CHARACTERIZATION OF RAB22B, AN ASTROGLIAENRICHED RAB GTPASE, AND ITS’ ROLE IN GOLGI AND POST-GOLGI MEMBRANE TRAFFIC NG, EE LING NATIONAL UNIVERSITY OF SINGAPORE 2008 CHARACTERIZATION OF RAB22B, AN ASTROGLIAENRICHED RAB GTPASE, AND ITS’ ROLE IN GOLGI AND POST-GOLGI MEMBRANE TRAFFIC NG, EE LING B. APP. SC. (HONS) (QUEENSLAND UNIVERSITY OF TECHNOLOGY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (BIOCHEMISTRY) DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE Acknowledgements Acknowledgements First and foremost, I would like to thank my supervisor for his unwavering support and guidance throughout the entire course of my studies. I would also like to extend my appreciation to Wang Ya, Lim Sharon and fellow colleagues for sharing of technical knowledge and support. Last but not least, I would like to extend my gratitude to my understanding and patient husband for standing by me during the period of my studies. i Contents Contents Contents pg. ii Abstract pg. v List of figures pg. viii List of units pg. xiii 1. Introduction 1.1 1.2 1.3 pg. An overview to Rab GTPases and membrane trafficking pg. 1.1.1 Regulators of Rab GTPases pg. 1.1.2 Effectors of Rab GTPases pg. Rab GTPases in the central nervous system (CNS) pg. 1.2.1 Rab GTPases in neuronal protein trafficking pg. 10 1.2.2 Rab GTPases in CNS development pg. 16 1.2.3 Rab GTPases in the regulation of synaptic transmission pg. 19 1.2.4 Rab GTPases in glia cells Aims and rationale of current work pg. 24 pg. 27 2. Materials and methods 2.1 Cell culture pg. 28 2.1.1 pg. 28 Preparation and maintenance of primary astrocytes, oligodendrocytes and neurons 2.1.2 Maintenance of mammalian cell lines pg. 32 ii Contents 2.2 Transfection of mammalian cell lines 2.2.1 Transfecting mammalian cell lines with expression pg. 33 constructs 2.2.2 Transfecting mammalian cell lines with pg. 35 si(small-interfering)RNA 2.2.3 Generation of Rab22A/Rab22B-expressing pg. 36 stable clones 2.3 Trafficking assays 2.3.1 Shiga toxin uptake and transport pg. 36 2.3.2 Transferrin (Tf) uptake and transport pg. 37 2.3.3 Epidermal growth factor (EGF) uptake and transport pg. 38 2.3.4 Monitoring vesicular stomatitis viral glycoprotein pg. 38 (VSVG) transport 2.4 2.5 Immunofluorescence assays and confocal microscopy 2.4.1 Immunohistochemistry pg. 39 2.4.2 Immunocytochemistry pg. 42 2.4.3 Confocal microscopy and imaging pg. 43 DNA and protein manipulation 2.5.1 Polymerase chain reaction (PCR) pg. 44 2.5.2 Bacterial and mammalian expression constructs pg. 45 2.5.3 Large-scale induction of bacterial fusion proteins pg. 51 2.5.4 Lysis of cultured cells or tissues pg. 52 2.5.5 Affinity pull-down assay pg. 53 iii Contents 2.5.6 Co-immunoprecipitation pg. 54 2.5.7 Western immunoblot pg. 55 2.5.8 MTS assay pg. 56 2.6 Animal immunization and antibody production pg. 57 2.7 Statistical analysis pg. 58 3. Results & discussions 3.1 Generation of a specific antibody against Rab22B and tissue pg. 59 distribution of Rab22B 3.2 Brain cell-type distribution of Rab22B pg. 67 3.3 Sub-cellular distribution of Rab22B pg. 77 3.4 Rab22B’s dynamics at the TGN pg. 88 3.5 Rab22B’s role in TGN and post-Golgi traffic pg. 107 3.6 Rab22B’s role in modulating EGFR trafficking and pg. 117 signaling 4. Conclusions and future perspectives 4.1 General conclusions pg. 134 4.2 Future perspectives and directions pg. 136 5. Bibliography pg. 142 6. Appendix A pg. 167 7. Appendix B pg. 170 - List of publications iv Abstract Abstract The small Rab GTPase, Rab22B, was first cloned in 1996 (Chen et al., 1996). However, other than the fact that it is brain-enriched, its’ brain expression profile, exact cellular and sub-cellular localization, and functions have remained uncharacterized. Specific antibodies against Rab22B, which does not cross react with its close paralogue Rab22A, were generated to help to answer the above questions. A tissue survey using specific antibodies against Rab22B confirms that Rab22B is indeed brain-enriched, but is also expressed in appreciable levels in the spleen and intestine. Immunohistochemistry analysis revealed that in the mouse embryonic brain, Rab22B labeling is found in nestin and RC2 epitope-positive fibers of the radial glia. In the mouse adult brain, Rab22B is rather specifically expressed in glial fibrillary acidic protein (GFAP)-positive astrocytes, but apparently undetectable in
III tubulin (TuJ)positive neurons and CNPase-positive oligodendrocytes. These observations were further confirmed with the staining of glia cells in culture. Detailed immunocytochemistry analysis revealed that the primary membrane localization of Rab22B is at the trans-Golgi network (TGN). Over-expression of the GTP-binding mutant Rab22B S20N disrupted the TGN localization of a dynamic marker, TGN46, which cycles between the TGN and the plasma membrane. Other TGN resident membrane protein such as syntaxin 16, and cis-Golgi markers such as GM130 and syntaxin 5, and the TGN/late endosome marker mannose 6-phosphate receptor (M6PR), are however not affected. These results demonstrate that the effect of Rab22B SN on v Abstract TGN traffic disruption affects a dynamic TGN marker rather than the more static residents. Rab22B’s apparent TGN localization and the ability to disrupt a specific marker seemed to indicate that it may be involved in trafficking in and/or out of the TGN. Further investigations illustrate that Rab22B is not involved in retrograde trafficking of some common endocytic cargoes such as transferrin and Shiga B toxin. However, overexpression of Rab22B S20N mutant inhibits the exit of the anterograde cargo vesicular stomatitis G protein (VSVG) out of the TGN Recent reports had also revealed that Rab22B shares its guanine nucleotide exchange factor, GAPex-5, with Rab5 (Lodhi et al., 2007). The silencing of GAPex-5 appears to mediate the trafficking of EGFR. In spite of being non-essential for endocytosis for some common cargoes, Rab22B appears to mediate EGFR trafficking. Rab22B silencing inhibited or delayed the trafficking of EGFR, as well as Texas-Red conjugated EGF, into the CD63-positive late endosomal/lysosomal compartment. Affinity pull-down assay and co-immunoprecipitation analysis show that Rab22B interacts with EGFR in a GTP-dependent manner. While the silencing of Rab22B does not affect the phosphorylation kinetics of the signaling intermediates downstream of EGFR, ERK1/2, dephosphorylation of ERK1/2 appears to be slower. This may also shed light on why the proliferation rates, monitored vi Abstract over 72 hours, were significantly increased in cells treated with Rab22B siRNA compared to scramble control RNA. In summary, my work revealed Rab22B as the first Rab GTPase that is specifically enriched in the brain astroglia lineage, and may play a role in regulating Golgi and post-Golgi trafficking. vii List of figures List of figures 1. Section 1; Fig. 1.1: Schematic diagram depicting the paths of membrane traffic within a typical eukaryotic cell. 2. Section 1.1; Fig. 1.2: Rab’s activities and functions is regulated by three classes of regulatory molecules, namely the guanine nucleotide exchange factor, the GTPase activating protein and guanine nucleotide dissociation inhibitor. 3. Section 1.2; Fig. 1.3: A schematic diagram depicting the localization and function of Rab GTPases in diverse neuronal processes (polarized neurite outgrowth/regeneration, axonal retrograde transport, postsypnaptic glutamate receptor trafficking and synaptic vesicle exocytosis) 4. Section 2.1.1; Fig. 2.1: Schematic diagram illustrating the procedures for isolation of astrocytes and oligodendrocytes from rat pups. 5. Section 2.4.3; Fig. 2.2: A typical image taken with the confocal microscope. 6. Section 2.5.1; Fig. 2.3: A typical picture of analysis performed with an agarose gel electrophoresis. 7. Section 2.5.2; Fig. 2.4: Vector map of pGEX-4T-1. 8. Section 2.5.2; Fig. 2.5: Vector map of pCI-Neo. 9. Section 2.5.3; Fig. 2.6: Picture showing SDS-PAGE analysis of fusion protein production. 10. Section 2.6; Fig. 2.7: Schematic diagram showing the process of animal immunization and antibody production. 11. Section 3.1; Fig. 3.1: Alignment of Rab22A and Rab22B amino acid sequences. 12. 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Beads were then washed and bound proteins eluted with SDSPAGE sample buffer, followed by Western immunoblot analysis with EEA1 antibody. 168 Appendix A Fig. A3: Over-expression Rab40C of affects the transport of Tf-FITC. Myc-tagged Rab40C WT and its’ mutants were over-expressed in HeLa cells. Following transfection (after 16-18 hours), the cells were subjected to a 18oC incubation to internalize transferrin-FITC and a further incubation at 37oC for 10 minutes. Cells were then fixed for immunofluorescence analysis with myc antibodies, followed by TxR-conjugated donkey anti-rabbit Ig. Bar = 10μm. 169 Appendix B 170 Appendix B 171 Appendix B Rab22B is expressed in CNS astroglia lineage and influences epidermal growth factor receptor trafficking – submitted to Molecular Membrane Biology for review Ee Ling Ng1, Fengyi Liang2, and Bor Luen Tang1,* Department of 1Biochemistry and 2Department of Anatomy Yong Loo Lin School of Medicine, National University of Singapore, Medical Drive, Singapore 117597. *Corresponding author: Phone: 65-5161040; Fax: 65-67791453; Email: bor_luen_tang@nuhs.edu.sg ------------------------------------------------------------------------- Running title: Rab22B is astroglia-enriched and modules EGFR Keywords: Rab22B, small GTPase, trans-Golgi network (TGN) 172 Appendix B Abstract The expression profile and functions of the brain-enriched Rab22B/Rab31 small GTPase had remained uncharacterized. Using specific antibodies against Rab22B, we found the protein to be exceptionally enriched in nestin and RC2-positive radial glia of embryonic mouse brain. In the adult brain, Rab22B is rather specifically expressed in glial fibrillary acidic protein (GFAP)-positive mature astrocytes, but is not clearly detectable in either CNPase-positive mature oligodendrocytes or
III-tubulin-positive neurons. In probing for specific functions of Rab22B, we found that Rab22B silencing in A431 cells resulted in abnormal trafficking of the epidermal growth factor receptor (EGFR) as well as Texas-Red-labeled EGF. Affinity pull-down assays and coimmunoprecipitation analysis indicated that Rab22b could associate with EGFR in a GTP-dependent manner. Rab22B is thus a Rab protein specifically expressed in the astrogolia lineage and may have a role in regulating EGFR trafficking. 173 [...]... in the form of lipoproteins Receptor-mediated endocytosis of receptor-ligand complexes also functions in regulating cellular signaling (Derby and Gleeson, 2007; Hanyaloglu and von Zastrow, 2008) 1.1 An overview of Rab GTPases and membrane trafficking The enormous flux of membrane traffic within a cell at any point of its existence necessitates stringent control of both the rate and specificity of traffic. .. II of the GTP-binding domains and similarly, mutant forms of Rabs can be generated by creating amino acid substitutions in the Region I (GDTGVDKS) and II (DXXGQ) of the GTP-binding domains At Region I, substitution of the serine (S) residue to an asparagine (N) residue (to be refered as a SN mutant) will render the Rab protein deficient in binding of GTP, which means that it will be permanently bound... general role in neuronal traffic and neurite outgrowth, Rab8 and Rab1 1 also regulate polarized transport processes in these specialized neural cell types 13 Introduction Early studies suggested that Rab6 (Shetty et al., 1998) and Rab8 associate with post -Golgi membranes sequentially at different stages during rhodopsin transport, and Rab8 may mediate the interaction of transport vesicles or membrane with... endosomes An important Rab protein that is involved in both these processes is Rab1 1 One primary function of Rab1 1 in non-neuronal cells is to control membrane trafficking through the recycling endosomes Dominant-negative Rab1 1A appears to inhibit apical recycling and basolateral-to-apical transcytosis in polarized Madin-Darby canine kidney (MDCK) cells (Wang et al., 2000) In the brain, Rab1 1 is localized... Rab3 8, Rab1 1, Rab1 , Rab8 and Rab3 ) (Buvelot et al., 2006) of eukaryotic Rabs and can be subgrouped into eight branches in a phylogenetic tree, with members on the same branch exhibiting similar patterns of cellular localization and function (Pereira-Leal and Seabra, 2001) The roughly six fold increase in terms of the number of Rabs from the unicellular yeast to human is more than a simple reflection of an. .. vesicle’s membrane Binding of Rab to a Rab effector will help to tether the vesicle to its appropriate target membrane and allows other membrane surface proteins such as SNAREs to interact, resulting in the docking of the vesicle to the target membrane When the Rab has fulfilled its function, GTP is hydrolyzed back to GDP Rabs have low intrinsic GTPase activity, which is enhanced by the GTPase activating... proteins In the cytosol, the GDP dissociation inhibitor binds to the GDP-bound Rab and inhibits the exchange of GDP for GTP, which would reactivate the Rab protein Bioinformatical analysis has revealed that Rab genes exhibit a rather strict phylogeny of homology and function The Rab proteins found in Saccharomyces cerevisiae had be grouped into 10 major subclasses (Rab5 , Rab7 , Rab6 , Rab4 , Rab2 8, Rab3 8,... three classes of regulatory molecules, namely the guanine nucleotide exchange factor, the GTPase activating protein and guanine nucleotide dissociation inhibitor Once Rab proteins are bound to a membrane of a vesicle via its lipid tail, the exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors Rabs bound to GTP are in the active conformation and can now interact with its effectors... regulation of synaptic transmission The Rab3 subfamily (Rab3 A, Rab3 B, Rab3 C, and Rab3 D) is arguably the most extensively investigated Rabs in terms of synaptic function The Rab3 proteins are expressed in brain as well as endocrine tissues and, in conjunction with their effectors such as rabphilin (Shirataki et al., 1993; Li et al., 1994) and Rim (Wang et al., 1997), function in regulated exocytosis and neurotransmitter... number of Rabs, such as Rab3 A, Rab8 and Rab2 3, have been shown to be brain -enriched and have documented roles in neurons (Geppert et al., 1997; Evans et al., 2003) Recent findings on the roles played by Rab GTPases, their regulatory modules and effectors in the central nervous system (CNS), will be briefly discussed and summarized below, with respect to anterograde and retrograde transport within the . CHARACTERIZATION OF RAB2 2B, AN ASTROGLIA- ENRICHED R AB GTPASE, AND ITS ROLE IN GOLGI AND POST -GOLGI MEMBRANE TRAFFIC NG, EE LING NATIONAL UNIVERSITY OF SINGAPORE 2008 CHARACTERIZATION. 2008 CHARACTERIZATION OF RAB2 2B, AN ASTROGLIA- ENRICHED R AB GTPASE, AND ITS ROLE IN GOLGI AND POST -GOLGI MEMBRANE TRAFFIC NG, EE LING B. APP. SC. (HONS) (QUEENSLAND UNIVERSITY OF TECHNOLOGY). of Rab GTPases and membrane trafficking The enormous flux of membrane traffic within a cell at any point of its existence necessitates stringent control of both the rate and specificity of