在神經裡，分子馬達蛋白經由體細胞運送突觸小泡到突觸的過程中，微管(MT)扮演一個重要的角色。我們對於突觸傳遞與神經貨物的搬運已有相當的了解，改變突觸傳遞會直接影響神經軸突的運輸。然而，我們對這些變化會如何更改MT上的轉譯後修飾(PTM)並不清楚。在這項研究中，我們調查了不同突變的秀麗桿線蟲，(帶有各類的突觸傳遞缺陷)，是如何影響軸突的微管蛋白密碼。我們使用了七種突變蟲，分別為dat-1(ok157), eat-4(ky5), unc17(e245), unc25(e156), tbh-1(n3247), tdc-1(ok914), tph-1(mg280)。利用廣泛的技術，例如分析軸突馬達蛋白與貨物的運動、以及西方墨點法(了解PTM表現量)、蟲體免疫染色(了解PTM在MT上的修飾程度)。
從我們的研究結果顯示，這些突觸傳遞缺陷的線蟲，其神經主要的馬達蛋白UNC-104明顯地被影響運動模式，此外，unc17(e245)突變蟲又有顯著乙酰化和酪胺酸化的增加。由於這些結果，我們選擇unc17(e245)作為我們進一步的研究方向，更深入了解UNC-17(編碼囊泡乙酰膽鹼傳輸蛋白，VAChT)如何影響微管PTM以及軸突運輸的深層機制。

Microtubules (MTs) play an important role in trafficking of synaptic vesicles transported by molecular motor proteins from the cell body to the synapse. The relation between neuronal cargo transport and synaptic transmission has been well documented. Changes in synaptic transmission has been shown to directly affect axonal transport, however, little knowledge exists on how these changes affect post-translational modification (PTM) of MTs which in turn alters neuronal transport. In this study, we investigated how C. elegans animals, carrying various mutations that cause various types of synaptic transmission defects, affect the “tubulin code” in axons.
In this study, we employed seven mutant strains with different synaptic transmission defects namely, dat-1(ok157), eat-4(ky5), unc17(e245), unc25(e156), tbh-1(n3247), tdc-1(ok914) and, tph-1(mg280), respectively. We then used a broad range of techniques such as analysis of motor and cargo motility in axons as well as whole worm immunostaining (to understand changes in PTM of axonal microtubules) as well as Western blot analysis (to understand whether PTM enzymes may be up or down regulated).
From our results, it is evident that the major transporter of synaptic vesicles UNC-104/KIF1A (a neuron specific kinesin-3) is largely affected in these synaptic transmission defective worms. Moreover, we found that microtubule acetylation as well as tyrosination significantly changed in unc-17(e245) mutants. Based on these results, future experiments are suggested to focus on investigating the deeper mechanisms on how UNC-17 (encoding for a vesicular acetylcholine transporter, VAChT) affects both PTM of microtubules as well as axonal transport.

1. Introduction P.1-6
1.1 UNC-104 and RAB-3 P.1-2
1.2 Synaptic transmission P.2-3
1.3 Overview of post-translational modification P.3-4
1.4 Acetylation of tubulin P.4
1.5 Tyrosination / detyrosination of tubulin P.5
1.6 ALM neurons P.5
1.7 Employed mutant strains P.6
1.8 Purpose of study P.6
2. Materials and Methods P.7-15
2.1 Nematode Growth Medium (NGM) agar plate P.7
2.2 M9 buffer P.7
2.3 LB medium P.7
2.4 Genomic DNA isolation buffer P.8
2.5 C. elegans maintenance P.8
2.6 C. elegans strains and plasmid P.8-10
2.7 Microinjection P.10-11
2.8 RNA interference (RNAi) by feeding P.11-12
2.9 Crossing of C. elegans P.12
2.10 Genotyping P.12-13
2.11 Motility analysis P.13
2.12 UNC-104 and its cargo RAB-3 distribution analysis P.14
2.13 Western blot analysis P.14
2.14 Immunostaining P.15
3. Result P.16-21
3.1 Genotyping of transgenic strains P.16
3.2 Effect of synaptic transmission defective worms on UNC-104’s motility P.17-18
3.3 UNC-104 particles distribution P.18
3.4 Acetylation and tyrosination levels are increased in unc-17(e245) worm
lysates from Western blot result P.19-20
3.5 RAB-3 cargo motility and distribution are comparable to UNC-104 motor
motility and distribution P.20
3.6 Whole mount immunostaining reveals different PTM of MTs in different
mutant backgrounds P.21
4. Discussion P.22-30
4.1 Effect of UNC-104 in various worms with synaptic transmission defects P.22-26
4.2 unc-17(e245) shows acute defect in motility P.26-27
4.3 Significant changes in PTM of MTs in unc-17(e245) P.27-29
4.4 Dynein opposed moving direction to kinesin-3 P.29-30
4.5 Future experiments and perspective P.30
5.Reference P.31-35
6.Figures P.36-47
7.Appendix P.48-51
8.Supplemental Figure P.52-55

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