Research Projects

Microtubule cytoskeleton are made of alpha/beta-tubulin heterodimers, which undergo variety of post-translational modifications (PTMs). The biochemical changes arising from tubulin PTMs regulate microtubule functions in a specific manner, widely known as tubulin code hypothesis. Our lab and several studies from the field have shown the biochemical evidence for tubulin code regulating molecular motors, MAP binding, cargo transport and microtubule architecture. However, a key limitation in studying ‘tubulin code’ is the lack of tools that can specifically mark tubulin PTMs in living cells. We previously developed the first microtubule PTM sensor, which can label specifically the tyrosinated microtubules in cells. Building on this success, in this proposal I aim to develop microtubule glutamylation specific sensors using yeast display libraries. Microtubule glutamylation modification occurs at the carboxy-terminal tails (CTTs) of both alpha- and beta-tubulin subunits and are highly enriched in brain, cilia and flagella. Excessive glutamylation has been linked to neurodegeneration and cancer, however their mechanism is still unclear. A family of glutamylases, Tubulin Tyrosine Ligase Like (TTLLs) catalyze polyglutamylation modifications. Characterization of glutamylases show that each TTLLs enzyme exhibits specificity towards alpha- or beta-tubulin CTT and can add unique glutamylation modification i.e., mono- or poly- or multiple glutamylation within the same tubulin CTT. The antibodies that detect microtubule glutamylation modification are inept in discriminating different glutamylation modification that can occur in a cell. To overcome this, in this proposal we will design different glutamylation epitopes and identify specific binders against each epitope. These glutamylation binders will be extensively validated using biochemical and cell biology methods for their application in studying microtubule glutamylation function in cells. The validated glutamylation binders will be used to characterize microtubule glutamylation patterns across different cell types using expansion coupled super-resolution microscopy. Further the glutamylation binders will also be applied to study the glutamylases or TTLL enzyme activity in real time. Together, the proposed study will provide tools to probe the function of microtubule glutamylation in cells and provide insights into the function of this enigmatic microtubule modification.