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SWC_20230508 Final dissertation.pdf (20.67 MB)

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In Vitro Reconstitution of Tubulin Polyglycylation

Cummings, Steven Willis
http://orcid.org/0000-0001-7435-7799
http://rave.ohiolink.edu/etdc/view?acc_num=osu1683551414456465

Abstract Details

2023, Doctor of Philosophy, Ohio State University, Chemical Engineering.
Microtubules are essential cytoskeletal polymers that are critically involved in diverse cellular processes ranging from intracellular transport to cell division and motility. To support these myriad functions, microtubules assemble chemically and morphologically diverse arrays. The axonemal microtubules in cilia and flagella are abundantly modified with chemically diverse posttranslational modifications that are important for cilia and flagella biogenesis, maintenance, motility, and signaling. Glycylation, the addition of variable numbers of glycines to internal glutamate residues in tubulin, is an understudied tubulin post-translational modification found almost exclusively in axonemal microtubules. Glycylation is mediated by enzymes of the Tubulin Tyrosine Ligase-Like (TTLL) family, of which there are three – TTLL3, 8, and 10 – in mammals. In cellulo and in vivo studies suggest that this modification is important for the biogenesis and stability of primary and motile cilia, and that the loss of glycylation may lead to male subfertility and increased cellular proliferation. However, the immense biochemical complexity of the microtubule cytoskeleton and its associated proteins makes it difficult to study the effects of individual tubulin post-translational modifications on specific microtubule effectors. Thus, in vitro reconstitution assays are necessary to elucidate the precise nature of these interactions. In this work, we characterize the TTLL8 and TTLL10 glycylase enzymes. Using tandem mass-spectroscopy we show that, unlike TTLL3 which preferentially monoglycylates β-tubulin tails, TTLL8 adds monoglycines at multiple internal glutamate positions on both α- and β-tubulin tails. TTLL10 elongates these monoglycines, generating long poly-glycine chains on both α- and β-tubulin. Using microscopy-based assays we show that monoglycylation is required for efficient TTLL10 binding to microtubules, and that polyglycylation suggests a self-limiting mechanism for glycine chain elongation. Furthermore, we present the characterization of phosphinic acid-based glycylase inhibitors which may enable future structural studies of glycylase enzyme active sites by X-Ray crystallography. Finally, we use in vitro reconstitution assays to study the effects of tubulin post-translational modifications – notably glycylation and glutamylation – on the motility of a ciliary motor protein, Kinesin-2 / KIF3AB.
Antonina Roll-Mecak (Advisor)
David Wood (Advisor)
Sarah Heissler (Committee Member)
Chalmers Jeffrey (Committee Member)
Eduardo Reategui (Committee Member)
Stephen Osmani (Committee Member)
148 p.
Biochemistry; Molecular Biology
Microtubule Tubulin Molecular Biology Biochemistry Cytoskeleton Enzyme Cell Protein

Recommended Citations

Citations

  • Cummings, S. W. (2023). In Vitro Reconstitution of Tubulin Polyglycylation [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1683551414456465

    APA Style (7th edition)

  • Cummings, Steven. In Vitro Reconstitution of Tubulin Polyglycylation. 2023. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1683551414456465.

    MLA Style (8th edition)

  • Cummings, Steven. "In Vitro Reconstitution of Tubulin Polyglycylation." Doctoral dissertation, Ohio State University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=osu1683551414456465

    Chicago Manual of Style (17th edition)

osu1683551414456465
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