臺灣博碩士論文加值系統

孕烯醇酮（Pregnenolone簡稱P5）是細胞合成類固醇過程中的第一個產物，也是一種神經類固醇。先前研究指出P5在神經系統中有重要功能，P5可以增進記憶與幫助神經細胞修復。同時P5也調控斑馬魚胚胎早期發育，若在發育過程中抑制P5合成，將會破壞細胞骨架中微管的穩定性並嚴重阻礙胚胎發育。但先前研究尚未明確驗証P5影響生理反應的作用機轉。本論文針對P5的功能與作用機制進行研究，証明P5經由結合並活化微管正端結合蛋白CLIP-170而促進微管生成與細胞遷移，進而控制斑馬魚胚胎發育。經由使用P5光活化探針技術與液相層析串聯質譜儀分析，本論文証明CLIP-170是P5結合蛋白。P5結合在CLIP-170蛋白中間的捲曲螺旋結構區段，當P5與CLIP-170結合，CLIP-170蛋白形態由折疊去活化態翻轉為線形活化態；活化態CLIP-170可與微管、微管結合蛋白p150Glued、LIS1結合並可促進微管生成。可証，P5與CLIP-170結合將增強CLIP-170功能。抑制斑馬魚胚胎中CLIP-170的表現則將破壞微管並阻礙胚胎發育，正如同抑制P5所造成之現象。而當斑馬魚胚胎中的P5被人為表現之P5結合胜肽捕捉而無法正確與CLIP-170結合，斑馬魚胚胎發育則被抑制。由此可知，P5與CLIP-170正確結合的重要性。再者，缺乏P5的老鼠細胞在遷移時，不斷改變遷移方向呈現原地繞圈現象，無法正確向前遷移。而P5也須經由CLIP-170才能發揮其促進微管生成與調節斑馬魚胚胎發育的功能。本論文研究結果証明P5與CLIP-170結合後改變CLIP-170結構並活化其功能，P5與CLIP-170共同增進微管生成並調控細胞遷移與胚胎發育。再者，缺乏P5亦會破壞細胞骨架中肌動蛋白纖維穩定性、延緩細胞遷移過程中細胞形態的轉變、降低胚胎胞吞作用的能力。將斑馬魚胚胎培養於含有P5的培養液中，則可提升胚胎對肌動蛋白阻斷劑cytochalasin B的耐受性，減低其對胚胎發育所造成之傷害。此研究結果証明P5可作用於穩固肌動蛋白纖維。綜合本論文研究結果，P5可經由穩定細胞骨架中微管與肌動蛋白纖維並促其生成進而增進細胞遷移與調控斑馬魚胚胎發育。

Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. This thesis shows that P5 promotes cell migration and microtubule polymerization by binding to a microtubule-plus-end-tracking cytoplasmic linker protein 1 (CLIP-170). CLIP-170 was captured from zebrafish embryonic extract by a P5 photoaffinity probe conjugated to diaminobenzophenone and identified by LC-MS/MS analysis. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150Glued, and LIS1; it also promoted CLIP-170-dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly migration during embryogenesis; overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. It shows that P5 activates CLIP-170 to promote microtubule polymerization and cell migration. Besides microtubules, P5 deficient embryos showed abnormal F-actin architectures and decreased endocytotic activity. P5 is required and sufficient for resistance to the F-actin disrupting agent. It shows that P5 stabilizes F-actin. Together, P5 promotes zebrafish embryonic movement by controlling both microtubule and F-actin.

摘要 --------------------------------------- I
Abstract ---------------------------------- II
Table of Contents ------------------------- III
List of Figures --------------------------- IV
Abbreviation ------------------------------ VI
Introduction ------------------------------ 1
Materials and Methods --------------------- 8
Results ----------------------------------- 16
Conclusion -------------------------------- 28
Discussion -------------------------------- 29
Future Direction -------------------------- 37
Reference --------------------------------- 38
Introduction Figures ---------------------- 47
Figures ----------------------------------- 52
Supplementary information ----------------- 100
Figure I. Basic structure of a steroid. 47
Figure II. Steroidogenic pathway. 48
Figure III. Neurosteroidogenic pathway. 49
Figure IV. Structures of pregnenolone (P5) and its
derivatives. 50
Figure V. Structure of CLIP proteins. 51

Figure 1. P5 cooperates with other proteins to promote MT
polymerization. 52
Figure 2. Strategy for identifying P5-binding proteins. 54
Figure 3. P5-NBPN possesses better labeling ability than P5- BP and functions as P5. 56
Figure 4. Clip1a is identified as the P5-binding candidate. 58
Figure 5. P5 interacts with CLIP-170. 60
Figure 6. P5-NBPN does not specifically label MAP2c, CLIP-
115, nor tau. 62
Figure 7. The middle region of the coiled-coil domain in
CLIP-170 is required and sufficient for P5
binding. 64
Figure 8. Ability of photoaffinity probes in labeling CLIP-
170 and promoting MT assembly. 66
Figure 9. Study of CLIP-170 conformation by TEM. 68
Figure 10. P5 extends CLIP-170 conformation. 70
Figure 11. P5 activates CLIP-170. 72
Figure 12. P5 promotes CLIP-170 location on MT plus-ends. 74
Figure 13. CLIP-170 promotes MT growth. 76
Figure 14. P5 promotes MT growth via CLIP-170 in Y1 cells.78
Figure 15. P5 sustains migration directionality. 80
Figure 16. Genomic structures and protein alignment of
zebrafish clip1a and clip1b. 82
Figure 17. Specificity of morpholinos against zebrafish
clip1a. 84
Figure 18. P5 and CLIP-170 coordinately regulate epiboly
movement during zebrafish development. 86
Figure 19. P5 is required for the F-actin-driving forces. 88
Figure 20. P5 stabilizes the F-actin architecture. 90
Figure 21. P5 is required for endocytosis activity. 92
Figure 22. Location of FITC-P5 in organelle and membrane
ruffles. 94
Figure 23. P5 activates CLIP-170 via changing CLIP-170
conformation. 96
Figure 24. P5 controls cytoskeleton to promote cell
migration and zebrafish development. 98

Information I. Synthesis of 7a-Hydroxypregnenolone by Dr.
Chao-Tsen Chen’s group in the Institute of
Chemistry of NTU. 100
Information II. Synthetic photoaffinity probes by Dr. Chao- Tsen Chen’s group. 101
Information III. Expression pattern of zebrafish clip1a and
clip1b, examined by Dr. Sok-Keng Tong. 102

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