自豬腦中純化出來的後突觸質密區(postsynaptic density，簡稱PSD)結構非常緊密，使得研究PSD內部的結構組成相當不容易。本實驗室在之前研究(Cheng, 2009)中表示，當神經細胞經由cold treatments時，會發現α、β-tubulins從dendritic spines中移出(exit)，而幾個PSD中的主要蛋白質: CaMKIIα、cDHC、MAP2，也會從dendritic spines中移出(exit)，這些變化影響著dendritic spines在型態上的改變，並暗示著這些蛋白質彼此之間可能具有交互作用；另外，在彭聖智的論文中也顯示，α-tubulin與CaMKIIα可能會跟其他蛋白質形成次級蛋白質複合體。在本篇論文裡，我們改良過去的方法，利用urea及thiourea將PSD結構打散，再利用免疫吸附法，確定α-tubulin或CaMKIIα所形成的次級蛋白質複合體是相似的，且不同於其他次級蛋白質複合體；並進一步的利用化學交聯劑DTSP，配合免疫吸附及對角線電泳的分析，我們發現在PSD中，可能與α-tubulin存在著直接交互作用的蛋白質有: β-tubulin、actin、CaMKIIα；可能與CaMKIIα直接交互作用的蛋白質為: CaMKIIβ、α-tubulin、β-tubulin。Actin不與CaMKIIα直接作用。另外，在cytosol裡，發現大部分的β-tubulin存在其中，並且與α-tubulin形成hetero-dimer，也可與其他蛋白質形成不同的聚合體；而CaMKIIα在cytosol的含量稀少，但可與α-tubulin形成聚合體，並且在PSD中也可發現相似的聚合體。於是，我們推測，大量存在於PSD的CaMKIIα，部分與α-tubulin形成交互作用，且可能干擾microtubule的形成。這些不同的蛋白質聚合體，意涵著不同的意義及功能，是如何牽涉到 protein trafficking並影響著dendritic spines的dynamic，將是未來需要進一步研究的課題。

Allison Daniel W., Chervin Adam S., Gelfand Vladimir I., Craig Ann Marie. (2000) Postsynaptic scaffolds of excitatory and inhibitory synapses in hippocampal neurons: Maintenance of core components independent of actin filaments and microtubules. J. Neurosci. 20, 4545-4554.

Baas P. W., Vidya Nadar C. and Myers K. A. (2006) Axonal transport of microtubules: the long and short of it. Traffic 7, 490–498.

Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Brocke, L., Chiang, L. W., Wagner, P. D., Shulman, H. (1999) Functional implications of the subunit composition of neuronal CaM kinase II. J. Biol. Chem. 274, 22713-22722.

Bredt DS, Ferris CD, Snyder SH. (1992) Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase; identification of flavin and calmodulin binding sites. J Biol Chem 267: 10976-10981.

Casadevall, N., Lacombe, C., Muller, O., Gisselbrecht, S. et al. (1991) Multimeric structure of the membrena erythropoietin receptor of murine erythroleukemia cells (Friend Cells). J. Biol. Chem. 266, 16015-16020

Chang Chia-Wei, Peng Sheng-Chih, Cheng Wen-Yao, Liu Szu-Heng, Cheng Huei-Hsuan, Huang San-Yuang, Chang Yen-Chung. (2007) Studying the protein-protein interactions in the post-synaptic density by immunoabsorption and chemical crosslinking means. Proteomics. 1, 1499-1512.

Cheng Hui-Hsuan, Huang Zu-Han, Lin Wei-Hsiang, Chow Wei-Yuan, Chang Yen-Chung (2009) Cold-induced exodus of postsynaptic proteins from dendritic spines. J Neurosci Res. 87(2),460-9.

Cheng, H. H., Liu, S. H., Lee, H. J., Lin, Y. S., Huang, Z. H., Hsu, C. I., Chen, Y. C., Chang, Y. C. (2006) The heavy chain of cytoplasmic dynein is a major protein component of the postsynaptic density fraction. J. Neurosci. Res. 84, 244-254.

Cotman CW, Banker G, Churchil L, Taylor D. (1974) Isolation of postsynaptic densities from rat brain. J Cell Biol 63: 441-455.

Dosemeci, A., Tao-Cheng, J. H., Vinade, L., Jaffe, H. (2006) Preparation of postsynaptic density fraction from hippocampal slices and proteomic analysis. Biochemical and Biophysical Research Communications 339, 687-694.

Erondu Ngozi E., Kennedy Mary B. (1985) Regional distribution of type II Ca2+/Calmodulin-dependent protein kinase in rat brain. J. Neurosci. 5, 3270-3277.

Fiala JC, Kirov SA, Feinberg MD, Petrak LJ, George P, Goddard CA and Harris KM. (2003) Timing of neuronal and glial ultrastructure disruption during brain slice preparation and recovery in vitro. J. Comp. Neurol. 465, 90-103.

Fukushima N, Furuta D, Hidaka Y, Moriyama R, Tsujiuchi T. (2009) Post-translational modifications of tubulin in the nervous system. J Neurochem. 109(3),683-93.

Gray EG, Westrum L, Burgoyne RD and Barron J. (1982) Synaptic organization and neuron microtubule distribution. Cell Tissue Res. 226, 579-588.

Hasegawa S, Furuichi T, Yoshida T, Endoh K, Kato K, Sado M, Maeda R, Kitamoto A, Miyao T, Suzuki R, Homma S, Masushige S, Kajii Y, Kida S. (2009) Transgenic up-regulation of alpha-CaMKII in forebrain leads to increased anxiety-like behaviors and aggression. Mol Brain. 2(1):6.

Hoelz, A., Nairn, A. C., Kuriyan, J. (2003) Crystal structure of a tetradecameric assembly of the association domain of Ca2+/calmodulin-dependent protein kinase II. Mol. Cell. 11, 1241-1251.

Jan Huei-Hsuan, Chen I-Tong, Tsai Yu-Yi, Chang Yen-Chung (2002) Structural roles of the zinc ions bound to the postsynaptic density. J. Neurochem. 82, 525-534.

Jordan, B. A., Fernholz, B. D., Boussac, M., Xu, C. et al. (2004) Identification and verification of novel rodent postsynaptic density proteins. Mol. Cell. Proteomics 3, 857-871.

Kandel ER, Schwartz JH, Jessell TM. (2000) Principles of neuronal science. Fourth edition. New York: McGraw-Hill. 210 p.

Kennedy MB. (1997) The postsynaptic density at glutamatergic synapses. Trends Neurosci 20: 264-268.

Kolb, S. J., Hudmon, A., Ginsberg, T. R., and Waxham, M. N. (1998) Identification of domains essential for the assembly of calcium/calmodulin-dependent protein kinase II holoenzymes. J. Biol. Chem. 273, 31555-31564.

Kolodziej, S. J., Hudmon, a., Waxham, M. N., and Stoops, J. K. (2000) Three dimensional reconstructions of calcium/calmodulin-dependent (CaM) kinase IIα and truncated CaM kinase IIα reveal a unique organization for its structural core and functional domains. J. Biol. Chem. 275, 14354-14359.

Lai S. L., Chiang S. F., Chen I. T., Chow W. Y. (1999) Interprotein disulfide bonds formed during isolation process tighten the structure of the postsynaptic density. J. Neurochem. 73, 2130-2138.

Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Li, K. W., Hornshaw, M. P., Van der Schors, R. C., Watson, R. et al. (2004) Proteomics analysis of rat brain postsynaptic density: implications of the diverse protein functionalgroups for the integration of synaptic physiology. J. Biol. Chem. 279, 987-1002.

Lisman J, Schulman H, Cline H. (2002) The molecular basis of CaMKII function in synaptic and behavioral memory. Nature Rev Neurosci 3: 175-190.

Lin YC, Redmond L. (2008) CaMKII{beta} binding to stable F-actin in vivo regulates F-actin filament stability. Proc Natl Acad Sci USA. 105(41),15791-6.

Lo Li-Ping, Liu Szu-Heng, Chang Yen-Chung (2007) Assembling microtubules disintegrate the postsynaptic density in vitro. Cell Motility and the Cytoskeleton. 64, 6-18.

O’Leary Heather, Lasda Erika, Bayer K. Ulrich (2006) CaMKIIβ association with the actin-cytoskeleton is regulated by alternative splicing. Mol. Biol. Cell. 17, 4656-4665.

Okamoto Ken-Ichi, Narayanan Radhakrishnan, Lee Sang H., Murata Kazuyoshi, Hayashi Yasunori. (2007) The role of CaMKII as an F-actin-bundling protein crucial for maintenance of dendritic spine structure. Proc. Natl. Acad. Sci. U.S.A. 104, 6418-6423.

Peng, J., Kim, M. J., Cheng, D., Duong, D. M. et al. (2004) Semiquantitative proteomic analysis of rat forebrain postsynapic density fractions by mass spectrometry. J. Biol. Chem. 279, 21003-21011.

Qoronfleh MW, Ren L, Emery D, Perr M and Kaboord B. (2003) Use of immunomatrix methods to improve protein-protein interaction detection. J. Biomed Biotechnol. 2003, 291-298.

Sanabria, Hugo; Steven, Kolodziej J.; Swulius, Matthew T.; Liu, Jun; Waxham, M. Neal. (2009) {beta}CaMKII regulates actin assembly and structure. Biophysical Journal 284(15),9770-80.

Satoh, K., Takeuchi, M., Oda, Y., Deguchi-Tawarada, M. et al. (2002) Identification of activity-regulated proteins in the postsynaptic density fraction. Genes Cells 7, 187-197.

Schneider C, Newman RA, Sutherland DR, Asser U and Greaves MF. (1982) A one-step purification of membrane proteins using a high efficiency immunomatrix. J Biol. Chem. 257, 10766-10769.

Shen, K., Teruel, M. N., Subramanian, K., Meyer, T. (1998) CaMKIIβ functions as an F-actin targeting module that localizes CaMKIIα/β heterooligomers to dendritic spines. Neuron 21, 593-606.

Strack S, Choi S, Lovinge DM, Colbran RJ. (1997b) Translocation of autophosphorylated calcium/calmodulin-dependent protein kinase II to the postsynaptic density. J Biol Chem 272: 13467-13470.

Vallano ML, Goldenring JR, Lasher RS, Delorenzo RJ. (1986) Association of calcium/calmodulin-dependent kinase with cytoskeletal preparations: phosphorylation of tubulin, neurofilament, and microtubule-associated proteins. Ann N Y Acad Sci. 466, 357-74.

Van Rossum D and Hanisch UK. (1999) Cytoskeletal dynamics in dendritic spines: Direct modulation by glutamate receptors? Trends Neurosci. 22, 290-295.

Walikonis RS, Jensen ON, Mann M, Provance DW, Jr., Mercer JA, Kennedy MB. (2000) Identification of proteins in the postsynaptic density fraction by mass spectrometry. J Neurosci 20: 4069-4080.

Walsh MJ, Kuruc N. (1992) The postsynaptic density: Constituent and associated proteins characterized by electrophoresis, immunoblotting, and peptide sequencing. J Neurochem 59: 667-678.

Wessel D. and Flugge U. I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141-143.

Westrum LE and Gray EG. (1976) Microtubules and membrane specializations. Brain Res. 105, 547-550.

Westrum LE and Gray EG. (1977) Microtubules associated with postsynaptic thickenings. J. Neurocytol. 6, 505-518.

Westrum LE, Jones DH, Gray EG and Barron J. (1980) Microtubules, dendritic spines and spine apparatuses. Cell Tissue Res. 208, 171-181.

Yamauchi T. (2002) Molecular constituents and phosphorylation-dependent regulation of the post-synaptic density. Mass Spectrom Rev 21:266–86.

Yoshimura Y, Yamauchi T. (1997) Phosphorylation-dependent reversible association of Ca2+/calmodulin-dependent protein kinase II with the postsynaptic densities. J Biol Chem 272: 26354-26359.

Yoshimura Y, Aoi C, Yamauchi T. (2000) Investigation of protein substrates of Ca2+/calmodulin-dependent protein kinase II translocated to the postsynaptic density. Mol Brain Res 81: 118-128.

Yoshimura, Y., Yamauchi, Y., Shinkawa, T., Taoka, M. et al. (2004) Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography tandem mass spectrometry. J. Neurochem. 88, 759-768.