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。它们受到高度多样化、丰富性和进化保守性的翻译后修饰。微管蛋白修饰水平和模式的破坏会导致癌症、神经病理和轴突再生缺陷。我们的长期目标是了解细胞如何利用微管蛋白异构体的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。尽管在30多年前就发现了微管蛋白，但人们对微管蛋白的化学和遗传复杂性的作用仍然难以理解。我的团队整合了生物物理学、蛋白质组学、结构生物学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)开发用于产生均一的工程化单一异构体重组未修饰的人微管蛋白的新方法(Vemu等，J.Biol。(2)重组同位素纯重组神经元微管蛋白的一级结构和动态不稳定性参数的测定(Vemu等人，J.Biol.(3)开发获得微管蛋白的生化平台，并定量定义翻译后修饰水平(Valenstein和Roll-Mecak，Cell 2016)，并使用该平台来(4)显示重要的微管调节因子--遗传性痉挛截瘫蛋白spastin对微管蛋白谷氨化的分级反应(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。我们最新的工作表明，具有不同异构体组成的微管表现出显著不同的动力学特性，并且这些特性可以通过不同的微管异构体组成按比例进行调节(Vemu等人，Mol.比奥尔。细胞，2017)。这表明，在形态发生和肿瘤形成过程中观察到的异构体表达的变化对微管动力学有直接影响，不需要通过反式效应分子来介导。使用我们的平台来生成定量定义的修饰微管以及重组工程人微管，我们将继续询问微管编码如何通过遗传变异和翻译后修饰来调节微管的基本生物物理属性以及与神经退行性疾病密切相关的分子马达和神经元图谱。

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); (3) 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 (4) show 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. Our most recent work shows that microtubules with different isoform compositions exhibit dramatically different dynamic properties and that these properties can be proportionally tuned by varying tubulin isoform composition (Vemu et al., Mol. Biol. Cell, 2017). This indicates that the changes in isoform expression observed during morphogenesis and tumorigensis have a direct impact on microtubule dynamics and do not need to be mediated in trans through effectors. Using our platform for generating quantitatively defined modified microtubules as well recombinant engineered human microtubules, we will continue to interrogate 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.