Date of Award
Doctor of Philosophy (PhD)
Loren E. Hough
Tubulin is the heterodimeric building block of microtubules. Regulation of tubulin in the cell occurs primarily through post-translational modifications on its two C-terminal tails. These intrinsically disordered tails are involved in mediating binding interactions between tubulin and microtubule-associated proteins as well as affecting larger scale microtubule dynamics. The work presented here uses the organism Tetrahymena thermophila to purify tubulin with varying degrees of post-translational modifications. The novel growth scheme developed here isotopically labels endogenous tubulin for study by nuclear magnetic resonance (NMR). The purification scheme yields predominantly unmodified and poly-glycylated tubulin as determined by mass spectrometry. We see evidence of two populations of the C-terminal tails, suggesting interaction with the tubulin surface and discrimination between different states of tubulin. Comparison to isolated C-terminal tail peptides attached to GST shows that the C-terminal tails attached to the dimer are sensitive to the tubulin surface as well as the post-translational modifications present. This suggests the importance of studying the C-terminal tails in the context of the dimer rather than as isolated peptides to understand the mechanism by which they aid in tubulin and microtubule regulation.
A number of T. thermophila strains have been developed to alter the modifications present on the C-terminal tails of tubulin. Using these strains, pools of differentially-modified tubulin have been purified and polymerization-competent. This work examines how the differences in modification state of the C-terminal tails affect the structure of the microtubule lattice and the overall flexibility of microtubules. Poly-glycylated microtubules are thinner in size by electron microscopy, but also stiffer overall than microtubules with lower levels of glycylation and increased glutamylation. The stiffness of these microtubules is also sensitive to slight changes in salt conditions. This work provides support for the hypothesis that the post-translational modifications of the C-terminal tails affect the structure and flexibility of microtubules as a possible means of regulating microtubule properties for different functions.
Wall, Kathryn Palma, "Investigating the Role of the Disordered C-Terminal Tails in Tubulin Dynamics" (2021). Biochemistry Graduate Theses & Dissertations. 2.
Available for download on Thursday, May 13, 2021