Molecular mechanisms for changes in microtubule dynamics

微管动力学变化的分子机制

• 批准号: 05454644
• 负责人: NISHIDA Eisuke
• 金额: $ 3.97万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454644/
• 关键词: Microtubule Savering Factor; Cell Cycle; Microtubule dynamics; M phase; 微小管結合蛋白質; 細胞分裂; MPF; 細胞骨格; Xenopus; Microtubule Savering Factor; Cell Cycle; Microtubule dynamics; M phase; 微小管結合蛋白質; 細胞分裂; MPF; 細胞骨格; Xenopus

An activity that severs stable microtubules has previously been detected in M phase extracts, but not in interphase extracts, of Xenopus eggs. We reported previously the identification and purification from Xenopus M phase eggs of a microtubule-severing factor. It is a homo-oligomeric protein composed of 56-kDa polypeptide subunit (p56) that can sever stable microtubules slowly. Protein microsequencing indicated that p56 is a previously unidentified protein. Recently, we found andther microtubule-severing activity from Xenopus M phase eggs and purified it to near homogeneity by sequential chromatography. The newly purified factor had a single polypeptide of 48-kDa (p48) , and severed microtubules very rapidly in an ATP-independent manner. It was found that microtubule severing induced by p48 is not accompanied by significant depolymerization of microtubules. These characteristics of p48 are clearly different from those of katanin, an ATPase that severs and disassembles stable microtubules, which has been identified in sea urchin eggs by Vale and co-workers. We produced anti-p48 antibody that reacts with p48 specifically in total cell lysates. The immunoblotting with this anti-p48 antibody revealed the universal existence of p48 in almost all tissues of Xenopus and rat. Furthermore, p48 which was purified by chromatographya on anti-p48 antibody-fixed beads exhibited a microtubule severing activity, confirming the identity of p48 as a severing factor. When purified p48 was reacted with cytoplasmic microtubule network emanating from centrosomes, rapid breaking of microtubules occurred. p48 was revealed to be EF1-alpha. These results suggest that EF1-alpha may function as a microtubule-severing factor in cells.

先前在M相提取物中检测到了稳定微管的活性，但在相间提取物中未检测到Xenopus卵。我们先前报道了微管 - 卵形因子的爪蟾M相卵的鉴定和纯化。它是一种由56 kDa多肽亚基（p56）组成的同型蛋白质，可以缓慢地切断可稳定的微管。蛋白质测序表明p56是先前未鉴定的蛋白质。最近，我们发现了从异爪蟾鸡蛋中的微管卵性活性，并通过顺序色谱法将其纯化至近似均匀性。新纯化的因子具有48 kDa（p48）的单一多肽，并且以ATP独立的方式非常快地切断微管。发现由P48诱导的微管切断不伴有微管的显着解聚。 p48的这些特征显然与katanin的特征不同，katanin的ATPase被瓦尔和同事在海胆和同事中鉴定出来，该ATPase已切断和分解稳定的微管。我们产生了在总细胞裂解物中与P48反应的抗P48抗体。使用这种抗P48抗体的免疫印迹揭示了p48在爪蟾和大鼠几乎所有组织中的普遍存在。此外，抗P48抗体固定珠在抗P48抗体固定珠上纯化的p48表现出微管的切断活性，证实了p48的身份为切断因子。当纯化的p48与从中心体产生的细胞质微管网络反应时，微管的快速破裂。 P48揭示为EF1-Alpha。这些结果表明，EF1-Alpha可能在细胞中起到微管为因素。