粒線體是一個具有動態平衡特性的胞器，會不斷的進行分裂與融合，配合上移動與清除，以達到動態網路。粒線體具有能量提供、存放離子甚至是調控細胞凋亡的功能。許多先前研究中，發現肌動蛋白會參與粒線體的動態平衡調控，但並不了解其作用機制。我們實驗室在先前已經找出會與粒線體分裂蛋白DRP1有交互作用的肌動蛋白調節蛋白CAP2，本篇研究的目標是以U2-OS 細胞釐清CAP2如何參與調控粒線體的動態平衡。我們利用CRISPR/Cas9 基因編輯技術在U-2 OS細胞株中剔除CAP2基因，會造成具有超長網絡狀粒線體型形態的細胞比例增加，指出CAP2確實會參與粒線體分裂的過程。在剔除CAP2後，同時也會發現細胞倍數增值所需的時間增加，而在模擬相似的粒腺體形態情況下，並無發現倍數增值時間增加。在粒線體活性測試中，包含粒線體的膜電位、細胞內的氧化自由基總量和細胞的單位時間耗氧量，發現剔除CAP2後並無明顯的變化，表示剔除CAP2造成的形態變化與粒線體的功能缺陷之關聯性仍有待釐清。綜合以上，我們指出CAP2確實會影響粒線體動態平衡尤其是在粒線體分裂的過程。

Mitochondria are highly dynamic organelles; fusion, fission, and trafficking, determine mitochondrial reticulum morphology. Previous studies have implied that actin cytoskeleton involved in mitochondria dynamics; however, the role of actin regulates mitochondria dynamics remains obscure. Genetic screen performed in our lab identified an actin regulatory protein, Adenylyl cyclase associated protein (CAP2), may involve in mitochondrial dynamics regulation. CAP2 is known to regulate actin dynamics by severing F-actin and recharging G-actin. To clarify the role of CAP2 in mitochondria dynamics, we applied CRISPR/Cas9 system to delete CAP2 in U-2 OS cells. We found mitochondrial morphology in CAP2 knockout cells was hyperfused compared to parental cells. In CAP2 knockout cells, actin filaments assembly was disrupted. The similar hyperfused mitochondrial morphology was found under the treatment of pharmacological compound that disrupted actin dynamics. Additionally, we found CAP2 knockout alters cell cycle progression independent of mitochondrial morphology. Furthermore, CAP2 deletion does not affects mitochondrial activity. Together, we suggested CAP2 acts as a mitochondrial pro-fission factor in U2-OS cells.

Abstract 2
中文摘要 3
Acknowledgement 4
Contents 5
Chapter 1. Introduction 8
1.1 Mitochondria are double-membrane organelles of cells, responsible for ATP production, and regulating other important biological functions. 8
1.2 Mitochondria dynamics regulates the mitochondrial morphology via fission, fusion, and trafficking. 9
1.3 Mitochondrial dynamics associates with mitochondrial quality control 10
1.4 Relationship of cell cycle and mitochondrial morphology is blurry 11
1.5 The actin filaments are suggested a component of mitochondrial fission 12
1.6 CAP is a multifunctional protein, including actin polymerization and severing, these functions regulate actin dynamics 12
1.7 Specific aim 13
Chapter 2. Material and Method 14
2.1 Cell culture 14
2.2 Transfection of plasmids into cells 14
2.3 Nuclear genome edited by CRISPR/Cas9 system 15
2.4 DNA and RNA extraction 16
2.5 T7E1 Surveyor assay 17
2.6 Reverse transcription 18
2.7 Real-time quantitative PCR 19
2.8 Immunofluorescence staining 19
2.9 Actin labeling 20
2.10 Plasmid construction 20
2.11 Protein extraction 21
2.12 Western bolt 21
2.13 Antibodies 22
2.14 Cell cycle synchronization 23
2.15 Cell cycle analysis 23
2.16 Measurement of mitochondrial membrane potential 24
2.17 Measurment of cellular reactive oxygen species (ROS) 25
2.18 Measurement of oxygen consumption rate 25
Chapter 3. Results 27
3.1 Deleting CAP2 gene in U-2 OS cells by CRISPR/Cas9 system 27
3.2 CAP2 deletion causes hyperfused mitochondria 28
3.3 CAP2 deletion disrupts actin dynamics 28
3.4 Disruption of actin polymerization affects mitochondrial morphology 29
3.5 CAP2 deletion prolongs G2/M phase period 30
3.6 Hyperfused mitochondria is not the key factor to alter cell cycle progression in CAP2-/- cells 31
3.7 CAP2 deletion does not alter mitochondrial activity 32
Chapter 4. Conclusion, Discussion and Perspective 34
4.1 CAP2 regulates mitochondrial dynamics through its function in actin dynamics regulation 34
4.2 CAP2 deletion delays cell cycle progression independent of altered mitochondrial dynamics 34
4.3 CAP2 deletion affects mitochondrial dynamics, but not mitochondrial activity. 35
4.4 Perspective 36
Chapter 5. Reference 38
List of Figures
Figure 1. The illustration of our project. 45
Figure 2. Verifying CAP2-/- strain generated by CRISPR/Cas9 system. 47
Figure 3. CAP2 deletion affects mitochondrial dynamics. 49
Figure 4. CAP2 deletion affects actin dynamics. 50
Figure 5. Disrupting actin dynamics affects mitochondrial dynamics. 53
Figure 6. CAP2 regulates cell cycle, particularly G2/M phase. 56
Figure 7. Cell cycle progression delayed in CAP2-/- is not caused by mitochondrial morphology. 60
Figure 8. CAP2 knockout does not affect mitochondrial activity. 63
Figure 9. the illustrations of our research 64

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