探討秀麗隱桿線蟲在dynein和dynactin變異下軸突中UNC-104的聚集與運輸

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研究生：	彭郁斐 Peng, Yu-Fei
論文名稱：	探討秀麗隱桿線蟲在dynein和dynactin變異下軸突中UNC-104的聚集與運輸 Axonal UNC-104 clustering and motility in dynein and dynactin C. elegans mutants
指導教授：	王歐力 Wagner, Oliver
口試委員:	張壯榮黃兆祺
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：	2013
畢業學年度：	102
語文別：	英文
論文頁數：	62
中文關鍵詞：	突觸囊泡運輸、線蟲、dynein 、dynactin
外文關鍵詞：	axonal transport, C. elegans, dynein, dynactin
相關次數：	點閱：3 下載：0
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神經，是一高極性細胞，由細胞本體，樹突，以及又長又細的軸突所構成。在軸突中，由微管和微絲這些細胞骨架構成的「走道」，用於運送突觸囊泡和前驅物等等的「貨物」。微管是帶有極性的纖維，其正端指向神經突觸，負端指向神經細胞本體。在微管上，分子馬達負責運送各式各樣的貨物。有兩大類的分子馬達，kinesin和dynein，負責微管的貨物運輸。Kinesin-3 (哺乳類動物中的KIF1A和線蟲中的 UNC-104)主要負責往微管的正端運送突觸囊泡，而dynein則是將其往回運送至負端。Kinesin和dynein的合作構成軸突中的雙向運輸。UNC-104是一單體分子馬達，必須要成為雙體才能作用，而dynein則是必須在連接蛋白dynactin的幫助下，才能作用。雖然目前已經有很多文獻針對軸突中基於kinesin和dynein雙向運動的囊泡進行研究，但我們對於其引發和調控的分子訊息，還是所知甚少。
本研究利用dynein和dynactin的突變株以及RNA干擾，研究線蟲中UNC-104的運動以及其聚集型態。我們的研究結果顯示，dynactin的突變株和RNA干擾會影響順行和逆行雙向的運動，這指出，dynactin在軸突中，對雙向的運動來說都很重要。Dynein heavy chain的突變株也會影響雙向的運動，其中，對順行運動的影響指出dynein和UNC-104有相互作用關係並調節其運動。dlc-1、dli-1、dyrb-1以及dylt-1的RNA干擾實驗也顯示出對順行逆行雙向運動皆有影響。我們進一步分析數據，歸納出dynein對長距離運動有較大的影響，而與順行相比，其影響逆行運動較深。而dyrb-1和dylt-1，不論在長或短距離下，都對分子馬達的運動持續性，有顯著的影響。總結來說，dynein對軸突中，順行以及逆行的運動都很重要。
本實驗在活體中研究dynein和dynactin的突變株以及RNA干擾，提供軸突雙向運輸中，分子馬達和支架蛋白共同合作並產生功能的線索，並顯示出dynein和dynactin對軸突雙向運輸都是不可缺的。

Neurons are highly polarized cells that consist of a cell body (soma), dendrites and a long and thin axon. In axons, cytoskeletal filaments (microtubules or microfilaments) serve as “tracks” for transport directed and processive transport of cargo (as synaptic vesicles and precursors). Microtubules are polarized filaments with their plus-ends directed to the synapse and their minus-ends directed towards the cell body of the neuron. Molecular motors carry different cargos along microtubules. There are two classes of molecular motors responsible for cargo transport along microtubules, the kinesin super family motors and cytoplasmic dynein. Kinesin-3 (KIF1A in mammals and UNC-104 in C. elegans) is the major transporter of synaptic vesicles with net movemetns towards plus-ends while dynein is able to transport cargo back towards the minus-ends. Kinesin collaborates with dynein for axonal bi-directional transports. UNC-104 is a monomeric motor that needs to dimerize to become processive while dynein needs an additional adaptor dynactin that facilitates processivity transport. Though kinesin and dynein-based bi-directional movement of vesicles has been well described in the literature, we still lack knowledge about molecular signals that trigger and regulate the specific type of axonal transport.
In this study, we use several dynein and dynactin mutant/knockdown strains to study the motility and cluster pattern of UNC-104 in C. elegans. Dynactin mutants and RNAi knockdown affect both anterograde and retrograde transport indicated that dynactin plays a role in both directions of axonal transport. Dynein heavy chain mutant also shows effects in both directions, while the effect on anterograde transport indicates that dynein directly interact with UNC-104 to regulate its motility. Knockdown of dlc-1, dli-1, dyrb-1, and dylt-1 mRNA shows as well defects in both anterograde and retrograde transport. By further analyzing the data, we conclude that dynein affects the bi-directional transport on longer run length, and the effect is more serious for retrograde movements. Dryb-1 and dylt-1 are important for motor persistency no matter in short or long ranges. Most of all, dynein is essential for both retrograde and anterograde axonal bi-directional movement.
In this study, mutating as well as knockdown of specific subunits of the dynein/dynactin complex in vivo provides important information how motors and scaffold proteins may cooperatively work and function in axonal bi-directional transport. Our study indicates that dynein and dynactin both play an important role in axonal bi-directional transport.

中文摘要    I
Abstract    II
Acknowledgement    IV
Table of contents    V
1    Introduction    1
1.1    C. elegans and its neuronal system    1
1.1.1    Introduction of C. elegans    1
1.1.2    Neuronal system of C. elegans    1
1.2    Molecular motors and bi-directional axonal transport    2
1.2.1    Axonal transport in neuron    2
1.2.2    Molecular motors in axonal transport    2
1.3    UNC-104    3
1.4    Dynein    4
1.5    Dynactin    5
1.6    Specific aim in this study    6
2    Materials and Methods    8
2.1    Reagents    8
2.1.1    Nematode Growth Medium agar plate    8
2.1.2    M9 buffer    8
2.1.3    Luria-Bertani medium    8
2.1.4    Luria-Bertani agar plate    8
2.2    C. elegans maintenance    9
2.3    C. elegans strains    9
2.3.1    Control strain    9
2.3.2    Mutation strain    9
2.3.3    RNAi sensitive strain    10
2.4    Crossing of C. elegans    10
2.4.1    Heat shock experiments    10
2.4.2    Crossing    11
2.5    Phenotype analysis    11
2.6    RNAi clone library    11
2.7    RNA interference (RNAi) by feeding    12
2.8    Motility analysis and cluster pattern    12
2.9    Image analysis and statistics    13
2.9.1    Motility analysis and statistics    13
2.9.2    Cluster pattern analysis and statistics    13
3    Results    14
3.1    Dynein and dynactin mutant affect UNC-104 motility in axonal bi-directional transport    14
3.2    Dnc-1 knockdown shows significant affect to UNC-104 motility while dynein heavy chain knockdown does not affect UNC-104 motility    15
3.3    Increasing the efficiency of RNAi    16
3.4    Dynein knockdown in different subunits affect axonal bi-directional transport    16
3.5    Time vs. distance diagrams show more distinct motor movement patterns    18
3.6    UNC-104 cluster analysis    19
3.7    Further analyzed dynein knockdown data reveals a deeper understanding to dynein in bi-directional transport    20
4    Discussion    22
4.1    The role of dynactin in axonal bi-directional transport    22
4.2    The role of dynein in axonal bi-directional transport    23
4.3    The role of other dynein subunits in axonal bi-directional transport    24
5    References    27
6    Tables    33
Table 1. Strains used in this study    33
Table 2. Punc-104::UNC-104::mRFP (e1265;mg366) phenotype analysis    34
Table 3. RNAi clone used in this study    35
Table 4. Cluster density and size in dynactin and dynein mutant worms    35
Table 5. Cluster density and size in dynactin and dynein knock-down worms    36
Table 4. Cluster density and size in dynein different subunit knock-down worms    36
7    Figures    37
Figure 1. The models of UNC-104, dynein, and dynactin used in this study    37
Figure 2. Possible models for bi-directional transport along microtubules    38
Figure 3. Distribution of UNC-104 motility    39
Figure 4. Kymographs from time-lapse images    40
Figure 5. UNC-104 motility is affected in dynein and dynactin mutantion worms    41
Figure 6. UNC-104 motility is affected in dynactin knockdown worms    43
Figure 7. UNC-104 motility is affected in both dynactin and dynein knockdown worms after doubling the IPTG concentration    45
Figure 8. UNC-104 motility is affected in different dynein subunits knockdown worms    48
Figure 9. Time vs. distance diagrams of motor movements in C. elegans neurons    51
Figure 10. Time vs. distance diagrams of motor movements in C. elegans neurons    52
Figure 11. Cluster pattern analysis in dynein and dynactin mutant worms    54
Figure 12. Cluster pattern analysis in dynein and dynactin knockdown worms    55
Figure 13. Plotting different motility parameters against each other for dynein knockdown data    57
8    Appendix    59
Appendix 1. Mechanosensory neurons in C. elegans    59
Appendix 2. Crossing scheme of Punc-104::UNC-104::mRFP (e1265) strain and GR1373    60
Appendix 3. Procedure of generating kymographs from time-lapse images and kymograph analysis.    61 
                                

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