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.Chem.，2016；Vemu et al.2020)；(3)证明具有不同异构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异构体组成来成比例地调节(Vemu等人，Mol.比奥尔。(4)开发了获得微管蛋白的生化平台，该平台具有定量定义的翻译后修饰水平(Valenstein和Roll-Mecak，Cell 2016)，并利用该平台来(5)展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。利用我们用于生成定量定义的修饰微管以及重组工程化人类微管的平台，我们目前正在研究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。具体地说，今年我们阐明了谷氨酰化是如何影响微管动力学的，谷氨酰化是一种长期与细胞中微管稳定性相关的修饰。来自不同领域的细胞生物学家使用反对这种修饰的抗体来替代细胞中稳定的微管。利用我们在获得差异谷氨酸化微管蛋白方面的最新进展以及微管动力学重建，我们表明，令人惊讶的是，谷氨酰化对微管生长起到负调节作用(Chen和Roll-Mecak，2023)，并且不稳定微管。因此，细胞内谷氨酰化微管的较高稳定性一定是由于反式作用。我们未来的努力将集中在识别招募到谷氨酸化微管的蛋白质组。综上所述，我们最近在谷氨酰化方面的工作，以及我们以前在酪氨酸化、去酪氨酸化和Delta2微管蛋白方面的工作证实，长期与细胞内微管稳定性增加相关的翻译后修饰都不能直接增加稳定性。因此，这些修饰起到信号的作用，以时间和空间控制的方式将效应器招募到微管。

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 glutamylation, a modification long associated with microtubule stability in cells affects microtubule dynamics. Cell biologists from diverse fields use antibodies against this modification as a proxy for stable microtubules in cells. Using our recent advances in obtaining differentially glutamylated tubulin coupled with microtubule dynamics reconstitution we showed that, surprisingly, glutamylation acts as a negative regulator of microtubule growth (Chen and Roll-Mecak, 2023)and does not stabilize microtubules. Thus, the higher stability of glutamylated microtubules in cells must be due to trans effects. Our future efforts will focus on identifying the proteome that is recruited to glutamylated microtubules. Taken together, our most recent work on glutamylation, as well as our previous work on tyrosination, detyrosination and Delta2 tubulin established that none of the posttranslational modifications long associated with increased microtubule stability in cells, confer increased stability directly. Thus, these modifications function as signals to recruit effectors in a temporally and spatially controlled manner to the microtubule.