過去20年， 線蟲成為模式生物並且頻繁地被用於神經中運輸蛋白 (Kinesins) 和動力蛋白(dynein) 的研究。為了研究線蟲的神經軸突中，以肌動蛋白為基礎 (actin-based) 運動的分子馬達(motors)中，是否存在與肌凝蛋白-V (myosin-V) 的同源蛋白，我們使用以hum-2啟動子驅動螢光蛋白分布的質體 (transcriptional plasmid of hum-2) 觀察能表現跟肌凝蛋白-V (myosin-V) 同源的hum-2蛋白的細胞。因此，我們以線蟲行為配合生物資訊分析HUM-2蛋白來解析肌凝蛋白在神經軸突中運輸的角色。我們使用化學趨向性 (chemotaxis)、咽頭吸吐速度(pumping rate)與存活度分析線蟲行為變化， 以及用分子運動分析來研究神經軸突的運輸變化。實驗結果顯示hum-2基因會在神經中表現， 並且會影響化學趨向性與咽頭吸吐速度，也參與突觸小泡 (synaptic vesicle) 的運輸。

C. elegans have been used as a model organism for studying kinesins and dynein in neurons. In an effort to evaluate whether or not a C. elegans homolog of myosin-V exists, and therefore to gain a better understanding of axonal actin-based motors, a transcriptional fusion of hum-2 (supposedly an orthologue of mammalian myosin-V) has been created to detect its cellular expression. Moreover, initial worm behavioral and bioinformatics analysis have been performed to further characterize the role of this myosin (hum-2) in C. elegans. Online bioinformatics resources revealed similar predicted protein domains in the hum-2 gene when compared with mammalian myosin-V. Further, I performed behavioral analyses like chemotaxis, pumping rate and survival assays in a hum-2 mutant worm that displayed a defect in its chemosensory signaling as well as pumping rate. For the study of axonal transport, I carried out motility analyses and observed UNC-104 in hum-2 RNAi as well as mutant background. Results implicate significant changes in velocity, persistency of movement, total run length, pausing and motor reversals. Deeper kymograph analysis revealed a decrease in UNC-104 activation after a pause. Also, interestingly interaction between motile and non-motile UNC-104 clusters partially rescued UNC-104 activation. Finally, results revealed hum-2 expression in neurons, and its pivotal role in chemosensory signaling, pharynx pumping rate and also synaptic vesicle transport and UNC-104 activation.

Introduction - 7 -
1 Myosin-V - 7 -
1.1 Myosins in C. elegans - 7 -
1.2 Myosin-V Introduction - 7 -
1.3 Myosin-V Structure - 8 -
1.4 Myosin-V functions in neurons - 9 -
2 KIF1A/UNC-104 motor - 10 -
3 Myosin-V and cargo co-transport on microtubules - 11 -
3.1 Overview - 11 -
3.2 Kinesin, dynein and myosin-V cargo adaptors - 12 -
3.3 Bidirectional cargo transport - 13 -
3.3.1 Tug-of-war - 13 -
3.3.2 Explaining the paradox of co-dependence - 14 -
4 Purpose of study - 15 -
Materials and Methods - 17 -
1 Reagents - 17 -
1.1 M9 buffer - 17 -
1.1 Nematode growth medium (NGM) agar plates - 17 -
1.3 LB Medium - 17 -
1.2 Genomic DNA isolation buffer - 18 -
2 Maintenance - 18 -
3 C. elegans strains and plasmids - 18 -
4 C. elegans Mating - 19 -
4.1 Male generation by heat shock - 19 -
4.2 Outcrossing - 20 -
5 Microinjection - 20 -
6 Behavioral analysis - 21 -
6.1 Chemotaxis assay - 21 -
6.2 Survival assay - 22 -
6.3 Pumping rate assay - 22 -
7 Mitochondria and UNC-104 distribution analysis - 22 -
8 Motility analysis - 23 -
Results - 25 -
1 HUM-2/myosin-V homology analysis - 25 -
2 Hum-2 expression pattern - 25 -
3 HUM-2 Behavioral Analysis - 27 -
4 Hum-2 knockdown affects the mitochondrial length in ALM neurons - 27 -
5 Hum-2 knockdown decreases RAB-3 anterograde velocity in ALM - 28 -
6 HUM-2 increases RAB-3 labeled vesicles activation from its paused state - 29 -
7 HUM-2 affects UNC-104 motility parameters in ALM - 29 -
8 HUM-2 increases UNC-104 activation from its paused state - 30 -
9 UNC-104 non-motile clusters rescue anterograde activation in hum-2(ok596) - 31 -
10 HUM-2 affects UNC-104 distribution - 32 -
11 HUM-2 affects small aggregates of UNC-104 more than big aggregates of UNC-104 - 32 -
Discussion - 35 -
Hum-2 expression pattern - 35 -
HUM-2 behavioral and organelle analysis - 36 -
HUM-2 and its effect on RAB-3 labeled Synaptic Vesicles transport - 37 -
HUM-2 and its effect on UNC-104 and axonal transport - 39 -
HUM-2 affects small cargo more than large cargo transport - 42 -
HUM-2’s role in the paradox of co-dependence - 43 -
Figures - 45 -
References - 71 -
Appendix - 81 -

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