臺灣博碩士論文加值系統

在真核細胞內，粒線體除合成ATP外，還有調控細胞內鈣離子濃度及細胞凋亡等重要功能。為因應細胞內局部區域對ATP與鈣離子緩衝的需求，細胞仰賴特殊的機制來調控粒線體的分布與運輸。由於神經細胞特殊的形態與代謝需求，粒線體在神經細胞內的分布與運輸尤具挑戰性。在神經細胞內，粒線體在細胞本體生成後，會被運送到細胞內高ATP需求的區域，如突觸跟生長錐(growth cone)來供應足夠的ATP。老化及損傷的粒線體則會被運送回細胞本體修復或分解。此類長距離的雙向運輸主要由細胞骨架中微管上的驅動蛋白(kinesin)和肌凝蛋白(dynein)達成，而短距離的粒線體運輸及固定則由微絲及微絲上的肌球蛋白(myosin)達成。雖同為細胞骨架的一員，因為不具極性和相關聯的動力蛋白，中間絲在粒線體運輸中所扮演的角色較不明朗。過去的研究指出，中間絲可能是粒線體在維持靜止狀態時所需的固著點，或者中間絲可能透過微管上的動力蛋白來影響粒線體運輸。
在此篇研究中，我們探討秀麗隱桿線蟲(C.elegans)的中間絲蛋白IFB-1在amphid神經細胞中對粒線體運輸的影響。Amphid為位於線蟲頭部左右兩側的感覺神經。我們建立了在amphid中帶有粒線體螢光蛋白標記的野生型與ifb-1突變線蟲，並利用縮時攝影(time-lapse imaging)記錄粒線體在線蟲蟲體及分離的線蟲神經細胞中的動態。我們發現IFB-1突變造成靜止狀態的粒線體比例增加，顯示IFB-1可能參與調控粒線體在靜止狀態和移動狀態間的平衡。此外，失去IFB-1其中一個isoform不僅降低粒線體運輸速率和粒線體改變方向的頻率，還增加粒線體停頓的頻率和粒線體每次移動所持續的時間。由此複雜的結果，我們推測IFB-1可能透過驅動蛋白或其相關分子來影響粒線體運輸。 

Mitochondria are essential for survival of eukaryotic cells since they are responsible for ATP synthesis, calcium buffering and apoptosis. Thus, specialized transport and anchoring mechanisms are necessary to distribute and position mitochondria in response to local needs for ATP and calcium buffering. Mitochondrial transport and positioning are particularly challenging in neurons because of the unique geometry and metabolic needs of neurons. In neurons, mitochondria are transported from the soma, where mitochondria biogenesis takes place, to ATP-demanding sites as synaptic terminals and growth cones. Mitochondria remain at these sites until they are transported back to the soma for recycling or degradation. This long-range bidirectional transport mainly depends on microtubules and microtubule-based motor proteins as kinesins and dynein, while short-range mitochondrial transport and docking mainly rely on actin and actin-based motors myosins. In contrast to actin and microtubules, intermediate filaments (IFs) do not possess polarity or associated motor proteins. Therefore, how IFs take part in mitochondrial positioning is relatively unclear. Previous studies suggest that IFs may serve as docking sites for mitochondria, or affect mitochondrial transport through microtubule-based motors.
In this study, we investigate the functions of IFB-1, a C. elegans IF protein, in the mitochondrial transport system of C. elegans amphid neurons. Amphids are sensory organs consisting of a pair of sensilla running along the lateral sides of the worm head. We established wild-type and ifb-1 mutant worms carrying fluorescently-labeled mitochondria in the amphid neurons, and recorded time-lapse images from these worms and their isolated neuronal cells. We found that mutations of IFB-1 lead to larger pools of stationary mitochondria, suggesting a role of IFB-1 in the balance of stationary phases and motile phases of mitochondrial transport. In addition, loss of one isoform of IFB-1 reduces mitochondrial transport velocities and frequencies of changes of directions, but increases pausing frequencies as well as moving persistencies. To explain these complex effects of IFB-1 on mitochondrial motility, we propose a model in which IFB-1 influences mitochondrial transport through affecting kinesin or kinesin-associated proteins.

摘要 2
Abstract 3
1 Introduction 8
1.1 Transport of mitochondria in neurons 8
1.1.1 Significance of proper mitochondrial positioning in neurons 8
1.1.2 Microtubule-based mitochondrial transport 8
1.1.3 Actin-based mitochondrial transport 10
1.1.4 Regulation of mitochondrial transport 11
1.1.5 Intermediate filaments and mitochondrial transport 12
1.2 C. elegans intermediate filament protein IFB-1 13
1.3 C. elegans amphid sensory neurons 13
2 Materials and Methods 15
2.1 Reagents 15
2.1.1 M9 buffer 15
2.1.2 Nematode growth medium (NGM) agar plates 15
2.1.3 Peptone-rich agar plates 15
2.1.4 Microinjection buffer 10X 16
2.1.5 Bleaching solution 16
2.1.6 Egg buffer 16
2.1.7 Chitinase solution 16
2.1.8 Neuronal cell culture medium 16
2.1.9 Lectin coating 17
2.2 Maintenance 17
2.3 DNA extraction from C. elegans 17
2.4 C. elegans strains 17
2.5 Plasmid cloning 18
2.6 Microinjection 18
2.7 Primary neuronal cell culture of C. elegans 19
2.8 Microscopy on whole worms 20
2.9 Microscopy on primary neuronal cell culture 20
2.10 Motility analysis and statistics 20
2.10.1 Generation of kymographs 20
2.10.2 Measurement mitochondrial length and density 20
2.10.3 Stationary, vibrating, and processive mitochondria 21
2.10.4 Analysis of mitochondrial motility 21
2.11 Structural and functional analysis of chemosensory neurons 22
2.11.1 Dye filling 22
2.11.2 Chemotaxis assay 22
3 Result 23
3.1 Cloning of Posm-5::Mito::GFP and generation of a transgenic C. elegans line 23
3.2 Mitochondrial motility in whole worms 24
3.2.1 Time-lapse imaging and generation of kymographs 24
3.2.2 Dosage of Posm-5::Mito::GFP does not influence mitochondrial motility 24
3.2.3 Mitochondrial motility is altered in ifb-1(ju71) worms 25
3.3 Observing mitochondrial length, density and motility at single cell level 26
3.3.1 Posm-5::Mito::GFP-expressing cells in primary neuronal cell cultures 26
3.3.2 Mitochondrial length and density in neurites of isolated neuronal cells 27
3.3.3 Three types of mitochondrial motility in cells 27
3.3.4 Motility analysis 28
3.4 Structural integrity of amphids in ifb-1 mutants 29
3.5 Functional integrity of amphids in ifb-1 mutants 29
4 Discussion 31
4.1 Neuronal IF proteins play roles in balancing mitochondria between different types of motions 31
4.2 IF proteins affect mitochondrial transport velocities 33
4.3 The effect of IFB-1 on mitochondrial pausing 34
4.4 The effect of IFB-1 on mitochondrial directional changes and moving persistencies 35
4.5 Future work 36
5 References 38
6 Figures 43
Figure 1. Neuronal mitochondrial transport and C. elegans amphids 44
Figure 2. Posm-5::Mito::GFP labels mitochondria in C. elegans sensory neurons 46
Figure 3. Posm-5::Mito::GFP expression in N2, ifb-1(ok1317), and ifb-1(ju71) worms. 48
Figure 4. Analysis of mitochondrial motility of N2, ifb-1(ok1317), and ifb-1(ju71) 49
Figure 5. Posm-5::Mito::GFP expression in primary neuronal cell culture and comparison of mitochondrial density and length in N2 and ifb-1 mutants 52
Figure 6. Mitochondrial motility in Posm-5::Mito::GFP-expressing isolated neurons 54
Figure 7. Structural and functional analysis of amphids in N2 and ifb-1 mutants 56
8 Appendix 57
8.1 Expression patterns, functions and available mutants of C. elegans IF proteins 57
8.2 Kymographs and motility analysis 58
8.2.1 Generating kymographs from time-lapse movies 58
8.2.2 Obtaining X and Y positions 59
8.2.3 Distinguishing three types of mitochondrial movements 60
8.2.4 Measuring mitochondrial length and mitochondrial density 62
8.3 Genotyping of transgenic ifb-1 mutants 63
8.3.1 Genotyping confirmed homozygosity for the ifb-1(ok1317) allele 63
8.3.2 Genotyping confirmed homozygosity for the ifb-1(ju71) allele 64
8.4 Primer sequences used in this study 65

WormBase WS220 16 Dec 2010 http://ws220.wormbase.org/

Altun, Z.F., Herndon, L.A., Crocker, C., Lints, R. and Hall, D.H. (ed.s) (2002-2010). WormAtlas.
Bargmann, C.I. (2006). Chemosensation in C. elegans. WormBook : the online review of C elegans biology, 1-29.
Bargmann, C.I., and Mori, I. (1997). Chemotaxis and Thermotaxis. In C elegans II, D.L. Riddle, T. Blumenthal, B.J. Meyer, and J.R. Priess, eds. (Cold Spring Harbor (NY)).
Boldogh, I.R., and Pon, L.A. (2007). Mitochondria on the move. Trends in cell biology 17, 502-510.
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.
Calkins, M.J., Manczak, M., Mao, P., Shirendeb, U., and Reddy, P.H. (2011). Impaired mitochondrial biogenesis, defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic degeneration in a mouse model of Alzheimer's disease. Human molecular genetics 20, 4515-4529.
Calkins, M.J., and Reddy, P.H. (2011). Amyloid beta impairs mitochondrial anterograde transport and degenerates synapses in Alzheimer's disease neurons. Biochimica et biophysica acta 1812, 507-513.
Carberry, K., Wiesenfahrt, T., Windoffer, R., Bossinger, O., and Leube, R.E. (2009). Intermediate filaments in Caenorhabditis elegans. Cell motility and the cytoskeleton 66, 852-864.
Chada, S.R., and Hollenbeck, P.J. (2004). Nerve growth factor signaling regulates motility and docking of axonal mitochondria. Curr Biol 14, 1272-1276.
Chang, D.T., Rintoul, G.L., Pandipati, S., and Reynolds, I.J. (2006). Mutant huntingtin aggregates impair mitochondrial movement and trafficking in cortical neurons. Neurobiol Dis 22, 388-400.
Chang, K.T., Niescier, R.F., and Min, K.T. (2011). Mitochondrial matrix Ca2+ as an intrinsic signal regulating mitochondrial motility in axons. Proc Natl Acad Sci USA 108, 15456-15461.
Chen, H., and Chan, D.C. (2009). Mitochondrial dynamics — fusion, fission, movement, and mitophagy — in neurodegenerative diseases. Hum Mol Genet 18, 169-176.
Chen, S., Owens, G.C., Crossin, K.L., and Edelman, D.B. (2007). Serotonin stimulates mitochondrial transport in hippocampal neurons. Mol Cell Neurosci 36, 472-483.
Chen, Y.M., Gerwin, C., and Sheng, Z.H. (2009). Dynein light chain LC8 regulates syntaphilin-mediated mitochondrial docking in axons. J Neurosci 29, 9429-9438.
Christensen, M., Estevez, A., Yin, X., Fox, R., Morrison, R., McDonnell, M., Gleason, C., Miller, D.M., 3rd, and Strange, K. (2002). A primary culture system for functional analysis of C. elegans neurons and muscle cells. Neuron 33, 503-514.
Detmer, S.A., and Chan, D.C. (2007). Functions and dysfunctions of mitochondrial dynamics. Nature reviews Molecular cell biology 8, 870-879.
Dixit, R., Ross, J.L., Goldman, Y.E., and Holzbaur, E.L. (2008). Differential regulation of dynein and kinesin motor proteins by tau. Science 319, 1086-1089.
Driscoll, M., and Kaplan, J. (1997). Mechanotransduction. In C elegans II, D.L. Riddle, T. Blumenthal, B.J. Meyer, and J.R. Priess, eds. (Cold Spring Harbor (NY)).
Gentil, B.J., Minotti, S., Beange, M., Baloh, R.H., Julien, J.P., and Durham, H.D. (2012). Normal role of the low-molecular-weight neurofilament protein in mitochondrial dynamics and disruption in Charcot-Marie-Tooth disease. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 26, 1194-1203.
Glater, E.E., Megeath, L.J., Stowers, R.S., and Schwarz, T.L. (2006). Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent. J Cell Biol 173, 545-557.
Haghnia, M. (2007). Dynactin is required for coordinated bidirectional motility, but not for dynein membrane attachment. Mol Biol Cell 18, 2081-2089.
Haycraft, C.J., Swoboda, P., Taulman, P.D., Thomas, J.H., and Yoder, B.K. (2001). The C. elegans homolog of the murine cystic kidney disease gene Tg737 functions in a ciliogenic pathway and is disrupted in osm-5 mutant worms. Development 128, 1493-1505.
Hirokawa, N. (1982). Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J Cell Biol 94, 129-142.
Hollenbeck, P.J., and Saxton, W.M. (2005). The axonal transport of mitochondria. J Cell Sci 118, 5411-5419.
Horiuchi, D., Barkus, R.V., Pilling, A.D., Gassman, A., and Saxton, W.M. (2005). APLIP1, a kinesin binding JIP-1/JNK scaffold protein, influences the axonal transport of both vesicles and mitochondria in Drosophila. Curr Biol 15, 2137-2141.
Inglis, P.N., Ou, G., Leroux, M.R., and Scholey, J.M. (2007). The sensory cilia of Caenorhabditis elegans. WormBook : the online review of C elegans biology, 1-22.
Jiménez-Mateos, E.M., González-Billault, C., Dawson, H.N., Vitek, M.P., and Avila, J. (2006). Role of MAP1B in axonal retrograde transport of mitochondria. Biochem J 397, 53-59.
Kaminsky, R., Denison, C., Bening-Abu-Shach, U., Chisholm, A.D., Gygi, S.P., and Broday, L. (2009). SUMO regulates the assembly and function of a cytoplasmic intermediate filament protein in C. elegans. Developmental cell 17, 724-735.
Kang, J.S. (2008). Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation. Cell 132, 137-148.
Karabinos, A., Schulze, E., Schunemann, J., Parry, D.A., and Weber, K. (2003). In vivo and in vitro evidence that the four essential intermediate filament (IF) proteins A1, A2, A3 and B1 of the nematode Caenorhabditis elegans form an obligate heteropolymeric IF system. Journal of molecular biology 333, 307-319.
Kardon, J.R., and Vale, R.D. (2009). Regulators of the cytoplasmic dynein motor. Nature reviews Molecular cell biology 10, 854-865.
Kocsis, E., Trus, B.L., Steer, C.J., Bisher, M.E., and Steven, A.C. (1991). Image averaging of flexible fibrous macromolecules: the clathrin triskelion has an elastic proximal segment. Journal of structural biology 107, 6-14.
Labouesse, M. (2006). Epithelial junctions and attachments. WormBook : the online review of C elegans biology, 1-21.
Leterrier, J.F., Rusakov, D.A., Nelson, B.D., and Linden, M. (1994). Interactions between brain mitochondria and cytoskeleton: evidence for specialized outer membrane domains involved in the association of cytoskeleton-associated proteins to mitochondria in situ and in vitro. Microscopy research and technique 27, 233-261.
Liu, J., Wang, P., He, L., Li, Y., Luo, J., Cheng, L., Qin, Q., Brako, L.A., Lo, W.K., Lewis, W., et al. (2011). Cardiomyocyte-Restricted Deletion of PPARbeta/delta in PPARalpha-Null Mice Causes Impaired Mitochondrial Biogenesis and Defense, but No Further Depression of Myocardial Fatty Acid Oxidation. PPAR research 2011, 372854.
Liu, Q.A., and Shio, H. (2008). Mitochondrial morphogenesis, dendrite development, and synapse formation in cerebellum require both Bcl-w and the glutamate receptor delta2. PLoS genetics 4, e1000097.
Macaskill, A.F. (2009). Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses. Neuron 61, 541-555.
MacAskill, A.F., and Kittler, J.T. (2010). Control of mitochondrial transport and localization in neurons. Trends Cell Biol 20, 102-112.
Magrane, J., and Manfredi, G. (2009). Mitochondrial function, morphology, and axonal transport in amyotrophic lateral sclerosis. Antioxidants &; redox signaling 11, 1615-1626.
Mironov, S.L. (2007). ADP regulates movements of mitochondria in neurons. Biophys J 92, 2944-2952.
Misko, A., Jiang, S., Wegorzewska, I., Milbrandt, J., and Baloh, R.H. (2010). Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex. J Neurosci 30, 4232-4240.
Morris, R.L., and Hollenbeck, P.J. (1993). The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth. J Cell Sci 104, 917-927.
Naisbitt, S. (2000). Interaction of the postsynaptic density-95/guanylate kinase domain-associated protein complex with a light chain of myosin-V and dynein. J Neurosci 20, 4524-4534.
Nekrasova, O.E., Mendez, M.G., Chernoivanenko, I.S., Tyurin-Kuzmin, P.A., Kuczmarski, E.R., Gelfand, V.I., Goldman, R.D., and Minin, A.A. (2011). Vimentin intermediate filaments modulate the motility of mitochondria. Molecular biology of the cell 22, 2282-2289.
Orr, A.L. (2008). N-terminal mutant huntingtin associates with mitochondria and impairs mitochondrial trafficking. J Neurosci 28, 2783-2792.
Perkins, L.A., Hedgecock, E.M., Thomson, J.N., and Culotti, J.G. (1986). Mutant sensory cilia in the nematode Caenorhabditis elegans. Developmental biology 117, 456-487.
Perrot, R., and Julien, J.P. (2009). Real-time imaging reveals defects of fast axonal transport induced by disorganization of intermediate filaments. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 23, 3213-3225.
Pierce-Shimomura, J.T., Faumont, S., Gaston, M.R., Pearson, B.J., and Lockery, S.R. (2001). The homeobox gene lim-6 is required for distinct chemosensory representations in C. elegans. Nature 410, 694-698.
Pilling, A.D., Horiuchi, D., Lively, C.M., and Saxton, W.M. (2006). Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons. Mol Biol Cell 17, 2057-2068.
Quintero, O.A. (2009). Human Myo19 is a novel myosin that associates with mitochondria. Curr Biol 19, 2008-2013.
Riddle, D.L., and Albert, P.S. (1997). Genetic and Environmental Regulation of Dauer Larva Development. In C elegans II, D.L. Riddle, T. Blumenthal, B.J. Meyer, and J.R. Priess, eds. (Cold Spring Harbor (NY)).
Russo, G.J. (2009). Drosophila Miro is required for both anterograde and retrograde axonal mitochondrial transport. J Neurosci 29, 5443-5455.
Saeki, S., Yamamoto, M., and Iino, Y. (2001). Plasticity of chemotaxis revealed by paired presentation of a chemoattractant and starvation in the nematode Caenorhabditis elegans. The Journal of experimental biology 204, 1757-1764.
Schafer, J.C., Winkelbauer, M.E., Williams, C.L., Haycraft, C.J., Desmond, R.A., and Yoder, B.K. (2006). IFTA-2 is a conserved cilia protein involved in pathways regulating longevity and dauer formation in Caenorhabditis elegans. Journal of cell science 119, 4088-4100.
Sheng, Z.H., and Cai, Q. (2012). Mitochondrial transport in neurons: impact on synaptic homeostasis and neurodegeneration. Nature reviews Neuroscience 13, 77-93.
Shi, P., Ström, A.L., Gal, J., and Zhu, H. (2010). Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport. Biochim Biophys Acta 1802, 707-716.
Strange, K., Christensen, M., and Morrison, R. (2007). Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies. Nature protocols 2, 1003-1012.
Suter, D.M., and Hollenbeck, P.J. (2012). How to get on the right track. Nature neuroscience 15, 7-8.
Tanaka, K., Sugiura, Y., Ichishita, R., Mihara, K., and Oka, T. (2011). KLP6: a newly identified kinesin that regulates the morphology and transport of mitochondria in neuronal cells. Journal of cell science 124, 2457-2465.
Tang, H.L., Lung, H.L., Wu, K.C., Le, A.H., Tang, H.M., and Fung, M.C. (2008). Vimentin supports mitochondrial morphology and organization. The Biochemical journal 410, 141-146.
Tien, N.W., Wu, G.H., Hsu, C.C., Chang, C.Y., and Wagner, O.I. (2011). Tau/PTL-1 associates with kinesin-3 KIF1A/UNC-104 and affects the motor's motility characteristics in C. elegans neurons. Neurobiology of disease 43, 495-506.
Trinczek, B., Ebneth, A., Mandelkow, E.M., and Mandelkow, E. (1999). Tau regulates the attachment/detachment but not the speed of motors in microtubule-dependent transport of single vesicles and organelles. J Cell Sci 112, 2355-2367.
Wagner, O.I. (2003). Mechanisms of mitochondria-neurofilament interactions. J Neurosci 23, 9046-9058.
Wang, X. (2011). PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell 147, 893-906.
Wang, X., and Schwarz, T.L. (2009). The mechanism of Ca2+-dependent regulation of kinesin-mediated mitochondrial motility. Cell 136, 163-174.
Winter, L., Abrahamsberg, C., and Wiche, G. (2008). Plectin isoform 1b mediates mitochondrion-intermediate filament network linkage and controls organelle shape. The Journal of cell biology 181, 903-911.
Woo, W.M., Goncharov, A., Jin, Y., and Chisholm, A.D. (2004). Intermediate filaments are required for C. elegans epidermal elongation. Developmental biology 267, 216-229.
Wozniak, M.J., Melzer, M., Dorner, C., Haring, H.U., and Lammers, R. (2005). The novel protein KBP regulates mitochondria localization by interaction with a kinesin-like protein. BMC cell biology 6, 35.