Microtubules from Cultured Cardiac Myocytes." Microscopy and Microanalysis 21, no. 2 (March 16, 2015): 334–42. http://dx.doi.org/10.1017/s1431927615000185.
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AbstractMicrotubules of cardiac myocytes depolymerize after a hypoxic insult or treatment with colchicine. However, little attention has been paid to quantifying changes in microtubule distribution when using fluorescent images. We converted fluorescence images of labeled microtubules in H9C2 cardiac myocytes to grayscale images, then filtered the images to remove any noise, and used grayscale histograms to quantify features of the images. The results show that parameters such as the mean, variance, skewness, kurtosis, energy, and entropy can be used to quantitatively describe the distribution of microtubules in cells. Quantitative characteristics of microtubule distribution were similar after culturing cells under hypoxic conditions or after treatment with colchicine. These results parallel those described for neonatal rat cardiac myocytes following ischemia and hypoxia. In addition, we provide a method for internal segmentation of the cells, which revealed that microtubular depolymerization was more evident near the cell membrane following hypoxia or colchicine treatment.
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Häussinger, Dieter, Barbara Stoll, Stephan vom Dahl, Panayiotis A. Theodoropoulos, Emmanuel Markogiannakis, Achille Gravanis, Florian Lang, and Christos Stournaras. "Effect of hepatocyte swelling on microtubule stability and tubulin mRNA levels." Biochemistry and Cell Biology 72, no. 1-2 (January 1, 1994): 12–19. http://dx.doi.org/10.1139/o94-003.
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Incubation of isolated rat hepatocytes under conditions known to induce cell swelling caused several alterations in microtubule physiology. As shown by immunofluorescence microscopy experiments in the absence and presence of triethyllead or colchicine (two well-established microtubule inhibitors), an apparent stabilization of the microtubule network became evident in hepatocytes exposed to hypotonic (190 mosmol/L) conditions. A similar stabilizing effect was also observed upon cell swelling induced by addition of insulin (100 nmol/L) or glutamine (10 mmol/L). The differential microtubule stabilities were not attributed to a differential incorporation of the antimicrotubular agents into hepatocytes as shown by [3H]colchicine-uptake experiments. The swelling-induced alterations of microtubules may contribute to the swelling-induced changes of liver cell function: in perfused rat liver it was found that the established inhibitory effect of hypotonic cell swelling on hepatic proteolysis was largely abolished in presence of colchicine. Tubulin mRNA levels increased by 1.9-, 2.1- and 2.7-fold in isolated hepatocytes being exposed for 120 min to hypotonic medium, insulin, or glutamine, respectively. The results suggest an involvement of microtubular structures in the regulation of liver metabolism in response to alterations of the cellular hydration state.Key words: microtubules, cell swelling, glutamine, gene expression, proteolysis.
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Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson. "Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport." Journal of Cell Biology 100, no. 5 (May 1, 1985): 1736–52. http://dx.doi.org/10.1083/jcb.100.5.1736.
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Native microtubules prepared from extruded and dissociated axoplasm have been observed to transport organelles and vesicles unidirectionally in fresh preparations and more slowly and bidirectionally in older preparations. Both endogenous and exogenous (fluorescent polystyrene) particles in rapid Brownian motion alight on and adhere to microtubules and are transported along them. Particles can switch from one intersecting microtubule to another and move in either direction. Microtubular segments 1 to 30 microns long, produced by gentle homogenization, glide over glass surfaces for hundreds of micrometers in straight lines unless acted upon by obstacles. While gliding they transport particles either in the same (forward) direction and/or in the backward direction. Particle movement and gliding of microtubule segments require ATP and are insensitive to taxol (30 microM). It appears, therefore, that the mechanisms producing the motive force are very closely associated with the native microtubule itself or with its associated proteins. Although these movements appear irreconcilable with several current theories of fast axoplasmic transport, in this article we propose two models that might explain the observed phenomena and, by extension, the process of fast axoplasmic transport itself. The findings presented and the possible mechanisms proposed for fast axoplasmic transport have potential applications across the spectrum of microtubule-based motility processes.
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Cao, Jingjing, Cui Lin, Huijuan Wang, Lun Wang, Niu Zhou, Yulan Jin, Min Liao, and Jiyong Zhou. "Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein." Journal of Virology 89, no. 5 (December 24, 2014): 2777–91. http://dx.doi.org/10.1128/jvi.03117-14.
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ABSTRACTMicrotubule transport of circovirus from the periphery of the cell to the nucleus is essential for viral replication in early infection. How the microtubule is recruited to the viral cargo remains unclear. In this study, we observed that circovirus trafficking is dependent on microtubule polymerization and that incoming circovirus particles colocalize with cytoplasmic dynein and endosomes. However, circovirus binding to dynein was independent of the presence of microtubular α-tubulin and translocation of cytoplasmic dynein into the nucleus. The circovirus capsid (Cap) subunit enhanced microtubular acetylation and directly interacted with intermediate chain 1 (IC1) of dynein. N-terminal residues 42 to 100 of the Cap viral protein were required for efficient binding to the dynein IC1 subunit and for retrograde transport. Knockdown of IC1 decreased virus transport and replication. These results demonstrate that Cap is a direct ligand of the cytoplasmic dynein IC1 subunit and an inducer of microtubule α-tubulin acetylation. Furthermore, Cap recruits the host dynein/microtubule machinery to facilitate transport toward the nucleus by an endosomal mechanism distinct from that used for physiological dynein cargo.IMPORTANCEIncoming viral particles hijack the intracellular trafficking machinery of the host in order to migrate from the cell surface to the replication sites. Better knowledge of the interaction between viruses and virus proteins and the intracellular trafficking machinery may provide new targets for antiviral therapies. Currently, little is known about the molecular mechanisms of circovirus transport. Here, we report that circovirus particles enter early endosomes and utilize the microtubule-associated molecular motor dynein to travel along microtubules. The circovirus capsid subunit enhances microtubular acetylation, and N-terminal residues 42 to 100 directly interact with the dynein IC1 subunit during retrograde transport. These findings highlight a mechanism whereby circoviruses recruit dynein for transport to the nucleus via the dynein/microtubule machinery.
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Segaar, Peter J. "Dynamics of the microtubular cytoskeleton in the green alga Aphanochaete magna (Chlorophyta). II. The cortical cytoskeleton, astral microtubules, and spindle during the division cycle." Canadian Journal of Botany 67, no. 1 (January 1, 1989): 239–46. http://dx.doi.org/10.1139/b89-033.
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The organization and ontogeny of microtubular structures associated with cell division in the green alga Aphanochaete magna have been analyzed using indirect immunofluorescence and transmission electron microscopy. The cortical cytoskeleton lining the longitudinal cell walls in dividing apical and side branch initiating cells depolymerizes at prophase, has completely disappeared at anaphase, and starts to repolymerize in the apex of the cell at the transition of mitosis and cytokinesis. At prophase, astral microtubule systems develop from the nuclear pole associated centriole complexes, and the nucleus becomes ensheathed by centriole-interconnecting astral microtubules. The asters disappear at metaphase–anaphase, reappear at telophase, and finally depolymerize at posttelophase. A star-shaped phycoplast develops at telophase, transforms into a planar array at posttelophase, and becomes associated with the new cross wall, while the cortical cytoskeleton lining the longitudinal cell walls reforms (resulting in the coexistence of two wall-lining microtubule assemblages having different origins). Finally, the phycoplast microtubules depolymerize. The presence of astral microtubule systems at prophase in green algae is discussed and the behaviour of the cortical cytoskeleton at cell division is compared with that in other plant cell systems.
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Uyeda, T. Q., and M. Furuya. "Evidence for active interactions between microfilaments and microtubules in myxomycete flagellates." Journal of Cell Biology 108, no. 5 (May 1, 1989): 1727–35. http://dx.doi.org/10.1083/jcb.108.5.1727.
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We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.
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Willingham, M. C. "Apoptosis And Resistance To Anti-Microtubule Agents." Microscopy and Microanalysis 4, S2 (July 1998): 1036–37. http://dx.doi.org/10.1017/s1431927600025307.
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Several clinically important anti-cancer agents exert their effects on tumor cells through interference with the function of microtubules. In addition to the Vinca alkaloids, such as vinblastine and vincristine, the taxanes, such as paclitaxel (Trade Name: Taxol), kill tumor cells through a microtubular target. Treatment with taxol leads to the inability of microtubules to depolymerize, leading to the formation of large intracellular microtubular bundles. In tumor cells that progress through the cell cycle, this leads to the inability of these cells to disassembly interphase microtubule networks and a failure to form functional mitotic spindles. These cells arrest in M phase, from which they eventually progress, either by the induction of apoptotic cell death, or by micronucleation and the formation of tetraploid cells. There is also the possibility that taxol has other effects on the regulation of genes or other systems to enhance cell killing, perhaps through lowering the threshold of cells to the induction of apoptotic cell death.
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Kok, J. W., K. Hoekstra, S. Eskelinen, and D. Hoekstra. "Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes." Journal of Cell Science 103, no. 4 (December 1, 1992): 1139–52. http://dx.doi.org/10.1242/jcs.103.4.1139.
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Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.
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Abbate, M., J. V. Bonventre, and D. Brown. "The microtubule network of renal epithelial cells is disrupted by ischemia and reperfusion." American Journal of Physiology-Renal Physiology 267, no. 6 (December 1, 1994): F971—F978. http://dx.doi.org/10.1152/ajprenal.1994.267.6.f971.
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Ischemia results in alterations in the integrity of the plasma membrane of renal epithelial cells, changes in cell polarity, and initiation of cell division. Because microtubules are implicated in these processes, we examined the effects of ischemia and reperfusion on the microtubular cytoskeleton in rat kidney. Major alterations in the microtubule network of S3 proximal tubules were detected after 40 min of ischemia followed by 1 h of reperfusion. There was fragmentation of microtubules and considerably less intense staining with antitubulin antibodies than with normal kidneys. Some thick ascending limbs of Henle close to medullary vascular bundles showed a variable loss of tubulin staining. After 24 and 48 h of reperfusion, tubulin labeling was again present in most proximal tubule cells in contact with the basement membrane but was not detectable in exfoliated cells. Numerous mitotic figures were present in kidneys 48 h after reperfusion. Kidneys subjected to 40 min of ischemia without reperfusion and contralateral kidneys studied after 1 h of reperfusion showed only mild microtubular disruption. Because of the established role of microtubules in the generation and maintenance of epithelial cell polarity, their loss may contribute to structural changes that occur after ischemia and reperfusion.
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Pyka, Janette, Aleksandra Glogowska, Henning Dralle, Cuong Hoang-Vu, and Thomas Klonisch. "Cytoplasmic Domain of proEGF Affects Distribution and Post-Translational Modification of Microtubuli and Increases Microtubule-Associated Proteins 1b and 2 Production in Human Thyroid Carcinoma Cells." Cancer Research 65, no. 4 (February 15, 2005): 1343–51. http://dx.doi.org/10.1158/0008-5472.can-04-2030.
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Apirakaramwong, A., Perayot Pamonsinlapatham, S. Techaarpornkul, Praneet Opanasopit, Suwannee Panomsuk, and S. Soksawatmaekhin. "Mechanisms of Cellular Uptake with Chitosan/DNA Complex in Hepatoma Cell Line." Advanced Materials Research 506 (April 2012): 485–88. http://dx.doi.org/10.4028/www.scientific.net/amr.506.485.
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Chitosan (CS) has a high potential for gene delivery into mammalian cells. However, its uptake mechanism is not well clarified. We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine), caveolae-mediated endocytosis (genistein), macropinocytosis (LY 29004 and wortmannin), microtubuli polymerization (nocodazole) and of membrane cholesterol recycle (methyl-β-cyclodextrin) on the transfection efficiency with CS/pEGFP complexes and on the internalization of CS/rhodamine-labeled pEGFP complexes by hepatoma cell line (Huh 7 cells). The transfection was blocked by nocodazole, genistein, and methyl-β-cyclodextrin, respectively. CS/DNA complexes internalization was clearly inhibited by genistein. We conclude that the complexes uptake predominantly by caveolin-mediated pathways. In addition, fluorescence colocalization studies with acidotropic probes, LysoSensor dye, illustrated that CS/DNA complexes are targeted to lysosomes for the degradation after internalization.
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SASSE, FLORENZ, HEINRICH SIEINMETZ, JÜRGEN HEIL, GERHARD HÖFLE, and HANS REICHENBACH. "Tubulysins, New Cytostatic Peptides from Myxobacteria Acting on Microtubuli. Production, Isolation, Physico-chemical and Biological Properties." Journal of Antibiotics 53, no. 9 (2000): 879–85. http://dx.doi.org/10.7164/antibiotics.53.879.
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Kenney, D. M., and R. W. Linck. "The cystoskeleton of unstimulated blood platelets: structure and composition of the isolated marginal microtubular band." Journal of Cell Science 78, no. 1 (October 1, 1985): 1–22. http://dx.doi.org/10.1242/jcs.78.1.1.
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Detergent-insoluble, marginal microtubular band (MB) cytoskeletons were isolated from unstimulated blood platelets after pretreatment with glycerol or with Taxol. MB cytoskeletons retained the shape of intact platelets and behaved in suspension as coherent structural units. The major structural component was a continuous coil of long microtubule(s), often with granular/amorphous material present in the centre; few typical actin filaments were observed. The coiled microtubules often had an amorphous surface coating, but no discrete inter-microtubule bridges were seen. Tubulin and actin (identified by immunochemical staining) were major polypeptides. None of the minor (greater than 10) polypeptide components comigrated with high molecular weight microtubule-associated proteins in brain tubulin. A novel polypeptide, resolved by two-dimensional electrophoresis and designated IEF-51K, was present in MB cytoskeletons in amounts approximately equivalent to each of the tubulin polypeptides. Evidence suggests that IEF-51K is a distinct, previously undescribed component of the platelet cytoskeletal system.
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Kallajoki, M., K. Weber, and M. Osborn. "Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome." Journal of Cell Science 102, no. 1 (May 1, 1992): 91–102. http://dx.doi.org/10.1242/jcs.102.1.91.
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The SPN antigen plays an essential role in mitosis, since microinjection of antibodies causes mitotic arrest. Here we show, by examination of the relative locations of SPN antigen, the centrosomal 5051 antigen and tubulin in normal mitotic, and in taxol-treated mitotic cells, that the SPN antigen is involved in organizing the microtubules of the spindle. The 210 kDa protein defined as SPN antigen relocates from the nuclear matrix to the centrosome at prophase, remains associated with the poles at metaphase and anaphase, and dissociates from the centrosomes in telophase. In taxol-treated mitotic cells, SPN staining shows a striking redistribution while 5051 antigen remains associated with centrosomes. SPN antigen is seen at the plasma membrane end of the rearranged microtubules. SPN antigen is always at the center of the multiple microtubule asters (5 to 20 per cell) induced by taxol, whereas 5051 again remains associated with the centrosomal complex (1 to 2 foci per cell). Microtubule nucleation is associated with the SPN antigen rather than with the 5051 antigen. Microinjection of SPN-3 antibody into taxol-treated mitotic PtK2 cells causes disruption of the asters as judged by tubulin staining of the same cells. Finally, SPN antigen extracted in soluble form from synchronized mitotic HeLa cells binds to, and sediments with, pig brain microtubules stabilized by taxol. This association of SPN antigen with microtubules is partially dissociated by 0.5 M NaCl but not by 5 mM ATP. Thus SPN antigen binds to microtubules in vitro and seems to act as a microtubular minus-end organizer in mitotic cells in vivo.
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Perou, C. M., and J. Kaplan. "Chediak-Higashi syndrome is not due to a defect in microtubule-based lysosomal mobility." Journal of Cell Science 106, no. 1 (September 1, 1993): 99–107. http://dx.doi.org/10.1242/jcs.106.1.99.
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Chediak-Higashi Syndrome is an autosomal recessive disorder that affects intracellular vesicle formation. The diagnostic feature of Chediak-Higashi Syndrome is the presence of ‘giant’ lysosomes clustered near the nucleus. Lysosome morphology in macrophages is maintained by microtubules and microtubule-based motors, such as kinesin. Dramatic changes in lysosome morphology can be induced by lowering cytoplasmic pH or by adding phorbol esters. When macrophages from beige mice (a murine homolog of Chediak-Higashi Syndrome) were subjected to these protocols they were able to alter their lysosomal distribution and morphology to the same degree as macrophages from control mice. These results indicate that lysosomes in Chediak cells are capable of interacting with the microtubule-based motor system, suggesting that the defective gene product is not an altered microtubular element involved in lysosomal movement.
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Sider, J. R., C. A. Mandato, K. L. Weber, A. J. Zandy, D. Beach, R. J. Finst, J. Skoble, and W. M. Bement. "Direct observation of microtubule-f-actin interaction in cell free lysates." Journal of Cell Science 112, no. 12 (June 15, 1999): 1947–56. http://dx.doi.org/10.1242/jcs.112.12.1947.
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Coordinated interplay of the microtubule and actin cytoskeletons has long been known to be crucial for many cellular processes including cell migration and cytokinesis. However, interactions between these two systems have been difficult to document by conventional approaches, for a variety of technical reasons. Here the distribution of f-actin and microtubules were analyzed in the absence of fixation using Xenopus egg extracts as an in vitro source of microtubules and f-actin, demembranated Xenopus sperm to nucleate microtubule asters, fluorescent phalloidin as a probe for f-actin, and fluorescent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive washing in flow chambers. F-actin-microtubule association was heterogenous within a given aster, such that f-actin is most abundant toward the distal (plus) ends of microtubules, and microtubules heavily labeled with f-actin are found in close proximity to microtubules devoid of f-actin. However, this distribution changed over time, in that 5 minute asters had more f-actin in their interiors than did 15 minute asters. Microtubule association with f-actin was correlated with microtubule bending and kinking, while elimination of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally significant. F-actin was also found to associate in a lengthwise fashion with microtubules in asters centrifuged through 30% sucrose, and microtubules alone (i.e. microtubules not seeded from demembranated sperm) centrifuged through sucrose, indicating that the association cannot be explained by flow-induced trapping and alignment of f-actin by aster microtubules. Further, cosedimentation analysis revealed that microtubule-f-actin association could be reconstituted from microtubules assembled from purified brain tubulin and f-actin assembled from purified muscle actin in the presence, but not the absence, of Xenopus oocyte microtubule binding proteins. The results provide direct evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule binding proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.
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XuHan, X., and A. A. M. Van Lammeren. "Microtubular configurations during endosperm development in Phaseolus vulgaris." Canadian Journal of Botany 72, no. 10 (October 1, 1994): 1489–95. http://dx.doi.org/10.1139/b94-183.
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Microtubular cytoskeletons in nuclear, alveolar, and cellular endosperm of bean (Phaseolus vulgaris) were analyzed immunocytochemically and by electron microscopy to reveal their function during cellularization. Nuclear endosperm showed a fine network of microtubules between the wide-spaced nuclei observed towards the chalazal pole. Near the embryo, where nuclei were densely packed, bundles of microtubules radiated from nuclei. They were formed just before alveolus formation and functioned in spacing nuclei and in forming internuclear, phragmoplast-like structures that gave rise to nonmitosis-related cell plates. During alveolus formation cell plates extended and fused with other newly formed walls, thus forming the walls of alveoli. Growing wall edges of cell plates exhibited arrays of microtubules perpendicular to the plane of the wall, initially. When two growing walls were about to fuse, microtubules of both walls interacted, and because of the interaction of microtubules, the cell walls changed their position. When a growing wall was about to fuse with an already existing wall, such interactions between microtubules were not observed. It is therefore concluded that interactions of microtubules of fusing walls influence shape and position of walls. Thus microtubules control the dynamics of cell wall positioning and initial cell shaping. Key words: cell wall, cellularization, endosperm, microtubule, Phaseolus vulgaris.
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Burkart, Graham M., and Ram Dixit. "Microtubule bundling by MAP65-1 protects against severing by inhibiting the binding of katanin." Molecular Biology of the Cell 30, no. 13 (June 15, 2019): 1587–97. http://dx.doi.org/10.1091/mbc.e18-12-0776.
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The microtubule-severing enzyme katanin (KTN1) regulates the organization and turnover of microtubule arrays by the localized breakdown of microtubule polymers. In land plants, KTN1 activity is essential for the formation of linearly organized cortical microtubule arrays that determine the axis of cell expansion. Cell biological studies have shown that even though KTN1 binds to the sidewalls of single and bundled microtubules, severing activity is restricted to microtubule cross-over and nucleation sites, indicating that cells contain protective mechanisms to prevent indiscriminate microtubule severing. Here, we show that the microtubule-bundling protein MAP65-1 inhibits KTN1-mediated microtubule severing in vitro. Severing is inhibited at bundled microtubule segments and the severing rate of nonbundled microtubules is reduced by MAP65-1 in a concentration-dependent manner. Using various MAP65-1 mutant proteins, we demonstrate that efficient cross-linking of microtubules is crucial for this protective effect and that microtubule binding alone is not sufficient. Reduced severing due to microtubule bundling by MAP65-1 correlated to decreased binding of KTN1 to these microtubules. Taken together, our work reveals that cross-linking of microtubules by MAP65-1 confers resistance to severing by inhibiting the binding of KTN1 and identifies the structural features of MAP65-1 that are important for this activity.
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Farrell, KW, MA Jordan, HP Miller, and L. Wilson. "Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling." Journal of Cell Biology 104, no. 4 (April 1, 1987): 1035–46. http://dx.doi.org/10.1083/jcb.104.4.1035.
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The length dynamics both of microtubule-associated protein (MAP)-rich and MAP-depleted bovine brain microtubules were examined at polymer mass steady state. In both preparations, the microtubules exhibited length redistributions shortly after polymer mass steady state was attained. With time, however, both populations relaxed to a state in which no further changes in length distributions could be detected. Shearing the microtubules or diluting the microtubule suspensions transiently increased the extent to which microtubule length redistributions occurred, but again the microtubules relaxed to a state in which changes in the polymer length distributions were not detected. Under steady-state conditions of constant polymer mass and stable microtubule length distribution, both MAP-rich and MAP-depleted microtubules exhibited behavior consistent with treadmilling. MAPs strongly suppressed the magnitude of length redistributions and the steady-state treadmilling rates. These data indicate that the inherent tendency of microtubules in vitro is to relax to a steady state in which net changes in the microtubule length distributions are zero. If the basis of the observed length redistributions is the spontaneous loss and regain of GTP-tubulin ("GTP caps") at microtubule ends, then in order to account for stable length distributions the microtubule ends must reside in the capped state far longer than in the uncapped state, and uncapped microtubule ends must be rapidly recapped. The data suggest that microtubules in cells may have an inherent tendency to remain in the polymerized state, and that microtubule disassembly must be induced actively.
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Cassimeris, L., C. L. Rieder, G. Rupp, and E. D. Salmon. "Stability of microtubule attachment to metaphase kinetochores in PtK1 cells." Journal of Cell Science 96, no. 1 (May 1, 1990): 9–15. http://dx.doi.org/10.1242/jcs.96.1.9.
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Kinetochore microtubules are known to be differentially stable to a variety of microtubule depolymerization agents compared to the non-kinetochore polar microtubules, but the dynamics of microtubule attachment to the kinetochore is currently controversial. We have examined the stability of kinetochore microtubules in metaphase PtK1 spindles at 23 degrees C when microtubule assembly is abruptly blocked with the drug nocodazole. Metaphase cells were incubated in medium containing 34 microM nocodazole for various times before fixation and processing either for immunofluorescence light microscopy or serial-section electron microscopy. Microtubules not associated with kinetochore fibers disappeared completely in less than 1 min. Kinetochore fibers persisted and shortened, as the spindle poles moved close to the chromosomes over a 10–20 min interval. During this shortening process, the number of kinetochore microtubules decreased slowly. The mean number of kinetochore microtubules was 24 +/− 5 in control cells and zero in cells incubated with nocodazole for 20 min. The half-time of microtubule attachment to the kinetochore was approximately 7.5 min. These results show that when microtubule assembly is blocked, kinetochore microtubules shorten more slowly and persist about 10 times longer than the labile polar microtubules. If kinetochore microtubules shorten by tubulin dissociation at their plus-ends like the non-kinetochore polar microtubules, then the microtubule surface lattice must be able to translocate through the kinetochore attachment site without frequent detachment occurring.
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Takemura, R., S. Okabe, T. Umeyama, Y. Kanai, N. J. Cowan, and N. Hirokawa. "Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau." Journal of Cell Science 103, no. 4 (December 1, 1992): 953–64. http://dx.doi.org/10.1242/jcs.103.4.953.
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We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.
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Vedrenne, Cécile, Dieter R. Klopfenstein, and Hans-Peter Hauri. "Phosphorylation Controls CLIMP-63–mediated Anchoring of the Endoplasmic Reticulum to Microtubules." Molecular Biology of the Cell 16, no. 4 (April 2005): 1928–37. http://dx.doi.org/10.1091/mbc.e04-07-0554.
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The microtubule-binding 63-kDa cytoskeleton-linking membrane protein (CLIMP-63) is an integral membrane protein that links the endoplasmic reticulum (ER) to microtubules. Here, we tested whether this interaction is regulated by phosphorylation. Metabolic labeling with 32P showed that CLIMP-63 is a phosphoprotein with increased phosphorylation during mitosis. CLIMP-63 of mitotic cells is unable to bind to microtubules in vitro. Mitotic phosphorylation can be prevented by mutation of serines 3, 17, and 19 in the cytoplasmic domain of CLIMP-63. When these residues are mutated to glutamic acid, and hence mimic mitotic phosphorylation, CLIMP-63 does no longer bind to microtubules in vitro. Overexpression of the phospho-mimicking mitotic form of CLIMP-63 in interphase cells leads to a collapse of the ER around the nucleus, leaving the microtubular network intact. The results suggest that CLIMP-63–mediated stable anchoring of the ER to microtubules is required to maintain the spatial distribution of the ER during interphase and that this interaction is abolished by phosphorylation of CLIMP-63 during mitosis.
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Ayscough, K., N. M. Hajibagheri, R. Watson, and G. Warren. "Stacking of Golgi cisternae in Schizosaccharomyces pombe requires intact microtubules." Journal of Cell Science 106, no. 4 (December 1, 1993): 1227–37. http://dx.doi.org/10.1242/jcs.106.4.1227.
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Fission yeast was treated with the anti-microtubule agent, thiabendazole. Cytoplasmic microtubules broke down with a half-time of less than 10 minutes followed closely by the unstacking of Golgi cisternae. The final product appeared to be single Golgi cisternae. No other organelle seemed to be affected by this treatment, which was completely reversible. The nda3 mutant strain has an altered beta-tubulin and its cytoplasmic microtubules are resistant to thiabendazole. The Golgi in this cold-sensitive mutant was unaffected by treatment at the permissive temperature but unstacked at the non-permissive temperature even in the absence of thiabendazole. Taken together these data show that disruption of the microtubular network can cause dissociation of Golgi cisternae. Newly synthesised acid phosphatase was transported and secreted to the same extent and with the same kinetics whether or not the Golgi was unstacked. The possible role of microtubules in Golgi stacking and the lack of effect on secretion are discussed.
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Italiano, Joseph E., Jennifer L. Richardson, Harald Schulze, Ksenija Drabek, Chloe Bulinski, Niels Galjart, Ramesh A. Shivdasani, John H. Hartwig, and Sunita R. Patel. "The Marginal Microtubule Coil in the Resting Blood Platelet Is a Dynamic Bipolar Array." Blood 106, no. 11 (November 16, 2005): 1653. http://dx.doi.org/10.1182/blood.v106.11.1653.1653.
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Abstract The discoid shape of the resting blood platelet is maintained by its marginal microtubule band. Structural studies have concluded that this band is composed of a single microtubule coiled 8-12 times around the cell periphery. To understand the dynamics of the microtubule coil, we took advantage of EB1 and EB3, proteins that highlight the ends of growing microtubules. Immunofluorescence microscopy with anti-EB1 revealed clear staining of numerous (8.7 +/− 2.0, range 4–12) comet-like dashes in the microtubule coil, suggesting the presence of several microtubule plus ends. Consistent with this observation, rhodamine-tubulin added to permeabilized platelets incorporates at multiple (7.9 +/−1.9) points throughout the microtubule coil. To visualize microtubule dynamics in platelets, we retrovirally directed megakaryocytes to express the microtubule plus-end marker EB3-GFP and isolated platelets released in these cultures. Fluorescence time-lapse microscopy of EB3-GFP-expressing resting platelets revealed multiple microtubule plus ends that grew in both clockwise and counterclockwise directions. Antibodies that recognize tyrosinated tubulin, which preferentially label newly assembled microtubules and not stable microtubules, stain the microtubule coil. These results indicate that resting platelets contain a bipolar array of microtubules that undergoes continuous assembly. When EB3-GFP-expressing platelets are activated with thrombin, the number of polymerizing microtubules increases dramatically and the microtubules grow into filopodia. Collectively, these results suggest that the marginal band of the resting blood platelet is highly dynamic, bipolar, and contains multiple microtubule plus ends. These ends are amplified in platelet activation and point towards the active edges of the cells and the tips of filopodia.
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Mogensen, M. M., and J. B. Tucker. "Taxol influences control of protofilament number at microtubule-nucleating sites in Drosophila." Journal of Cell Science 97, no. 1 (September 1, 1990): 101–7. http://dx.doi.org/10.1242/jcs.97.1.101.
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Control of protofilament number has been investigated using Drosophila wings at a stage when 15-protofilament microtubules assemble under normal conditions. Microtubule nucleation still progressed at the usual microtubule-nucleating sites in the presence of taxol. However, provided taxol was introduced before microtubule nucleation began, few microtubules with 15 protofilaments assembled. Most microtubules were composed of 12 protofilaments (a previously undetected value for Drosophila) or 13 protofilaments (which is the value for microtubules in most eukaryotic cells). Unexpectedly, a comparatively mild challenge to control of nucleation (in vitro wing culture) also promoted assembly of 13-protofilament microtubules. Hence, the microtubule-nucleating sites may possess a relatively labile control specifying 15 protofilaments superimposed upon that for maintaining 13-protofilament fidelity.
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Straube, Anne, Gerd Hause, Gero Fink, and Gero Steinberg. "Conventional Kinesin Mediates Microtubule-Microtubule Interactions In Vivo." Molecular Biology of the Cell 17, no. 2 (February 2006): 907–16. http://dx.doi.org/10.1091/mbc.e05-06-0542.
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Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.
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Saoudi, Yasmina, Rati Fotedar, Ariane Abrieu, Marcel Dorée, Jürgen Wehland, Robert L. Margolis, and Didier Job. "Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells." Journal of Cell Biology 142, no. 6 (September 21, 1998): 1519–32. http://dx.doi.org/10.1083/jcb.142.6.1519.
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Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.
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Infante, A. S., M. S. Stein, Y. Zhai, G. G. Borisy, and G. G. Gundersen. "Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap." Journal of Cell Science 113, no. 22 (November 15, 2000): 3907–19. http://dx.doi.org/10.1242/jcs.113.22.3907.
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Many cell types contain a subset of long-lived, ‘stable’ microtubules that differ from dynamic microtubules in that they are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). Elevated Glu tubulin does not stabilize the microtubules and the mechanism for the stability of Glu microtubules is not known. We used detergent-extracted cell models to investigate the nature of Glu microtubule stability. In these cell models, Glu microtubules did not incorporate exogenously added tubulin subunits on their distal ends, while >70% of the bulk microtubules did. Ca(2+)-generated fragments of Glu microtubules incorporated tubulin, showing that Glu microtubule ends are capped. Consistent with this, Glu microtubules in cell models were resistant to dilution-induced breakdown. Known microtubule end-associated proteins (EB1, APC, p150(Glued) and vinculin focal adhesions) were not localized on Glu microtubule ends. ATP, but not nonhydrolyzable analogues, induced depolymerization of Glu microtubules in cell models. Timelapse and photobleaching studies showed that ATP triggered subunit loss from the plus end. ATP breakdown of Glu microtubules was inhibited by AMP-PNP and vanadate, but not by kinase or other inhibitors. Additional experiments showed that conventional kinesin or kif3 were not involved in Glu microtubule capping. We conclude that Glu microtubules are stabilized by a plus-end cap that includes an ATPase with properties similar to kinesins.
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Ondrej, Vladan, Emilie Lukásová, Martin Falk, and Stanislav Kozubek. "The role of actin and microtubule networks in plasmid DNA intracellular trafficking." Acta Biochimica Polonica 54, no. 3 (August 23, 2007): 657–63. http://dx.doi.org/10.18388/abp.2007_3239.
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This work is focused on the function of the microtubule and actin networks in plasmid DNA transport during liposomal transfection. We observed strong binding of plasmid DNA-lipid complexes (lipoplexes) to both networks and directional long-range motion of these lipoplexes along the microtubules. Disruption of either of these networks led to the cessation of plasmid transport to the nucleus, a decreased mobility of plasmids, and accumulation of plasmid DNA in large aggregates at the cell periphery. Our findings show an indispensable but different role of both types of cytoskeleton, actin and microtubular, in the processes of gene delivery.
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Cravchik, A., D. Reddy, and A. Matus. "Identification of a novel microtubule-binding domain in microtubule-associated protein 1A (MAP1A)." Journal of Cell Science 107, no. 3 (March 1, 1994): 661–72. http://dx.doi.org/10.1242/jcs.107.3.661.
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Several microtubule-associated proteins (MAPs) have been shown to bind to microtubules via short sequences with repeated amino acids motifs. A microtubule-binding domain has hitherto not been defined for the adult brain microtubule-associated protein 1A (MAP1A). We have searched for a microtubule-binding domain by expressing different protein regions of MAP1A in cultured cell lines using cDNA constructs. One construct included an area with homology to the microtubule-binding domain of MAP1B (Noble et al. (1989) J. Cell Biol. 109, 437–448), but this did not bind to microtubules in transfected cells. Further investigation of other areas of MAP1A revealed a protein domain, capable of autonomously binding to microtubules, which bears no homology to any previously described microtubule-binding sequence. This MAP1A domain is rich in charged amino acids, as are other mammalian microtubule-binding domains, but unlike them has no identifiable sequence repeats. Whereas all previously described mammalian microtubule-binding domains are basic, this novel microtubule-binding domain of MAP1A is acidic. The expression of this polypeptide in cultured cell lines led to a rearrangement of the microtubules in a pattern distinct from that produced by MAP2 or tau, and increased their resistance to treatment with the microtubule depolymerising agent nocodazole. When the MAP1A microtubule-binding domain was co-expressed in cultured cell lines together with MAP2c, the MAP1A microtubule-binding domain was able to bind to the MAP2c-induced microtubule bundles. These results suggest that different microtubule-binding sequences have a common ability to stabilise microtubules but differ in their influence on microtubule arrangement in the cell. This may be significant in neurons, where microtubule-associated proteins with different microtubule-binding sequences are expressed in different cell compartments and at different times during development.
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Sasse, Florenz, Heinrich Steinmetz, Juergen Heil, Gerhard Hoefle, and Hans Reichenbach. "ChemInform Abstract: Tubulysins, New Cytostatic Peptides from Myxobacteria Acting on Microtubuli. Production, Isolation, Physicochemical and Biological Properties." ChemInform 32, no. 6 (February 6, 2001): no. http://dx.doi.org/10.1002/chin.200106163.
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Vorobjev, I. A., T. M. Svitkina, and G. G. Borisy. "Cytoplasmic assembly of microtubules in cultured cells." Journal of Cell Science 110, no. 21 (November 1, 1997): 2635–45. http://dx.doi.org/10.1242/jcs.110.21.2635.
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The origin of non-centrosomal microtubules was investigated in a variety of animal cells in culture by means of time-lapse digital fluorescence microscopy. A previous study (Keating et al. (1997) Proc. Nat. Acad. Sci. USA 94, 5078–5083) demonstrated a pathway for formation of non-centrosomal microtubules by release from the centrosome. Here we show a parallel pathway not dependent upon the centrosome. Correlative immunostaining with anti-tubulin antibodies and electron microscopy established that apparent free microtubules observed in vivo were not growing ends of long stable microtubules. Free microtubules appeared spontaneously in the cytoplasm and occasionally by breakage of long microtubules. Estimates of the frequencies of free microtubule formation suggest that it can be a relatively common rather than exceptional event in PtK1 cells and may represent a significant source of non-centrosomal microtubules. The observation of free microtubules permitted analysis of the microtubule minus end. Unlike the plus end which showed dynamic instability, the minus end was stable or depolymerized. Breakage of long microtubules generated nascent plus and minus ends; the nascent minus end was generally stable while the plus end was always dynamic. The stability of microtubule minus ends in vivo apparently provides the necessary condition for free microtubule formation in the cytoplasm. Parameters of the dynamic instability of plus ends of free microtubules were similar to those for the distal ends of long microtubules, indicating that the free microtubules were not exceptional in their dynamic behavior. Random walk analysis of microtubule end dynamics gave apparent diffusion coefficients for free and long microtubules which permitted an estimate of turnover half-times. The results support the concept that, in PtK1 cells, a pathway other than plus end dynamics is needed to account for the rapidity of microtubule turnover.
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Liu, Jun, Laura Wetzel, Ying Zhang, Eiji Nagayasu, Stephanie Ems-McClung, Laurence Florens, and Ke Hu. "Novel Thioredoxin-Like Proteins Are Components of a Protein Complex Coating the Cortical Microtubules of Toxoplasma gondii." Eukaryotic Cell 12, no. 12 (July 19, 2013): 1588–99. http://dx.doi.org/10.1128/ec.00082-13.
Full textAbstract:
ABSTRACT Microtubules are versatile biopolymers that support numerous vital cellular functions in eukaryotes. The specific properties of microtubules are dependent on distinct microtubule-associated proteins, as the tubulin subunits and microtubule structure are exceptionally conserved. Highly specialized microtubule-containing assemblies are often found in protists, which are rich sources for novel microtubule-associated proteins. A protozoan parasite, Toxoplasma gondii , possesses several distinct tubulin-containing structures, including 22 microtubules closely associated with the cortical membrane. Early ultrastructural studies have shown that the cortical microtubules are heavily decorated with associating proteins. However, little is known about the identities of these proteins. Here, we report the discovery of a novel protein, TrxL1 (for T hio r edo x in- L ike protein 1), and an associating complex that coats the cortical microtubules. TrxL1 contains a thioredoxin-like fold. To visualize its localization in live parasites by fluorescence, we replaced the endogenous TrxL1 gene with an mEmeraldFP-TrxL1 fusion gene. Structured illumination-based superresolution imaging of this parasite line produced a detailed view of the microtubule cytoskeleton. Despite its stable association with the cortical microtubules in the parasite, TrxL1 does not seem to bind to microtubules directly. Coimmunoprecipitation experiments showed that TrxL1 associates with a protein complex containing SPM1, a previously reported microtubule-associated protein in T. gondii . We also found that SPM1 recruits TrxL1 to the cortical microtubules. Besides SPM1, several other novel proteins are found in the TrxL1-containing complex, including TrxL2, a close homolog of TrxL1. Thus, our results reveal for the first time a microtubule-associated complex in T. gondii .
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Lu, Wen, Michael Winding, Margot Lakonishok, Jill Wildonger, and Vladimir I. Gelfand. "Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes." Proceedings of the National Academy of Sciences 113, no. 34 (August 10, 2016): E4995—E5004. http://dx.doi.org/10.1073/pnas.1522424113.
Full textAbstract:
Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.
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Schulze, E., D. J. Asai, J. C. Bulinski, and M. Kirschner. "Posttranslational modification and microtubule stability." Journal of Cell Biology 105, no. 5 (November 1, 1987): 2167–77. http://dx.doi.org/10.1083/jcb.105.5.2167.
Full textAbstract:
We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after injection of biotin-tubulin were all posttranslationally modified; furthermore dynamic microtubules were all unmodified. We therefore conclude that in these cells the stable, acetylated, and detyrosinated microtubules represent the same subset of the cells' total network. Posttranslational modification, however, is not a prerequisite for microtubule stability and vice versa. Potorous tridactylis kidney cells have no detectable acetylated microtubules but do have a sizable subset of stable ones, and chick embryo fibroblast cells are extensively modified but have few stable microtubules. We conclude that different cell types can create specific microtubule subsets by modulating the relative rates of posttranslational modification and microtubule turnover.
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He, Liu, Lotte van Beem, Berend Snel, Casper C. Hoogenraad, and Martin Harterink. "PTRN-1 (CAMSAP) and NOCA-2 (NINEIN) are required for microtubule polarity in Caenorhabditis elegans dendrites." PLOS Biology 20, no. 11 (November 17, 2022): e3001855. http://dx.doi.org/10.1371/journal.pbio.3001855.
Full textAbstract:
The neuronal microtubule cytoskeleton is key to establish axon-dendrite polarity. Dendrites are characterized by the presence of minus-end out microtubules. However, the mechanisms that organize these microtubules with the correct orientation are still poorly understood. Using Caenorhabditis elegans as a model system for microtubule organization, we characterized the role of 2 microtubule minus-end related proteins in this process, the microtubule minus-end stabilizing protein calmodulin-regulated spectrin-associated protein (CAMSAP/PTRN-1), and the NINEIN homologue, NOCA-2 (noncentrosomal microtubule array). We found that CAMSAP and NINEIN function in parallel to mediate microtubule organization in dendrites. During dendrite outgrowth, RAB-11-positive vesicles localized to the dendrite tip to nucleate microtubules and function as a microtubule organizing center (MTOC). In the absence of either CAMSAP or NINEIN, we observed a low penetrance MTOC vesicles mislocalization to the cell body, and a nearly fully penetrant phenotype in double mutant animals. This suggests that both proteins are important for localizing the MTOC vesicles to the growing dendrite tip to organize microtubules minus-end out. Whereas NINEIN localizes to the MTOC vesicles where it is important for the recruitment of the microtubule nucleator γ-tubulin, CAMSAP localizes around the MTOC vesicles and is cotranslocated forward with the MTOC vesicles upon dendritic growth. Together, these results indicate that microtubule nucleation from the MTOC vesicles and microtubule stabilization are both important to localize the MTOC vesicles distally to organize dendritic microtubules minus-end out.
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Tirnauer, Jennifer S., Sonia Grego, E. D. Salmon, and Timothy J. Mitchison. "EB1–Microtubule Interactions in Xenopus Egg Extracts: Role of EB1 in Microtubule Stabilization and Mechanisms of Targeting to Microtubules." Molecular Biology of the Cell 13, no. 10 (October 2002): 3614–26. http://dx.doi.org/10.1091/mbc.e02-04-0210.
Full textAbstract:
EB1 targets to polymerizing microtubule ends, where it is favorably positioned to regulate microtubule polymerization and confer molecular recognition of the microtubule end. In this study, we focus on two aspects of the EB1–microtubule interaction: regulation of microtubule dynamics by EB1 and the mechanism of EB1 association with microtubules. Immunodepletion of EB1 from cytostatic factor-arrested M-phaseXenopus egg extracts dramatically reduced microtubule length; this was complemented by readdition of EB1. By time-lapse microscopy, EB1 increased the frequency of microtubule rescues and decreased catastrophes, resulting in increased polymerization and decreased depolymerization and pausing. Imaging of EB1 fluorescence revealed a novel structure: filamentous extensions on microtubule plus ends that appeared during microtubule pauses; loss of these extensions correlated with the abrupt onset of polymerization. Fluorescent EB1 localized to comets at the polymerizing plus ends of microtubules in cytostatic factor extracts and uniformly along the lengths of microtubules in interphase extracts. The temporal decay of EB1 fluorescence from polymerizing microtubule plus ends predicted a dissociation half-life of seconds. Fluorescence recovery after photobleaching also revealed dissociation and rebinding of EB1 to the microtubule wall with a similar half-life. EB1 targeting to microtubules is thus described by a combination of higher affinity binding to polymerizing ends and lower affinity binding along the wall, with continuous dissociation. The latter is likely to be attenuated in interphase. The highly conserved effect of EB1 on microtubule dynamics suggests it belongs to a core set of regulatory factors conserved in higher organisms, and the complex pattern of EB1 targeting to microtubules could be exploited by the cell for coordinating microtubule behaviors.