Function of kinetochore proteins in post-mitotic neurons

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Project Summary Microtubules are both critical structural elements and the tracks for long-range transport. They are much more stable in neurons than other cells, and, surprisingly, the regulatory machinery associated with kinetochores in mitosis is required for this stability. KMN network proteins link the kinetochore to dynamic microtubules in mitosis and were recently shown to impact cell shape in post-mitotic mouse, worm and fly neurons. Preliminary data in this proposal indicates they function to suppress microtubule dynamics in neurons. A similar role in regulation of neuronal microtubule dynamics was found for the regulatory Chromosome Passenger Complex (CPC) and Spindle Assembly Checkpoint (SAC) proteins. How and where these mitotic proteins act in mature neurons to control microtubule stability will be investigated. Aim 1 What pathway is used by kinetochore proteins to control microtubule plus end number in neurons? When levels of kinetochore proteins are reduced in post-mitotic Drosophila neurons, more microtubule plus ends are observed in dendrites, but not axons. Upregulated microtubule severing or nucleation could account for the increase in plus end number. Minus ends generated by severing are recognized by Patronin, so the number of growing Patronin-tagged minus ends will be used to distinguish between these two possibilities. Analysis of genetic interactions will also be used to determine whether kinetochore proteins suppress nucleation or severing. In neurons increased nucleation is linked to neuroprotection while severing precedes degeneration, so is it important to understand which is regulated by kinetochore proteins. Aim 2. Where and how are KMN proteins localized in neurons? Pilot studies have demonstrated that three different KMN network proteins localize to puncta in the neuronal cell body. The identity of these punctate tether sites will be determined. Most likely candidates are the Golgi complex and lysosomes as these organelles are localized predominantly in the cell body. Aim 3. Do kinetochore proteins sense microtubule plus end arrival in the cell body? The final goal in this proposal is to test whether kinetochore proteins function analogously in neurons and mitotic cells. In early mitosis the KMN network cooperates with CPC and SAC proteins to sense microtubule arrival at the kinetochore. After microtubule arrival the CPC and SAC change localization. In neurons these proteins could work together to detect number of microtubules growing into the cell body from dendrites. To test this hypothesis the number of microtubules entering from dendrites will be increased and decreased while monitoring kinetochore protein localization in the cell body. Kinetochore proteins represent major new regulators of neuronal microtubule behavior. This initial exploration of how and where they function in neurons will provide a strong foundation for understanding their contribution to life after mitosis.