Visualization of interactions between molecular motors and their adaptors in the nervous system of C. elegans using the BiFC assay

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研究生：	孟卡廉諾 Juan David Moncaleano
論文名稱：	Visualization of interactions between molecular motors and their adaptors in the nervous system of C. elegans using the BiFC assay 藉由螢光雙分子雜交技術研究線蟲神經系統中分子馬達和其連接蛋白的交互作用
指導教授：	王歐力 Oliver Wagner
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2010
畢業學年度：	99
語文別：	英文
論文頁數：	74
中文關鍵詞：	線蟲、分子馬達、神經、蛋白交互作用、螢光雙分子雜交技術、神經系統
外文關鍵詞：	C. elegans, Molecular motor, Neuron, Protein interaction, Protein complementation, Nervous system
相關次數：	點閱：2 下載：0
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Neurons are highly polarized cells whose function depends on the targeted delivery of proteins, vesicle cargos, membranous organelles and mRNAs to specific locations in axons and dendrites. This process is accomplished by molecular motors that move cargos along microtubule tracks. Defects in axonal transport have an important role in the pathogenesis of various neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington disease, while in Alzheimer’s disease and Parkinson’s disease, the accumulation of cargo is a hallmark. Although the mechanisms that regulate molecular motors are not well understood, the role of adaptors that coordinate cargo-bound motors and bidirectional transport that occurs when opposite polarity motors attached to the same cargo, have been described as important levels of regulation. Using an interactome map that include predicted interactions, we identified UNC-16(JIP3) and DNC-1(p150GLUED) as binding partners of the major synaptic vesicle transporter UNC-104(KIF1A) in C. elegans. UNC-16 has been known to regulate the vesicle transport in C. elegans, acting as a scaffold protein, binding the light chain of kinesin-1 (KLC-2) and JNK signaling components. However a direct interaction between UNC-104 and UNC-16 has not been reported. In addition we were also interested in studying the interaction between UNC-104 and DNC-1 (major subunit of dynactin), since dynactin is an important adaptor of the minus-end directed molecular motor dynein. We applied the novel Bimolecular Fluorescence Complementation (BiFC) assay to visualize interactions between molecular motors and adaptors in living C. elegans. In this assay, interacting partners are fused to complementary fragments of a fluorescent protein, which is reconstituted if the interaction between the partners occurs. With this method we were able to visualize the following interactions in living worm and at sub-cellular level: UNC-104/UNC-16, UNC-104/DNC-1, UNC-16/KLC-2, and UNC-16/DNC-1. Interestingly, these interaction complexes showed different sub-cellular distributions in the neuron, which provide evidence of the important role of adaptor proteins in molecular motors regulation. To identify novel regulators of these interaction complexes, we already used gamma irradiation to integrate the extrachromosomal arrays in the BiFC worms generated by microinjection and intend to combine the BiFC assay with EMS mutagenesis and RNAi screening.

神經細胞是高度極化的細胞，這種極化的特性來自於神經樹突、軸突位置中各類蛋白、囊泡載體、帶膜類胞器、以及mRNA等特定分子的運輸。而運輸過程是藉著分子馬達將載體沿著微小管形成的軌道運送。已知神經軸突運輸的缺陷在各類神經退化疾病中扮演重要的角色，諸如肌萎縮性側索硬化症 (ALS)、亨丁頓氏舞蹈症、阿滋海默症、和帕金森氏症等疾病中載體的不正常堆積都是顯著的現象。雖然目前對於分子馬達的調節機制仍然不甚清楚，但連接蛋白(adaptors)能協調載體-分子馬達複合體以及當走向相反的分子馬達接在同一個載體時所造成的雙向運動，且過去文獻中也肯定連接蛋白本身具有相當的調節功能。此外，利用生物資訊程式可以用來預測蛋白質的交互作用網路。我們確認UNC-16(JIP3) 和 DNC-1(p150GLUED) 是線蟲主要的突觸囊泡運輸分子馬達UNC-104(KIF1A)的結合蛋白。UNC-16 蛋白已知在線蟲中調節囊泡運輸時扮演鷹架蛋白(scaffold protein)的角色，它能與kinesin-1 (KLC-2) 的輕鏈及JNK訊號蛋白結合。然而，探討UNC-104和 UNC-16之間直接的交互作用的研究成果目前還沒有被發表過。此外，我們也對研究UNC-104 和 DNC-1 (dynactin的次單位)間的交互作用深感興趣。Dynactin 是負極走向分子馬達dynein的連接蛋白。我們利用螢光雙分子雜交技術 (Bimolecular Fluorescence Complementation )去觀察在活體線蟲中的分子馬達和間接蛋白的交互作用。這種分析是以不同蛋白個別接上一個結構上互補的螢光分子序列，當蛋白有交互作用時，互補的螢光分子結構會相結合形成完整的螢光分子。利用這種方法，我們可以直接觀察到在活體線蟲或線蟲細胞中的UNC-104/UNC-16, UNC-104/DNC-1, UNC-16/KLC-2, and UNC-16/DNC-1的交互作用。有趣的是，在細胞層次上，我們發現這些交互作用的分子在神經細胞中有不同的分布型態，這個發現提供了證據說明間接蛋白在分子馬達調節功能上扮演重要的角色。為了確認由此技術觀察到的調節分子，我們利用γ-ray 去照射表現BiFC螢光的線蟲，使當初利用顯微注射後以染色體外陣列(extrachromosomal array) 形式存在蟲體中的外來質體能夠插入染色體，同時將使用EMS突變劑及RNAi篩選的方式來搭配BiFC 的分析。

Table of Contents
Abstract    I
摘要    III
1    Introduction
1.1    Molecular motors and directional transport in neurons
1.2    Regulation of bidirectional transport by tug-of-war and coordinated activity
1.3    Studying protein-protein interactions using the BiFC assay
1.4    A novel approach for the identification of components of neuronal precursor complexes
1.5    Identification of probable novel UNC-104 interaction partners using an interactome map
2    Materials and methods
2.1    Reagents
2.1.1    Bleaching solution
2.1.2    Egg buffer
2.1.3    Chitinase solution
2.1.4    Neuronal cell culture medium
2.1.5    PBS 10X
2.1.6    DAPI staining solution
2.1.7    M9 buffer
2.1.8    LB medium
2.1.9    E. coli OP50 liquid culture
2.1.10    Nematode Growth Medium (NGM)
2.1.11    Microinjection buffer (10X)
2.1.12    5X SDS loading buffer
2.1.13    Transfer buffer
2.1.14    Tris-buffered saline with Tween-20 (TBST)
2.1.15    Antibodies for Western blotting
2.1.16    Lysation buffer
2.2    Constructs used for BiFC experiments
2.3    C. elegans strains
2.3.1    Positive control
2.3.2    Negative controls
2.3.3    UNC-16 interaction with KLC-2
2.3.4    UNC-16 interaction with UNC-104
2.3.5    UNC-16 interaction with DNC-1
2.3.6    UNC-104 interaction with DNC-1
2.4    Microinjection
2.5    Primary neuronal cell culture of C. elegans and DAPI staining
2.6    Detection of BiFC fusion proteins in negative controls by Western blot analysis and PCR on genomic DNA
2.7    Whole worm microscopy, quantification of fluorescent expression in isolated cells and motility analysis
2.8    Gamma rays integration of extrachromosomal arrays
3    Results
3.1    Setting-up of BiFC constructs
3.2    Transgenic lines and positive and negative controls
3.3    In vivo BiFC assay experiments reveal a biased expression pattern of UNC-16/KLC-2 in neurons
3.4    In vivo BiFC assay experiments reveal that UNC-16 interacts with UNC-104 in a biased distribution
3.5    In vivo BiFC assay experiments reveal that UNC-16 interacts with a regulator of Dynein (DNC-1) in neurons
3.6    In vivo BiFC assay experiments reveal a dynamic behavior of UNC-104/DNC-1 in neurons
3.7    Integration of extrachromosomal arrays
4    Discussion
4.1    Distribution of UNC-16/KLC-2 and UNC-16/UNC-104 interaction complexes
4.2    Distribution of UNC-16/DNC-1 interaction complexes
4.3    Distribution of UNC-104/DNC-1 interaction complexes
4.4    Further prospects
5    References
6    Figures
Figure 1. Motors and adaptors used in this study
Figure 2. Models of bidirectional transport
Figure 3. Venus fluorescent protein and the BiFC assay
Figure 4. BiFC experimental set up
Figure 5. C. elegans interactome sub-network
Figure 6. Interactions visualized using the BiFC assay
Figure 7. Gateway cloning of BiFC pairs
Figure 8. Constructs used for the BiFC assay
Figure 9. Negative controls used in this study.
Figure 10. Visualization of the UNC-16/KLC-2 interaction in living C. elegans.
Figure 11. Visualization of the UNC-16/KLC-2 interaction in a primary neuronal cell culture of the BiFC worm
Figure 12. Visualization of the UNC-16/UNC-104 interaction in living C. elegans
Figure 13. Visualization of the UNC-16/UNC-104 interaction in a primary neuronal cell culture of the BiFC worm
Figure 14. Visualization of the UNC-16/DNC-1 interaction in living C. elegans.
Figure 15. Visualization of the UNC-16/DNC-1 interaction in a primary neuronal cell culture of the BiFC worm
Figure 16. Visualization of the UNC-104/DNC-1 interaction in living C. elegans.
Figure 17. Visualization of the UNC-104/DNC-1 interaction in a primary neuronal cell culture of the BiFC worm
Figure 18. Motility properties of the UNC-104/DNC1 BiFC complex in primary neuronal cell culture.
Figure 19. Summary of fluorescence intensity of primary neuronal cell culture of BiFC worms as a function of distance from the cell body
Figure 20. Summary of expression of BiFC pairs in isolated neurons
7    Appendix
Appendix 1. Primer sequences used in this study
Appendix 2. BiFC constructs used in this study
Appendix 3. Strains used in this study
Appendix 4. Rescue experiment of CB1265 mutant strain.
Appendix 5. Kymograph analysis.
Appendix 6. UNC-104::GFP expression in C. elegans.
Appendix 7. Comparison of the UNC-104/DNC-1 complex anterograde motility with the reported UNC-104 anterograde motility in cell culture

                                

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