Readout of the tubulin code by cellular effectors

微管是细胞形态发生、细胞分裂和细胞内运输所必需的聚合物。它们具有高度多样性、丰富性和进化上保守的翻译后修饰。 微管蛋白修饰水平和模式的破坏会导致癌症、神经病理学和轴突再生缺陷。我们的长期目标是了解细胞如何使用微管蛋白异构体多样性和翻译后修饰来调节微管的结构和动力学，以及它们与分子马达和微管相关蛋白（MAP）的相互作用。虽然在三十多年前发现，但对微管蛋白的化学和遗传复杂性的作用的理解仍然难以捉摸。我的团队整合了生物物理学、蛋白质组学、结构和细胞生物学的技术和概念，以解决微管细胞生物学中的这一基本问题。 我的实验室在实现这些目标方面取得了重大进展。这些包括：（1）开发用于产生同质工程化单一同种型重组未修饰人微管蛋白的新方法（Vemu et al.，J. Biol. Chem.，（2016年）;（2）重组同位素纯的重组神经元微管蛋白的第一结构和动态不稳定性参数的测定（Vemu等，J. Biol. Chem.，二〇一六年; Vemu等人，2020）;（3）证明具有不同同种型组成的微管表现出不同的动态特性，并且这些特性可以通过改变微管蛋白同种型组成来成比例地调节（Vemu等人，摩尔Cell，2017）。(4)开发用于获得具有定量限定水平的翻译后修饰的微管蛋白的生物化学平台（Valenstein和Roll-Mecak，Cell 2016）和使用该平台来（5）显示重要的微管调节剂，遗传性痉挛性截瘫蛋白spastin，对微管蛋白谷氨酰化的分级反应（Valenstein和Roll-Mecak，Cell 2016），从而为微管蛋白编码假说提供了强有力的支持。使用我们的平台，用于产生定量定义的修饰微管以及重组工程化的人微管，我们目前正在研究如何微管蛋白代码，通过遗传变异和翻译后修饰，调节微管的基本生物物理特性以及分子马达和神经元MAP与神经退行性疾病的强烈参与。 具体来说，今年我们阐明了两个α-微管蛋白翻译后修饰如何调节细胞中的微管动力学。20世纪80年代后期，Kirschner和Borisy小组的开创性工作表明，周转缓慢的微管富含两种丰富的α-微管蛋白翻译后修饰，即脱酪氨酸和Delta 2微管蛋白，而动态的快速周转微管富含酪氨酸。 从那时起，来自不同领域的细胞生物学家使用针对这些修饰的抗体作为细胞中稳定和动态微管的代表。然而，这些修饰如何影响内在微管动力学特性以及它们在细胞中的差异稳定性背后的机制的基本问题仍然没有答案。利用我们在重组微管蛋白工程结合微管动力学重建和分子动力学模拟方面的最新进展，我们表明酪氨酸化、去酪氨酸化和Delta 2微管具有不可区分的动力学参数，并且它们在细胞中的差异稳定性是效应子特异性募集的结果（Chen et al，2021）。 我们发现，酪氨酸定量调谐微管效应器CLIP-170的正端微管和CLIP-170的梯度招聘与尖端跟踪蛋白EB 1的酪氨酸化微管协同作用，选择性地增加其动态。因此，我们的工作表明，调节剂的修饰依赖性募集可以产生具有不同动态特性的微管亚群。作为合作努力的一部分，我们还表明细胞中的ER分布受微管蛋白谷氨酰化的调节（Zhang et al.，2021年）。因此，我们的工作奠定了基础，阐明微管功能的微管蛋白代码在细胞中的调节。

Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. They are subject to highly diverse, abundant and evolutionarily conserved post-translational modifications. Disruption of tubulin modification levels and patterns leads to cancers, neuropathologies and defective axonal regeneration. Our long-term goal is to understand how cells use tubulin isoform diversity and posttranslational modifications to regulate the structure and dynamics of microtubules as well as their interactions with molecular motors and microtubule associated proteins (MAPs). Although discovered over thirty years ago, an understanding of the roles of the chemical and genetic complexity of tubulin has remained elusive. My group integrates techniques and concepts from biophysics, proteomics, structural and cell biology to address this fundamental problem in microtubule cell biology. My laboratory has made significant progress towards these goals. These include: (1) development of novel methods for generating homogenous engineered single isoform recombinant unmodified human tubulin (Vemu et al., J. Biol. Chem., 2016); (2) determination of the first structure and dynamic instability parameters of recombinant isotopically pure recombinant neuronal tubulin (Vemu et al., J. Biol. Chem., 2016; Vemu et al. 2020); (3) demonstration that microtubules with different isoform compositions exhibit different dynamic properties and that these properties can be proportionally tuned by varying tubulin isoform composition (Vemu et al., Mol. Biol. Cell, 2017).(4) development of a biochemical platform for obtaining tubulin with quantitatively defined levels of posttranslational modifications (Valenstein and Roll-Mecak, Cell 2016) and use of this platform to (5) showing the graded response of an important microtubule regulator, the hereditary spastic paraplegia protein spastin, to tubulin glutamylation (Valenstein and Roll-Mecak, Cell 2016) thus furnishing strong support for the tubulin code hypothesis. Using our platform for generating quantitatively defined modified microtubules as well recombinant engineered human microtubules, we are currently investigating how the tubulin code, both through genetic variation and posttranslational modifications, regulates the basic biophysical properties of microtubules as well as molecular motors and neuronal MAPs with strong involvement in neurodegenerative disorders. Specifically, this year we elucidated how two alpha-tubulin posttranslational modifications regulate microtubule dynamics in cells. Pioneering work in the late 1980s from the Kirschner and Borisy groups showed that microtubules with slow turnover are enriched in two abundant alpha-tubulin posttranslational modifications, detyrosination and Delta2 tubulin, while dynamic, fast turnover microtubules are enriched in tyrosination. Since then, cell biologists from diverse fields use antibodies against these modifications as proxies for stable and dynamic microtubule in cells. However, the fundamental question of how these modifications affect intrinsic microtubule dynamic properties and what the mechanism behind their differential stability in cells has remained unanswered. Using our recent advances in recombinant tubulin engineering coupled with microtubule dynamics reconstitution and molecular dynamics simulations we showed that tyrosinated, detyrosinated and Delta2 microtubules have indistinguishable dynamic parameters and that their differential stability in cells is a result of the specific recruitment of effectors (Chen et al,, 2021). We found that tyrosination quantitatively tunes the graded recruitment of the microtubule effector CLIP-170 to the plus-end of microtubules and CLIP-170 acts synergistically with the tip-tracking protein EB1 on tyrosinated microtubules to selectively increase their dynamicity. Thus, our work showed that modification-dependent recruitment of regulators can generate microtubule subpopulations with distinct dynamic properties. As part of a collaborative effort, we also showed that the ER distribution in the cell is regulated by tubulin glutamylation (Zhang et al., 2021). Our work thus lays the foundation for the elucidation of regulation of microtubule functions by the tubulin code in cells.