| Summary: | 碩士 === 國立交通大學 === 生物科技學系 === 108 === Microtubules (MTs) are indispensable for neuronal developmental processes, such as migration, polarization, and maturation. In the post-mitotic neurons, non-centrosomal MT-organizing center (ncMTOC) plays an important role in regulating neuronal MT nucleation. Our lab has discovered that TPX2 is an important component of the ncMTOC in neurons, and found that the small GTPase Ran may regulate the activity of TPX2 in neurons. In this work, I attempted to confirm that Ran GTPase can indeed regulate non-centrosomal MT formation in neurons. Since Ran cycles between the active GTP-bound form (RanGTP) and the inactive GDP-bound form (RanGDP), I regulate the level of RanGTP/GDP by expressing the Ran Guanine nucleotide Exchange Factor (RanGEF) or Ran GTPase-Activating Protein (RanGAP) in the neuron. Because the major RanGEF in neurons (RCC1) binds to the chromatin and resides in the nucleus, I modified RCC1 so that it can serve as a cytoplasmic RanGEF. I then confirmed the activity of this modified RCC1 by examining its intracellular localization and its affinity towards the RanGDP-mimic mutant (RanT24N). Finally, we expressed the modified RCC1 or RanGAP1 in dissociated neurons, and found that non-centrosomal MTs in neurons behave the way as we anticipated. Cytoplasmic RCC1 expression increases non-centrosomal MT nucleation while RanGAP1 expression results in the opposite. These results confirm that Ran GTPase regulates non-centrosomal MT nucleation in post-mitotic neurons.
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