多種神經性中間絲蛋白 (neuronal intermediate filament) 之缺陷已知與神經系統疾病有關，例如帕金森氏症 (PD) 與肌萎縮性脊髓側索硬化症 (ALS)。科學家時常應用模式生物，例如斑馬魚、果蠅、與線蟲，來探討人類疾病之病理。然而，在秀麗隱桿線蟲 (Caenorhabditis elegans) 中是否存在神經絲蛋白 (neurofilament) 的同源基因 (homolog) 則尚未明朗。本研究參酌生物資訊工具 WormBase BLASTP 對於人類基因 neurofilament heavy polypeptide (nefh) 之同源性演算結果，旨在探討相應之線蟲同源基因 temporarily assigned gene-63 (tag-63)。我們建立了轉錄融合 (transcriptional reporter) 的 tag-63 轉殖蟲株，發現 tag-63 的基因表現模式與泛神經元蛋白標記 UNC-104 有部分重合。此外，我們定序及遠交 (outcrossing) 缺失突變蟲株 VC275 tag-63(ok471)，將其分別配種 (crossing) 於轉譯融合 (translational reporter) 的 UNC-104/KIF1A (kinesin-3 motors) 與 SNB-1/VAMP1 (synaptobrevin-1 synaptic vesicles) 轉殖蟲株。先藉由基因分型 PCR 與西方墨點法來確認配種成功後，再藉由活體線蟲感覺神經元 ALM 之縮時攝影，來量測 tag-63(ok471) 對於 motor UNC-104 與相應的cargo SNB-1 快速軸突運輸 (fast axonal transport) 之影響。分析發現，tag-63(ok471) 顯著地藉由差異性調控 UNC-104 與 SNB-1 的運輸參數，導致活化逆行 (retrograde) 運輸機制且抑制順行 (anterograde) 運輸機制。進一步分析發現，TAG-63 突變造成 UNC-104 與 SNB-1 在順行運輸而非逆行運輸中形成了相似的運輸動態 (motility scheme)。基於以上實驗結果，我們提出嶄新的假說：神經絲蛋白同源基因 TAG-63 之所以影響秀麗隱桿線蟲快速軸突運輸，可能是藉由 TAG-63 與運輸複合體 (motor-cargo complex) 的暫時交互作用所致。

Various neuronal intermediate filament defects are associated with neurological disorders, such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). To study how human diseases develop, scientists frequently employ model organisms, such as zebrafish, fruit flies, and worms. However, whether a neurofilament homolog exists in the nematode Caenorhabditis elegans remains unclear. In this study, we investigated a candidate gene, temporarily assigned gene-63 (tag-63), which we hypothesized to be a homolog to human neurofilament heavy polypeptide (nefh) according to WormBase BLASTP. We established a stable transgenic worm line expressing the tag-63 transcriptional reporter and found that tag-63 expresses in some neurons showing partial co-occurrence with the pan-neuronal reporter UNC-104. Additionally, we sequenced and outcrossed the deletion mutant strain VC275 tag-63(ok471) and separately crossed it with the translational reporters UNC-104/KIF1A (kinesin-3 motors) and SNB-1/VAMP1 (synaptobrevin-1 synaptic vesicles). We verified the crossing through genotyping PCR and Western blotting and examined anterograde motor UNC-104 and corresponding cargo SNB-1 fast axonal transport dynamics in the mechanosensory neuron ALM by using time-lapse imaging in live worms. Intriguingly, we found that worms carrying the TAG-63 mutation significantly activate the retrograde machinery and deactivate the anterograde machinery of UNC-104 and SNB-1 by acting on differential transport parameters. Moreover, the TAG-63 mutation causes similar motility schemes between UNC-104 and SNB-1 in anterograde but not retrograde transport directions. Based on these results, we propose a novel model that neurofilament homolog TAG-63 affecting fast axonal transport in C. elegans via transient interactions between TAG-63 and the motor-cargo complex.

1. Introduction 1
1.1. Human neurological disorders 1
1.2. Intermediate filament neurofilaments 2
1.3. Fast axonal transport machinery 3
1.4. Related research studies in Caenorhabditis elegans 5
1.5. Research questions in this study 7

2. Materials & Methods 9
2.1. Caenorhabditis elegans strains & Maintenance 9
2.2. Cloning, Microinjection, & Microscopy 9
2.3. Outcrossing, Crossing, & Genotyping 10
2.4. Protein extraction & Western blot 11
2.5. Worm fixation & Immunofluorescence 12
2.6. Motility imaging & Statistical analysis 14
2.7. Bioinformatics analysis 15

3. Results 17
3.1. TAG-63 homology and identity 17
3.2. Expression patterns of Ptag-63::GFP worms 18
3.3. Sequencing, outcrossing, and crossing of VC275 tag-63(ok471) worms 19
3.4. Western blot of wild-type and tag-63(ok471) worms by anti-NEFH an-tibodies 22
3.5. Transport motilities of UNC-104::mRFP in wild-type and tag-63(ok471) worms 24
3.6. Transport motilities of SNB-1::mRFP in wild-type and tag-63(ok471) worms 26
3.7. Transport dynamics of UNC-104 and SNB-1 in wild-type and tag-63(ok471) worms 27

4. Discussion 29
4.1. Overview of transport dynamics 29
4.2. Modeling of transport dynamics 31
4.3. Analysis of transport dynamics 32
4.4. Analysis of bioinformatics about 34
4.5. Outlook of the study 35

5. Acknowledgements 37

6. References 38

7. Figures 48
Figure 1. Protein homology, gene structure, and gene expression profile of tag-63 in C. elegans. 51
Figure 2. Expression pattern analysis of Ptag-63::GFP and Punc-104::UNC-104::mRFP stable transgenic worms. 54
Figure 3. Ptag-63::GFP partially co-occurs with Punc-104::UNC-104::mRFP in some neurons of stable transgenic worms. 57
Figure 4. Sequencing, outcrossing, crossing, and genotyping of the worm strain VC275 tag-63(ok471) with respective reporter strains Punc-104::UNC-104::mRFP and Punc-86::SNB-1::mRFP. 61
Figure 5. Western blot of wild-type worm and mutant worm tag-63(ok471) by monoclonal anti-NEFH mouse antibody (Sigma). 63
Figure 6. Motility analysis of the reporter Punc-104::UNC-104::mRFP in wild-type and tag-63(ok471) worms. 66
Figure 7. Motility analysis of the reporter Punc-86::SNB-1::mRFP in wild-type and tag-63(ok471) worms. 69
Figure 8. Directionality dynamics analysis and motility data summary of the reporters Punc-104::UNC-104::mRFP and Punc-86::SNB-1::mRFP in wild-type and tag-63(ok471) worms. 72
Figure 9. Summary and modeling of TAG-63 filament knockout effects on fast axonal transport of UNC-104 and SNB-1. 74

8. Appendixes 76
Appendix 1. Introduction to human diseases, neurofilaments, fast axonal transport, and related Caenorhabditis elegans topics. 82
Appendix 2. Experimental materials, scientific software, bioinformatics tools, and motility analysis procedures used in this study. 88
Appendix 3. Ptag-63::GFP expression pattern of continuous z-axis scanning. 93
Appendix 4. Immunofluorescence and Western blot of wild-type worms and mutant worms tag-63(ok471) by polyclonal anti-NEFH rabbit antibody (Abcam). 96
Appendix 5. Bioinformatics analysis of TAG-63 identity and relations in Caenorhabditis elegans. 99

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