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First published online 5 December 2006
doi: 10.1242/jcs.03302

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A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer

The Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK

View larger version (67K): [in this window] [in a new window]   Fig. 1. RNAi loss-of-function screen to identify sorting nexins involved in retromer function. (A) Images obtained from the ArrayScan II 96 well-based, wide-field fluorescence microscopic imaging system depicting the normal steady-state enrichment of the CI-MPR in the TGN, and a redistribution of the CI-MPR into peripherally dispersed cytosolic punctae in cells suppressed for SNX1, SNX5 and SNX6. Enlarged insets, show cells scored as `normal CI-MPR staining'(), cells with `dispersed CI-MPR staining' (). (B) Data collected from >150 cells per SMARTpool, showing cells with a dispersed CI-MPR staining. Apart from the known retromer components VPS26A, VPS29 and VPS35, SNX5 and SNX6 show a high percentage of cells with altered CI-MPR staining. Transfection of cells with SNX15 or with SNX18 siRNA led to a large decrease in the number of cells due to cell death. indicates remaining cells that showed strong morphological changes and abnormal Golgi organization. contr. indicates cells treated with a scrambled siRNA. /. indicates untransfected control.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Knock-down of SNX5 and SNX6 as shown by RT-PCR. Transfection of HeLa cells with individual SNX5 siRNA, SNX6 siRNA or respective SMARTpools led to a strong reduction of RNA levels as shown by RT-PCR. (-) control sample without template; (contr.) indicates template from the control cells treated with a scrambled siRNA; (D1, D2, D3, D4) individual siRNAs for SNX5 or SNX 6; (Pool) SMARTpool. For all further experiments in this study, we selected SNX5 D1 and SNX6 D2 siRNA, although similar results were obtained with the other siRNAs or the pools (see supplementary material Fig. S2). GAPDH-specific PCR primers were used as control amplicon.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Confocal imaging confirms the redistribution of CI-MPR into early endosomes in SNX5- and/or SNX6-suppressed HeLa cells. Suppression of SNX5 and SNX6 in HeLa cells was by transfection with gene-specific siRNA; cells were stained for EEA1 and the CI-MPR, and analysed by confocal microscopy. Bars, 20 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 4. RNAi-mediated suppression of SNX5 induces fragmentation of the Golgi complex. Suppression of SNX5 alone or SNX5 and SNX6 together leads to morphological changes of the trans-Golgi (stained with TGN46) and the cis-Golgi (stained with GM130), whereas single suppression of SNX1 or SNX6 alone does not appear to have a major effect on TGN organization. Compare also with Fig. 5A for these changes of the TGN morphology. Bars, 20 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 5. RNAi-mediated suppression of SNX5 and/or SNX6 perturbs the kinetics of retromer-mediated endosome-to-TGN transport of CD8-CI-MPR. (A) Images depicting the kinetics of delivery of cell-surface-labelled CD8-CI-MPR (green) to the TGN46-labelled TGN (red) in control or SNX5- and/or SNX6-suppressed HeLaM cells. Images are projections of eight confocal sections and are representative of >50 cells imaged for each condition. Bar, 20 µm. (B) Quantification of the kinetics of cell-surface-labelled CD8-CI-MPR transport to the TGN46-labelled TGN in HeLaM cells expressed as percent colocalization between the two labels, normalised to control cells. Data were obtained as outlined in Materials and Methods from n>50 cells for each time point, and are expressed as the mean ± s.d.

View larger version (48K): [in this window] [in a new window]   Fig. 6. Localization of SNX5 and SNX6 using GFP-tagging or immunostaining of endogenous protein. (A-C) Endogenous SNX6 shows a high degree of colocalization with the early endosomal marker EEA1 (A) and with SNX1 (B), whereas little colocalization was observed with the late endosomal marker LAMP1 (C). Similar results were obtained with GFP-tagged SNX6 (D and data not shown). (E,F) GFP-SNX5 also showed a high degree of colocalization with SNX1 (F), but little with EEA1 (E). Bars, 20 µm.

View larger version (127K): [in this window] [in a new window]   Fig. 7. Live cell imaging of HeLa cells co-expressing mRFP-SNX1 and GFP-SNX5 or SNX6. (A,B) SNX1 colocalises with SNX5 (A) or SNX6 (B) on the same vesicles (arrows) and also on tubules emanating from these vesicles (arrowheads) and on pinched-off tubules, suggesting a cooperation between SNX1 and SNX5, and between SNX1 and SNX6 in tubule formation.

View larger version (45K): [in this window] [in a new window]   Fig. 8. SNX6 co-immunoprecipitates with endogenous SNX1. (A) HeLa cells were transiently transfected with Flag-tagged constructs encoding either full-length SNX5 or SNX6. After 48 hours incubation, cell lysates were incubated with anti-Flag antibodies as described in Materials and Methods. The resultant immunoprecipitates were resolved by SDS-PAGE prior to western blotting with anti-SNX1 antibodies. Whereas endogenous SNX1 strongly co-precipitates with overexpressed SNX6, only an insignificant amount is co-precipitated with Flag-SNX5 (B,C) Endogenous SNX5 or SNX6 were immunoprecipitated and probed for co-immunoprecipitation with SNX1. A fraction of endogenous SNX1 is stably associated with SNX6. IgG HC, heavy chain of mouse IgG antibody; P, pellet; S, supernatant.

View larger version (62K): [in this window] [in a new window]   Fig. 9. Silencing of SNX5 and SNX6 leads to a decrease of global SNX1 levels. (A) Whereas suppression of SNX5 or SNX6 both impair SNX1 levels, their double knock-down almost completely eliminates SNX1 in HeLa cells. (B) To confirm that this effect was not due to off-targeting effects by SNX5 or SNX6 siRNA, RT-PCR was performed and SNX1 mRNA levels were compared with control cells that had been transfected with a scrambled siRNA. No difference in SNX1 mRNA was found between the control and SNX5 or SNX6 knock-down cells, suggesting that the reduction of SNX1 level is a post-transcriptional phenomenon, most probably due to the degradation of SNX1 in the absence of SNX5 and SNX6. ß-actin was used as a loading control for western blots; GAPDH was used as a control in RT-PCR.

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