A major deficit in our understanding of microtubule function is a lack of knowledge of how tubulin post translational modifications such as glutamylation impact microtubule dynamics. Altered glutamylation is associated with human ciliopathies and neurodegeneration therefore there is a critical need to identify the specific contributions glutamylation makes to modulating microtubule properties. Studies that have focused on individual glutamylating or deglutamylating enzymes have revealed functions for glutamylation in the maintenance of cilia stability and intracellular transport, but the effect on the microtubule dynamics remains obscure. The overall objective of the proposed study, is to determine the contribution tubulin glutamylation makes to regulating microtubule properties and the stability of the microtubule-based centriole. Preliminary findings show that glutamylation aids tolerance of cold and colchicine, stressors that destabilize the microtubules. The central hypothesis guiding the proposed work is that tubulin glutamylation modulates microtubule dynamics to tailor microtubule stability to cellular needs, including tolerance of stressors. The rationale underlying this research is that by determining how microtubule properties are modulated by glutamylation, the contribution glutamylation makes, within the context of the tubulin code, to maintaining normal cytoskeletal function will be identified. To test the central hypothesis and, attain the overall objective, three specific aims will be addressed: 1) Determine the contribution glutamylation makes to centrosome stability. 2) Identify the mechanism by which glutamylation increases tolerance of microtubule stressors. 3) Determine how hyperglutamylation causes fertility defects. C. elegans mutants with reduced or hyper- glutamylated microtubules have been generated and light microscopic analyses of these worms will be used to dissect the function of glutamylation.
Under aim one the focus will be on examining centrosome integrity and function by fixed and live microscopy.
Under aims two and three analyses will investigate microtubule dynamics using EBP-2::GFP. The proposed research is innovative because genetically ablating glutamylation will allow the full suite of its functions to be elucidated in an intact multicellular organism. Completion of these aims will illuminate the role of glutamylation in modulating microtubule properties and clarify how it impacts centrosome function. This will be significant because it will allow the contribution glutamylation makes to the tubulin code with respect to regulating microtubule dynamics to be identified. Ultimately this knowledge has the potential to enhance our understanding of how normal microtubule function is impaired resulting in human disease.
RESEARCH NARRATIVE The proposed research is relevant to public health because changes to microtubule function are associated with acquired resistance to some cancer chemotherateutics, and also with human diseases such as ciliopathies and neural degeneration. Glutamylation is found on discrete microtubule populations in many tissues, yet we do not fully understand how it modulates microtubule dynamics to impact their function. The proposed project is relevant to NIGMS?s mission because enhancing our understanding of microtubule regulation will lay the foundation for advances in disease diagnosis and improved treatment strategies.
