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

彭郁斐(Yu-Fei Peng)

指導教授 ： 王歐力

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2013年)

摘要

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。

關鍵字

突觸囊泡運輸 ； 線蟲 ； dynein ； dynactin

並列摘要

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.

並列關鍵字

axonal transport ； C. elegans ； dynein ； dynactin

參考文獻

(1998). Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012-2018.

Antinone, S.E., and Smith, G.A. (2006). Two modes of herpesvirus trafficking in neurons: membrane acquisition directs motion. Journal of virology 80, 11235-11240.

Bargmann, C.I. (2001). High-throughput reverse genetics: RNAi screens in Caenorhabditis elegans. Genome biology 2, REVIEWS1005.

Berezuk, M.A., and Schroer, T.A. (2007). Dynactin enhances the processivity of kinesin-2. Traffic 8, 124-129.

Blangy, A., Arnaud, L., and Nigg, E.A. (1997). Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150. The Journal of biological chemistry 272, 19418-19424.