| Budget Amount *help |
¥26,260,000 (Direct Cost: ¥20,200,000、Indirect Cost: ¥6,060,000)
Fiscal Year 2010: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2009: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2008: ¥18,720,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥4,320,000)
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| Research Abstract |
Microtubules are dynamic polymers made from α/β-tubulin dimers, and are crucial for various cellular events such as cell division, motility or organelle transport. Since the discovery of γ-tubulin, attention has been focused upon its role as a microtubule nucleator at the centrosome. However, recent observations revealed that spindle microtubules can also be generated by non-centrosomal pathways. A prior RNAi screen in Drosophila S2 cells identified 8 novel genes (Dgt2-9) that are required for localizing γ-tubulin to spindle microtubules. We showed that the Dgt proteins interact, forming a stable complex, which we named "augmin". Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequence on mitotic spindle formation and function, causing reduced kinetochore-fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identified the human augmin complex of 8 subunits and showed that it in
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teracts with the γ-tubulin ring complex (γ-TuRC). A link between augmin and γ-TuRC is likely critical for these functions, since mutants of augmin or γ-TuRC that attenuate their interaction did not restore function in vivo. Our results suggested that γ-tubulin's most important mitotic function may lie within the spindle, where augmin and γ-tubulin function cooperatively to amplify the number of microtubules. In human cells, we also showed that augmin-dependent de novo microtubule generation in the interchromosomal region during anaphase is important for central spindle formation. Generation of interchromosomal microtubules and subsequent formation of the central spindle occurred independently of pre-anaphase microtubules or centrosomal microtubule nucleation. Based upon these results, a new model for central spindle assembly was proposed. Finally, we identified a novel protein "Sentin" that is localized at the growing end of microtubules via binding to EB1 protein. Sentin depletion in Drosophila S2 cells, similar to EB1 depletion, resulted in the increase in microtubule pausing and led to the formation of shorter spindles, without displacing EB1 from growing microtubules. We demonstrated that Sentin's association with EB1 is critical for its plus-end localisation and function; furthermore, the EB1 phenotype was rescued by expressing an EBN-Sentin fusion protein in which the C-terminal cargo-binding region of EB1 was replaced with Sentin. These results indicated that EB1 promotes dynamic microtubule behavior by recruiting the cargo protein Sentin to the microtubule tip. Less
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