粒線體為細胞內主要提供能量的胞器，維持細胞正常生長功能。粒線體為高度動態的胞器，透過不斷地分裂及融合形成網路形態。阿茲海默症及帕金森斯症等神經退化性疾病與粒線體動態平衡失衡有高度相關。根據先前的研究，細胞骨架系統參與粒線體分裂的過程，我們實驗室已經找到與粒線體分裂蛋白Drp1有交互作用的肌動蛋白調節蛋白CAP2，此蛋白具有調控肌動蛋白動態平衡的功能。本篇研究主要在深入探討CAP2參與粒線體動態平衡的過程機制。我們實驗室先前已利用CRISPR/Cas9基因剔除系統在U2OS細胞中剔除CAP2 基因。在剔除CAP2基因細胞中我們發現較長網絡狀的粒線體形態，以及不規則聚集的肌動蛋白形態。剔除CAP2基因同時也影響粒線體的狀態及功能活性，例如：粒線體膜電位降低、基礎耗氧量提高以及使粒線體移動速度變慢。同時我們也發現高基礎耗氧量伴隨電子傳遞鏈蛋白質組成增加。最後我們在剔除CAP2基因的細胞中將CAP2蛋白專一性的標定在粒線體外膜造成粒線體形態呈現碎裂狀。藉由這些實驗結果，CAP2確實會透過聚合及調控肌動蛋白影響粒線體動態平衡以及功能。

Mitochondria serve as energy factory that can produce most of the cellular energy to maintain regular physical function in cells. Intriguingly, mitochondria are highly dynamic organelles. Mitochondrial morphology can be changed through reaching balance of fission and fusion process. Both fission and fusion are important to maintain mitochondrial function. Many neurodegenerative diseases, such as, Alzheimer's disease and Parkinson's disease, are closely associated with disrupted mitochondria dynamics. The regulation of mitochondrial morphology involves cytoskeleton. Actin has been indicated to play an important role in mitochondrial fission. We identified an actin-regulatory protein Srv2/CAP2 that can interact with mitochondrial fission GTPase Dnm1/DRP1. Cyclase associated protein (CAP2) was known to regulates actin dynamics by severing F-actin and recharging G-actin. To elucidate how does CAP2 regulates mitochondria dynamics, we generated CAP2 knockout in U2OS cells by CRISPR/Cas9 system. Here we report that CAP2 deletion caused hyperfused mitochondria and aggregated actin filaments. Additionally, CAP2 knockout caused reduced mitochondrial membrane potential and reduced mitochondrial dynamics rate. Furthermore, CAP2-/- cells had higher oxygen consumption and more oxidative phosphorylation complex. We examined the effects of artificially targeting CAP2 to mitochondria. The results showed that targeting CAP2 to mitochondria causes more fragmented mitochondria and complements mitochondrial phenotypes in CAP2-/- cells. Together, our results suggested that CAP2 acts as a pro-fission protein that mediates mitochondrial morphology and activity through actin assembly.

致謝 I
中文摘要 II
Abstract III
Contents IV
Chapter1. Introduction 1
1.1 Mitochondria are responsible for cellular energy production through oxidative phosphorylation. 1
1.2 Mitochondria dynamics includes fusion and fission process. 1
1.3 Mitochondrial dynamics is important for maintaining organelle function. 3
1.4 ER-mitochondria contact sites define the position where division occur. 3
1.5 The actin participates in mitochondrial fission process. 4
1.6 CAP is a multifunctional protein which regulates actin dynamics. 5
1.7 Specific aim 6
Chapter 2. Material and Method 7
2.1 Cell culture 7
2.2 Plasmid transfection 7
2.3 Immunofluorescence 7
2.4 Actin labeling 8
2.5 Protein extraction and Western blotting 8
2.6 Antibodies 9
2.7 Measurement of mitochondrial membrane potential by flow cytometry 9
2.8 Measurement of mitochondrial membrane potential by JC-1 staining 10
2.9 Measurement of mitochondrial oxygen consumption rate 10
2.10 Measurement of succinate dehydrogenase activity 10
Chapter 3. Results 11
3.1 CAP2 knock out in U2OS caused hyperfused mitochondrial network. 11
3.2 Loss of CAP2 affects actin filaments assembly. 11
3.3 The structure of endoplasmic reticulum is altered in CAP2-/- cells 12
3.4 The fission process is inhibited by CAP2 deletion. 12
3.5 CAP2 is required for maintaining mitochondrial membrane potential. 13
3.6 CAP2 deletion cells have higher basal respiration rate. 14
3.7 CAP2 alters the components of mitochondrial oxidative phosphorylation complex. 15
3.8 Targeting CAP2 to mitochondria increases mitochondria fragmentation. 15
3.9 The effects of CAP2 knocked out on mitochondrial dynamics factors does not depend on altering protein levels of dynamic factors. 16
Chapter 4 Discussion, conclusion and perspective 17
4.1 Discussion 17
4.1.1 CAP2 deletion causes the mitochondria and actin morphology changed. 17
4.1.2 CAP2 deletion affects ER/mitochondria contact site that may related to calcium homeostasis 17
4.1.3 CAP2 deletion affects mitochondrial activities and components of OXPHOS complexes. 18
4.1.4 NTom20-CAP2 overexpression causes fragmented mitochondria. 19
4.1.5 CAP2 deletion had minimal impacts on mitochondrial dynamic factor protein level. 19
4.2 Conclusion 19
4.3 Perspective 20
Reference 21
Figures 24
Figure 1. CAP2 deletion affects mitochondrial dynamics and this phenotype can be compensated by re-transfection CAP2. 25
Figure 2. Loss of CAP2 affected actin filaments assembly. 27
Figure 3. The structure of endoplasmic reticulum is loose in CAP2-/- cells 29
Figure 5. CAP2 is required for maintaining mitochondrial membrane potential. 32
Figure 6. CAP2 deletion cells had higher basal respiration than wild type. 34
Figure 7. CAP2 deletion alters the components of OXPHOS complex 35
Figure. 8 Design the plasmid which can target CAP2 to mitochondria. 36
Figure. 9 NTom20-CAP2 overexpression causes more fragmented mitochondria. 38
Figure 10. The effects of CAP2 knocked out on mitochondrial dynamics does not depend on altering protein levels of dynamic factors 39
Figure 10. CAP2 knocked out affected short form Opa1 processing. 40

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