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1 July 2000 Nucleation of Astral-shaped Microtubules from Latex Beads Conjugated with MTOG Proteins
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Abstract
A model system for the formation of astral-shaped microtubules (Mts) consisting of Latex beads (diameter of 0.2 μm), a protein fraction (p51) comprised of MTOGs (microtubule-organizing granules) and tubulin was established. The Latex beads were first incubated with p51 in the presence of GTP at 0°C, then the purified tubulin dimer fraction was added, resulting in the formation of an aster-like structure observed by dark-field microscopy. On preincubation of the Latex beads with GDP instead of GTP, the asters did not form. Unhydrolyzable GTP analogues such as GTP-γS and GMP-PNP promoted aster formation as did GTP as observed by dark-field microscopy. Polylysine, as representative of basic polymers capable of binding to the surface of the Latex beads, promoted spontaneous Mt assembly, and eventually an aster-like structure without Latex beads in the center formed. Further analyses made by measuring the optical density of the aster-forming system produced the following results. 1) preincubation of the Latex beads with GTP or GMP-PNP supported Mt assembly from the beads showing profiles typical for a sitedirected assembly without the lag phase. 2) GTP-γS and GDP inhibited the turbidity increase of the system, causing a decrease in both the initial velocity and the level of steady state of Mt assembly. 3) Anti-p51 monoclonal antibody (HP1) substantially inhibited the aster formation, while anti-γ-tubulin antibody only slightly inhibited assembly.
Hikoichi Sakai, Ayako Maruno, Natsuki Sugawara, Kyoko Takahashi, Chihiro Hoshi, Akiko Nakamura, Risako Nakamura, Naeko Shinozaki, Mamiko Sato and Masako Osumi "Nucleation of Astral-shaped Microtubules from Latex Beads Conjugated with MTOG Proteins," Zoological Science 17(5), (1 July 2000). https://doi.org/10.2108/zsj.17.609
Received: 20 September 1999; Accepted: 1 November 1999; Published: 1 July 2000
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