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First published online 23 November 2004
doi: 10.1242/jcs.01561

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Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma membrane following EGF stimulation

Ana Merino-Trigo^1,*, Markus C. Kerr², Fiona Houghton¹, Anna Lindberg², Christina Mitchell³, Rohan D. Teasdale² and Paul A. Gleeson^1,

¹ The Russell Grimwade School of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria, 3010, Australia
² Institute for Molecular Bioscience and Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland, 4072, Australia
³ Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia

View larger version (16K): [in a new window]   Fig. 1. SNX5 PX domain colocalizes with the full-length SNX5 protein. HeLa cells were co-transfected with the full-length SNX5 protein (FLAG-SNX5FL) and a construct of SNX5 containing the PX domain (GFP-SNX5PX). After 12 hours of transfection, cells were fixed, permeabilized and stained with anti-FLAG monoclonal antibody followed by goat anti-mouse IgG-Alexa568 conjugate. Confocal images were collected with identical iris settings. Superimposed images (Merge) reveal regions of colocalization. Plasma membrane staining is due to non-specific staining with the anti-FLAG monoclonal antibody. Bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 2. Intracellular localization of human SNX5 PX domain in transfected HeLa cells. (A) HeLa cells were transiently transfected with GFP-SNX5PX and after 12 hours of transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100, and co-stained for EEA1, Lamp-1 and SNX1. EEA1 was detected with human anti-EEA1 antibodies followed by human anti-Ig Alexa568, and Lamp-1 and SNX1 were detected with mouse monoclonal antibodies and goat anti-mouse IgG-Alexa568 conjugate. (B) EEA1 was detected with human anti-EEA1 antibodies and FITC-goat anti-human IgG and SNX1 as for A. Control incubations demonstrated no cross-reactivity between the anti-Ig conjugates and the irrelevant primary antibody. Confocal images were collected with identical iris settings. Superimposed images (Merge) reveal regions of colocalization. Inserts show regions of the cell at higher magnification. Bar, 10 µm.

View larger version (26K): [in a new window]   Fig. 3. Localization of GFP-SNX5FL fusion protein in stably transfected HEK293 cells. (A) HEK293 cells stably expressing a GFP-SNX5FL fusion protein were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100, and co-stained for EEA1 with human anti-EEA1 antibodies followed by human anti-Ig Alexa568. (B) High power image showing enlarged GFP-SNX5FL-positive structures that also stain for EEA1. Note the lack of colocalization of the two molecules within these structures. Confocal images were collected with identical iris settings. Superimposed images (Merge) reveal regions of colocalization. Bar, 10 µm (A) and 5 µm (B).

View larger version (56K): [in a new window]   Fig. 4. Colocalization of GFP-SNX5FL fusion protein in HEK293 cells with the fluid-phase marker, Texas-Red dextran. HEK293 cells stably expressing the GFP-SNX5FL fusion protein were incubated in Texas-Red dextran (100-200 µg/ml) for 15 minutes at 37°C as described in Materials and Methods. Cells were fixed in 4% paraformaldehyde. Confocal images were collected with identical iris settings. Superimposed images (Merge) reveal regions of colocalization. Inserts show regions of the cell at higher magnification. Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 5. Endosomal localization of PX domain of SNX5 requires the activity of a wortmannin sensitive PI 3-kinase. (A) HeLa cells were transfected with GFP-SNX5PX, and after 12 hours cell monolayers were serum-starved for 3 hours. Cells were either untreated or treated with 100 nM wortmannin for 2 hours at 37°C and fixed in 4% paraformaldehyde. (B) HEK293 cells expressing GFP-SNX5FL were either untreated or treated with 100 nM wortmannin for 20 minutes at 37°C and fixed in 4% paraformaldehyde. Bar, 10 µm.

View larger version (48K): [in a new window]   Fig. 6. SNX5 partially colocalizes with the PtdIns(3)P probe, GFP-FYVEEEA1. (A) HeLa cells were transfected with FLAG-SNX5FL and GFP-FYVEEEA1. After 24 hours, cell monolayers were permeabilized and stained with anti-FLAG monoclonal antibody followed by goat anti-mouse IgG-Alexa568 conjugate. (B) HeLa cells transfected with GFP-FYVEEEA1 as above and stained for EEA1 with mouse anti-EEA1 antibodies followed by anti-Ig Alexa568. Confocal images were collected with identical iris settings. Superimposed images (Merge) reveal regions of colocalization. Bar, 10 µm.

View larger version (26K): [in a new window]   Fig. 7. The PX domain of SNX5 binds to PtdIns(3)P and PtdIns(3,4)P2. The ability of SNX5PX domain to bind different phosphoinositides was analysed using a liposome binding assay. 3H-labelled liposomes were prepared as described containing either PtdIns(3)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(3,4,5)P3 or no PI, as indicated. Sonicated liposomes (100 µl) were incubated for 30 minutes at room temperature with 20 µg GST or purified GST-SNX5PX fusion protein immobilized on glutathione-Sepharose beads. After incubation, the beads were washed three times and collected by centrifugation. The radioactivity in both pellets and supernatant was determined. The assay was performed in triplicate and the standard deviations are shown.

View larger version (43K): [in a new window]   Fig. 8. SNX5 associates with the plasma membrane following EGF stimulation of transfected HeLa cells. HeLa cells were transfected with constructs encoding GFP-SNX5PX or GFP-SNX5FL. Untransfected HeLa cells were also grown under the same conditions. After 12 hours, cell monolayers were serum-starved for 3 h and either left unstimulated or stimulated with human EGF (100 ng/ml) for the indicated times, fixed and processed for fluorescence. The distribution of GFP-SNX5PX and GFP-SNX5FL was directly visualized by confocal microscopy. Untransfected cells were stained with anti-SNX1 monoclonal antibody followed by goat-anti-mouse IgG conjugated to Alexa568. Bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 9. SNX5 associates with the plasma membrane following EGF stimulation of HEK293 cells expressing GFP-SNX5FL. Stable HEK293 cells stably expressing GFP-SNX5FL were serum-starved for 3 h and either left unstimulated or stimulated with human EGF (20 ng/ml) for the indicated times, fixed and processed for fluorescence. Bar, 10 µm.

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