Readout of the tubulin code by cellular effectors

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

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.