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研究生: 羅麗萍
Li-Ping Lo
論文名稱: 豬腦後突觸質密區與微管間作用之研究
指導教授: 張兗君
Yen-Chung Chang
口試委員:
學位類別: 碩士
Master
系所名稱: 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年: 2005
畢業學年度: 93
語文別: 中文
論文頁數: 79
中文關鍵詞: 豬腦後突觸質密區微管
外文關鍵詞: PSD, postsynaptic density, microtubule
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  • Postsynaptic density (PSD)為後突觸膜下方,由超過三十多種蛋白質聚集而成的圓盤狀細緻結構,由其組成蛋白質與所在的位置推測,PSD與神經傳導 (neurotransmission)及突觸的可塑性 (synaptic plasticity)有極為密切的關連。在樹突小刺 (dendritic spine) 中,PSD位於突觸小刺的頭部頂點,正對前突觸活化區域處。過去許多對於PSD組成蛋白的研究發現,PSD的組成中含有大量的tubulin及其他與microtubule相關的蛋白質,且於PSD的結構中發現與microtubule有密切關係的PSDSU結構,然而卻極少在樹突小刺中,PSD的附近觀察到microtubule的存在。我們實驗室首先利用混濁度及離心沉澱實驗證明PSD與microtubule有能力聚合在一起形成MT•PSD complex,且MT•PSD complex的形成可能使得PSD的結構發生改變。並更進一步以穿透式電子顯微鏡觀察證明PSD的確可與microtubule接合。當microtubule形成時,PSD的結構被改變,異於典型PSD緊密堅實的結構,PSD形成較為鬆散的結構和microtubule相接。觀察其接合點發現,PSD可以形成特殊的構造接在microtubule邊緣或跨越其上,這些構造不只連結了PSD與microtubule,更影響microtubule的生長方向,且可能幫助穩定microtubule或調控microtubule生長。


    謝誌 摘要 壹、 前言.......................................1 貳、 材料與方法.................................13 (一) 藥品與材料.................................13 (二) 純化PSD ...................................14 (三) 透析.......................................18 (四) 純化microtubule proteins...................19 (五) 蛋白質濃度測定.............................22 (六) 混濁度分析法...............................22 (七) 離心沉降分析法.............................23 (八) 凝膠電泳分析...............................24 (九) Coomassie Blue染色法.......................25 (十) 銀染色法 ..................................25 (十一) 穿透式電子顯微鏡樣品置備...................26 參、 結果.......................................28 (一) 混濁度實驗部分.............................28 (二) 離心沉降實驗...............................31 (三) 電子顯微鏡觀察.............................37 肆、 討論.......................................41 伍、 參考文獻...................................47 圖表集..............................................59 附錄................................................79

    Apperson, M.L., Moon, I.S. and Kennedy, M.B. (1996) Characterization of densin-180, a new brain-specific synaptic protein of the O-sialoglycoprotein family. J. Neurosci. 16: 6839-6852.
    Beesley, P.W., Mummery, R. and Tibaldi, J. (1995) N-cadherin is a major glycoprotein component of isolated rat forebrain postsynaptic densities. J Neurochem. 64: 2288-2294.
    Berne, J.B., (1974) Interpretation of the light scattering from long rods. J. Mol. Biol. 89: 755-758.
    Blitz, A.L. and Fine, R.E. (1974) Muscle-like contractile proteins and tubulin in synaptosomes. Proc Natl Acad Sci U S A. 71: 4472-4476.
    Brenman, J.E., Chao, D.S., Gee, S.H., McGee, A.W., Craven, S.E., Santillano, D.R., Wu, Z., Huang, F., Xia, H., Peters, M.F., Froehner, S.C. and Bredt, D.S.. (1996) Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell. 84: 757-767.
    Caceres, A., Binder, L.I., Payne, M.R., Bender, P., Rebhun, L. and Steward, O. Differential subcellular localization of tubulin and the microtubule-associated protein MAP2 in brain tissue as revealed by immunocytochemistry with monoclonal hybridoma antibodies. J Neurosci. 4: 394-410.
    Caceres, A., Payne, M.R., Binder, L.I. and Steward, O. (1983) Immunocytochemical localization of action and microtubule-associated protein MAP2 in dendritic spines. Proc. Natl. Acad. Sci. USA. 80: 1738-1742.
    Cajal S.R. (1911) Histologie du systeme nerveux de homee et des vertrbres, Paris: Maloine.
    Carlin, R.K., Grab, D.J. and Siekevitz, P. (1982) Postmortem accumulation of tubulin in postsynaptic density preparations. J. Neurochem. 38: 94-100.
    Carlin, R.K, Siekevitz, P. (1984) Characterization of Na+-independent GABA and flunitrazepam binding sites in preparations of synaptic membranes and postsynaptic densities isolated from canine cerebral cortex and cerebellum. J Neurochem. 43: 1011-1007.
    Carr, D.W., Stofko-Hahn, R.E., Fraser, I.D., Cone, R.D. and Scott, J.D.. (1992) Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79. J Biol Chem. 267: 16816-19823.
    Chicurel, M.E. and Harris, K.M. (1992) Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and their synaptic relationships with mossy fiber boutons in the rat hippocampus. J Comp Neurol. 325: 169-182.
    Cohen, R.S., Blomberg, F., Berzins, K. and Siekevitz, P. (1977) The structure of postsynaptic densities isolated from dog cerebral cortex. J. Cell Biol. 74: 181-203.
    Cotman, C.W., Banker, G., Churchill, L. and Taylor, D. (1974) Isolation of postsynaptic densities from rat brain. J Cell Biol. 63: 441-455.
    Desmond, N.L. and Weinberg, R.J. (1998) Enhanced expression of AMPA receptor protein at perforated axospinous synapses. Neuroreport. 9: 857-860.
    Dosemeci, A, Reese, T.S., Petersen, J. and Tao-Cheng, J.H. (2000) A novel particulate form of Ca2+/calmodulin-dependent [correction of Ca2+/CaMKII-dependent] protein kinase II in neurons. J Neurosci. 20: 3076-3084.
    Dosemeci, A. and Reese, T.S. (1993) Inhibition of endogenous phosphatase in a postsynaptic density fraction allows extensive phosphorylation of the major postsynaptic density protein. J Neurochem. 61: 550-555.
    Faddis, B.T., Hasbani, M.J. and Goldberg, M.P. (1997) Calpain activation contributes to dendritic remodeling after brief excitotoxic injury in vitro. J Neurosci. 17: 951-959.
    Fagg, G.E. and Matus, A. (1984) Selective association of N-methyl aspartate and quisqualate types of L-glutamate receptor with brain postsynaptic densities. Proc. Natl. Acad. Sci. USA. 81: 6876-6880.
    Gaskin, F., Cantor, C.R. and Shelanski, M.C., (1974) Turbidimetric studies of the in Vitro assembly and disassembly of porcine neurotubules. J. Mol. Boil. 89: 737-758.
    Geinisman, Y., deToledo-Morrell, L. and Morrell, F. (1991) Induction of long-term potentiation is associated with an increase in the number of axospinous synapses with segmented postsynaptic densities. Brain Res. 566: 77-88.
    Geinisman, Y., Morrell, F. and de Toledo-Morrell, L. (1987) Synapses on dendritic shafts exhibit a perforated postsynaptic density. Brain Res. 422: 352-356.
    Harris, K.M. and Kater, S.B. (1994) Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function. Annu Rev Neurosci. 17: 341-371.
    Hayashi, Y., Ishida, A., Katagiri, H., Mishina, M., Fujisawa, H., Manabe, T. and Takahashi, T. (1997) Calcium- and calmodulin-dependent phosphorylation of AMPA type glutamate receptor subunits by endogenous protein kinases in the post-synaptic density. Brain Res Mol Brain Res. 46: 338-342.
    Hering, H. and Sheng, M. (2001) Dendritic spines: structure, dynamics and regulation. Nat Rev Neurosci. 2: 880-888.
    Heukeshoven, J. and Dernick, R. (1985) Simplified method for silverstaining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis. 6: 103-112.
    Hollmann, M. and Heinemann, S. (1994) Cloned glutamate receptors. Annu Rev Neurosci. 17: 31-108.
    Hunt, C.A., Schenker, L.J. and Kennedy, M.B.. (1996) PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses. J Neurosci. 16: 1380-1388.
    Ichtchenko, K., Hata, Y., Nguyen, T., Ullrich, B., Missler, M., Moomaw, C. and Sudhof, T.C.. (1995) Neuroligin 1: a splice site-specific ligand for beta-neurexins. Cell. 81: 435-443.
    Johnson, K.A., and Borisy, G.G., (1977) Kinetic analysis of microtubule self-assembly. J. Mol. Biol. 117: 1-31.
    Kaech, S., Fischer, M., Doll, T., and Matus, A. (1997) Isoform specificity in the relationship of actin to dendritic spines. J Neurosci. 17: 9565-9572.
    Kelly, P.T. and Cotman, C.W. (1978) Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide. J Cell Biol. 79: 173-183.
    Kennedy. M. B. (1993) The postsynaptic density. Curr. Opin. Neurobiol. 3: 732–737.
    Kennedy, M.B., Bennett, M.K. and Erondu, N.E. (1983) Biochemical and immunochemical evidence that the “major postsynaptic density protein” is a calmodulin-dependent protein kinase. Proc. Natl. Acad. Sci. USA. 80: 7357-7361.
    Kim, E., Cho, K.O., Rothschild, A. and Sheng, M.. (1996) Heteromultimerization and NMDA receptor-clustering activity of Chapsyn-110, a member of the PSD-95 family of proteins. Neuron. 17: 103-113.
    Kim, T.W., Wu, K., Xu, J.L. and Black, I.B. (1992) etection of dystrophin in the postsynaptic density of rat brain and deficiency in a mouse model of Duchenne muscular dystrophy. Proc Natl Acad Sci U S A. 89: 11642-11644.
    Kunau, W.H., Dommes, V. and Schulz, H. beta-oxidation of fatty acids in mitochondria, peroxisomes, and bacteria: a century of continued progress. Prog Lipid Res. 34: 267-342.
    Luscher, C., Nicoll, R.A., Malenka, R.C. and Muller, D. (2000) Synaptic plasticity and dynamic modulation of the postsynaptic membrane. Nat Neurosci. 3: 545-550.
    Laemmli, U.K., (1970) Cleavage of structure proteins during assembly of the head of the bacteriophage T4. Nature. 227: 680-685.
    Lai, S.L., Ling, S.C., Kuo, L.H., Shu, Y.C., Chow, W.Y. and Chang, Y.C. (1998) Characterization of granular particles isolated from postsynaptic densities.J Neurochem. 71: 1694-1701.
    Loganzo, F., Discafani, C.M., Annable. T., Beyer, C., Musto, S., Hari, M., Tan, X., Hardy, C., Hernandez, R., Baxter, M., Singanallore, T., Khafizova, G., Poruchynsky, M.S., Fojo, T., Nieman, J.A., Ayral-Kaloustian, S., Zask, A., Andersen, R.J., Greenberger, L.M., (2003) A synthetic analogue of the tripeptide hemiasterlin, is a potent antimicrotubule agent that circumvents P-glycoprotein-mediated resistance in vitro and in vivo. Cancer Res. 63: 1838-1845.
    Lin, C.T., Dedman, J.R., Brinkley, B.R. and Means, A.R. (1980) Localization of calmodulin in rat cerebellum by immunoelectron microscopy. J Cell Biol. 85: 473-480.
    Markham, J.A. and Fifkova, E. (1986) Actin filament organization within dendrites and dendritic spines during development. Brain Res. 392: 263-269.
    Matus, A., Pehling, G., Ackermann, M. and Maeder J. (1980) Brain postsynaptic densities: the relationship to glial and neuronal filaments. J Cell Biol. 87: 346-359.
    Matus, A. (2000) Actin-based plasticity in dendritic spines. Science. 290: 754-758.
    Matus, A., Ackermann, M and Pehling, G. (1981) Regularity and differentiation within the structure of brain postsynaptic densities. J. Neurocytology. 10: 889-896.
    Matus, A., Ackermann, M., Pehling, G., Byers, H.R. and Fujiwara, K. (1982) High actin concentrations in brain dendritic spines and postsynaptic densities. Proc Natl Acad Sci U S A. 79: 7590-7594.
    Matus, A.I. and Walters, B.B. (1975) Ultrastructure of the synaptic junctional lattice isolated from mammalian brain. J Neurocytol. 4: 369-375.
    Monahan, J.B. and Michel, J. (1987) Identification and characterization an N-methyl-D-aspartata-specific L-[3H]glutamate recognition site in synaptic plasma membranes. J. Neurochem. 48: 1699-1708.
    Moon, I.S., Apperson, M.L. & Kennedy, M.B. (1994) The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B. Proc. Natl. Acad. Sci. USA. 91: 3954–3958.
    Moon, R.T. and McMahon, A.P. (1990) Generation of diversity in nonerythroid spectrins. Multiple polypeptides are predicted by sequence analysis of cDNAs encompassing the coding region of human nonerythroid alpha-spectrin. J Biol Chem. 265: 4427-4433.
    Müller, B. M., Kistner, U., Beh, R. W., Cases, L. C., Becker, B., Gundelfinger, E. D. and Garner, C. C. (1995) Moleuclar characterization and spatial distribution of SAP97, anovel presynaptic protein homologous to SAP90 and the Drosophila dics-large tumor suppressor protein. J. Neurosci. 15: 2354-2366.
    Müller, B. M. Kistener, U., Kindler, S., Chung, W. J., Kuhlendahl, S., Fenster, S. D., Lau, L. F., Veh, R. W., Huganir, R. L., Gundelfinger, E. D., Garner, C.C. (1996) Sap102, a novel postsynaptic protein that interacts with NMDA receptor complexes in vivo. Neuron. 17: 255-265.
    Nusser, Z., Mulvihill, E., Streit, P. and Somogyi, P. (1994) Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization. Neuroscience. 61: 421-427.
    Nusser, Z., Lujan, R., Laube, G., Roberts, J.D., Molnar, E. and Somogyi, P. (1998) Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus. Neuron. 21: 545-559.
    Peng, J., Kim, M.J., Cheng, D., Duong, D.M., Gygi, S.P. and Sheng, M. (2004) Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry. J Biol Chem. 279: 21003-21011.
    Pin, J.P. and Duvoisin, R. (1995) The metabotropic glutamate receptors: structure and functions. Neuropharmacology. 34: 1-26.
    Ramõn, y. and Cajal, S. (1888) Estructura de los centros nerviosos de la saves. Rev. Tnm. Histol. Norm. Pat.
    Sabatini, B. L., and Svoboda, K. (2000) Analysis of calcium channels in single spines using optical fluctuation analysis. Nature 408: 589-593.
    Sheng, M.. (2001) Molecular organization of the postsynaptic specialization. Proc. Natl. Acad. Sci. U.S.A. 98: 7058–7061.
    Sheng M. and Sala C. (2001) PDZ domains and the organization of supramolecular complexes. Annu. Rev. Neurosci. 24: 1–29.
    Shepherd GM. (1996) The dendritic spine: a multifunctional integrative unit. J Neurophysiol. 75: 2197-2210.
    Siekevitz, P. (1985) The postsynaptic density: a possible role in long-lasting effects in the central nervous system. Proc Natl Acad Sci U S A. 82:3494-3498.
    Sorra, K.E. and Harris, K.M. (2000) Overview on the structure, composition, function, development, and plasticity of hippocampal dendritic spines. Hippocampus. 10: 501-511.
    Sorra, K.E., Fiala, J.C. and Harris, K.M. Critical assessment of the involvement of perforations, spinules, and spine branching in hippocampal synapse formation. J Comp Neurol. 398: 225-240.
    Spacek, J. and Harris, K.M. (1997) Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendritic spines of the immature and mature rat. J Neurosci. 17: 190-203.
    Strader, C.D., Pickel, V.M., Joh, T.H., Strohsacker, M.W., Shorr, R.G., Lefkowitz, R.J. and Caron, M.G. (1983) Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain. Proc Natl Acad Sci U S A. 80: 1840-1844.
    Suzuki, T., Okumura-Noji, K., Tanaka, R., Ogura, A., Kyoko, N., Kudo, Y. and Tada, T. (1993) Characterization of protein kinase C activities in postsynaptic density fractions prepared from cerebral cortex, hippocampus, and cerebellum. Brain Res. 619: 69-75.
    Suzuki, T. and Okumura-Noji. K. (1995) NMDA receptor subunits epsilon 1 (NR2A) and epsilon 2 (NR2B) are substrates for Fyn in the postsynaptic density fraction isolated from the rat brain.Biochem Biophys Res Commun. 216: 582-588.
    Suzuki, T., Mitake, S. and Murata, S. (1999)Presence of up-stream and downstream components of a mitogen-activated protein kinase pathway in the PSD of the rat forebrain. Brain Res. 840: 36-44.
    Thomas, U. (2000) Modulation of synaptic signalling complexes by Homer proteins. J Neurochem. 81: 407-413.
    Toni, N., Buchs, P.A., Nikonenko, I., Povilaitite, P., Parisi, L. and Müller, D. (2001) Remodeling of synaptic membranes after induction of long-term potentiation. J Neurosci. 21: 6245-6251.
    Toni, N., Buchs, P.A., Nikonenko, I., Bron, C.R. and Muller, D. (1999) LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature. 402: 421-425.
    Van, Rossum, D., and Hanisch, U.K.(1999) Cytoskeletal dynamics in dendritic spines: direct modulation by glutamate receptors? Trends Neurosci. 22: 290-295.
    Verdier-Pinard, P., Gares. M., Wright M. (1999) Differential in vitro association of vinca alkaloid-induced tubulin spiral filaments into aggregated spirals. Biochem Pharmacol. 58: 959-971.
    Walsh, M.J. and Kuruc, N. (1992) The postsynaptic density: constituent and associated proteins characterized by electrophoresis, immunoblotting, and peptide sequencing. J Neurochem. 59: 667-678.
    Walters, B.B. and Matus, A.I. (1975) Tubulin in postynaptic junctional lattice. Nature. 257: 496-498.
    Westrum, L.E., Jones, D.H., Gray, E.G. and Barron, J. (1980) Microtubules, Dendritic Spines and Spine Apparatuses. Cell Tissue Res. 208: 171-181.
    Wolf, M., Burgess, S., Misra, U.K. and Sahyoun, N. (1986) Postsynaptic densities contain a subtype of protein kinase C. Biochem Biophys Res Commun. 140: 691-698.
    Wiuiams, R.C. Jr. and Lee, J.C. (1982) Preparation of tubulin from brain. Methods in Enzymology. 85: 376-385.
    Wu, K., Nigam, S. K., Ledous, M., Huang, Y. Y., Aoki, C. and Siekevitz, P. (1992) Occurrence of the alpha subunits of G proteins in cerebral cortex synaptic membrane and postsynaptic density fractions: modulation of ADP-ribosylation by Ca2+/calmodulin. Proc. Natl. Acad. Sci. USA. 89: 8686-8690.

    Wu, K., Carlin, R., Sachs, L. and Siekevitz, P. (1985) Existence of a Ca2+-dependent K+ channel in synaptic membrane and postsynaptic density fractions isolated from canine cerebral cortex and cerebellum, as determined by apamin binding. Brain Res. 360: 183-194.
    Wyszynski, M., Lin, J., Rao, A., Nigh, E., Beggs, A.H., Craig, A.M. and Sheng, M. (1997) Competitive binding of alpha-actinin and calmodulin to the NMDA receptor. Nature. 385: 439-442.
    Yamauchi, T. (2002) Molecular constituents and phosphorylation dependent regulation of the post-synaptic density. Mass Spectrometry Rev. 21: 266–286.
    Yuste, R. and Bonhoeffer, T. (2004) Genesis of dendritic spines: insights from ultrastructural and imaging studies. Nat Rev Neurosci. 5:24-34.
    Ziff, E.B. (1997) Enlightening the postsynaptic density. Neuron. 19: 1163-1174.
    賴森林 (1998) 後突觸質密區結構之研究–蛋白質間雙硫鍵的形成與影響, 國立清華大學生命科學所碩士論文.
    李慧貞 (2002) 後突觸質密區中新鑑定的蛋白質–PSD500之研究, 國立清華大學生命科學所碩士論文.

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