細胞內有各式各樣的膜狀胞器或囊泡，以便分工執行各種生理活動，同時也作為離子儲存的場所，以維持胞內離子恆定。多數膜狀構造多在1 um以下，並藉由分裂與融合機制，維持其動態平衡。然而一般螢光顯微鏡無法提供適當解析度，以觀察這些膜狀構造的動態變化。在本實驗中，我們在顯微鏡相機成像前，加裝一組圓柱狀透鏡，改變螢光亮點在不同聚焦面的成像形狀，再以函數計算，還原此亮點在空間中正確的位置，其精確度可達0.1 um。我們先以H2O2刺激大鼠腎上腺髓質嗜鉻細胞瘤細胞 (PC12 cell)，提高胞內鋅離子濃度，以觸發自噬體的產生，並以綠螢光蛋白修飾的微管相關蛋白1A/1B輕鏈3 (Microtubule-associated protein 1A/1B-light chain 3, LC3) 標記自噬體。結果顯示我們能成功追蹤到自噬體的形成與增大，且觀測到的自噬體平均大小成長為41.0 ± 8.5％。由於鋅離子恆定的重要性，以鋅離子螢光染劑，標記初級培養的大鼠胚胎皮質神經細胞中的鋅離子分布，發現在一些細胞中有高濃度的鋅離子聚集，以鋅離子螯合劑處理後，這些聚集亮點的型態迅速萎縮且螢光強度逐漸消散，其形態大小平均下降47.3 ± 8.3％，而螢光強度平均下降了32.0 ± 3.5％。顯示鋅離子對於維持含鋅顆粒的完整性很重要。有些研究報導溶小體 (lysosome) 也會聚集鋅離子，且溶小體大小與前述鋅離子聚集點相當，因此我們以溶小體染劑處理神經細胞，再加上鋅離子螯合劑，結果顯示溶小體的型態以及螢光強度也會快速萎縮，大小平均下降59.0 ± 6.9％，而螢光強度平均下降44.3 ± 7.0％。顯示鋅離子對於維持溶小體的完整性更為重要。為進一步了解鋅離子螯合劑所造成的溶小體形態變化，與其分裂融合機制的關係，我們先以管化 (tubulation) 抑制劑處理細胞以抑制其分裂，發現溶小體在鋅離子螯合劑處理後，其形態以及螢光消散的程度明顯地降低，大小平均下降16.0 ± 4.6％，而螢光強度平均下降29.3 ± 4.6％。顯示了鋅離子螯合所導致的萎縮和溶小體管化有很大的關係，抑制管化可以阻止鋅離子螯合所導致的萎縮。這些結果顯示鋅離子對維持溶小體的動態平衡扮演重要的角色，缺乏鋅離子，會加速溶小體的分裂，導致其變小。因此我們所用的技術，可以穩定追蹤胞內相關膜狀構造的動態變化，以探討相關生理功能。

Cells utilize various membrane-enclosed vesicles to execute different physiological activities. These vesicles are intracellular stores of ions to maintain the ionic homeostasis in the cells. Most membrane-enclosed structures are smaller than 1 um and their dynamic equilibrium is maintained by fission and fusion. However, an ordinary fluorescence microscope doesn’t have enough resolution to monitor the dynamic changes of these membrane-enclosed structures. In this study, we introduced a 3D tracking microscopic technique with a cylindrical lens before the camera to produce astigmatism. With this technique, the shape of a fluorescent particle varies in different focal planes. Recording the shape and orientation of the fluorescent particle and calculating the correlating mathematical functions, we can determine the location of the fluorescent particle with the accuracy of ~100 nm. First, we stimulated PC12 cell with H2O2 to escalate the concentration of zinc ions in the cell and accelerate the formation of autophagosomes. Meanwhile, we also tagged a well-known autophagosome marker of the microtubule-associated protein 1A/1B-light chain 3 (LC3-Ⅱ) with green fluorescent proteins (GFP). The experimental results showed that we could successfully monitor the formation and enlargement of autophagosomes. The average enlargement of the autophagosomes is 41.0 ± 8.5％. Moreover, we monitored the distribution of zinc ions in cultured neuron cells with the aid of zinc fluorescent dye. We found intracellular zinc aggregation in some cells. However, after the zinc chelator treatment, these fluorescent aggregation points dispersed dramatically with the reduction of morphological size by 47.3 ± 8.3％ and the fluorescence intensity by 32.0 ± 3.5％, indicating the importance of zinc for maintaining the completeness of zinc-containing puncta. It has been reported that zinc aggregates in lysosomes with the size of the lysosomes similar to the zinc aggregation points. Therefore, we labeld the lysosomes with lysotracker and monitored their morphological changes after zinc chelation. The result observed in lysosomes is similar to the zinc aggregation points with the dramatic reduction of morphological size by 59.0 ± 6.9％and fluorescence intensity by 44.3 ± 7.0％, indicating that zinc plays crucial roles for maintaining the completeness of lysosomes. To further understand the morphological changes of lysosomes caused by zinc chelation and their relationships between fission and fusion, we treated the cells with a tubulation inhibitor to inhibit the lysosomal fission. After the zinc chelation, the measured shrinkage of lysosomes was insignificant with the decreases of morphological size only by 16.0 ± 4.6％ and fluorescence intensity by 29.3 ± 4.6％, revealing the relation of lysosomal tubulation to its morphological changes after zinc chelation. The inhibition of tubulation can prevent the shrinkage of lysosomes caused by zinc chelation. These results showed that zinc ions play a vital role in maintaining the dynamic equilibrium of lysosomes, in which zinc deficiency will accelerate the fission of lysosome and lead to the shrinkage of lysosomes. Finally, our 3D tracking microscopic technique allowed us to monitor the dynamic changes of intracellular membrane-enclosed structures and explore the related physiological activities

口試委員會審定書……………………………………………………………………………..…………Ⅰ
誌謝……………………………………………………………………………………………………….….…..Ⅱ
中文摘要…………………………………………………………………………..…………………………..Ⅲ
Abstract ……………………………………………………………………………………….………………..Ⅴ
目錄……………………………………………………………………………………….……………………...Ⅷ
圖目錄…………………………………………………..…………………………….……………………..…Ⅹ
表目錄…………………………………………………..…………………………….…………………...…XII
第一章 序論…………………..……………………………………………….……………………………. 1
1. 巨自噬作用的重要性…………………………………..….……………………………... 1
1.1. 蛋白質分解系統………………………………….………………………………..1
1.2. 自噬作用和疾病的關聯…………………………………….………………….4
1.3. 微管相關蛋白1A/1B輕鏈3………………………………….……………..6
1.4. 自噬體的產生速率、大小及數量………………………………………..8
1.5. 自噬體和溶小體的關係……………….……………………………….………8
2. 溶小體的重要性……………………………………………………………………………..11
2.1. 溶小體的生理作用…………………………….………………………………..11
2.2. 溶小體相關的疾病……………………………….……………………………..14
3. 鋅離子的生理功用………………………………………………………………………...14
3.1. 鋅離子對人體的生理重要性……………………………………………...14
3.2. 鋅離子的生化特性………………………………………….………………..…15
3.3. 鋅離子在人體內的調節與恆定…………………………………………..17
4. 三維追蹤顯微技術……………………………………………………………………..….20
5. 研究動機……………………………….………………………………………………………..22
第二章 實驗方法………………………………………….……………………………….……………23
1. 螢光顯微鏡………………………………….…………………………………………………23
2. 細胞培養與處理……………………….……………………………………………………..23
第三章 實驗結果與討論…………………………………….………………………………………25
1. 三維追蹤顯微技術……………………………….………………………………………….25
2. 氧化壓力下自噬體的形成與形變……………………………….…………………..28
3. 鋅離子螯合導致含鋅顆粒的消散………………………………….…………….….30
4. 鋅離子螯合造成溶小體萎縮………………………….………………………………..34
5. 抑制溶小體管化可阻止鋅離子螯合所導致的萎縮…………………………39
6. 討論………………………….………………………………………………………………………42
第四章 結論……………………………….……………………………………………………………….44
參考文獻…………………………………….………………………………………………………………..45

1.Nedelsky, N.B., P.K. Todd, and J.P. Taylor, Autophagy and the ubiquitin-proteasome system: Collaborators in neuroprotection. Biochimica Et Biophysica Acta-Molecular Basis of Disease, 2008. 1782(12): p. 691-699.
2.Tanida, I., Autophagosome Formation and Molecular Mechanism of Autophagy. Antioxidants & Redox Signaling, 2011. 14(11): p. 2201-2214.
3.Eskelinen, E.L. and P. Saftig, Autophagy: A lysosomal degradation pathway with a central role in health and disease. Biochimica Et Biophysica Acta-Molecular Cell Research, 2009. 1793(4): p. 664-673.
4.Feng, Y.C., et al., Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation. Autophagy, 2016. 12(4): p. 648-658.
5.Xie, Z.P., U. Nair, and D.J. Klionsky, Atg8 controls phagophore expansion during autophagosome formation. Molecular Biology of the Cell, 2008. 19(8): p. 3290-3298.
6.Hikita, H., S. Sakane, and T. Takehara. Mechanisms of the autophagosome-lysosome fusion step and its relation to non-alcoholic fatty liver disease. Liver Research 2018 [cited 2 3]; 120-124].
7.Itakura, E., C. Kishi-Itakura, and N. Mizushima, The Hairpin-type Tail-Anchored SNARE Syntaxin 17 Targets to Autophagosomes for Fusion with Endosomes/Lysosomes. Cell, 2012. 151(6): p. 1256-1269.
8.Tai, H.C. and E.M. Schuman, Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction. Nature Reviews Neuroscience, 2008. 9(11): p. 826-838.
9.Zoncu, R., et al., mTORC1 Senses Lysosomal Amino Acids Through an Inside-Out Mechanism That Requires the Vacuolar H+-ATPase. Science, 2011. 334(6056): p. 678-683.
10.Yu, L., et al., Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature, 2010. 465(7300): p. 942-U11.
11.Zhou, J., et al., Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion. Cell Research, 2013. 23(4): p. 508-523.
12.I, M., Organelles observed: lysosomes. Science, 1989: p. 853-854.
13.Xu, H.X. and D.J. Ren, Lysosomal Physiology, in Annual Review of Physiology, Vol 77, D. Julius, Editor. 2015. p. 57-80.
14.Kambe, T., et al., THE PHYSIOLOGICAL, BIOCHEMICAL, AND MOLECULAR ROLES OF ZINC TRANSPORTERS IN ZINC HOMEOSTASIS AND METABOLISM. Physiological Reviews, 2015. 95(3): p. 749-784.
15.J., R., Etudes clinique sur la vegetation. Annales des Scienceas Naturelle: Botanique, 1869. 11: p. 93–299.
16.Prasad, A.S., CLINICAL MANIFESTATIONS OF ZINC-DEFICIENCY. Annual Review of Nutrition, 1985. 5: p. 341-365.
17.Hambidge, M., Human zinc deficiency. Journal of Nutrition, 2000. 130(5): p. 1344S-1349S.
18.Maret, W. and H.H. Sandstead, Zinc requirements and the risks and benefits of zinc supplementation. Journal of Trace Elements in Medicine and Biology, 2006. 20(1): p. 3-18.
19.Takeda, A. and H. Tamano, Insight into zinc signaling from dietary zinc deficiency. Brain Research Reviews, 2009. 62(1): p. 33-44.
20.Sandstead, H.H., Human Zinc Deficiency: Discovery to Initial Translation. Advances in Nutrition, 2013. 4(1): p. 76-81.
21.McCord, M.C. and E. Aizenman, The role of intracellular zinc release in aging, oxidative stress, and Alzheimer''s disease. Frontiers in Aging Neuroscience, 2014. 6.
22.Bonilha, V.L., Age and disease-related structural changes in the retinal pigment epithelium. Clinical Ophthalmology, 2008. 2: p. 413-424.
23.Julien, S., et al., Zinc Deficiency Leads to Lipofuscin Accumulation in the Retinal Pigment Epithelium of Pigmented Rats. Plos One, 2011. 6(12).
24.Erie, J.C., et al., Reduced Zinc and Copper in the Retinal Pigment Epithelium and Choroid in Age-related Macular Degeneration. American Journal of Ophthalmology, 2009. 147(2): p. 276-282.
25.Smailhodzic, D., et al., Zinc Supplementation Inhibits Complement Activation in Age-Related Macular Degeneration. Plos One, 2014. 9(11).
26.Broun, E.R., et al., EXCESSIVE ZINC INGESTION - A REVERSIBLE CAUSE OF SIDEROBLASTIC ANEMIA AND BONE-MARROW DEPRESSION. Jama-Journal of the American Medical Association, 1990. 264(11): p. 1441-1443.
27.DA, S. and F. AM, The insulin and the zinc content of normal and diabetic pancreas. J Clin Invest, 1938. 17: p. 725-728.
28.Miller, J., A.D. McLachlan, and A. Klug, Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes (Reprinted from EMBO Journal, vol 4, pg 1609-1614, 1985). Journal of Trace Elements in Experimental Medicine, 2001. 14(2): p. 157-169.
29.Maret, W., Zinc and sulfur: A critical biological partnership. Biochemistry, 2004. 43(12): p. 3301-3309.
30.Keilin, D. and T. Mann, Carbonic anhydrase. Nature, 1939. 144: p. 442-443.
31.Kochanczyk, T., A. Drozd, and A. Krezel, Relationship between the architecture of zinc coordination and zinc binding affinity in proteins - insights into zinc regulation. Metallomics, 2015. 7(2): p. 244-257.
32.Sensi, S.L., et al., Zinc in the physiology and pathology of the CNS. Nature Reviews Neuroscience, 2009. 10(11): p. 780-U38.
33.Frederickson, C.J., J.Y. Koh, and A.I. Bush, The neurobiology of zinc in health and disease. Nature Reviews Neuroscience, 2005. 6(6): p. 449-462.
34.Tamaki, M., et al., The diabetes-susceptible gene SLC30A8/ZnT8 regulates hepatic insulin clearance. Journal of Clinical Investigation, 2013. 123(10): p. 4513-4524.
35.Ghafghazi, T., M.L. McDaniel, and P.E. Lacy, ZINC-INDUCED INHIBITION OF INSULIN-SECRETION FROM ISOLATED RAT ISLETS OF LANGERHANS. Diabetes, 1981. 30(4): p. 341-345.
36.Hirano, T., et al., Roles of zinc and zinc signaling in immunity: Zinc as an intracellular signaling molecule, in Advances in Immunology, Vol 97, F.W. Alt, et al., Editors. 2008. p. 149-176.
37.Taylor, K.M., et al., Protein Kinase CK2 Triggers Cytosolic Zinc Signaling Pathways by Phosphorylation of Zinc Channel ZIP7. Science Signaling, 2012. 5(210).
38.Aras, M.A. and E. Aizenman, Redox Regulation of Intracellular Zinc: Molecular Signaling in the Life and Death of Neurons. Antioxidants & Redox Signaling, 2011. 15(8): p. 2249-2263.
39.Haase, H., et al., Zinc Signals Are Essential for Lipopolysaccharide-Induced Signal Transduction in Monocytes. Journal of Immunology, 2008. 181(9): p. 6491-6502.
40.Kitamura, H., et al., Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function. Nature Immunology, 2006. 7(9): p. 971-977.
41.Brautigan, D.L., P. Bornstein, and B. Gallis, PHOSPHOTYROSYL-PROTEIN PHOSPHATASE - SPECIFIC-INHIBITION BY ZN-2+. Journal of Biological Chemistry, 1981. 256(13): p. 6519-6522.
42.Hojyo, S., et al., The Zinc Transporter SLC39A14/ZIP14 Controls G-Protein Coupled Receptor-Mediated Signaling Required for Systemic Growth. Plos One, 2011. 6(3).
43.Velazquez-Delgado, E.M. and J.A. Hardy, Zinc-mediated Allosteric Inhibition of Caspase-6. Journal of Biological Chemistry, 2012. 287(43): p. 36000-36011.
44.Csermely, P., et al., ZINC CAN INCREASE THE ACTIVITY OF PROTEIN KINASE-C AND CONTRIBUTES TO ITS BINDING TO PLASMA-MEMBRANES IN LYMPHOCYTES-T. Journal of Biological Chemistry, 1988. 263(14): p. 6487-6490.
45.Haase, H. and W. Maret, Intracellular zinc fluctuations modulate protein tyrosine phosphatase activity in insulin/insulin-like growth factor-1 signaling. Experimental Cell Research, 2003. 291(2): p. 289-298.
46.Huber, K.L. and J.A. Hardy, Mechanism of zinc-mediated inhibition of caspase-9. Protein Science, 2012. 21(7): p. 1056-1065.
47.MJ, J., Physiology of Zinc: General Aspects. Zinc in Human Biology, 1989: p. 1-14.
48.Barnett, J.P., et al., Allosteric modulation of zinc speciation by fatty acids. Biochimica Et Biophysica Acta-General Subjects, 2013. 1830(12): p. 5456-5464.
49.Haase, H. and L. Rink, Zinc signals and immune function. Biofactors, 2014. 40(1): p. 27-40.
50.Park, J.G., et al., New Sensors for Quantitative Measurement of Mitochondrial Zn2+. Acs Chemical Biology, 2012. 7(10): p. 1636-1640.
51.Qin, Y., et al., Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn2+ with genetically encoded sensors. Proceedings of the National Academy of Sciences of the United States of America, 2011. 108(18): p. 7351-7356.
52.Chabosseau, P., et al., Mitochondrial and ER-Targeted eCALWY Probes Reveal High Levels of Free Zn2+. Acs Chemical Biology, 2014. 9(9): p. 2111-2120.
53.Fukada, T. and T. Kambe, Molecular and genetic features of zinc transporters in physiology and pathogenesis. Metallomics, 2011. 3(7): p. 662-674.
54.Huang, B., et al., Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science, 2008. 319(5864): p. 810-813.
55.Matias, A.C., T.M. Manieri, and G. Cerchiaro, Zinc Chelation Mediates the Lysosomal Disruption without Intracellular ROS Generation. Oxidative Medicine and Cellular Longevity, 2016.