SNX27 is important for postnatal development in mice

SNX27 IS IMPORTANT FOR POSTNATAL DEVELOPMENT IN MICE CAI LEI INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 SNX27 IS IMPORTANT FOR POSTNATAL DEVELOPMENT IN MICE CAI LEI (B.SC.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS This work is directed and supervised by Professor HONG Wan Jin. Appreciated for his instructions from the experiment planning to results explanation and data discussion in helping me finish this research. Also thanks a lot for offering me the opportunities to enrich my work in the internal and external collaborations. Meanwhile, I would like to acknowledge Professors Walter HUNZIKER and CAO Xin Min, as members of my committee meeting, gave a lot of suggestions and comments on the whole process. I would like to acknowledge Dr. Brendon HANSON for his help during the initiation of this project; Dr. Vadim ATLACHKINE and Miss HUANG Cai Xia for their great contributions on generating of the SNX27-null mice; Dr. GUO Ke, LI Jie in IMCB for teaching me histological assays; Dr. WANG Cheng Chun for his suggestions on the in vivo examinations and reviewing the project; Dr. LOO Li Shen for teaching me the Live Cell Imaging assays. I thank past and present members of HWJ lab: THAM Mae Lan Jill, ZHANG Xiao Qian, WANG Tuan Lao, CHAN Siew Wee, Sofie VAN HUFFEL, LIU Ning Sheng, LI Hong Yu, TRAN Thi Ton Hoai, ONG Yan Shan, TAN Yik Loo, LIM Chun Jye and CHONG Yaan Fun for the weekly discussion and sharing of cell lines, DNA constructs and experiment protocols. With their kind help, I can get so many exciting results to promote the research. I am so grateful to have all the friends in IMCB who share my days of excitements and despairs along these years, making it a cherishable memory. At last, I would leave my best thanks to my parents. It is their unremitting encouragements and supports that make me overcome all the difficulties. I TABLE OF CONTENTS ABSTRACT LIST OF TABLES LIST OF FIGURES ABBREVIATIONS CHAPTER 1: INTRODUCTION 1.1 Membrane transport 1.2 Molecular mechanisms of membrane transport 1.2.1 Adaptors and coat proteins 1.2.2 SNARE proteins 1.2.3 Tethering factors 1.2.4 Small G proteins 1.2.5 Lipids 1.3 The PX domain 1.4 Sorting Nexin family 1.4.1 SNX1 and retromer complex 1.4.2 SNX3 1.4.3 SNX4 1.4.4 SNX5/SNX6 1.4.5 SNX9 1.4.6 SNX13 1.4.7 SNX15 1.4.8 SNX16 1.4.9 SNX17 1.4.10 SNX23 1.4.11 SNX27 1.5 Application of RNAi in cell biology 1.6 Targeted gene replacement as a tool to study the functions of mammalian genes 1.7 Rationale of the work CHAPTER 2: MATERIALS AND METHODS 2.1 cDNA cloning II 2.2 Expression and purification of GST-fusion proteins 2.3 Immunization of rabbits and affinity purification of antibodies 2.4 Antibodies used in the study 2.5 Cell lines used in the study 2.6 Transient expression 2.7 SDS-PAGE 2.8 Coomassie blue staining 2.9 Immunoprecipitation and Western blot 2.10 Indirect immunoflurescence microscopy 2.11 Protein/lipid overlay assay 2.12 Short hairpin RNA-mediated knockdown of SNX27 2.13 Retroviral infection 2.14 EGF internalization 2.15 EGF stimulation and EGFR degradation 2.16 Cell surface biotinylation and stripping 2.17 Yeast two-hybrid assays 2.18 Generation of the SNX27-/- mice 2.19 Mouse genotyping 2.20 DNA extraction 2.21 Histological analysis and immuno-staining 2.22 Tissue immunoblot analysis 2.23 Mice urinary and blood chemistry studies III 2.24 Isolation and culture of mouse collecting duct cells 2.25 Hippocampal cultures and transfection 2.26 Apoptosis assay CHAPTER 3: CELL BIOLOGICAL CHARIATERAZATION OF 00000000000SNX27 3.1 Identification of SNX27 and gene cloning 3.2 SNX27 is targeted to the early endosome 3.3 PX domain is required for endosomal localization of SNX27 3.4 The PX domain mediates direct interaction with PI(3)P 3.5 Generation of rabbit anti-SNX27 polyclonal antibody and characterization endogenous SNX27 3.6 Endogenous SNX27 is preferentially localized to the early endosome 3.7 Endosomal association of SNX27 is dependent on PI 3-kinase activity 3.8 SNX27 tissue expression pattern 3.9 Discussion CHAPTER 4: FUNCTIONAL CHARACTERIZATION OF SNX27 00USING KNOCKOUT MICE 4.1 Generation of SNX27 deficient mice 4.1.1 Construction of a targeting vector and genotype analysis 4.1.2 Obtaining of SNX27 null mutants 4.2 Analysis of SNX27 deficient mice 4.2.1 Birth defects of SNX27-/- mice 4.2.2 SNX27 deficiency results in mouse growth retardation and postnatal lethality 4.2.3 SNX27-/- mice displayed striking abnormalities in the kidney development 4.2.4 Kidney morphological study in SNX27-/- mice revealed the IV delayed nephron maturation and papillary atrophy 4.3 Reducing milk competition can rescue some SNX27-/- pups 4.4 The kidneys of surviving SNX27-/progressive medulla degeneration 4.5 Urine chemistry analysis of one-month-old SNX27-/- mice 4.6 SNX27 may functionally regulate surface AQP2 levels mice displayed 4.7 Other phenotypes 4.7.1 Male SNX27-/- mice are infertile 4.7.2 Some SNX27 null mice showed severe brain atrophy 4.7.3 Altered expression of Kir3 channels in SNX27-/- mice 4.8 Discussion CHAPTER 5: SNX27 REGULATES THE ENDOCYTOSIS OF 00EGFR 5.1 ShRNA-mediated knockdown endocytosis of EGFR of SNX27 inhibited 5.2 Depletion of SNX27 inhibited lysosomal degradation of EGFR 5.3 Direct visualization of EGFR endocytosis using live imaging technique 5.4 Discussion cell 0IDENTIFICATION OF NMDA RECEPTOR-2C AS A SNX27INTERACTING PROTEIN CHAPTER 6: 00000000 6.1 Gal4 based yeast two-hybrid screening for SNX27 interacting proteins 6.2 Direct PDZ domain mediated interaction of SNX27 with NR2c 6.3 The PDZ binding motif is critical for the interaction of NR2c with SNX27 V 6.4 SNX27 co-localizes with NR2c in rat hippocampal neurons 6.5 Increased NR2c expression in the SNX27-/- mice brains 6.6 Discussion CHAPTER 7: CONCLUSIONS AND FUTURE PERSPECTIVES REFERENCES APPENDICES VI ABSTRACT Sorting nexin 27 (SNX27) is a newly identified member of the SNX family that is characterized by the presence of an evolutionarily conserved Phox (PX) domain. SNX27 is targeted to the early endosome by the interaction of its PX domain with phosphatidylinositol-3-phosphate (PI(3)P). In order to study the physiological function of SNX27, we have produced mice homozygous for a null mutation of SNX27 by gene targeting. Generally, SNX27-/- mice displayed severe growth retardation and all died within weeks of age. Retardation of growth was seen in most organs as they are all smaller than those in the wild-type littermates. As the kidney undergoes extensive postnatal growth and morphogenesis, we have focused on the defect of kidney development in SNX27-/- mice. SNX27-/- mice showed a clear delay in nephron maturation and developed pronounced lesions in the kidney. The urine chemistry analysis of SNX27-/- mice revealed highly upregulated osmolality and proteinuria. These lesions were accompanied by increased levels of AQP2 in the collecting ducts. In addition, RNAi-mediated knockdown of SNX27 in A431 cells was found to inhibit endocytosis of EGFR. Furthermore, through yeast two-hybrid screening, we identified a novel SNX27 interacting protein, the N-methyl-D-aspartate (NMDA) receptor 2c (NR2c). This interaction was mediated by the binding of the PDZ domain of SNX27 with the C-terminal PDZ-binding motif of NR2c. The level of NR2c was found to be increased in SNX27-/- mice, implying that SNX27 may function to regulate endosomal sorting of NR2c for lysosomal degradation. These results suggest that SNX27 may regulate endosomal sorting of membrane proteins VII containing PDZ-binding motif and its absence may alter the trafficking of these proteins, leading to survival and organ developmental defects in mice. Taken together, the research presented in this thesis reveals the basic biochemical and cell biological properties of SNX27 and its important role in maintaining mouse postnatal growth and survival.     130 Kirchhausen, T. (2000a). Clathrin. Annu Rev Biochem 69, 699-727. Kirchhausen, T. (2000b). Three ways to make a vesicle. Nat Rev Mol Cell Biol 1, 187-198. Kiuchi-Saishin, Y., Gotoh, S., Furuse, M., Takasuga, A., Tano, Y., and Tsukita, S. (2002). Differential expression patterns of claudins, tight junction membrane proteins, in mouse nephron segments. J Am Soc Nephrol 13, 875-886. Kohan, D.E. (2008). Progress in gene targeting: using mutant mice to study renal function and disease. Kidney Int 74, 427-437. Kornfeld, S. (1992). Structure and function of the mannose 6-phosphate/insulinlike growth factor II receptors. Annu Rev Biochem 61, 307-330. Krauss, M., Kinuta, M., Wenk, M.R., De Camilli, P., Takei, K., and Haucke, V. (2003). ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma. J Cell Biol 162, 113124. Kurten, R.C., Cadena, D.L., and Gill, G.N. (1996). Enhanced degradation of EGF receptors by a sorting nexin, SNX1. Science 272, 1008-1010. Laemmli, U.K. (1970), Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685. Lau, C.G., and Zukin, R.S. (2007). NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders. Nat Rev Neurosci 8, 413-426. Lawe, D.C., Chawla, A., Merithew, E., Dumas, J., Carrington, W., Fogarty, K., Lifshitz, L., Tuft, R., Lambright, D., and Corvera, S. (2002). Sequential roles for       131 phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1. J Biol Chem 277, 8611-8617. Le Borgne, R. and Hoflack B. (1998). Protein transport from the secretory to the endocytic pathway in mammalian cells. Biochimica Et Biophysica Acta 1404(1-2): 195-209. Lee, J., Retamal, C., Cuitino, L., Caruano-Yzermans, A., Shin, J.E., van Kerkhof, P., Marzolo, M.P., and Bu, G. (2008). Adaptor protein sorting nexin 17 regulates amyloid precursor protein trafficking and processing in the early endosomes. J Biol Chem 283, 11501-11508. Lee, M. C., Miller, E. A., Goldberg, J., Orci, L. and Schekman, R. (2004). Bidirectional protein transport between the ER and Golgi. Annu.Rev. Cell Dev Biol 20, 87–123. Lelievre-Pegorier, M., Vilar, J., Ferrie, M., Moreau, E., Freund, N., Gilbert, T. and Merlet-Benichou, C. (1998). Mild vitamin A deficiency leads to inborn nephron deficit in the rat. Kidney Int 54, 1455–1462. Lemmon, M.A. (2003). Phosphoinositide recognition domains. Traffic 4, 201-213. Liewen, H., Meinhold-Heerlein, I., Oliveira, V., Schwarzenbacher, R., Luo, G., Wadle, A., Jung, M., Pfreundschuh, M., and Stenner-Liewen, F. (2005). Characterization of the human GARP (Golgi associated retrograde protein) complex. Exp Cell Res 306, 24-34. Lin, Q., Lo, C.G., Cerione, R.A., and Yang, W. (2002). The Cdc42 target ACK2 interacts with sorting nexin (SH3PX1) to regulate epidermal growth factor receptor degradation. J Biol Chem 277, 10134-10138.       132 Lu, H., Sun, T.X., Bouley, R., Blackburn, K., McLaughlin, M., and Brown, D. (2004). Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2 Am J Physiol Renal Physiol 286, F233-F243. Lu, J., Garcia, J., Dulubova, I., Sudhof, T.C., and Rizo, J. (2002). Solution structure of the Vam7p PX domain. Biochemistry 41, 5956-5962. Lundmark, R., and Carlsson, S.R. (2003). Sorting nexin participates in clathrinmediated endocytosis through interactions with the core components. J Biol Chem 278, 46772-46781. Lundmark, R., and Carlsson, S.R. (2004). Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9. J Biol Chem 279, 42694-42702. Lunn, M.L., Nassirpour, R., Arrabit, C., Tan, J., McLeod, I., Arias, C.M., Sawchenko, P.E., Yates, J.R., 3rd, and Slesinger, P.A. (2007). A unique sorting nexin regulates trafficking of potassium channels via a PDZ domain interaction. Nat Neurosci 10, 1249-1259. Lupashin, V., and Sztul, E. (2005). Golgi tethering factors. Biochim Biophys Acta 1744, 325-339. MaCaulay, S.L., Stoichevska, V., Grusovin, J., Gough, K.H., Castelli, L.A., and Ward, C.W. (2003). Insulin stimulates movement of sorting nexin between cellular compartments: a putative role mediating cell surface receptor expression and insulin action. Biochem J 376, 123-134. MacNeil, A.J, Mansour, M. and Pohajdak, B. (2007). Biochemi and Bioph Res Comm 359, 848–853. Mallard, F., Tang, B.L., Galli, T., Tenza, D., Saint-Pol, A., Yue, X., Antony, C., Hong, W., Goud, B., and Johannes, L. (2002). Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform. J Cell Biol 156, 653-664.       133 Malinow R. and Malenka R. C. (2002). AMPA receptor trafficking and synaptic plasticity. Annu Rev Neurosci 25, 103–126. Maric, K., Oksche, A., and Rosenthal, W. (1998). Aquaporin-2 expression in primary cultured rat inner medullary collecting duct cells. Am. J. Physiol. 275, F796-F801. Maxfield, F.R., and McGraw, T.E. (2004). Endocytic recycling. Nat Rev Mol Cell Biol 5, 121-132. Mayor, S., and Pagano, R.E. (2007). Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 8, 603-612. McNiven, M.A., and Thompson, H.M. (2006). Vesicle formation at the plasma membrane and trans-Golgi network: the same but different. Science 313, 1591-1594. Merino-Trigo, A., Kerr, M.C., Houghton, F., Lindberg, A., Mitchell, C., Teasdale, R.D., and Gleeson, P.A. (2004). Sorting nexin is localized to a subdomain of the early endosomes and is recruited to the plasma membrane following EGF stimulation. J Cell Sci 117, 6413-6424. Miki, H., Okada, Y., and Hirokawa, N. (2005). Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol 15, 467-476. Molloy, S.S., Anderson, E.D., Jean, F., and Thomas, G. (1999). Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. Trends Cell Biol 9, 28-35. Moritz, K.M. and Wintour, E.M. (1999). Functional development of the meso and metanephros. Pediatr Nephrol 13, 171–178. Mukherjee, S., Ghosh, R.N., and Maxfield, F.R. (1997). Endocytosis. Physiol Rev 77, 759-803.       134 Murray, R.Z., Wylie, F.G., Khromykh, T., Hume, D.A., and Stow, J.L. (2005). Syntaxin and Vti1b form a novel SNARE complex, which is up-regulated in activated macrophages to facilitate exocytosis of tumor necrosis Factor-alpha. J Biol Chem 280, 10478-10483. Neiss, W. F. (1982).Histogenesis of the loop of Henle in the rat kidney. Anat. Embryol. 164, 315-330. Nichols, B.J., and Lippincott-Schwartz, J. (2001). Endocytosis without clathrin coats. Trends Cell Biol 11, 406-412. Nielsen, S., Frøkiaer, J., Marples, D., Kwon, T.H., Agre, P. and Knepper, M.A. (2002). Aquaporins in the kidney: from molecules to medicine. Physiol Rev 82, 205244. Nielsen, S., Kwon, T.H., Frøkiaer, J. and Agre, P. (2007). Regulation and dysregulation of aquaporins in water balance disorders. J Intern Med 261, 53-64. Nothwehr, S.F., Ha, S.A., and Bruinsma, P. (2000). Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains withVps35p. J Cell Biol 151,297-310. Otsuki, T., Kajigaya, S., Ozawa, K. and Liu, J.M. (1999). SNX5, a new member of the sorting nexin family, binds to the Fanconi anemia complementation group A protein. Biochem Biophys Res Commun 265, 630-635. Parks, W.T., Frank, D.B., Huff, C., Renfrew Haft, C., Martin, J., Meng, X., de Caestecker, M.P., McNally, J.G., Reddi, A., Taylor, S.I., et al. (2001). Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases. J Biol Chem 276, 19332-19339. Parton, R.G., and Simons, K. (2007). The multiple faces of caveolae. Nat Rev Mol Cell Biol 8, 185-194.       135 Pasqualato, S., Renault, L., and Cherfils, J. (2002). Arf, Arl, Arp and Sar proteins: a family of GTP-binding proteins with a structural device for 'front-back' communication. EMBO Rep 3, 1035-1041. Patki, V., Virbasius, J., Lane, W.S., Toh, B.H., Shpetner, H.S., and Corvera, S. (1997). Identification of an early endosomal protein regulated by phosphatidylinositol 3kinase. Proc Natl Acad Sci U S A 94, 7326-7330. Pearlson, G.D., Warren, A.C., Starkstein, S.E., Aylward, E.H., Kumar, A.J., Chase, G.A., and Folstein, M.F. (1990). Brain atrophy in 18 patients with Down syndrome: a CT study. AJNR Am J Neuroradiol 11, 811-816. Perez-Victoria, F.J., Mardones, G.A., and Bonifacino, J.S. (2008). Requirement of the Human GARP Complex for Mannose 6-phosphate-receptor-dependent Sorting of Cathepsin D to Lysosomes. Mol Biol Cell 19, 2350-2362. Peter B.J., Kent H.M., Mills I.G., Vallis Y., Butler P.J, Evans P.R., McMahon H.T., (2004) BAR domains as sensors of membrane curvature: the amphiphysin BAR structure, Science 303 495–499. Pfeffer, S.R. (2007). Unsolved mysteries in membrane traffic. Annu Rev Biochem 76, 629-645. Phillips, S.A., Barr, V.A., Haft, D.H., Taylor, S.I., and Haft, C.R. (2001). Identification and characterization of SNX15, a novel sorting nexin involved in protein trafficking. J Biol Chem 276, 5074-5084. Ponting, C.P. (1996). Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains? Protein Sci 5, 2353-2357. Puertollano, R., Aguilar, R.C., Gorshkova, I., Crouch, R.J., and Bonifacino, J.S. (2001). Sorting of mannose 6-phosphate receptors mediated by the GGAs. Science 292, 1712-1716.       136 Puertollano, R., van der Wel, N.N., Greene, L.E., Eisenberg, E., Peters, P.J., and Bonifacino,J.S. (2003). Morphology and dynamics of clathrin/GGAl-coated carriers budding from the trans-Golgi network. Mol Biol Cell 14, 1545-1557. Rabouille, C., and Klumperman, J. (2005). Opinion: The maturing role of COPI vesicles in intra-Golgi transport. Nat Rev Mol Cell Biol 6: 812-817. Raiborg, C., Bache, K.G., Mehlum, A., and Stenmark, H. (2001). Function of Hrs in endocytic trafficking and signalling. Biochem Soc Trans 29, 472-475. Rameh L. E. and Cantley, L. C. (1999).The role of phosphoinositide 3-kinase lipid products in cell function. J. Biol. Chem. 274, 8347–8350. Rincon, E., Santos, T., Avila-Flores, A., Albar, J.P., Lalioti, V., Lei, C., Hong, W., and Merida, I. (2007). Proteomics identification of sorting nexin 27 as a diacylglycerol kinase zeta-associated protein: new diacylglycerol kinase roles in endocytic recycling. Mol Cell Proteomics 6, 1073-1087. Robinson, M. S. (1994). The role of clathrin, adaptors and dynamin in endocytosis.Current Opinion in Cell Biology , 538-44. Robinson, M.S. (2004). Adaptable adaptors for coated vesicles. Trends Cell Biol 14, 167-174. Robinson, M.S., and Bonifacino, J.S. (2001). Adaptor-related proteins. Curr Opin Cell Biol 13,444-453. Roche, K.W., Standley, S., McCallum, J., Dune Ly, C., Ehlers, M.D., and Wenthold, R.J. (2001). Molecular determinants of NMDA receptor internalization. Nat Neurosci 4, 794-802.       137 Rojas, R., Kametaka, S., Haft, C.R., and Bonifacino, J.S. (2007). Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors. Mol Cell Biol 27, 1112-1124. Rojek, A., Fuchtbauer, E., Kwon, T., Frøkiær, J. and Nielsen, S. (2006). Severe urinary concentrating defect in renal collecting duct-selective AQP2 conditionalknockout mice PNAS 15, 6037–6042. Rothman, J.E. (1994). Mechanisms of intracellular protein transport. Nature 372, 55– 63. Rothman, J.E., and Wieland, F.T. (1996). Protein sorting by transport vesicles. Science 272, 227-234. Rubino, M., Miaczynska, M., Lippe, R., and Zerial, M. (2000). Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes. J Biol Chem 275, 3745-3748. Sandholzer, U., von Figura, K., and Pohlmann, R. (2000). Function and properties of chimeric MPR 46-MPR 300 mannose 6-phosphate receptors. J Biol Chem 275, 14132-14138. Sasaki, S., Kuwahara, M, Yamashita, Y., and Marumo, F. (2000). Structure and function of AQP2. Nephrol Dial Transplant. 15, 21-22. Schlessinger, J. (2004). Common and distinct elements in cellular signaling via EGF and FGF receptors. Science 306, 1506-1507. Schwarz, D.G., Griffin, C.T., Schneider, E.A., Yee, D., and Magnuson, T. (2002). Genetic analysis of sorting nexins and reveals a redundant and essential function in mice. Mol Biol Cell 13, 3588-3600.       138 Scott, D. B., Michailidis, I., Mu, Y., Logothetis, D. and Ehlers, M. D. (2004). Endocytosis and degradative sorting of NMDA receptors by conserved membraneproximal signals. J Neurosci 24, 7096–7109. Seaman, M.N. (2004). Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J Cell Biol 165, 111-122. Seaman, M.N., McCaffery, J.M., and Emr, S.D. (1998). A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast. J Cell Biol 142, 665681. Seet, L.F. and Hong, W. (2001). Endofin, an Endosomal FYVE Domain Protein Biol Chem 276, 42445–42454. J Seet, L.F. and Hong, W. (2006). The Phox (PX) domain proteins and membrane traffic Biochim Biophys Acta 1761, 878-896. Seyberth, H. W., Rascher, W., Schweer, H., Kuhl, P. G., Mehls, O. and Scharer, K. (1985). Congenital hypokalemia and hypercalciuria in preterm infants: a hyperprostaglandinuric tubular syndrome different from Bartter syndrome J Pediatr. 107, 694–701. Simmen, T., Honing, S., Icking, A., Tikkanen, R., and Hunziker, W. (2002). AP-4 binds basolateral signals and participates in basolateral sorting in epithelial MDCK cells. Nat Cell Biol 4, 154-159. Simonsen, A., Lippe, R., Christoforidis, S., Gaullier, J.M., Brech, A., Callaghan, J., Toh, B.H.,Murphy, C., Zerial, M., and Stenmark, H. (1998). EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 394, 494-498. Stahelin, R.V., Ananthanarayanan, B., Blatner, N.R., Singh, S., Bruzik, K.S., Murray, D., and Cho, W. (2004). Mechanism of membrane binding of the phospholipase D1 PX domain. J Biol Chem 279, 54918-54926.       139 Stockinger, W., Sailler, B., Strasser, V., Recheis, B., Fasching, D., Kahr, L., Schneider, W.J., and Nimpf, J. (2002). The PX-domain protein SNX17 interacts with members of the LDL receptor family and modulates endocytosis of the LDL receptor. EMBO J 21, 4259-4267. Stokes, J.B., Grupp, C., and Kinne, R.K. (1987). Purification of rat papillary collecting duct cells: functional and metabolic assessment. Am. J. Physiol. 253, F251F262. Strochlic, T. I., Setty, T. G., Sitaram, A. and Burd, C. G. (2007).Grd19/Snx3p functions as a cargo-specific adapter for retromer-dependent endocytic recycling. J. Cell Biol. 177, 115–12 . Shi, H., Rojas, R., Bonifacino, J.S., and Hurley, J.H. (2006). The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain. Nat Struct Mol Biol 13, 540-548. Snyder, E.M., Nong, Y., Almeida, C.G., Paul, S., Moran, T., Choi, E.Y., Nairn, A.C., Salter, M.W., Lombroso, P.J., Gouras, G.K., et al. (2005). Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8, 1051-1058. Spitzer, A. (2003). Twenty-one years of developmental nephrology: the kidney then and now. Pediatr Nephrol 18, 165-173. Sun, T. X., Van Hoek, A., Huang, Y., Bouley, R., McLaughlin, M. and Brown, D. (2002). Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin. Am J Physiol Renal Physiol. 282, F998–F1011. Szoka, P. R., Gallagher, J. F. and Held, W. A. (1980). In vitro synthesis and characterization of precursors to the mouse major urinary proteins. J Biol Chem 255,1367–1373. Sztul, E., and Lupashin, V. (2006). Role of tethering factors in secretory membrane traffic. Am J Physiol Cell Physiol 290, C11-26.       140 Takahashi, N., Chernavvsky, D.R., Gomez, R.A., Igarashi, P., Gitelman, H.J., and Smithies, O. (2000). Uncompensated polyuria in a mouse model of Bartter's syndrome. Proc Natl Acad Sci U S A 97, 5434-5439. Tang, B.L., Wang, Y., Ong, Y.S., and Hong, W. (2005). COPII and exit from the endoplasmic reticulum. Biochim Biophys Acta 1744, 293-303. Theos, A.C., Tenza, D., Martina, J.A., Hurbain, I., Peden, A.A., Sviderskaya, E.V., Stewart,A., Robinson, M.S., Bennett, D.C., Cutler, D.F., Bonifacino, J.S., Marks, M.S., and Raposo,G. (2005). Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting fromendosomes to melanosomes. Mol Biol Cell 16, 5356-5372. Tisher, C. C. and Madsen, K. M. (1996). Anatomy of the kidney. The Kidney. 5th ed. 3-71. Traer, C. J. Rutherford A C., Palmer K J., Wassmer T, Oakley J, Attar N, Carlton J G., Kremerskothen J, Stephens DJ.and. Cullen P J. (2007).Sorting nexin-4 co-ordinates endosomal sorting of transferrin receptor with dyneinmediated transport into the endocytic recycling compartment. Nature Cell Biol. 9, 1370–1380. Traub, L.M. (2005). Common principles in clathrin-mediated sorting at the Golgi and the plasma membrane. Biochim Biophys Acta 1744, 415-437. Tovar, K.R., and Westbrook, G.L., (1999). The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J. Neurosci. 19, 4180–4188. Ungar, D., Oka, T., Krieger, M., and Hughson, F.M. (2006). Retrograde transport on the COG railway. Trends Cell Biol 16, 113-120. Vanhaesebroeck, B., Leevers, S. J., Panayotou, G. and Waterfield, M. D. (1997). Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem. Sci. 22, 267–272.       141 Vicinanza, M., D'Angelo, G., Di Campli, A., and De Matteis, M.A. (2008). Phosphoinositides as regulators of membrane trafficking in health and disease. Cell Mol Life Sci 65, 2833-2841. Washbourne, P., Bennett, J. E. and McAllister, A. K. (2002). Rapid recruitment of NMDA receptor transport packets to nascent synapses. Nature Neurosci. 5, 751–759. Wassmer, T., Attar, N., Bujny, M.V., Oakley, J., Traer, C.J., and Cullen, P.J. (2007). A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer. J Cell Sci 120, 45-54. Wenk, M.R., and De Camilli, P. (2004). Protein-lipid interactions and phosphoinositide metabolism in membrane traffic: insights from vesicle recycling in nerve terminals. Proc Natl Acad Sci U S A 101, 8262-8269. Wenthold, R.J., Prybylowski, K., Standley, S., Sans, N. and Petralia, R.S. (2003).Trafficking of NMDA receptors. Annu Rev Pharmacol Toxicol 43, 335–358. Whitley, P., Reaves, B.J., Hashimoto, M., Riley, A.M., Potter, B.V., and Holman, G.D. (2003). Identification of mammalian Vps24p as an effector of phosphatidylinositol 3,5-bisphosphate-dependent endosome compartmentalization. J Biol Chem 278, 38786-38795. Wienholds, E., and Plasterk, R.H. (2005). MicroRNA function in animal development. FEBS Lett 579, 5911-5922. Wientjes, F.B., Reeves, E.P., Soskic, V., Furthmayr, H., and Segal, A.W. (2001). The NADPH oxidase components p47(phox) and p40(phox) bind to moesin through their PX domain. Biochem Biophys Res Commun 289, 382-388. Worby, C.A., and Dixon, J.E. (2002). Sorting out the cellular functions of sorting nexins. Nat Rev Mol Cell Biol 3, 919-931.       142 Xu, Y., Hortsman, H., Seet, L., Wong, S.H., and Hong, W. (2001a). SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P. Nat Cell Biol 3, 658-666. Xu,Y., Seet, L.F., Hanson, B., and Hong, W.(2001b). Biochem J 360, 513-530. Yang, B., Zhao, D., Qian, L., and Verkman, A.S. (2006). Mouse model of inducible nephrogenic diabetes insipidus produced by floxed aquaporin-2 gene deletion. Am J Physiol Renal Physiol 291, F465-472. Yarar, D., Waterman-Storer, C.M., and Schmid, S.L. (2007). SNX9 couples actin assembly to phosphoinositide signals and is required for membrane remodeling during endocytosis. Dev Cell 13, 43-56. Zheng, B., Ma, Y.C., Ostrom, R.S., Lavoie, C., Gill, G.N., Insel, P.A., Huang, X.Y., and Farquhar, M.G. (2001). RGS-PX1, a GAP for GalphaS and sorting nexin in vesicular trafficking. Science 294, 1939-1942. Zheng, B., Tang, T., Tang, N., Kudlicka, K., Ohtsubo, K., Ma, P., Marth, J.D., Farquhar, M.G., and Lehtonen, E. (2006). Essential role of RGS-PX1/sorting nexin 13 in mouse development and regulation of endocytosis dynamics. Proc Natl Acad Sci U S A 103, 16776-16781. Zhou, C.Z., de La Sierra-Gallay, I.L., Quevillon-Cheruel, S., Collinet, B., Minard, P., Blondeau, K., Henckes, G., Aufrere, R., Leulliot, N., Graille, M., et al. (2003). Crystal structure of the yeast Phox homology (PX) domain protein Grd19p complexed to phosphatidylinositol-3-phosphate. J Biol Chem 278, 50371-50376. Zhu, Y., Doray, B., Poussu, A., Lehto, V.P., and Kornfeld, S. (2001). Binding of GGA2 to the lysosomal enzyme sorting motif of the mannose 6-phosphate receptor. Science 292, 1716-1718.       APPENDICES 1. Phosphate-Buffered Saline (PBS), pH 7.4 Final concentration(for liter) 137 mM NaCl 2.7 mM KCl 10 mM Na2HPO4 mM KH2PO4 8g 0.2 g 1.44 g 0.24 g Top up with H2O to liter. 2. LB Medium (Luria Bertani Medium) For liter: Trypone Yeast extract NaCl 10g 5g 10g Adjust the pH to 7.0 with 5M NaOH (~0.2 ml). 3. LB Agar Agar powder Sodium chloride Tryptone Yeast extract 15 g 5g 10 g 5g Top up to liter with deionized water and adjust pH to ~7.0. Autoclave and pour about 25 ml per petri dish. 4. Bacteria Freezing Medium 30% (v/v) sterile glycerol in LB medium 5. Cell Freezing Solution 10% DMSO in FBS (Filter through 0.20 μm filter unit before use.)   143     6. DNA Electrophoresis buffers TAE Electrophoresis Buffer TAE Working concentration 1X 40 mM Trisacetate mM EDTA Stock solution/Liter 50X 242 g of Tris base 57.1 ml of glacial acetic acid 100 ml of 0.5 M EDTA (pH 8.0) 7. Agarose Gel Resolution % Gel 0.5 0.7 1.0 1.2 1.5 Optimum Resolution for Linear DNA (kb) 30 to 1.0 12 to 0.8 10 to 0.5 to 0.4 to 0.2 8. 6X DNA Gel-loading Buffers Buffer Type I (for larger size DNA) II(for small size DNA) 6X Buffer (50 ml) 30% glycerol (15 ml of glycerol stock) 0.3% bromophenol blue (0.15 g) mM EDTA (100 μl of 0.5 M EDTA, pH 8.0) 50% glycerol (25 ml of glycerol stock) 0.25% xylene cyanol FF (0.125 g) 9. Electrophoresis Buffer 1X Working concentration 25 mM Tris-Cl 250 mM glycine 0.1% SDS   5X Stock solution/Liter 15.1 g of Tris base 94 g of glycine (electrophoresis grade) 50 ml of 10% SDS (electrophoresis grade) 144     145 10. SDS- Polyacrylamide Gel Electrophoresis of Protein 11. Coating Glass Coverslips POLYLYSINE AND POLYORNITHINE Nearly all types of cells adhere to these polymers of basic amino acids. They are particularly useful for the culture of CNS neurons. The L- or D-isomers can be used for cell attachment, however, the D-isomer may be preferred because it is not subject to breakdown by proteases released by cells. a. Prepare polylysine or polyornithine (MW of 30,000 - 70,000) at 0.1-1 mg/ml in 0.15 M borate buffer (pH 8.3). Filter sterilize. b. Add enough solution to pool over surface of sterile glass coverslip. c. Incubate 2-24 hours at room temperature. d. Aspirate solution and wash coverslips times with media or PBS. e. Immediately add cell suspension or growth media.   [...]... al., 2003; Vicinanza et al., 2008) This lipid is involved in modulating early and late stages of endosome function through its interaction with a variety of functional protein domains, including the Phox (PX) domain and the FYVE domain (Lemmon, 2003; Birkeland and Stenmark, 2004) For example, the FYVE domain containing protein EEA1 is involved in modulating early endosomal dynamics by regulating homo-... Fig.3.7 SNX27 co-localizes with EEA1 Fig.3.8 Wortmannin-sensitive endosomal association of SNX27 Fig.3.9 SNX27 is expressed in multiple mouse tissues Fig.3.10 Detection of SNX27 protein expression in mouse tissue samples by indirect immunofluorescence Fig.4.1 Schematic drawing of SNX27 genomic fragment, targeting vector and the screening for positive ES clones Fig.4.2 SNX27 protein is undetectable in SNX27- /-... p47phox is also able to interact with moesin, a protein that links F-actin to the plasma membrane (Wientjes et al., 2001) Thus, PX domain not only acts as a lipid binding platform, but is also a protein-protein interacting domain 1.4 Sorting Nexin family       15 Sorting nexins (SNXs) are a sub-group of peripheral membrane proteins containing a conserved SNX-PX domain that targets SNX to endosomes (Worby... SNX15 SNX27 SNX16 SNX28 SNX17, SNX31 SNX29 SNX21 PX domain PDZ domain RA domain TM domain Coiled-coil MIT domain BAR domain RGS domain SH3 domain FERM domain TPR domain FHA domain Kinesin motor RhoGAP domain PXA domain   Fig 1.4: Domain architecture of the mammalian sorting nexins The key domains are shown at the lower panel         17 SNX1 is the prototype member of the SNX family It was identified in. .. been identified, including Ent3p (Friant et al., 2003), hVps24p (Whitley et al., 2003), WIPI49 (Jeffries et al., 2004) and sorting nexin 1 (SNX1) (Cozier et al., 2002) 1.3 The PX domain The Phox (PX) domain is a phosphoinositide binding domain involved in targeting proteins to endosome membranes This domain averaging 120 amino acids in length was first identified through the analysis of two of the... PX domain containing proteins have a proline rich motif (PXXP) in the middle, such as p47phox PXXP is characterized as SH3 domain binding motif (Ponting, 1996; Babior, 1999) Hiroaki et al demonstrated that p47phox is able to bind its own C-terminal SH3 domain through this proline rich motif (Hiroaki et al., 2001) Furthermore, Segal’s laboratory reported PX domain of p47phox is also able to interact... proteins from one-month-old mice Fig.4.16 Increased AQP2 expression in SNX27- /- kidneys Fig.4.17 Histological comparison of testis and brains from wild-type and SNX27- /- mice Fig.4.18 Protein expression levels of Kir3 channel in the brains of newborn SNX27- /- and wild-type pups Fig.5.1 Knockdown of SNX27 in A431 cells inhibited the endocytosis of EGFR Fig.5.2 Depletion of SNX27 inhibited the lysosomal... Direct visualization of EGF endocytosis by live cell imaging Fig.6.1 Characterization of the SNX27- NR2c interaction using the yeast two-hybrid assay mice exhibited significant XI Fig.6.2 The PDZ binding motif is critical for the interaction of NR2c with SNX27 Fig.6.3 SNX27 is partially overlapped with NR2c in primary cultured neurons Fig.6.4 The increased NR2c expression levels in SNX27- /- mice Fig.7.1... H&E staining of PN14 mouse kidney sections Fig.4.10 H&E staining of PN20 mouse kidney sections Fig.4.11 Enhanced apoptosis at PN20 in SNX27- /- kidneys Fig.4.12 The blood glucose level and image of rescued SNX27- /- mouse Fig.4.13 Rescued SNX27- /- mice kidneys displayed progressive medullar degeneration Fig.4.14 Comparison of urinalysis data from one-month-old mice Fig.4.15 Analysis of urine proteins from...VIII LIST OF TABLES Table 1: List of DNA plasmid constructs made for this study Table 2: List of used primary antibodies in this study Table 3: Water intake and urine extraction of one-month-old female mice Table 4: List of SNX27 interacting candidates IX LIST OF FIGURES Fig.1.1 Schematic drawing of membrane trafficking pathways Fig.1.2 Schematic drawing of membrane transport through . two-hybrid screening for SNX27 interacting proteins 6.2 Direct PDZ domain mediated interaction of SNX27 with NR2c 6.3 The PDZ binding motif is critical for the interaction of NR2c with SNX27 V. SNX27 IS IMPORTANT FOR POSTNATAL DEVELOPMENT IN MICE CAI LEI INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 SNX27 IS IMPORTANT. drawing of SNX27 genomic fragment, targeting vector and the screening for positive ES clones. Fig.4.2 SNX27 protein is undetectable in SNX27 -/- mice. Fig.4.3 Birth defect of SNX27 -/- mice.