4D map of the tubulin code in the human neuron

生命依赖于微管上细胞货物的有效和有组织的运输，微管是由α/β-微管蛋白组成的聚合物。没有细胞类型比神经元更依赖于这个过程，神经元需要支持间隔超过1米的隔间之间的快速，有效的通信。因此，神经元过程是密集的细胞环境之一，微管间距接近20 nm，支持生存和最佳功能所需的大量连续双向细胞交通。神经元已经进化出组织和区分这些微管轨道的手段，通过功能化微管蛋白与化学多样的翻译后修饰。一个新兴的范例是，这些修饰作为微管蛋白代码来调节微管动力学、微管相关蛋白和微管马达的力学和募集。该提案利用了我们团队在微管蛋白编码（Roll-Mecak，NINDS）和高分辨率细胞成像（Hari Shroff，NIBIB和AIM）方面的专业知识，通过开发扩展显微镜与单分子成像相结合的方法，在人类神经元中生成微管蛋白编码及其读者的完整单分子分辨率4D图谱。自该奖项成立以来，我们成功地完善了扩增方案，我们验证了与扩增显微镜兼容的抗体，并开发了对这些样品进行成像所需的方案，其中包括显微镜升级，以提高样品稳定性并提高相机灵敏度和分辨率。我们开发的方法将广泛适用于其他需要纳米级分辨率和分子对比度的结构问题。

Life depends on the efficient and organized transport of cellular cargo on microtubules, polymers composed of alpha/beta-tubulins. No cell type is more dependent on this process than the neuron which needs to support fast, efficient communication between compartments that can be more than 1 m apart. As a result, neuronal processes are one of the densest cellular environments, with microtubules spaced as close as 20 nm that support the large continuous volume of bidirectional cellular traffic necessary for survival and optimal function. Neurons have evolved means to organize and differentiate these microtubule tracks by functionalizing tubulin with chemically diverse posttranslational modifications. An emerging paradigm is that these modifications act as a tubulin code to regulate microtubule dynamics, mechanics and recruitment of microtubule-associated proteins and microtubule-based motors. This proposal leverages the expertise of our groups on the tubulin code (Roll-Mecak, NINDS) and high-resolution cellular imaging (Hari Shroff, NIBIB and AIM) to generate a complete single-molecule resolution 4D map of the tubulin code and its readers in the human neuron by developing methods for expansion microscopy coupled with single molecule imaging. Since the inception of this award we succeeded in refining the expansion protocols, we validated antibodies that are compatible for expansion microscopy and developed protocols necessary for imaging these samples, which included a microscope upgrade to improve sample stability and increase camera sensitivity and resolution. The methods we develop will be broadly applicable to other structural problems that require nanoscale resolution and molecular contrast.