Principal Investigator/Program Director(Last, First, Middle): Hough, Loren E. Microtubules (MTs) made from ↵- tubulin heterodimers are important for cell migration, long range transport, and cell division. A primary site of tubulin regulation is the C-terminal tails (CTTs). Major questions remain about the molecular mechanism of CTT function and regulation. CTTs affect microtubule length dynamics and mechanical properties even though they contribute only a small percentage of the binding interface between adjacent dimers. CTTs are a major site of tubulin post-translational modification (PTM) which regulates tubulin's binding interactions. PTM of the CTTs alters the processivity of motor proteins1, 2 and the affinity of proteins which affect MT stability (e.g. MCAK, CLIP-170)3, 4 and proteins which stabilize MTs (e.g. tau).5 Despite their importance, there are few molecular probes of CTT behavior. Because CTTs are flexible, they are typically undetectable in electron microscopy or x-ray crystallography studies. NMR is the best approach for determining the molecular mechanism of CTT regulation of MT polymerization, mechanics, binding and regulation by PTMs. However, standard techniques for incorporation of heavy isotopes have not worked for tubulin—the proteins have not yet been made in prokaryotic systems, and tubulin typically down regulates its own expression, making over-expression difficult in eukaryotic systems. We developed a method to produce heavy isotope labeled tubulin for study by NMR, allowing for a detailed study of the molecular mechanism of CTT function. Building on this breakthrough, we will study the role of CTT polyglycylation in MT regulation. The addition of glycine residues to glutamate side chains occurs on both tubulin CTTs. First identified as one of the two major poly-modifications on tubulin, polyglycylation is associated with particularly stable MTs, especially axonemes in mobile cilia.6 Mutations that decrease polyglycylation are associated with reduced stability of cilia, reduced numbers of cilia, increases in cell proliferation, and cancer. However, it is not known why polyglycylation causes these effects. PTMs can operate through a variety of mechanisms: modification of the overall charge distribution,7 alteration of binding interfaces,8 induction of structural changes through allosteric mechanisms,9 and modulation of disordered protein ensembles.10 We will combine NMR, binding assays, molecular simulation, and cell biology to test potential mechanisms of CTT function. We will study two focused questions: 1. How does polyglycylation affect MT polymerization dynamics and stiffness? The presence of the CTT affects MT polymerization dynamics and mechanical properties, suggesting that the CTTs of one dimer interact with other dimers in the MT lattice.11,12 We will purify heavy isotope-labeled tubulin with varying degrees of polyglycylation using mutations in TTLL3-family proteins and mutations in the tubulin CTT at polyglycy...