探究酵母菌中葡萄糖缺乏影響粒線體動態平衡的機制

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研究生：	黃信瑋 Huang, Hsin Wei
論文名稱：	探究酵母菌中葡萄糖缺乏影響粒線體動態平衡的機制 Elucidation of the Mechanisms of Glucose Starvation on Mitochondria Dynamics in Yeast Cells
指導教授：	張壯榮 Chang,Chuang Rung
口試委員:	廖泓鈞高茂傑
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	50
中文關鍵詞：	葡萄糖不足、粒線體動態平衡、白藜蘆醇、細胞衰老
外文關鍵詞：	glucose starvation, mitochondrial dynamics, senescence, resveratrol
相關次數：	點閱：1 下載：0
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粒線體是個高度動態的胞器，持續地進行融合和分裂，面對不同的環境，粒線體透過調控融合、分裂的平衡，進而改變粒線體的型態。粒線體動態平衡跟粒線體的完整性和功能有關。哺乳類細胞在糖不足的情況下，粒線體分裂減少，使得粒線體型態變長。在酵母菌細胞面對葡萄糖不足的情況下也會使得粒線體呈現較長的型態，但確切的機制還不明確。為了探討在葡萄糖不足情況下，粒線體型態改變的關係，本篇論文因此對不同酵母菌菌株在葡萄糖不足情況下粒線體型態的變化深入探究。在我的實驗中，發現在葡萄糖不足的情況下粒線體型態發生改變跟SNF1這條訊息傳導路徑有關。酵母菌在葡萄糖不足的狀況下，SNF1會使參與粒線體分裂的蛋白減少，使得粒線體型態變長。除此之外，我們先前的研究指出在衰老的酵母菌細胞中，粒線體型態呈現較多的碎片狀，因此酵母菌細胞的衰老狀況也會影響粒線體的型態，加入白藜蘆醇可以改變這個現象，我的實驗結果顯示，白藜蘆醇並不是透過SNF1和TOR1這兩條路徑來改變粒線體型態。

Mitochondria are highly dynamic organelles that constantly undergo fission/fusion processes in response to environment. The dynamics of mitochondria determines mitochondrial integrity and function. In mammalian cells, starvation leads to unopposed mitochondrial fusion and drives mitochondrial elongation. In yeast, glucose starvation also induces mitochondrial hyperfusion. However, the mechanism remains unclear. To clarify the relationship between glucose starvation and mitochondria morphology, I examined the mitochondrial dynamics by classifying morphology in WT, Δsnf1 and Δtor1 strains in nutrient-deprived medium. I found that SNF1 pathway involved in mitochondrial hyperfusion under glucose starvation condition. In addition, mitochondrial fusion and fission balance is also affected by senescence. Mitochondrial fragmentation was presented in yeast senescent cells and resveratrol can mitigate this phenomenon. My results demonstrated that that resveratrol reduces ratio cells with fragmented mitochondria is not through SNF1 or TOR1 pathway.

致謝    I
中文摘要    II
Abstract    III
CONTENTS    IV
LIST OF FIGURES    VI
Chapter 1 Introduction    1
1.1    Mitochondria are dynamic organelles.    1
1.2    Mitochondrial fusion and fission proteins.    1
1.3    Mitochondria morphology changed under starvation condition by adjusting fusion/fission balance.    2
1.4    Snf1 regulates carbon sources metabolism in yeast.    3
1.5    Tor1 controls growth in response to nutrients in yeast.    3
1.6    Mitochondria play important role in aging process.    4
1.7    Resveratrol affects mitochondria in senescent cells.    5
1.8    Specific aim    6
Chapter 2 Materials and Methods    7
2.1 Yeast strains and medium    7
2.2 Experiment procedures    7
2.2.1 Yeast transformation    7
2.2.2 Yeast cells switch to glucose starvation medium    8
2.2.3 Senescent yeast cells sorting    8
2.2.4 Fixation of yeasts to observe the mitochondrial morphology    9
2.2.5 Yeast genomic DNA extraction    10
2.2.6 Yeast RNA extraction    11
2.2.7 Reverse transcription polymerase chain reaction    12
2.2.8 Real time PCR    12
2.2.9 Mitochondrial membrane potential detection    13
2.2.10 Flow cytometry    14
Chapter 3 Results    15
3.1 WT, Δsnf1 and Δtor1 show the similar mitochondrial morphology in log phase.    15
3.2 Glucose starvation induces mitochondrial elongation in yeast.    15
3.3 SNF1 affects mitochondria dynamics under glucose starvation condition.    16
3.4 Mitochondrial fragmentation present in glucose starvation late stage.    17
3.5 Glucose starvation altered FIS1 gene expression level in Δsnf1 cells.    18
3.6 DNM1 gene expression is upregulated in response to glucose starvation.    18
3.7 FZO1 gene expression level is elevated inΔsnf1.    19
3.8 The mtDNA copy numbers are not affected by glucose starvation.    19
3.9 Mitochondrial membrane potential in Δsnf1 cells is increased greater than WT cells under glucose starvation.    20
3.10 Mitochondrial fragmentation was present in senescent yeast cells.    21
3.11 Resveratrol reduces mitochondria fragmentation in senescent cells.    21
3.12 Deletion of SNF1 and TOR1 has no effect on resveratrol in senescent cells.    22
Chapter 4 Conclusion and discussion    23
4.1 Conclusion    23
4.2 Discussion    23
4.2.1 SNF1 pathway plays a key role in glucose starvation early stage.    23
4.2.2 ∆snf1cells were sensitive to switch to fresh medium.    24
4.2.3 The translocation of Dnm1 to mitochondria is also important.    25
4.2.4 Glucose starvation elevated mitochondrial membrane potential.    25
Chapter 5 Perspective    26
Reference    27


 
LIST OF FIGURES
Fig. 1. The machinery of mitochondrial fusion and fission in yeast…………..31
Fig.2. The mitochondrial morphology in yeast…………………………………32
Fig. 3. Mitochondrial morphology shows no different in WT, Δsnf1 and Δtor1 cells in log phase…………………………………………………………………..33
Fig. 4. The flow chart of glucose starvation experiment…………………….....34
Fig. 5. Glucose starvation induces mitochondria hyperfusion in WT cells and ∆tor1 cells…………………………………………………………………………..36
Fig. 6. Mitochondria hyperfusion was not found in ∆snf1 cells………………..38
Fig. 7. Ratio cells with fragmented mitochondria increased under glucose starvation 4hr………………………………………………………………………40
Fig. 8. FIS1 gene expression level changed in ∆snf1 during glucose starvation.41
Fig. 9. DNM1 gene expression is upregulated under glucose starvation……….42
Fig. 10. FZO1 gene expression level increased inΔsnf1 cells under starvation43
Fig. 11. Mitochondrial DNA copy numbers are not affected under glucose starvation 2hr……………………………………………………………………….44
Fig. 12. Mitochondrial membrane potential increased under glucose starvation. …………………………………………………………………………..45
Fig. 13. Ratio of cells with fragmented mitochondria increased in senescent cells. …………………………………………………………………………………46
Fig. 14. Resveratrol treatment reduced fragmented mitochondria in senescent cells…………………………………………………………………………………..47
Fig. 15. Resveratrol reduces fragmented mitochondria in Δsnf1 and Δtor1 senescent cells……………………………………………………………………….49


                                

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