ジャーナル: Structure / 年: 2023 タイトル: Doublet microtubule inner junction protein FAP20 recruits tubulin to the microtubule lattice. 著者: Mamata Bangera / Archita Dungdung / Sujana Prabhu / Minhajuddin Sirajuddin / 要旨: Doublet microtubules of eukaryotic cilia and flagella are made up of a complete A- and an incomplete B-tubule that are fused together. Of the two fusion points, the outer junction is made of ...Doublet microtubules of eukaryotic cilia and flagella are made up of a complete A- and an incomplete B-tubule that are fused together. Of the two fusion points, the outer junction is made of tripartite tubulin connections, while the inner junction contains non-tubulin elements. The latter includes flagellar-associated protein 20 (FAP20) and Parkin co-regulated gene protein (PACRG) that together link the A- and B-tubule at the inner junction. While structures of doublet microtubules reveal molecular details, their assembly is poorly understood. In this study, we purified recombinant FAP20 and characterized its effects on microtubule dynamics. We use in vitro reconstitution and cryo-electron microscopy to show that FAP20 recruits free tubulin to the existing microtubule lattice. Our cryo-electron microscopy reconstruction of microtubule:FAP20:tubulin complex reveals the mode of tubulin recruitment by FAP20 onto microtubules, providing insights into assembly steps of B-tubule closure during doublet microtubule formation.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 298 K / 装置: FEI VITROBOT MARK IV 詳細: GMPCPP MT seeds in warm BRB80 was applied to the grid followed by second application of pre-warmed solution containing FAP20 and tubulin. After an incubation time of 1 minute, grid was ...詳細: GMPCPP MT seeds in warm BRB80 was applied to the grid followed by second application of pre-warmed solution containing FAP20 and tubulin. After an incubation time of 1 minute, grid was blotted for 3 seconds before plunging into liquid ethane..
詳細
Goat brain tubulin was polymerized with 1mM GMPCPP at 310K for 2 hours, spun on warm 50 percent BRB80 sucrose cushion and resuspended in warm BRB80 buffer.