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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.016709

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SNX9 regulates tubular invagination of the plasma membrane through interaction with actin cytoskeleton and dynamin 2

¹ Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, South Korea
² Department of Pathology, College of Physicians and Surgeons, Columbia University Medical Center, New York, NY, USA
³ Department of Neuroscience, Okayama University, Okayamashi, Japan
⁴ Deparment of Pathology, Chosun University, Gwangju 501-759, South Korea
⁵ Department of Biochemistry, Dongguk University, Gyeongju 780-714, South Korea

View larger version (53K): [in this window] [in a new window]   Fig. 1. SNX9 has a functional BAR domain and induces massive membrane tubulation. (A) Multiple sequence alignment of the BAR domains in SNX9 and other proteins with Clustal X. Conserved residues are highlighted with the following color code: yellow, hydrophobic; green, polar; blue, basic; red, acidic. Secondary structure of rAmphiphysin2 was determined from the crystal structure and that of SNX9 was predicted using online program NPS@ (http://npsa-pbil.ibcp.fr). (B) COS-7 cells were transfected with GFP-SNX9 and time-lapse imaging was performed 20 hours after transfection. Images were captured every 5 seconds for 5 minutes. High-magnification views are of the regions enclosed in rectangles. Arrowheads indicate the ends of tubules that underwent elongation. Scale bars: 30 µm (low magnification) and 3 µm (high magnification). (C) Domain structures of SNX9 and its deletion mutants. Tubulation ability is indicated. (D) COS-7 cells were transfected with GFP-tagged SNX9 or SNX9-SH3 and tubule formation and cellular localization were observed. Scale bars: 30 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 2. SNX9-induced tubules are elongated from the plasma membrane. (A-C) COS-7 cells expressing GFP-tagged SNX9 was stained with 2 µM CM-DiI (A), 2 µM cholera toxin B Alexa Fluor 594 (B) or 15 µM FM5-95 (C) for 10 minutes on ice and fixed for observation. SNX9-induced tubules (green) and membrane marker stained plasma membranes (red) were mostly colocalized at cell peripheries. High-magnification views are of the regions enclosed by rectangles. Scale bars: 30 µm (low magnification) and 3 µm (insets). (D) Tubules grown from the bottom of the cell in contact with the coverslip were imaged by TIRF microscopy. Arrowheads indicate tubules elongated from SNX9-positive spots at the plasma membrane. Insets are high-magnification views of the regions enclosed in rectangles. Scale bars: 30 µm (low magnification) and 8 µm (insets).

View larger version (56K): [in this window] [in a new window]   Fig. 3. SNX9 exhibits phospholipid binding and powerful lipid tubulating activity in vitro. (A) Nitrocellulose filters spotted with various phospholipids (100 pmol of each phospholipid as indicated in this figure) were incubated with purified 0.5 µg/ml of GST-SNX9 and the binding of each protein to the specific phospholipid was detected with an anti-SNX9 antibody. (B) Synthetic PE and PC liposomes supplemented with 10% of the indicated lipid were incubated with SNX9, sedimented and stained with Coomassie Blue. Three independent experiments were performed and the protein intensity was measured. S, supernatant; P, pellet. (C) Negative stain electron microscopy of liposome composed of a brain lipid extract after addition of recombinant SNX9. Scale bar: 300 nm. (D) Lipid droplets are rehydrated to form a lipid bilayer sheet with solution containing either GST-alone or GST-tagged SNX9 during microscopic observation. Time-lapse images of GST-control (top) and GST-tagged SNX9 (bottom) using differential interference contrast (DIC) microscopy. Dotted lines indicate the edges of membrane sheet. Scale bars: 40 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 4. SNX9 binds to dynamin 2 and N-WASP through its SH3 domain and colocalizes on tubules. (A) Pull-down assay from HEK 293T cells with purified GST fusion of SNX9, SNX9-SH3, SNX9-SH3 or GST-alone. The pull-downs were analyzed by Coomassie Blue staining. Arrow indicates major interacting protein predicted to be dynamin 2. Immunoblotting using anti-dynamin 2 or anti-N-WASP antibody was then performed. (B) COS-7 cells were cotransfected with Flag-tagged SNX9 or SNX9-SH3 and GFP-tagged N-WASP or GFP-tagged dynamin 2, and immunoprecipitation (IP) was carried out with anti-Flag antibody, followed by immunoblotting (IB) with anti-Flag or anti-GFP antibody. TCL, total cell lysates. (C,D) Dual-color simultaneous time-lapse imaging of SNX9 or SNX9-SH3 with dynamin 2 or N-WASP. GFP- or mRFP-tagged SNX9 or SNX9-SH3 was cotransfected with mRFP-tagged dynamin 2 or GFP-tagged N-WASP, respectively. Live dual-color imaging was performed 24 hours after transfection. High-magnification views are of the regions enclosed in rectangles. Scale bars: 10 µm (low magnification), 2 µm (insets).

View larger version (37K): [in this window] [in a new window]   Fig. 5. SNX9 binds to the Arp2/3 complex via its LC domain. (A) COS-7 cell lysates were immunoprecipitated with anti-Arp3 antibody and immunoblotted (IB) with anti-SNX9 antibody. (B) Schematic diagrams of the GST-tagged truncated LC domains of SNX9 constructs. (C) In vitro binding assays were carried out with purified GST-SNX9 protein fragments (full-length, SH3, LC or PXBAR) and Arp2/3 complex. The bound protein was verified using anti-Arp3 antibody. (D) In vitro binding assays were carried out with various truncated mutant of LC domain and the bound protein was immunoblotted with anti-Arp3 antibody. The VCA domain of N-WASP (GST-VCA) was used as a positive control for Arp2/3 binding. (E) Comparison of the SNX9 A-like domain with the A-like domains of N-WASP, WASP, Scar, Cortactin, Myo1b and ActA. Black box with an asterisk indicates conserved tryptophan residue and gray boxes indicate conserved aspartate or glutamate residues. (F) Endogenous SNX9 and Arp2/3 complex in Cos-7 cells were stained with anti-SNX9 antibody and anti-Arp2/3 antibody, respectively. High-magnification views of the regions enclosed by rectangles are shown below each image. Arrows indicate where two proteins were juxtaposed or overlap. Scale bars: 30 µm (low magnification), 5 µm (high magnification).

View larger version (67K): [in this window] [in a new window]   Fig. 6. Antagonistic effects of N-WASP and dynamin 2 on SNX9-induced tubulation. (A,B) When high levels of GFP-tagged N-WASP or mRFP-tagged dynamin 2 were coexpressed with GFP- or mRFP-tagged SNX9 in COS-7 cells, tubular invaginations were significantly reduced or even absent in many cases. (C,D) Coexpression with N-WASP-VCA, a mutant that cannot interact with the Arp2/3 complex, failed to affect tubulation by SNX9. Likewise, coexpression of SNX9 with dynamin 2 K44A, a GTPase-deficient mutant, abolished the antagonistic effect of dynamin 2 on tubulation. (E-H) Representative pictures from various combinations of SNX9 or SNX9-SH3 with dynamin2 or N-WASP. Scale bars: 30 µm. (I) Quantitative analysis of tubule length in various experimental schemes. The length of tubules was measured manually using a MetaMorph software. Three rectangular areas (20x20 µm) near the periphery of each cell were drawn, and within these areas, the tubules that were well separated from other tubules were selected and their lengths were measured in the x-y dimension. Three independent experiments were performed and the data were pooled and normalized against those of full-length SNX9 with mRFP empty vector. Error bar indicates s.d. n=50 cells. *P<0.05, significantly different from SNX9-GFP or -mRFP control or each other, ANOVA and Tukey's HSD post hoc test for several different groups and Student's t-test for two different groups.

View larger version (49K): [in this window] [in a new window]   Fig. 7. Perturbation of actin dynamics enhances tubule formation by SNX9. (A) COS-7 cells were transfected with low levels of GFP-SNX9, which does not induce membrane tubulation (left). After addition of latrunculin B (1 µM), tubular invaginations can be observed within a few seconds (right). Insets are high-magnification views and below are time-lapse images of the regions enclosed in rectangles. (B) Cells expressing low levels of GFP-SNX9 were imaged before and after the addition of jasplakinolide (2 µM). Scale bar: 30 µm; 2 µm for insets and time-lapse images. (C) COS-7 cells expressing GFP-tagged SNX9 was stained with 15 µM FM5-95 (C) for 10 minutes on ice and treated with latrunculin B. SNX9-induced tubules (green) and FM5-95-stained plasma membranes (red) were mostly colocalized. High-magnification views are of the regions enclosed in rectangles. Scale bars: 30 µm (low magnification), 5 µm (insets).

View larger version (82K): [in this window] [in a new window]   Fig. 8. SNX9 interacts via its PX domain with PtdIns(4)P-5-kinases I, Iβ and I, which regulate the tubulation activity of SNX9 and whose kinase activity is increased by SNX9. (A) COS-7 cell lysates were immunoprecipitated with anti-SNX9 antibody and immunoblotted (IB) with anti-SNX9 antibody or anti-PtdIns(4)P-5-kinase I antibody. TCL, total cell lysates. (B) In vitro GST-pull down assay was performed with purified GST-fused SNX9 and its truncated mutants (2 µg each) with His-tagged PtdIns(4)P-5-kinase I (200 ng). (C) COS-7 cells were cotransfected with GFP-tagged SNX9, SNX9-PX or SNX9-PX with HA-tagged PtdIns(4)P-5-kinase I, Iβ or I and immunoprecipitation (IP) was carried out with anti-GFP antibody, followed by immunoblotting (IB) with anti-HA or anti-GFP antibody. (D) Representative images of cells that were cotransfected with GFP-tagged SNX9 and HA-tagged PtdIns(4)P-5-kinase I, Iβ or empty vector. When HA-tagged PtdIns(4)P-5-kinase I or Iβ were coexpressed with low levels of GFP-tagged SNX9 in COS-7 cell, tubular invaginations were significantly increased around the cell periphery. (E) The kinase dead mutant of PtdIns(4)P-5-kinase I (D309N/R427Q) failed to increase tubulation by low levels of GFP-tagged SNX9 in COS-7 cells. (F) Coexpression of mRFP-tagged SNX9 with GFP-tagged 5-phosphatase domain of synaptojanin inhibits tubule formation even with high levels of SNX9 expression. Scale bars: 30 µm (low magnification), 2 µm (insets). (G) Recombinant PtdIns(4)P-5-kinase I-90 (PIPK) was pre-incubated with two concentrations of SNX9 and its activity measured using a PtdIns(4,5)P2-radiolabeling assay. At 1.0 µM SNX9, the kinase activity of PIPK was not significantly increased, but at 5.0 µM, there was a significant 2.5-fold increase in activity (**P<0.0001). (H) PtdIns(4)P-5-kinase I (both the 87 kDa and 90 kDa isoforms) was immunoprecipitated from rat brain cytosol and pre-incubated with two concentrations of SNX9 and its activity assayed as in G. At both 1.0 and 5.0 µM SNX9, the immunoprecipitated kinase showed a twofold (**P<0.001) and fivefold (**P<0.05) significant increase in kinase activity, respectively.

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