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• W2078264101 abstract "Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of full-length SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis. Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of full-length SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis. Sorting nexin 9 (SNX9), 2The abbreviations used are: SNX9sorting nexin 9SH3Src homology domain 3PXPhox homologyDIVdays in vitroPMSFphenylmethylsulfonyl fluorideBARBin/Amphiphysin/RvsPBSphosphate-buffered salineBSAbovine serum albuminGFPgreen fluorescent proteinGSTglutathione S-transferasePRDproline-rich domainANOVAanalysis of varianceLC-MS/MSliquid chromatography-tandem mass spectrometrysiRNAshort interfering RNAspHsynaptopHluorinmRFPmonomeric red fluorescent proteinAPaction potential. also known as SH3PX1, is a member of the sorting nexin superfamily characterized by the presence of a phospholipid-binding motif, the PX domain. Sorting nexin family proteins contribute to protein sorting in cells by their ability to bind specific lipids and to form protein-protein complexes. SNX9, initially identified as a protein interacting with the metalloproteases MDC9 and MDC15 (1Howard L. Nelson K.K. Maciewicz R.A. Blobel C.P. J. Biol. Chem. 1999; 274: 31693-31699Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), is composed of an N-terminal Src homology 3 domain, a low complexity region, a PX domain, and a C-terminal Bin/Amphiphysin/Rvs (BAR) domain (2Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 3Lundmark R. Carlsson S.R. J. Biol. Chem. 2004; 279: 42694-42702Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 4Yeow-Fong L. Lim L. Manser E. FEBS Lett. 2005; 579: 5040-5048Crossref PubMed Scopus (42) Google Scholar). It forms a complex with dynamin-2 and regulates the recruitment of dynamin-2 to the membrane (5Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar). It also enhances the assembly of dynamin and increases its GTPase activity (6Soulet F. Yarar D. Leonard M. Schmid S.L. Mol. Biol. Cell. 2005; 16: 2058-2067Crossref PubMed Scopus (161) Google Scholar). Other endocytic molecules, namely AP-2 (adaptor protein complex 2) and clathrin, also bind to the low complexity region of SNX9 in a cooperative manner (2Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Through these interactions, SNX9 plays an important role in clathrin-mediated endocytosis in non-neuronal cells (2Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 6Soulet F. Yarar D. Leonard M. Schmid S.L. Mol. Biol. Cell. 2005; 16: 2058-2067Crossref PubMed Scopus (161) Google Scholar). sorting nexin 9 Src homology domain 3 Phox homology days in vitro phenylmethylsulfonyl fluoride Bin/Amphiphysin/Rvs phosphate-buffered saline bovine serum albumin green fluorescent protein glutathione S-transferase proline-rich domain analysis of variance liquid chromatography-tandem mass spectrometry short interfering RNA synaptopHluorin monomeric red fluorescent protein action potential. Dynamin is centrally involved in clathrin-mediated endocytosis (7Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (584) Google Scholar, 8Sever S. Curr. Opin. Cell Biol. 2002; 14: 463-467Crossref PubMed Scopus (112) Google Scholar). It self-assembles around the necks of invaginated clathrin-coated pits and releases vesicles from the membrane via GTP hydrolysis (9Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1040) Google Scholar). It is composed of several domains. The N-terminal nucleotide-binding domain is responsible for GTP hydrolysis, and the C-terminal proline-rich domain (PRD) links it to several SH3 domain-containing proteins such as Grb2, amphiphysin, and endophilin (10Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (484) Google Scholar, 11Okamoto P.M. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1997; 272: 11629-11635Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 12Solomaha E. Szeto F.L. Yousef M.A. Palfrey H.C. J. Biol. Chem. 2005; 280: 23147-23156Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The central pleckstrin homology domain controls its binding to membrane phospholipids (13Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and a coiled-coil domain (also called the GTPase effector domain) is involved in its self-assembly and in regulating its GTPase activity. The affinity between the pleckstrin homology domain of dynamin and lipids is not high enough to translocate dynamin from the cytosol to the plasma membrane (14Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (617) Google Scholar). Rather, truncation of the PRD of dynamin blocks endocytosis because of mislocation of the protein (13Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) suggesting that it is the interaction of the PRD with SH3-containing domains of other endocytic molecules that is the key to the correct localization and functioning of dynamin. Because of the mechanistic similarity between clathrin-mediated endocytosis in non-neuronal cells and synaptic vesicle endocytosis, SNX9 is suspected of having a role in synaptic vesicle endocytosis. However, the presence of SNX9 in neuronal cells and the functional interplay between it and its presynaptic binding partners in synaptic vesicle endocytosis have never been studied. Here we report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons where it associates with dynamin-1 as well as N-WASP, and that it plays a regulatory role in synaptic vesicle endocytosis. Because SNX9 can be dimerized, and binds dynamin at one end and N-WASP at the other, our data raise the possibility that it links actin dynamics and synaptic vesicle endocytosis. GST Pulldown Assays—The GST-SNX9, GST-SNX9-SH3, GST-SNX9ΔSH3, and GST-virgin vector plasmids were transformed into Escherichia coli BL-21, and the transformants were cultured in LB medium supplemented with ampicillin. After overnight induction with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside at 25 °C, the cultures were sonicated in lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 20 mm Tris, pH 8.0, 150 mm NaCl, 1 mm MgCl2, 1 mm EGTA, 0.1 mm PMSF) and centrifuged for 15 min at 12,000 rpm, and the supernatants were incubated with glutathione-agarose-4B beads (Amersham Biosciences) at 4 °C for 30 min. After washing three times with lysis buffer, the beads were incubated for 2 h at 4 °C with a brain lysate in lysis buffer. The beads were then washed extensively with lysis buffer and analyzed by SDS-PAGE and immunoblotting. Co-immunoprecipitation—To detect SNX9 binding to dynamin-1 in vivo, COS-7 cells were transfected with FLAG-SNX9 and FLAG-SNX9-ΔSH3 together with GFP-dynamin-1 using Liopofectamine-2000 (Invitrogen). The cells were washed twice with cold PBS and extracted for 1 h at 4 °C in a modified RIPA buffer (50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1% Nonidet P-40, 1 mm sodium orthovanadate, 1 mm PMSF, 10 mm leupeptin, 1.5 mm pepstatin, and 1 mm aprotinin). They were then clarified by centrifugation at 12,000 rpm for 10 min, and protein concentrations were determined with a Bradford protein assay reagent kit (Bio-Rad). Samples containing 1 mg of total protein were immunoprecipitated for 4 h with anti-FLAG antibody, followed by an additional 3 h at 4 °C with protein G-Sepharose beads (Amersham Biosciences). The immunoprecipitates were extensively washed with lysis buffer and subjected to SDS-PAGE and immunoblot analysis with anti-FLAG and anti-GFP antibodies. In-gel Digestion and Peptide Sample Preparation—The SDS-polyacrylamide gels were silver-stained, and protein bands were excised. The resulting samples were washed three times with a 1:1 (v/v) solution of acetonitrile/deionized water for 10 min, dehydrated with 100% acetonitrile, washed with a 1:1 (v/v) solution of 100% acetonitrile, 100 mm ammonium bicarbonate, and dried using a SpeedVac. Then they were reduced with 10 mm tris(2-carboxyethyl)phosphine hydrochloride in 0.1 m ammonium bicarbonate at 56 °C for 45 min and alkylated with 55 mm iodoacetamide in 0.1 m ammonium bicarbonate at room temperature for 30 min. The above washing step was repeated on the alkylated samples, which were dried, soaked in sequencing-grade trypsin solution (500 ng) on ice for 45 min, and immersed in 100 μl of 50 mm ammonium bicarbonate, pH 8.0, at 37 °C for 14-18 h. The resulting peptides were extracted sequentially by agitation for 20 min with 45% acetonitrile in 20 mm ammonium bicarbonate, 45% acetonitrile in 0.5% trifluoroacetic acid, and 75% acetonitrile in 0.25% trifluoroacetic acid. The extracts containing tryptic peptides were pooled and evaporated under vacuum. Micro-LC-MS/MS Analysis and Protein Data Base Search—In gel digested proteins were loaded onto fused silica capillary columns (100-μm inner diameter, 360-μm outer diameter) containing 8 cm of 5-μm particle size Aqua C18 reverse-phase column material. The columns were placed in line with an Agilent HP 1100 quaternary LC pump, and a splitter system was used to achieve a flow rate of 250 nl/min. Buffer A (5% acetonitrile and 0.1% formic acid) and buffer B (80% acetonitrile and 0.1% formic acid) were used to make a 90-min gradient. The gradient profile started with 5 min of 100% buffer A, followed by a 60-min gradient from 0 to 55% buffer B, a 25-min gradient from 55 to 100% buffer B, and a 5-min gradient of 100% buffer B. Eluted peptides were directly electrosprayed into an LTQ linear ion trap mass spectrometer (ThermoFinnigan, Palo Alto, CA) by applying 2.3 kV of DC voltage. Data-dependent scans consisting of one full MS scan (400-1400 m/z) and five data-dependent MS/MS scans were used to generate MS/MS spectra of the eluted peptides. A normalized collision energy of 35% was used throughout data acquisition. MS/MS spectra were searched against an NCBI rat protein sequence data base using Bioworks version 3.1 and Sequest Cluster System (14 nodes). DTASelect was used to filter the search results, and the following Xcorr values were applied to the different charge states of peptides: 1.8 for singly charged peptides, 2.2 for doubly charged peptides, and 3.2 for triply charged peptides. Fragment ions in each MS/MS spectrum were manually assigned to confirm the data base search results. Cell Culture—E-18 primary rat hippocampal neurons were prepared as described (15Chang S. De Camilli P. Nat. Neurosci. 2001; 4: 787-793Crossref PubMed Scopus (127) Google Scholar). Briefly, hippocampi were dissected from embryonic day 18 Sprague-Dawley fetal rats, dissociated with papain, and triturated with a polished half-bore Pasteur pipette. Cells (250,000) in minimum Eagle's medium supplemented with 0.6% glucose, 1 mm pyruvate, 2 mm l-glutamine, 10% fetal bovine serum, and antibiotics were plated on poly-d-lysine-coated glass coverslips in a 60-mm Petri dish. Four hours after plating, the medium was replaced with basal media Eagle's (Invitrogen) supplemented with 2% B-27, 10 mm HEPES, and 0.5 mm pyruvate or Neurobasal (Invitrogen) supplemented with 2% B-27, 0.5 mm l-glutamine. 4 μm of 1-β-d-cytosine-arabinofuranoside (Ara-C, Sigma) was added as needed. Transfection—Neurons were transfected using calcium-phosphate (15Chang S. De Camilli P. Nat. Neurosci. 2001; 4: 787-793Crossref PubMed Scopus (127) Google Scholar). Briefly, synaptopHluorin (spH) alone or with either FLAG-tagged full-length SNX9 or its truncated variants were transfected at 10 DIV for the endocytosis assays. The spH and SNX9 constructs were cotransfected in a ratio of 1:2-1:5. After the endocytosis assays, the cells were fixed and doubly stained with anti-GFP and anti-FLAG antibodies to confirm cotransfection, and only immunopositive neurons were included in the analysis. The amount of spH construct transfected was fixed for constant fluorescent signals. Immunoblot Analysis—Hippocampal neurons (∼3,000,000) were plated on 100-mm tissue culture dishes coated with poly-d-lysine and grown for 3, 7, 14, or 21 days. They were lysed in a lysis buffer (1% SDS, 1 mm sodium orthovanadate, 10 mm NaF, 10 mm Tris-HCl, pH 7.4, 1 mm PMSF, 10 mm leupeptin, 1.5 mm pepstatin, and 1 mm aprotinin) and scratched out, boiled for 5 min, and clarified by centrifugation at 12,000 rpm for 10 min. Protein concentrations were measured with a bicinchoninic acid protein assay reagent kit (Pierce). Constant amounts of proteins were separated on SDS-PAGE and transferred to poly-vinylidene difluoride membranes (Bio-Rad). The membranes were blocked for 1 h with 5% nonfat dry milk in TBS/T (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 0.1% Tween 20), after which they were incubated with the respective primary antibodies, anti-SNX9, or anti-dynamin-1 (ABR, Golden, CO), and then with horseradish peroxidase-conjugated anti-rabbit IgG (Jackson ImmunoResearch). The antigen-antibody complexes were detected with enhanced chemiluminescence (ECL) reagents (Amersham Biosciences). Blots were stripped by heating to 60 °C for 30 min in a stripping buffer (100 mm β-mercaptoethanol, 2% SDS, and 62.5 mm Tris-HCl, pH 6.7) and re-probed with anti-tubulin (Sigma) as a control. Immunocytochemistry—Immunocytochemistry was performed as described previously (16Kim Y. Kim S. Lee S. Kim S.H. Kim Y. Park Z.Y. Song W.K. Chang S. J. Neurosci. 2005; 25: 9515-9523Crossref PubMed Scopus (23) Google Scholar). Cells were fixed in 4% formaldehyde, 4% sucrose, PBS for 15 min, permeabilized for 5 min in 0.25% Triton X-100, PBS and blocked for 30 min in 10% BSA, PBS at 37 °C. The cells were incubated with primary antibodies, 3% BSA, PBS for 2 h at 37 °C or overnight at 4 °C, washed in PBS, and incubated with secondary antibodies, 3% BSA, PBS for 45 min at 37 °C. Primary antibodies used were as follows: anti-SNX9 (provided by Dr. Sven R. Carlsson, Umeå University, Sweden, or Santa Cruz Biotechnology, Santa Cruz, CA), anti-synaptophysin, anti-synaptobrevin2, anti-synaptotagmin, anti-synaptojanin (Synaptic Systems, Göttingen, Germany), anti-GFP (Abcam, Cambridge, UK), anti-FLAG (Sigma), anti-dynamin-1 (ABR), anti-Hudy2 (Upstate Biotechnology, Lake Placid, NY), anti-GFAP (Chemicon, Temecula, CA), and anti-type β-III tubulin (Chemicon). Secondary antibodies were obtained from Jackson ImmunoResearch. SynaptopHluorin Endo-exocytosis Assay—Coverslips were mounted in a perfusion/stimulation chamber equipped with platinum-iridium field stimulus electrodes (EC-S-10, LCI, Seoul, Korea) on the stage of an Olympus IX-71 inverted microscope (Olympus, Tokyo, Japan) with 40 × 1.0 or 60 × 1.4 NA oil lenses. Cells were continuously perfused at room temperature with Tyrode solution (119 mm NaCl, 2.5 mm KCl, 2 mm CaCl2, 2mm MgCl2, 25 mm HEPES, 30 mm glucose, pH 7.4). 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione and 50 μm of dl-2-amino-5-phosphonovaleric acid were added to the Tyrode solution to reduce spontaneous activity and prevent recurrent excitation during stimulation. Time-lapse images were acquired every 10 s for 4 min using a CoolSNAP-ES CCD camera (Roper Scientific, Tucson, AZ) driven by MetaMorph Imaging software (Universal Imaging Corp., West Chester, PA) with a GFP optimized filter set (Omega Optical, Brattleboro, VT). From the 4th frame, cells were stimulated (1 ms, 20-50 V, bipolar) for 30 s at 20 Hz or 30 s at 10 Hz using a Grass SD9 stimulator (Grass-Telefactor, West Warwick, RI). Quantitative measurements of the fluorescence intensity at individual boutons were obtained by averaging a selected area of pixel intensities using MetaMorph software. Individual regions were selected by hand, and rectangular regions of interest were drawn around the synaptic boutons, and average intensities were calculated. Large puncta, typically representative of clusters of smaller synapses, were rejected during the selection procedure. The center of intensity of each synapse was calculated to correct for any image shift over the course of the experiment. Fluorescence was expressed in intensity units that correspond to fluorescence values averaged over all pixels within the region of interest. Light from a mercury lamp was shuttered using a VMM1 Unibilitz shutter (Vincent Associates, Rochester, NY). Net fluorescence changes were obtained by subtracting the average intensity of the first four frames (F0) from the intensity of each frame (Ft) for individual boutons. They were then normalized to the maximum fluorescence intensity (Fmax - F0) and averaged. The decay of fluorescence was fitted with a single exponential. All fitting was done using individual error bars to weight the fit, using SigmaPlot 6.0. In some experiments where fluorescence decay deviated from single exponential behavior, we obtained the best fitting single exponential function from the early portion of the decay. Data are presented as means ± S.E. For the rescue experiments, FLAG-SNX9 was coexpressed with HA-dynamin-1 or Myc-N-WASP; FLAG-SNX9-ΔBAR was coexpressed with HA-dynamin-1 or Myc-N-WASP, and FLAG-SNX9-SH3 was coexpressed with HA-dynamin-1-PRD. For the exocytosis assays, control neurons expressing spH, or neurons expressing FLAG-SNX9, FLAG-SNX9-ΔBAR, or FLAG-SNX9-SH3 with spH, were preincubated with bafilomycin A1 for 60 s and stimulated for 30 s at 10 Hz. Net fluorescence changes were obtained by subtracting the average intensity of the first four frames (F0) from the intensity of each frame (Ft) for individual boutons, then normalizing to the maximum fluorescence intensity (Fmax - F0), and averaging. bafilomycin A1 (Calbiochem) was dissolved in Me2SO to 0.2 mm and diluted to a final concentration of 0.5 μm prior to the experiments. Bafilomycin was applied throughout the fluorescence measurements. Expression of each construct was confirmed by retrospective immunostaining with specific antibodies (rabbit anti-Myc, mouse anti-FLAG, and rat-anti-HA antibodies), and only immunopositive neurons were included in the analysis. Statistical analysis was carried out with SigmaStat (Systat Software, Point Richmond, CA). For multiple conditions, we compared means by analysis of variance (ANOVA) followed by Tukey's HSD post hoc test or Fisher's LSD test (depending on the number of groups). FM 4-64 Endocytosis Assay—FM 4-64 was used at a concentration of 15 μm in the above Tyrode solution. Pools of synaptic vesicles were labeled during electrical stimulation for 30 s at 10 Hz in the presence of FM 4-64. After 10 min of washing in dye-free Tyrode, images were taken, and neurons were stimulated for 2 min at 10 Hz to unload the FM 4-64. A fully unloaded image was then taken. Net fluorescence changes were obtained by subtracting the intensity of the unloaded image from the intensity of the loaded image. A 15-min rest period was inserted between the end of the first unloading stimulus train and the start of the second loading stimulus. Images were acquired using a CoolSNAP-ES CCD camera driven by MetaMorph Imaging software with a FM 4-64 optimized filter set (Omega Optical). Statistical analysis was carried out using SigmaStat (Systat Software). Data are presented as means ± S.E. RNA Interference—SNX9-specific siRNAs were designed from the rat SNX9 cDNA sequence acquired by Blast search, targeting to the region of nucleotides 1183-1203 (siRNA 1, gi|2985617) and 1365-1385 (siRNA 2, gi|2942879). A pair of complementary oligonucleotides was synthesized separately with the addition of an ApaI site at the 5′ end and an EcoRI site at the 3′ end. The forward primer sequences were 5′-ATAGAACAGAAGTGTGACGTTCAAGAGACGTCACACTTCTGTTCTATTTTTTT-3′ and 5′-GGAGAGACGGACCTTAACATTCAAGAGATGTTAAGGTCCGTCTCTCCTTTTTT-3′) (the underlined letters are the SNX9-siRNA sequences). The annealed cDNA fragment was cloned into the ApaI-EcoRI sites of pSilencer 1.0-U6 vector (Ambion, Austin, TX) modified by inserting an mRFP tag at the C terminus. The knockdown efficiency of the siRNA was tested in Rat-1 cells of fibroblast origin. Hippocampal neurons were transfected with SNX9-specific siRNAs using calcium phosphate. SNX9 Is Expressed in Cultured Hippocampal Neurons and Its Expression Increases with Developmental Stage—A previous Northern blot analysis showed that SNX9 was expressed in many tissues (1Howard L. Nelson K.K. Maciewicz R.A. Blobel C.P. J. Biol. Chem. 1999; 274: 31693-31699Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To see whether it is expressed in cultured hippocampal neurons, we performed a Western blot analysis using an SNX9 antibody. We used three different culture conditions to test the expression of SNX9 as follows: a pure glial cell culture obtained by2hof treatment with 200 μm of glutamate to remove neurons; a serum-free Neurobasal culture completely depleted of glial cells with Ara-C, and a Neurobasal culture without Ara-C treatment, containing mostly neurons and only a few glial cells. The glial cells expressed SNX9 as expected. The hippocampal neurons also expressed considerable amounts of SNX9, and expression increased as the neurons matured (Fig. 1, B-D). SNX9 Is Present at the Presynaptic Terminals of Hippocampal Neurons Where It Binds to Dynamin-1—To study the distribution and subcellular localization of SNX9 in the neurons, we performed immunocytochemistry. SNX9 was found to be present diffusely all along the neurites, and formed many clusters in the axons, and the majority of it colocalized with the presynaptic vesicle markers synaptobrevin2, synaptophysin, and synaptotagmin (Fig. 2, A-I). Because SNX9 contains an SH3 domain and is present at presynaptic nerve terminals, it could bind PRD-containing proteins. Dynamin-1 is the major SH3-containing protein in the presynaptic compartment. To test whether SNX9 and dynamin-1 bind to each other via an SH3-PRD interaction, we performed a micro-LC-MS/MS analysis to identify SNX9-interacting proteins (Fig. 3A). Brain lysates were immunoprecipitated with an SNX9 antibody, and SDS-polyacrylamide gels were silver-stained. After in-gel digestion, micro-LC-MS/MS, and a protein data base search, dynamin-1 was identified as one of the endogenous binding partners (Fig. 3A). Binding of SNX9SH3 to dynamin-1 was as strong as that of endophilin-SH3 (Fig. 3B). We carried out a series of GST pulldown assays. Dynamin-1 was found to interact with full-length SNX9 and with its SH3 domain, but not with SNX9 lacking the SH3 domain (SNX9-ΔSH3) (Fig. 3, B and E). Co-immunoprecipitation analysis of lysates of cells overexpressing FLAG-SNX9 and GFP-dynamin-1 confirmed that SNX9 associated with dynamin-1 in vivo in an SH3 domain-dependent interaction (Fig. 3F). We did not observe any significant change in this interaction in response to high KCl (Fig. 3G). Interaction of SNX9 with dynamin-1 was further confirmed by immunocytochemistry. Neurons were doubly stained with SNX9 and dynamin-1 antibodies. Although SNX9 exhibited rather diffuse cytosolic staining, it colocalized with dynamin-1 throughout the neurites (Fig. 3I). SNX9 Interacts with N-WASP and Synaptojanin—N-WASP is the most potent and best characterized activator of actin nucleation by the Arp2/3 complex (17Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (555) Google Scholar), and it is also enriched at nerve terminals, where it links the dynamin-mediated processes of endocytosis to rearrangements of the actin cytoskeleton within nerve terminals. Drosophila SNX9 (dSH3PX1) is known to interact with the Drosophila orthologue of WASP (18Worby C.A. Simonson-Leff N. Clemens J.C. Kruger R.P. Muda M. Dixon J.E. J. Biol. Chem. 2001; 276: 41782-41789Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We also showed that SNX9 interacts with N-WASP using micro-LC-MS/MS analysis (Fig. 3A). Although we could not detect N-WASP in GST pulldown followed by Coomassie staining, immunoblotting with anti-N-WASP antibody revealed that N-WASP interacted with full-length SNX9 and with its SH3 domain, but not with SNX9 lacking the SH3 domain (SNX9-ΔSH3) (Fig. 3E). Co-immunoprecipitation of lysates of cells overexpressing FLAG-SNX9 and GFP-N-WASP cells confirmed that SNX9 indeed interacts with N-WASP in vivo in an SH3 domain-dependent interaction (Fig. 3F). Previous study showed that SNX9 interacts with synaptojanin (4Yeow-Fong L. Lim L. Manser E. FEBS Lett. 2005; 579: 5040-5048Crossref PubMed Scopus (42) Google Scholar). Synaptojanin is a polyphosphoinositide phosphatase implicated in synaptic vesicle recycling and vesicle trafficking (19Cremona O. Di Paolo G. Wenk M.R. Luthi A. Kim W.T. Takei K. Daniell L. Nemoto Y. Shears S.B. Flavell R.A. McCormick D.A. De Camilli P. Cell. 1999; 99: 179-188Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). Synaptojanin, however, was not detected in Coomassie staining (Fig. 3B) and only faintly visible in silver staining (Fig. 3C, arrow), suggesting very weak binding. The interaction of synaptojanin with SH3 domain of SNX9 further confirmed by immunoblotting with anti-synaptojanin antibody (Fig. 3C). SNX9 contains a single SH3 domain at its N terminus, by which it interacts with dynamin-1 and N-WASP. This raised the possibility that it dimerizes. Fig. 3H shows that SNX9 indeed dimerizes and that it does so via its BAR domain. Thus, our results suggest that SNX9 can dimerize, thus binding dynamin-1 and N-WASP simultaneously and coordinating clathrin-mediated endocytosis with the actin cytoskeleton. Overexpression of SNX9 Impairs Clathrin-mediated Synaptic Vesicle Endocytosis—We investigated whether the interaction between SNX9 and dynamin-1 could be involved in synaptic vesicle endocytosis. To measure synaptic vesicle endocytosis in the cultured hippocampal neurons we used synaptopHluorin (spH) (Fig. 4A). spH is a VAMP-2/synaptobrevin-2 fused with a pH-sensitive variant of GFP. The fluorescence of spH is quenched once synaptic vesicles are endocytosed and re-acidified (from external pH ∼7.4 to an internal pH of a synaptic vesicle ∼5.5). It has been proved that re-acidification is not a rate-limiting step; thus the fluorescence change to synapto-pHluorin reliably reflects the kinetics of endocytosis (20Sankaranarayanan S. Ryan T.A. Nat. Cell Biol. 2000; 2: 197-204Crossref PubMed Scopus (343) Google Scholar). To investigate the effect of SNX9 on endocytosis, FLAG-tagged SNX9 or truncated SNX9 variants were cotransfected with spH into hippocampal neurons. We confirmed by retrospective immunostaining with anti-FLAG antibody at the end of each experiment that the neurons expressing spH also expressed FLAG-tagged SNX9 or the truncated variants of SNX9. When neurons were stimulated electrically (600 action potentials/20 Hz), the fluorescence intensity of individual spH boutons increased rapidly, reached a peak, and then decayed with an exponential time course (Fig. 4A). In boutons expressing SNX9, synaptic vesicle endocytosis occurred much more slowly than in nonexpressing boutons (τ = 182.4 ± 28.9 for SNX9 expression; τ = 39.2 ± 3.4 for control; Fig. 4, A, C and D). In previous studies, inhibition of endocytosis was observed with the SH3-containing truncated variant (SNX9-ΔBAR) (2Lundmark R. Carlsson S.R. J. Biol. Chem. 2003; 278: 46772-46781Abstract" @default.
• W2078264101 created "2016-06-24" @default.
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• W2078264101 creator A5007952395 @default.
• W2078264101 creator A5026497101 @default.
• W2078264101 creator A5030586134 @default.
• W2078264101 creator A5046016624 @default.
• W2078264101 creator A5063342017 @default.
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• W2078264101 date "2007-09-01" @default.
• W2078264101 modified "2023-10-16" @default.
• W2078264101 title "Sorting Nexin 9 Interacts with Dynamin 1 and N-WASP and Coordinates Synaptic Vesicle Endocytosis" @default.
• W2078264101 cites W1498241934 @default.
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• W2078264101 cites W1575892864 @default.
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• W2078264101 cites W2013191689 @default.
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• W2078264101 cites W2015353371 @default.
• W2078264101 cites W2016441216 @default.
• W2078264101 cites W2029500247 @default.
• W2078264101 cites W2038212389 @default.
• W2078264101 cites W2045499828 @default.
• W2078264101 cites W2048597699 @default.
• W2078264101 cites W2051410189 @default.
• W2078264101 cites W2053610637 @default.
• W2078264101 cites W2056189730 @default.
• W2078264101 cites W2068269402 @default.
• W2078264101 cites W2078748790 @default.
• W2078264101 cites W2080579127 @default.
• W2078264101 cites W2087799476 @default.
• W2078264101 cites W2094419120 @default.
• W2078264101 cites W2106140806 @default.
• W2078264101 cites W2108264768 @default.
• W2078264101 cites W2108852246 @default.
• W2078264101 cites W2123553276 @default.
• W2078264101 cites W2125663667 @default.
• W2078264101 cites W2136203552 @default.
• W2078264101 cites W2149902705 @default.
• W2078264101 cites W2155904732 @default.
• W2078264101 cites W2157920366 @default.
• W2078264101 cites W2160776727 @default.
• W2078264101 cites W2164909338 @default.
• W2078264101 cites W4233015538 @default.
• W2078264101 cites W68973889 @default.
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