Department of Biomedical Engineering, The Pennsylvania State University, University Park, PA 16802.
Intercollege Graduate Degree Program in Bioengineering, The Pennsylvania State University, University Park, PA 16802.
Proc Natl Acad Sci U S A. 2019 Apr 9;116(15):7314-7322. doi: 10.1073/pnas.1815823116. Epub 2019 Feb 25.
The biochemical basis of microtubule growth has remained elusive for over 30 years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make direct, single-molecule measurements of tubulin association and dissociation rates. We detect two populations of transient dwell times and determine via binding-interface mutants that they are distinguished by the formation of one interprotofilament bond. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data and, furthermore, predicts plus-end tapering. Overall, we provide the most direct and complete experimental quantification of how microtubules grow to date.
尽管微管生长的生化基础对于细胞分裂和相关的化疗策略至关重要，但 30 多年来一直难以捉摸。在这里，我们结合干涉散射显微镜和重组微管蛋白来监测单个微管蛋白与生长中的微管尖端结合和解离。我们进行了直接的、单分子的微管蛋白结合和解离速率测量。我们检测到两种短暂停留时间的种群，并通过结合界面突变体确定它们是由一个原纤维间键的形成来区分的。应用计算模型，我们发现与原纤维上的强相互作用的缓慢结合动力学最能再现我们的数据，此外，还预测了正极端变细。总的来说，我们提供了迄今为止最直接和完整的微管生长实验定量。
