Properties of the interphase microtubule organizing centers (MTOCs) of CHO cells were investigated using indirect immunofluorescence staining technique.Microtubule proteins were isolated from swine brain by three cyc...Properties of the interphase microtubule organizing centers (MTOCs) of CHO cells were investigated using indirect immunofluorescence staining technique.Microtubule proteins were isolated from swine brain by three cycles of assembly-disassembly- 6S tubulin was purified by chromatograph y through cellulose phosphate p-11 column. Rabbits were immunized with the purified 6S tubulin and the anti-6S tubulin antiserum thus prepared was used in the indirect immunofluorescence staining of nuclei With double thymidine block method, we obtained cells at G1/S, S and G2 phases of cell cycle and isolated nuclei from cells of each pltase, Immunofluorescence staining of MTOCs could hardly he detected in the Triton X-100 treated cells nor in nuclei of any phase after preincubation with purified 6S tubulin alone. Yet,if the Triton X-100 treated cells or nuclei were incubated, in the presence of 5 mM ATP, with maturation promoting factor (MPF)isolated from mature oocytes of Xenopus for 40 minutes before the addition of the 6S tubulin, specifically stained MTOCs were clearly demonstrated in S and G2 nuclei Furthermore, microbubules could be observed radiating from the MTOCs in late S and G2 nuclei. However, alkaline pHospHatase treatment of the nuclei which had been incubated with the activated MPF prohibited the staining of MTOCs, and MPF isolated from premature oocytes failed to contribute to the immunostaining of MTOCs. Our results thus suggest that the transformation of kinetochores into MTOCs during cell cycle requires activated MPF and phosphorylation of proteins.展开更多
Sporogenesis in the hepatic Marchantia polymorpha L. provides an outstanding example of the pleiomorphic nature of the plant microtubule organizing center （MTOC）. Microtubules are nucleated from γ-tubuUn in MTOCs t...Sporogenesis in the hepatic Marchantia polymorpha L. provides an outstanding example of the pleiomorphic nature of the plant microtubule organizing center （MTOC）. Microtubules are nucleated from γ-tubuUn in MTOCs that change form during mitosis and meiosis. Following entry of cells into the reproductive pathway of sporogenesis, successive rounds of mitosis give rise to packets of 4-16 sporocytes. Mitotic spindles are organized at discrete polar organizers （POs）, a type of MTOC that is unique to this group of early divergent land plants. An abrupt and radical transformation in microtubule organization occurs when sporocytes enter meiosis： POs are lost and γ-tubulin is closely associated with surfaces of two large elongated plastids that subsequently divide into four. Migration of the four plastid MTOCs into a tetrahedral arrangement establishes the future spore domains and the division polarity of meiosis. As is typical of many bryophytes, cones of microtubules from the four plastid MTOCs initiate a quadripolar microtubule system （QMS） in meiotic prophase. At this point a transformation in the organization of the MTOCs occurs. The γ-tubulin detaches from plastids and forms a diffuse spheroidal pole in each of the spore domains. The plastids, which are no longer MTOCs, continue to divide. The diffuse MTOCs continue to nucleate cones of microtubules during transformation of the QMS to a bipolar spindle. Following meiosis I, γ-tubulin is associated with nuclear envelopes, and the spindles of meiosis II are organized from diffuse MTOCs at the tetrad poles. At simultaneous cytokinesis, radial microtubule systems are organized at nuclear envelope MTOCs in each of the tetrad members.展开更多
In most kinds of animal cells, the centrosome serves as the main microtubule organizing center （MTOC） that nucleates microtubule arrays throughout the cytoplasm to maintain cell structure, cell division and intracel...In most kinds of animal cells, the centrosome serves as the main microtubule organizing center （MTOC） that nucleates microtubule arrays throughout the cytoplasm to maintain cell structure, cell division and intracellular transport. Whereas in epithelial cells, non-centrosomal MTOCs are established in the apical domain for generating asymmetric microtubule fibers and cilia in epithelial cells for the organ morphogenesis during embryonic development. However, the mechanism by which MTOCs localize to the apical domain in epithelial cells remains largely unknown. Here, we show that Mid liplb has a close interaction with γ-tubulin protein, the central component of MTOC, and modulates lumen opening of the neural tube, gut, intestine, and kidney of zebrafish. Knockdown or dominant negative effect of Mid 1ip 1 b resulted in failure of lumen formation of the organs as aforementioned. Moreover, the non-centrosomal MTOCs were unable to orientate to the apical domain in Midliplb knockdown epithelial cells, and the centrosomal MTOCs were inaccurately placed in the apical domain, resulting in defective formation of asymmetric microtubules and misplacement of cilia in the apical domain. These data uncover a molecule that controls the proper localization of MTOCs in the apical domain in epithelial cells for organ morphogenesis during embryonic development.展开更多
文摘Properties of the interphase microtubule organizing centers (MTOCs) of CHO cells were investigated using indirect immunofluorescence staining technique.Microtubule proteins were isolated from swine brain by three cycles of assembly-disassembly- 6S tubulin was purified by chromatograph y through cellulose phosphate p-11 column. Rabbits were immunized with the purified 6S tubulin and the anti-6S tubulin antiserum thus prepared was used in the indirect immunofluorescence staining of nuclei With double thymidine block method, we obtained cells at G1/S, S and G2 phases of cell cycle and isolated nuclei from cells of each pltase, Immunofluorescence staining of MTOCs could hardly he detected in the Triton X-100 treated cells nor in nuclei of any phase after preincubation with purified 6S tubulin alone. Yet,if the Triton X-100 treated cells or nuclei were incubated, in the presence of 5 mM ATP, with maturation promoting factor (MPF)isolated from mature oocytes of Xenopus for 40 minutes before the addition of the 6S tubulin, specifically stained MTOCs were clearly demonstrated in S and G2 nuclei Furthermore, microbubules could be observed radiating from the MTOCs in late S and G2 nuclei. However, alkaline pHospHatase treatment of the nuclei which had been incubated with the activated MPF prohibited the staining of MTOCs, and MPF isolated from premature oocytes failed to contribute to the immunostaining of MTOCs. Our results thus suggest that the transformation of kinetochores into MTOCs during cell cycle requires activated MPF and phosphorylation of proteins.
文摘Sporogenesis in the hepatic Marchantia polymorpha L. provides an outstanding example of the pleiomorphic nature of the plant microtubule organizing center （MTOC）. Microtubules are nucleated from γ-tubuUn in MTOCs that change form during mitosis and meiosis. Following entry of cells into the reproductive pathway of sporogenesis, successive rounds of mitosis give rise to packets of 4-16 sporocytes. Mitotic spindles are organized at discrete polar organizers （POs）, a type of MTOC that is unique to this group of early divergent land plants. An abrupt and radical transformation in microtubule organization occurs when sporocytes enter meiosis： POs are lost and γ-tubulin is closely associated with surfaces of two large elongated plastids that subsequently divide into four. Migration of the four plastid MTOCs into a tetrahedral arrangement establishes the future spore domains and the division polarity of meiosis. As is typical of many bryophytes, cones of microtubules from the four plastid MTOCs initiate a quadripolar microtubule system （QMS） in meiotic prophase. At this point a transformation in the organization of the MTOCs occurs. The γ-tubulin detaches from plastids and forms a diffuse spheroidal pole in each of the spore domains. The plastids, which are no longer MTOCs, continue to divide. The diffuse MTOCs continue to nucleate cones of microtubules during transformation of the QMS to a bipolar spindle. Following meiosis I, γ-tubulin is associated with nuclear envelopes, and the spindles of meiosis II are organized from diffuse MTOCs at the tetrad poles. At simultaneous cytokinesis, radial microtubule systems are organized at nuclear envelope MTOCs in each of the tetrad members.
基金supported by the National Basic Research Program of China (to X.M.,2015CB942800)the Nature Science Foundation of China (to H. X. 31671502)
文摘In most kinds of animal cells, the centrosome serves as the main microtubule organizing center （MTOC） that nucleates microtubule arrays throughout the cytoplasm to maintain cell structure, cell division and intracellular transport. Whereas in epithelial cells, non-centrosomal MTOCs are established in the apical domain for generating asymmetric microtubule fibers and cilia in epithelial cells for the organ morphogenesis during embryonic development. However, the mechanism by which MTOCs localize to the apical domain in epithelial cells remains largely unknown. Here, we show that Mid liplb has a close interaction with γ-tubulin protein, the central component of MTOC, and modulates lumen opening of the neural tube, gut, intestine, and kidney of zebrafish. Knockdown or dominant negative effect of Mid 1ip 1 b resulted in failure of lumen formation of the organs as aforementioned. Moreover, the non-centrosomal MTOCs were unable to orientate to the apical domain in Midliplb knockdown epithelial cells, and the centrosomal MTOCs were inaccurately placed in the apical domain, resulting in defective formation of asymmetric microtubules and misplacement of cilia in the apical domain. These data uncover a molecule that controls the proper localization of MTOCs in the apical domain in epithelial cells for organ morphogenesis during embryonic development.
