藉由螢光雙分子雜交技術研究線蟲神經系統中蛋白質複合體之交互作用

• Main Authors: Hsu, Chih-Chun, 許智鈞
• Other Authors: Oliver I. Wagner
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/84697988696512442890

碩士 === 國立清華大學 === 分子與細胞生物研究所 === 98 === Neurons are specialized cells involved in impulse-conducting mechanisms with long extensions named axon and dendrites. Cargos as neuronal precursors as well as neurotransmitters synthesized in soma need to be transported long distances to the synapse. Molecular motors as kinesins and dynein accomplish this important task while the regulation of these cellular machines remains largely unknown. A recent study has shown that the cargo itself (liprin-alpha/SYD-2) can regulate kinesin (KIF1A/UNC-104) motility. However, a direct interaction between SYD-2 and UNC-104 in the living animal still needs to be proven. To approach this interesting question, we use a novel method BiFC (Bimolecular Fluorescence Complementation). This method allows us to detect protein-protein interaction in living cells by fusing proteins with fluorescent protein complementary fragments which can form functional fluorescence complexes, thus enabling us to investigate the physical interaction between two proteins in the living animal. We use a native, pan-neuronal promoter (pUnc104) to drive gene expressions in the nervous system of C. elegans and have investigated the following interaction partners: UNC-104/UNC-104 and SYD-2/UNC-104. Transgenic lines of worms expressing full lengths constructs were successfully generated by microinjection. The importance for investigating UNC-104/UNC-104 interaction lies in the current model of kinesin-3 activation-hypothesis: UNC-104 exists as a monomer in its inactive state, while during activation, UNC-104 undergoes a cargo-induced dimerization process. With the BiFC method we can now visualize and analyze the distribution and motility of constitutive UNC-104/UNC-104 dimers only. Research in process also includes EMS mutagenesis and genome-wide RNAi screen on our newly generated BiFC worms to identify novel regulators or suppressors involved in protein complex formation.