Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Cell. 2017 Jun 1;169(6):1066-1077.e10. doi: 10.1016/j.cell.2017.05.028.
Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C. elegans proteins. We found that macromolecular crowding drives assembly of the key PCM scaffold protein SPD-5 into spherical condensates that morphologically and dynamically resemble in vivo PCM. These SPD-5 condensates recruited the microtubule polymerase ZYG-9 (XMAP215 homolog) and the microtubule-stabilizing protein TPXL-1 (TPX2 homolog). Together, these three proteins concentrated tubulin ∼4-fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins.
中心体是非膜结合的隔间，它可以起始微管阵列。它们由纳米级别的中心粒组成，周围是微米级的、动态的蛋白质组装，称为中心粒周围物质（PCM）。为了研究 PCM 如何形成起始微管的球形隔间，我们使用重组 C. elegans 蛋白在体外重建了依赖 PCM 的微管起始。我们发现，大分子拥挤促使关键的 PCM 支架蛋白 SPD-5 组装成球形凝聚物，这些凝聚物在形态和动力学上与体内的 PCM 相似。这些 SPD-5 凝聚物招募了微管聚合酶 ZYG-9（XMAP215 同源物）和微管稳定蛋白 TPXL-1（TPX2 同源物）。这三种蛋白质一起将微管蛋白浓缩约 4 倍，足以在体外重建微管星状体的起始。我们的结果表明，体内的 PCM 是一种选择性的相，通过微管效应蛋白在局部浓缩微管蛋白来组织微管阵列。
