Readout of the tubulin code by cellular effectors

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

Microtubule cytoskeleton disruptions are a hallmark of neurodegenerative disease. Microtubules, and their tubulin component, are subject to many posttranslational modifications. These modifications regulate the activity of molecular motors kinesin and dynein, as well as microtubule associated proteins such as tau, associated with Alzheimer's and frontotemporal dementia. Disregulation of tubulin modification levels and patterns leads to cancers, neuropathologies and defective axonal regeneration. My group integrates techniques and concepts from biophysics, proteomics, structural and cell biology to uncover how cells use tubulin isoforms and posttranslational modifications to regulate the structure and dynamics of microtubules as well as their interactions with molecular effectors. My laboratory has made significant contributions towards understanding the mechanism of tubulin modifications and their role in regulating microtubule effectors, while also developing tools to study microtubule biology that have been adopted by the field. 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; Cummings et al. eLife 2025) and use of this platform to (5) 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. (6) Reconstituting polyglycylation in vitro for the first time and using differentially glycylated microtubules to show that the microtubule severing enzyme katanin is inhibited by glycylation as a function of the number of posttranslationally added glycines and that glutamylation and glycylation have antagonistic effects on microtubule severing (Szczesna et al, Dev. Cell 2022). 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 showed that (1) tau binding to the microtubule is regulated by tubulin glutamylation and that glutamylation facilitates kinesin motility; (2) we developed tools for proteomic profiling of glutamylation in living cells. These revealed that glutamylation is associated with almost all major pathways in the cell; (3) we developed and characterized a system for high-throughput axonal imaging of induced pluripotent stem cell-derived human i3Neurons. In combination with facile genetic engineering in i3Neurons this system provides a powerful tool to study human neurons and cytoskeletal perturbations in neuronal disease. We are currently using this system to study the effects of several tubulin posttranslational modifications on axonal transport.