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研究生:陳安琪
研究生(外文):An-Chi Chen
論文名稱:運用高時空解析度之3維時序顯微影像分析研究細胞分裂時粒線體動態
論文名稱(外文):Studies on Mitochondrial Dynamics during Cell Division by 3D+t Imaging with High Temporal and Spatial resolution
指導教授:林崇智林崇智引用關係
指導教授(外文):Chung-Chih Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學系暨基因體科學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:85
中文關鍵詞:粒線體動態變化細胞有絲分裂粒線體分配粒線體質量控制
外文關鍵詞:mitochondrial dynamicsmitosismitochondrial partitionmitochondrial quality control
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粒線體是個高度動態變化的胞器,它會不斷的進行融合、分裂來達到動態平衡。粒線體的品質管控對於粒線體型態的維持、粒線體DNA的完整性、粒線體氧化壓力(ROS)的反應有很大的關係,當細胞分裂時,不只有遺傳物質DNA合成需要高度管控,產生完整無誤的遺傳物質,均勻無誤送到子細胞當中。細胞中的粒線體也需要進行品管,讓健康的粒線體正確地被分配到子細胞。失能的粒線體,或是錯誤的粒線體分配,有可能停止細胞週期進行,或造成子細胞的異質性與影響子細胞生存。
過去本實驗室以AcGFP標記粒線體基質蛋白並使用轉盤式共軛焦顯微鏡來拍攝3D + T影像,最後用microP-3D軟體量化粒線體形態。結果發現當細胞進入M期,粒線體的體積與整體螢光強度都下降,顯示有細胞週期專一的粒線體降解現象。因粒線體融合成條狀造成數量下降,若以隨機分配方式分配到子細胞,可能會分配產生誤差而造成分配不均勻。因此探討以何種分配機制,成為重要的研究議題。另外,因為過去本實驗室以綠螢光標定粒線體,而綠螢光強度會因為pH值有影響,因此要先確認M期是否進行大量粒線體裂解,而此裂解是否會影響細胞之正常分裂。本研究將針對M期粒線體之型態變化過程的可能機制,以及確認M期粒線體降解是否影響細胞分裂,這兩方面進行研究。
在第一個研究主題想了解M期螢光強度下降是否為pH所影響,因此使用不會隨著pH值而在螢光上有變化的紅螢光蛋白DsRed-mito做為驗證,發現紅螢光蛋白的螢光強度會在M期下降,但下降的程度只有AcGFP-mito的六成,顯示粒線體在M期其基質有酸化與降解的現象。並且發現當細胞遇到分裂災難時,粒線體螢光蛋白的螢光強度並不會在前期減少,顯示粒線體的降解與細胞質分裂正常與否有關。但目前仍未釐清有絲分裂前期粒線體螢光減少與細胞分裂災難的因果關係。

在第二個部分,本實驗室過去發現粒線體在有絲分裂時會呈現產能較高的長條狀型態來暫時提供能量讓細胞順利分裂,並在分裂溝呈現拉直並產生斷裂面的現象。但轉盤式共軛焦顯微鏡之時間解析度不夠好,所以無法得知粒線體分配之形變過程。因此本研究使用Bessel-beam light sheet 顯微鏡,以每秒拍攝一個細胞83層三維立體影像的速度,紀錄粒線體在cytokinesis是如何被分配到兩顆子細胞,目前我們得到的影像判斷,粒線體分配出現三種模式:擾動、斷裂點直接斷開、先拉直,接著在斷裂點變細最後才斷裂。雖然時間與空間解析度都已大幅提升,但目前只能用肉眼觀察、手動標記粒線體走向,要確認此一現象,還需要拍攝更多影像,研究出追蹤單一粒線體動態提升分析速度,來增加觀察之細胞數。
總結本研究發現,粒線體在進入M期有大量降解現象,在M期結束到細胞進入下一個 細胞週期時,粒線體蛋白質大量合成。M期粒線體降解,與細胞質分裂成功有高度的關聯性。在M期粒線體呈長條狀,會讓粒線體能在分裂溝沿著分裂方向直線來回位移,在細胞質分裂後期,長條粒線體以非傳統粒線體斷裂方式被拉斷。粒線體來回運動,也與細胞質分裂成功與否有關連。整個研究顯示,粒線體在M期品管與形狀,讓粒線體可以有能量來回運動,讓粒線體能夠均勻分配。粒線體不能運動,可能會造成細胞質分裂的異常。

Mitochondrial morphology is dynamic and controlled by fusion and fission.
Mitochondrial quality control has a lot to do with maintaining mitochondrial morphology, mitochondrial DNA integrity, and reaction of oxidative stress (ROS).
During cell division, not only genetic materials but also organelles are evenly distributed to daughter cells. Mitochondria also needs to be quality control and the healthy mitochondria will correctly assign to the daughter cells. The wrong distribution or dysfunctional mitochondria is possible to stop the cell cycle, cause heterogeneity and survival issues of daughter cells.
In our lab previous studies, we use AcGFP to label mitochondrial matrix proteins and acquire 3D + T cell imaging to acquire time lapsed images of 3D mitochondrial dynamics during cell division by spinning-disc confocal microscopy and quantify mitochondrial morphology by use of 3D Micro-P. Our previous results support our argument that mitochondria undergo partial fission to keep some mitochondrial tubules for enough energy supply and totally mitochondrial fluorescent intensity decrease at M phase. It shown cell cycle-specific mitochondrial degradation phenomenon. Because of mitochondrial fusion into tubulated, resulting decline in the number. If the distribution to daughter cells were random, it would assign errors caused by uneven distribution. So explore what distribution mechanisms is certainly important research topic. In addition, our laboratory used green fluorescent protein(GFP) to label mitochondrial matrix proteins. But the intensity of GFP is pH-sensitive. In this study, results are divided into two parts. We investigated the possible mechanisms for mitochondrial partition patterns at M phase, also confirmed mitochondrial degradation at M phase whether or not affected cell division.

The first question is whether the decrease of fluorescence intensity at M phase will be affect by pH. We use pH- insensitive red fluorescent protein(RFP) DsRed-mito as verification. That still observed the same results. We found that RFP intensity is decrease at M phase, but the degree of decline just 60 percent of AcGFP-mito. It means that matrix of mitochondria at M pahse is degradation and acidification. Moreover, mitotic catastrophe and reduction of mitochondrial protein fluorescence intensity, both was happened at the same time. Mitotic catastrophe is an event in which a cell can’t through mitosis being two daughter cells. But we still not clarify the causal relationship between mitochondrial fluorescence reduce at prophase and mitotic catastrophe.
In the second part, our laboratory found that mitochondria will elongate to higher productivity at mitosis. The mitochondria straighten and break into a fracture surf at cleavage furrow. But the spatial resolution of spinning-disc confocal microscopy is not good enough. Therefore, this study using Bessel-beam light sheet microscope to acquire time lapsed 3D images with 83 step/s in one stack, recording mitochondria how to distributed to two daughter cells in cytokinesis. To determine from the results, the mitochondrial distribution occur three modes: just disturbance, breaking at division furrow, straightened tapering after straightening and finally broken. Although the temporal and spatial resolution are significantly improved. Currently we can only observe with the naked eye, to manually tag mitochondria. To confirm this phenomenon, we need to take more images to looking for an effective analytical procedure for tracking single object.

目錄 I
中文摘要 III
Abstract V
中英文對照表 VII
緒論 1
一、粒線體的動態平衡與其生理意義 1
(一) 粒線體動態變化 1
(二) 粒線體融合機制 1
(三) 粒線體分裂機制 2
二、粒線體的遺傳(mitochondrial inheritence) 3
(一) 酵母菌模式(yeast model) 3
(二) 哺乳動物模式(mammalian model) 3
三、參與粒線體運輸的蛋白質 5
四、粒線體品質管控(Mitochondrial quality control) 6
實驗目的與假說 7
材料與方法 9
一、實驗材料與藥品 9
二、研究方法 11
(一) 細胞培養 11
(二) 質體轉形(Transformation) 11
(三) 質體DNA純化 12
(四) 細胞轉染(Transfection) 13
(五) 病毒感染 (Transduction) 15
(六) 極限稀釋法(Limited dilution) 15
(七) 螢光顯微影像擷取 15
(八) 細胞週期同步之粒線體型態拍攝 19
(九) 粒線體螢光亮度分析 19
(十) 3D粒線體型態分類 19
(十一) 3D粒線體與細胞核型態之數值分析 20
實驗結果 21
一、篩選各種穩定表達的中國倉鼠卵巢細胞株 21
二、細胞同步化 22
(一) 飢餓法(starvation) 22
(二) Nocodazole 22
(三) Thymidine 23
三、M phase的粒線體品質管控與生合成 24
(一) Mito-GFP的螢光亮度變化 24
(二) pDsRed-mito的螢光亮度變化 25
(三) 細胞周期的粒線體螢光強度變化 26
(四) 單一觀察粒線體降解現象(Pulse and Chase) 26
四、粒線體的分配與動態 27
(一) 低時間解析度之長程拍攝 27
(二) 高時間解析度之短程拍攝 27
(三) 高時間與空間解析度之短程拍攝 28
結論與討論 29
參考文獻 31
圖表說明 35
圖1 :利用螢光顯微鏡觀察成功轉染與感染的細胞 37
圖2 :Nocodazole造成細胞週期停滯,去除藥物後產生有絲分裂災難 38
圖3 :Thymidine細胞同步化後,細胞能夠完成有絲分裂 40
圖4 :Mito-GFP的螢光亮度變化 41
圖5 :pDsRed-mito的螢光亮度變化 43
圖6 :整個細胞周期的Mito-GFP螢光亮度變化 44
圖7 :時間解析度需要提升 45
圖8 :提高時間解析度 46
圖9 :提高時間解析度 47
圖10:細胞於分裂溝粒線體斷裂可能的機制模型圖 48
圖11:不同時間的粒線體subtype比例變化 49
圖12:有絲分裂時粒腺體分配到子細胞可能發生之機制示意圖 50
附錄 51
附錄一、粒線體動態與細胞的生理功能 51
附錄二、哺乳類細胞之粒線體融合與分裂參與的蛋白質 52
附錄三、Retrograde Actin Cable Flow (RACF)參與在芽殖酵母細胞分裂時的粒線
體質量控制 53
附錄四、粒線體的三種質量控制模型 54
附錄五、有絲分裂時粒線體在分裂溝有清楚斷裂 55
附錄六:microP-3D影像處理示意圖 56
附錄七、傳統螢光顯微鏡與光佈螢光顯微鏡的差別 57
附錄八、Bessel beam light sheet microscopy介紹 58
附錄九、Bessel beam light sheet microscopy鏡頭設置示意圖 59
附錄十、Spinning disc confocal拍攝粒線體時序影像之亮度計算方法 60
附錄十一、Bessel beam light sheet拍攝粒線體時序影像之microP-3D分析 64
附錄十二、用Bessel beam light sheet拍攝影像來切割單一粒線體物件的方法 73


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