微管是神經細胞中含量最高的細胞骨架，而微管的正確生成對於神經系統的發育也非常重要。目前已知神經細胞中微管的生成依靠著非中心體組織中心聚核而成。我們實驗室近期發現TPX2是神經細胞中一種非中心體組織聚核蛋白，而Ran GTPase似乎是它的上游調節因子。此外，使神經細胞大量表現持續活化態或顯性抑制態的Ran突變體會影響神經突末端非中心體微管聚核。由於Ran參與核質運輸系統，如果活化或抑制神經細胞中所有的Ran，可能會導致神經存活率大幅下降或產生神經型態缺陷。所以在這篇論文中，我們試圖運用光遺傳系統達到在時間上與空間特定位置調控Ran的活性，我們在這稱此系統為RanTRAP，我們並以此系統研究Ran對於神經細胞的非中心體微管聚核的影響。首先，我們驗證了RanTRAP的光活化能力，並測試出在海拉細胞中局部進行光活化的最佳條件。其次，我們證實了在黑暗環境下，RanTRAP可以屏障Ran的功能，而在光活化Ran後可被釋放到細胞質中，並恢復其功能。最後，我們發現在神經突特定位置局部增加持續活化態Ran的濃度，可以促進該區域的非中心體微管聚核。這些結果代表Ran GTPase參與了神經細胞的非中心體微管聚核。此光遺傳RanTRAP系統在未來將使我們能夠進一步探討非中心體微管對神經型態的影響。

Microtubule is the most abundant cytoskeleton in neurons and the generation of microtubules is essential for the proper development of the nervous system. It has been shown that the generation of microtubules in post-mitotic neurons depends on nucleation by acentrosomal microtubule-organizing centers (MTOCs). Previous studies in our lab found that TPX2 is an acentrosomal nucleator in neurons and Ran GTPase appears to be its upstream regulator. In addition, overexpressing the constitutively active or dominant negative Ran mutants in neurons influences acentrosomal microtubule nucleation at the neurite tips. Since Ran participates in nucleocytoplasmic transport, globally activating or inactivating Ran may result in neuronal survival and morphogenetic defects. In this thesis work, we attempted to establish an optogenetic tool called RanTRAP that is capable of spatiotemporally controlling the activation of Ran, to study the acentrosomal microtubule nucleation in neurons. First, we validated the photoactivation capability of constructed RanTRAP and optimized the locally exposure condition in HeLa cells. Second, we confirmed that RanTRAP sequesters Ran mutants in the dark while allowing Ran mutants to be released into the cytosol upon local photoactivation. Finally, we found that locally increasing the concentration of a constitutively active Ran mutant promotes acentrosomal microtubule nucleation in the photoactivated sites in neurons. These results indicate that Ran GTPase is involved in acentrosomal microtubule nucleation in neurons. This optogenetic RanTRAP system will enable us to further examine the effect of acentrosomal microtubules on neuronal morphology in the future.

摘要 I
Abstract II
Abbreviation VIII
I. Introduction 1
1.1 Neuronal morphogenesis is regulated by the microtubule cytoskeleton 1
1.2 The importance of ascentrosomal microtubule organizing center in neurons 2
1.3 The regulation of TPX2 by Ran GTPase in microtubule nucleation 3
1.4 Ran GTPase affect acentrosomal microtubule formation in neurons 3
1.5 The optogenetic LOVTRAP system 4
1.6 Specific aim 5
II. Materials 6
2.1 Mouse strain 6
2.2 Bacterial strains 6
2.3 Cell lines 6
2.4 Plasmids 6
2.5 Primers 7
2.6 Restriction enzymes 8
2.7 Commercial material list 8
2.8 Buffers and solutions 10
2.9 Antibodies 14
2.9.1 Primary antibodies 14
2.9.2 Secondary antibodies 15
III. Methods 16
3.1. Mammalian expression plasmid construction 16
3.1.1 pTriEx-mCherry-ZDK1-RanQ69L/T24N 16
3.1.2 pTriEx-NTOM20-mVenus-LOV2-I539E 17
3.1.3 pTriEx-mVenus-LOV2wt 18
3.1.4 pTriEx-NTOM20-LOV2wt/I539E/C450A 18
3.1.5 pCAG-KPNB1-EGFP 19
3.2 Bacterial transformation 20
3.3 Immunoblot 20
3.4 Cell cultures 21
3.4.1 HeLa cell culture 21
3.4.2 Primary neuron culture 21
3.5 Preparing poly-L-lysine-coated coverslips or α-dish 22
3.6 Lipofectamine transfection 22
3.6.1 Lipofectamine transfection of HeLa cells 22
3.6.2 Lipofectamine transfection of primary neuron 23
3.7 Cell fixations 23
3.7.1 Formaldehyde fixation 23
3.7.2 Extracting cytosolic protein before cell fixation (pre-extraction) 23
3.8 Indirect Immunofluorescence staining (IF) 24
3.9 Mitotracker staining 24
3.10 HeLa cells synchronization by double thymidine arrest 25
3.11 Synchronizing HeLa cells by nocodazole 25
3.12 Images acquisition 25
3.13 Photoactivation 26
3.14 Images analysis 27
3.15 Statistical analysis 28
IV. Results 29
4.1 Optimization of the condition for LOVTRAP system in HeLa cells 29
4.1.1 Confirming the locolization of LOVTRAP proteins in HeLa cells 29
4.1.2 Optimizing transfection condition of LOVTRAP plasmids in HeLa cells 29
4.1.3 Examine the minimal irradiation to locally photoactivate LOVTRAP system in HeLa cells 30
4.1.4 Examine the dynamic of release mCherry-ZDK after locally photoactivate LOVTRAP system in HeLa cells 31
4.1.5 Optimizing light irradiation regime to locally photoactivate LOVTRAP system in HeLa cells 31
4.2 Constructing and validating photoactivatable RanTRAP in HeLa cells 32
4.2.1 Constructing RanTRAP system 32
4.2.2 Examining the localization of mCherry-ZDK-Ran in HeLa cells 32
4.2.3 Examining the trapping and photoactivatable capability of RanTRAP in HeLa cells 33
4.2.4. Functional validation of RanTRAP system in HeLa cells 33
4.2.5 Combining RanTRAP and the microtubule plus-end dynamics assay in HeLa cells 35
4.3 Optimizing and utilizing the photoactivatable RanTRAP system in neurons to study acentrosomal microtubule nucleation 35
4.3.1 NTOM20 target Ran protein to mitochondria along the neurite 35
4.3.2 Optimizing transfection condition of RanTRAP plasmids in neurons 36
4.3.3 Locally phototactive RanTRAP system in neurons 36
4.3.4 Combining RanTRAP and the microtubule plus-end dynamics assay in neurons 37
4.3.5 Locally producing RanGTP promotes acentrosomal microtubule nucleation along the neurite 37
V. Discussion 39
5.1 An optogenetic platform RanTRAP system to study acentrosomal microtubule nucleation 39
5.2 The filamentous distribution of mCherry-ZDK protein in HeLa cells 40
5.3 The perinuclear clustering distribution of mitochondria in RanTRAP expressing HeLa cells 40
5.4 RanTRAP sequester Ran mutant function in the dark by sterically blocking 40
5.5 The application of RanTRAP system in neuronal morphogenesis 41
VI. Reference 42
VII. Figures 45
VIII. Appendix 68

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