粒線體是一個高度動態的胞器，在細胞中持續地進行分裂與融合而展現出不同的型態。粒線體的動態平衡會隨外在環境的變化而做出改變，在細胞的生理調控中扮演相當重要的角色。最近的研究指出酵母菌在葡萄糖不足的狀況下，其粒線體會呈現較長的型態。然而，葡萄糖缺乏下之粒線體動態平衡調控機制目前尚未被了解。在酵母菌中，Snf1對於調控細胞適應糖分缺乏的環境相當關鍵。因此，我們推測Snf1會參與葡萄糖缺乏下粒線體動態平衡的調控。為了驗證我們的假設，我們對低糖狀態下的粒線體進行型態的分類以了解其動態平衡的狀態。另外，我們也測定了粒線體於低糖下的膜電位與耗氧率，藉以了解粒線體於低糖下的活性狀態。我們發現∆snf1酵母菌需要較長的反應時間來調節其在葡萄糖缺乏下的粒線體型態改變。再者，低糖狀態下的粒線體膜電位迅速的下降，並在葡萄糖缺乏後的三十分鐘內，逐漸地回升並達到穩定的平衡狀態，但在缺少Snf1的細胞中，其低糖下粒線體膜電位恢復的能力將會降低。此外，我們還發現在葡萄糖缺乏下粒線體的耗氧速率上升，然而∆snf1酵母菌耗氧量上升的程度與WT相比增加較少。我們的研究結果顯示Snf1的確參與了葡萄糖缺乏下粒線體動態平衡的調控。進一步的釐清酵母菌中葡萄糖缺乏與SNF1訊息傳導路徑間的關聯，可幫助我們更加了解真核細胞中養分缺乏下的細胞調控機制。

Mitochondria play essential roles in cells. These highly dynamic organelles constantly undergo morphological changes among fragmented, tubular and hyperfused reticules by the balance of fission and fusion events. Mitochondrial dynamics respond to external environmental changes. Recent studies showed that glucose starvation induced mitochondrial elongation. However, the mechanisms regulating mitochondria dynamics under glucose starvation still remain elusive. In yeast, the Snf1 protein kinase plays a key role in the regulation of adaptation to glucose limitation. We hypothesized Snf1 is involved in regulating mitochondrial dynamics under glucose starvation condition. To examine our hypothesis, we classify mitochondrial morphology to study mitochondrial dynamics changes under glucose starvation, and we also analyzed mitochondrial membrane potentials (∆ψ_m) and oxygen consumption rate (OCR) to determine mitochondrial activity under glucose starvation. We found ∆snf1 yeast cells take longer response time to adjust glucose starvation. The ∆ψ_m of WT and ∆snf1 promptly showed a drop, and then increased gradually to a steady state within 30 minutes after glucose starvation initiated. However, the recovery ability of ∆ψ_m would be reduced in SNF1 deleted cells. Besides that, the OCR of yeast would increase in glucose starvation condition; but the ∆snf1 cells showed less increase under glucose starvation as compared to WT. Our findings reveal that Snf1 does participate in the regulation of mitochondrial dynamics under glucose starvation. Further clarification of the correlation between glucose starvation and SNF1 pathway is crucial for better understanding of the mechanisms promoting cell survival under nutrient-limiting conditions.

口試委員審定書…………………………………………………... #
致謝………………………………………………………………………... i
ABSTRACT………………………………………………….......... ii
中文摘要…………………………………………………………... iii
CONTENTS……………………………………………………..... iv
LIST OF FIGURES……………………………………………..... vii
LIST OF TABLE………………………………………………...... viii


Chapter 1. Introduction……………………………………………………………………………………………………………………………1
1.1 Mitochondria play essential roles in eukaryotic cells…………………………………1
1.1.1 Mitochondria produce energy through the process of cellular respiration………………………………………………………………………………………………………………………………………………………………….1
1.1.2 Mitochondrial dynamics control mitochondrial morphology and maintain mitochondrial function…..………………………………………………………………………………………………………2
1.1.3 Mitochondrial dynamics is mediated by fission/ fusion proteins……………………………………………………………………………………………………………………………………………………………………………4
1.1.4 Mitochondrial dynamics is important for mitochondrial homeostasis and quality control………………………………………………………………………………………………………………………………………………5
1.1.5 Dysregulation of mitochondrial dynamic is linked to neurodegenerative disease………………………………………………………………………………………………………………………………6
1.2 Glucose starvation induces mitochondria elongation…………………………………………7

1.3 Snf1 plays a key role in the utilization of alternative carbon sources in yeast………………………………………………………………………………………………………………………………………………………7
1.4 Specific Aim………………………………………………………………………………………………………………………………………………9

Chapter 2. Materials and Methods…………………………………………………………………………………………………10
2.1 Yeast strains and plasmid…………………………………………………………………………………………………………10
2.2 Experiment protocol for glucose starvation……………………………………………………………10
2.3 Mitochondrial morphology observation and classification…………………………11
2.4 Microfluidic perfusion system………………………………………………………………………………………………11
2.5 Mitochondrial membrane potential detection……………………………………………………………12
2.6 Measurements of oxygen consumption rate assay……………………………………………………13

Chapter 3. Results………………………………………………………………………………………………………………………………………14
3.1 Glucose starvation results in slower growth rate in yeast……………………14
3.2 Mitochondria turn hyperfused under glucose starvation in yeast…………………………………………………………………………………………………………………………………………………………………………………14
3.2.1 Snf1 is critical for morphological changes of mitochondria during glucose starvation……………………………………………………………………………………………………………………………………………………………………15
3.2.2 Fzo1 is required for morphology shift of mitochondria under glucose starvation.……………………………………………………………………………………………………………………………………………17
3.2.3 Glucose starvation-induced mitochondrial elongation remains in the absence of fission/fusion proteins…………………………………………………………………………………………18
3.3 Microfluidic system facilitates a stable medium exchange, and reduces the concerns of composition changes in medium.………………………………………………19

3.4 The effects of glucose starvation on mitochondria membrane potential………………………………………………………………………………………………………………………………………………………………………21
3.4.1 Deletion of SNF1 reduces mitochondrial membrane potential recovery under glucose starvation………………………………………………………………………………………………………21
3.4.2 The absence of Dnm1 and Fzo1 does not affect mitochondrial membrane potential changes under glucose starvation……………………………………………………23
3.5 ∆snf1 shows less increase in mitochondrial oxygen consumption under glucose starvation as compared to WT………………………………………………………………………………………………23

Chapter 4. Discussions……………………………………………………………………………………………………………………………26
4.1 Snf1 is required for mitochondria to respond to glucose starvation……………………………………………………………………………………………………………………………………………………………………26
4.2 Regulations of mitochondrial dynamics and membrane potential under glucose starvation may involve different signaling pathway…………………………………………………………………………………………………………………………………………………………………………27
4.3 Mitochondrial membrane potential changes can promptly reflect the effect of glucose depletion.……………………………………………………………………………………………………………………28
4.4 Low cell density cultures exclude the impact of nutrients insufficient and waste accumulation…………………………………………………………………………………………………29
4.5 Cells cultured under microfluidic perfusion system displayed different mitochondrial morphologies………………………………………………………………………………………………29

Chapter 5. Conclusions and Prospective…………………………………………………………………………………31
Reference……………………………………………………………………………………………………………………………………………………………………32

Aung-Htut, M.T., Y.T. Lam, Y.-L. Lim, M. Rinnerthaler, C.L. Gelling, H. Yang, M. Breitenbach, and I.W. Dawes. 2013. Maintenance of mitochondrial morphology by autophagy and its role in high glucose effects on chronological lifespan of Saccharomyces cerevisiae. Oxidative medicine and cellular longevity. 2013.
Baker, B.M., and C.M. Haynes. 2011. Mitochondrial protein quality control during biogenesis and aging. Trends in biochemical sciences. 36:254-261.
Baloyannis, S.J. 2006. Mitochondrial alterations in Alzheimer's disease. Journal of Alzheimer's disease. 9:119-126.
Bossy-Wetzel, E., M.J. Barsoum, A. Godzik, R. Schwarzenbacher, and S.A. Lipton. 2003. Mitochondrial fission in apoptosis, neurodegeneration and aging. Current opinion in cell biology. 15:706-716.
Brandt, T., L. Cavellini, W. Kühlbrandt, and M.M. Cohen. 2016. A mitofusin-dependent docking ring complex triggers mitochondrial fusion in vitro. Elife. 5:e14618.
Bratic, I., and A. Trifunovic. 2010. Mitochondrial energy metabolism and ageing. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 1797:961-967.
Brown, W.M., M. George, and A.C. Wilson. 1979. Rapid evolution of animal mitochondrial DNA. Proceedings of the National Academy of Sciences. 76:1967-1971.
Carlson, M. 1998. Regulation of glucose utilization in yeast. Current opinion in genetics & development. 8:560-564.
Carlson, M. 1999. Glucose repression in yeast. Current opinion in microbiology. 2:202-207.
Chen, H., and D.C. Chan. 2009. Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases. Human molecular genetics. 18:R169-R176.
Chen, H., A. Chomyn, and D.C. Chan. 2005. Disruption of fusion results in mitochondrial heterogeneity and dysfunction. Journal of Biological Chemistry. 280:26185-26192.
Deng, H., M.W. Dodson, H. Huang, and M. Guo. 2008. The Parkinson's disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila. Proceedings of the National Academy of Sciences. 105:14503-14508.
Detmer, S.A., and D.C. Chan. 2007. Functions and dysfunctions of mitochondrial dynamics. Nature reviews Molecular cell biology. 8:870-879.
Frey, T.G., and C.A. Mannella. 2000. The internal structure of mitochondria. Trends in biochemical sciences. 25:319-324.
Gomes, L.C., G. Di Benedetto, and L. Scorrano. 2011. During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nature cell biology. 13:589-598.
Hardie, D.G. 2007. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nature reviews Molecular cell biology. 8:774-785.
Jiang, R., and M. Carlson. 1996. Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes & development. 10:3105-3115.
Johnston, M. 1999. Feasting, fasting and fermenting: glucose sensing in yeast and other cells. Trends in Genetics. 15:29-33.
Kim, J.-H., A. Roy, D. Jouandot, and K.H. Cho. 2013. The glucose signaling network in yeast. Biochimica et Biophysica Acta (BBA)-General Subjects. 1830:5204-5210.
Kim, J.-S., B. Kim, H. Lee, S. Thakkar, D.M. Babbitt, S. Eguchi, M.D. Brown, and J.-Y. Park. 2015. Shear stress-induced mitochondrial biogenesis decreases the release of microparticles from endothelial cells. American Journal of Physiology-Heart and Circulatory Physiology. 309:H425-H433.
Koshiba, T., S.A. Detmer, J.T. Kaiser, H. Chen, J.M. McCaffery, and D.C. Chan. 2004. Structural basis of mitochondrial tethering by mitofusin complexes. Science. 305:858-862.
Lee, P.J., N. Ghorashian, T.A. Gaige, and P.J. Hung. 2007. Microfluidic system for automated cell-based assays. JALA: Journal of the Association for Laboratory Automation. 12:363-367.
Lee, P.J., N.C. Helman, W.A. Lim, and P.J. Hung. 2008. A microfluidic system for dynamic yeast cell imaging. Biotechniques. 44:91-95.
Liesa, M., and O.S. Shirihai. 2013. Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure. Cell metabolism. 17:491-506.
Liu, Y., G. Fiskum, and D. Schubert. 2002. Generation of reactive oxygen species by the mitochondrial electron transport chain. Journal of neurochemistry. 80:780-787.
Ludin, K., R. Jiang, and M. Carlson. 1998. Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences. 95:6245-6250.
Ludovico, P., F. Sansonetty, and M. Côrte-Real. 2001. Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry. Microbiology. 147:3335-3343.
Martínez-Reyes, I., L.P. Diebold, H. Kong, M. Schieber, H. Huang, C.T. Hensley, M.M. Mehta, T. Wang, J.H. Santos, and R. Woychik. 2016. TCA cycle and mitochondrial membrane potential are necessary for diverse biological functions. Molecular cell. 61:199-209.
Matsushima, Y., and L.S. Kaguni. 2012. Matrix proteases in mitochondrial DNA function. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms. 1819:1080-1087.
Mishra, P., and D.C. Chan. 2014. Mitochondrial dynamics and inheritance during cell division, development and disease. Nature reviews Molecular cell biology. 15:634-646.
Molina, A.J., J.D. Wikstrom, L. Stiles, G. Las, H. Mohamed, A. Elorza, G. Walzer, G. Twig, S. Katz, and B.E. Corkey. 2009. Mitochondrial networking protects β-cells from nutrient-induced apoptosis. Diabetes. 58:2303-2315.
Mozdy, A., J. McCaffery, and J. Shaw. 2000. Dnm1p GTPase-mediated mitochondrial fission is a multi-step process requiring the novel integral membrane component Fis1p. The Journal of cell biology. 151:367-380.
Ni, H.-M., J.A. Williams, and W.-X. Ding. 2015. Mitochondrial dynamics and mitochondrial quality control. Redox biology. 4:6-13.
Otsuga, D., B.R. Keegan, E. Brisch, J.W. Thatcher, G.J. Hermann, W. Bleazard, and J.M. Shaw. 1998. The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast. The Journal of cell biology. 143:333-349.
Perry, C.G., D.A. Kane, I.R. Lanza, and P.D. Neufer. 2013. Methods for assessing mitochondrial function in diabetes. Diabetes. 62:1041-1053.
Perry, S.W., J.P. Norman, J. Barbieri, E.B. Brown, and H.A. Gelbard. 2011. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques. 50:98.
Pesta, D., and E. Gnaiger. 2012. High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Mitochondrial bioenergetics: methods and protocols:25-58.
Rambold, A.S., B. Kostelecky, N. Elia, and J. Lippincott-Schwartz. 2011. Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. Proceedings of the National Academy of Sciences. 108:10190-10195.
Rolland, F., J. Winderickx, and J.M. Thevelein. 2001. Glucose-sensing mechanisms in eukaryotic cells. Trends in biochemical sciences. 26:310-317.
Salvioli, S., A. Ardizzoni, C. Franceschi, and A. Cossarizza. 1997. JC‐1, but not DiOC6 (3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨ changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS letters. 411:77-82.
Sjöstrand, F.S., and V. Hanzon. 1954. Membrane structures of cytoplasm and mitochondria in exocrine cells of mouse pancreas as revealed by high resolution electron microscopy. Experimental cell research. 7:393-414.
Sugioka, R., S. Shimizu, and Y. Tsujimoto. 2004. Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis. Journal of Biological Chemistry. 279:52726-52734.
Tait, S.W., and D.R. Green. 2012. Mitochondria and cell signalling. J Cell Sci. 125:807-815.
Twig, G., A. Elorza, A.J. Molina, H. Mohamed, J.D. Wikstrom, G. Walzer, L. Stiles, S.E. Haigh, S. Katz, and G. Las. 2008. Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. The EMBO journal. 27:433-446.
van der Bliek, A.M., Q. Shen, and S. Kawajiri. 2013. Mechanisms of mitochondrial fission and fusion. Cold Spring Harbor perspectives in biology. 5:a011072.
Wai, T., and T. Langer. 2016. Mitochondrial dynamics and metabolic regulation. Trends in Endocrinology & Metabolism. 27:105-117.
Wang, H., P.J. Lim, M. Karbowski, and M.J. Monteiro. 2008. Effects of overexpression of huntingtin proteins on mitochondrial integrity. Human molecular genetics. 18:737-752.
Westermann, B., and W. Neupert. 2000. Mitochondria‐targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae. Yeast. 16:1421-1427.
Wilson, W.A., S.A. Hawley, and D.G. Hardie. 1996. Glucose repression/derepression in budding yeast: SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP: ATP ratio. Current Biology. 6:1426-1434.
Youle, R.J., and A.M. Van Der Bliek. 2012. Mitochondrial fission, fusion, and stress. Science. 337:1062-1065.