神經元由細胞體、軸突以及樹突組成，為一傳送神經訊號之特化細胞。許多於細胞體內合成之前驅物如神經傳導物質需被kinesin、dynein等分子馬達長距離的運送至突觸以發揮應有功能。然而，神經前驅物長距離運輸的調控機制仍有大部分未被詳細的研究。近期的研究指出一被運輸的蛋白SYD-2/liprin-α可以調控kinesin (KIF1A/UNC-104)的移動活性。於此我們利用新技術，螢光雙分子雜交技術 (Bimolecular Fluorescence Complementation)來檢驗SYD-2和UNC-104之間直接交互作用關係。此一技術的原理為融合兩蛋白至兩段互補之螢光蛋白片段，如果兩蛋白之間有直接的交互作用，則互補之螢光蛋白片段會相互結合並且重新組成有活性的螢光複合體。借由此螢光雙分子雜交技術，我們可於活體測試兩蛋白質之間直接的交互作用關係。我們在此於線蟲(C. elegans)系統中，以全神經系統之啟動子(pUnc-104)以及完整的基因構築質體，之後借由基因選殖送進質體並以顯微注射技術來測試活體線蟲中下列蛋白質之間的交互作用關係: UNC-104/UNC-104；SYD-2/UNC-104。成功表現螢光之線蟲則進一步的分析螢光表現位置以及信號強度。UNC-104為表現於神經系統之分子馬達，屬於kinesin-3的一員。一般假說認為不活化的UNC-104為單體。然而當UNC-104接觸貨物如神經前驅物複合體(Synaptic vesicles)時，UNC-104會相互結合形成雙體並開始運輸貨物。然而UNC-104單體、雙體之間的轉換及調控機制仍有許多爭議之處。因此藉由雙分子螢光互補技術，我們可以針對雙體UNC-104研究其分布以及運動活性借此探討此一轉換機制。同時，我們也正在進行針對UNC-104/UNC-104；SYD-2/UNC-104此新基因轉殖線蟲的多單點突變(EMS mutagenesis)以及基因體等級的RNAi 實驗(RNAi screen)並且期望能發現新的調控蛋白參與UNC-104/UNC-104或SYD-2/UNC-104間的交互作用。

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.

1 Introduction 1
1.1 Regulation of molecular motors in the nervous system 1
1.2 Presynaptic proteins complex as cargos of motors in neuronal cells 2
1.3 BiFC assay 3
1.4 UNC-104/KIF1A 4
1.5 SYD-2/ liprin-α 4
1.6 Novel method to identify protein-protein interaction in a Living organism 5
2 Materials and Methods 8
2.1 Reagents: 8
2.1.1 Bleaching buffer: 8
2.1.2 Egg buffer: 8
2.1.3 Chitinase solution: 8
2.1.4 Neuronal cell culture medium: 9
2.1.5 M9 buffer: 9
2.2 Constructs of BiFC assay 9
2.3 Maintenance of C elegans 9
2.3.1 NGM (Nematode Growth Medium) agar plates: 10
2.3.2 Enriched peptone agar plate: 10
2.4 C. elegans strains 10
2.4.1 N2: 10
2.4.2 CB1265: 11
2.4.3 UNC-104::GFP (e1265) II : 11
2.4.4 bJUN::VN173/bFOS::VC155 N2: 11
2.4.5 VN173::SYD-2/UNC-104::VC155 (e1265) II: 12
2.4.6 UNC-104::VN173/UNC-104::VC155 (e1265) II: 12
2.4.7 VN173::SYD-2/VC155::UNC-104(e1265) II: 12
2.5 Microinjection of BiFC pairs: 13
2.5.1 Microinjection needles: 13
2.5.2 Injection pads: 13
2.5.3 Injection buffer (10X): 14
2.5.4 Procedure of microinjection: 14
2.6 Primary neuronal cultures of C. elegans 14
2.6.1 Synchronization of worm cultures 15
2.6.2 Egg isolation 15
2.6.3 Preparation of dissociated embryonic cells 16
2.6.4 Filtration of dissociated embryo cells 16
2.6.5 Setting up cultures 16
2.7 Transfection of primary neuronal culture in C. elegans 17
2.8 EMS mutagenesis 17
2.9 Microscopy and data quantification 18
3 Result 19
3.1 Set-up of BiFC shuttle constructs 19
3.2 Aggregation of bFOS/bJUN complex in nucleus 20
3.3 In vivo BiFC assay experiments reveal a biased expression pattern of SYD-2/UNC-104 in neurons 21
3.4 In vivo BiFC assay experiments reveal that UNC-104/UNC-104 complexes are homogeneously expressed in neurons 23
3.5 Biased distribution of UNC-104/UNC-104; SYD-2/UNC-104 complexes in terminal ends of axons 24
4 Discussion 25
4.1 Distribution of SYD-2/UNC-104 interaction complexes 25
4.2 Distribution of UNC-104/UNC-104 interaction complexes 26
4.3 Further prospects 27
5 References 29
6 Figures 32
7 Appendixes 49

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