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First published online June 5, 2007
doi: 10.1242/10.1242/jcs.003111

Journal of Cell Science 120, 2010-2021 (2007)
Published by The Company of Biologists 2007
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The retromer component sorting nexin-1 is required for efficient retrograde transport of Shiga toxin from early endosome to the trans Golgi network

Miriam V. Bujny1, Vincent Popoff2, Ludger Johannes2,* and Peter J. Cullen1,*

1 The Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK
2 Traffic and Signaling Laboratory, UMR144 Curie/Centre National de la Recherche Scientifique, Institut Curie, Paris, France


Figure 1
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  		Fig. 1. Shiga toxin B-subunit is transported via SNX1-positive endosomes to the TGN. Fluorescence-labeled Shiga toxin B-subunit (FITC-STxB, green channel) was surface-bound to HeLa cells at 4°C and subsequently allowed to internalize for the indicated times at 37°C or 19.5°C; cells were fixed and immunolabeled for endogenous SNX1 (Alexa594, red channel) and imaged using confocal microscopy. Maximum projections of eight to ten optical z-slices (480 nm z-separation) are shown. (A) Cells were fixed without incubation at 37°C (0 minutes). (B) After 10 minutes at 37°C, the Shiga toxin B-subunit extensively colocalized with the SNX1-positive endosomal structures (yellow color in merged images). Note the subset of FITC-STxB tubules negative for SNX1 (arrowheads in B and C). (C) Shiga toxin B-subunit accumulated in SNX1-positive endosomes when incubated at 19.5°C for 1 hour. When incubated for longer times at 37°C (D,E), it displayed a TGN-like enrichment at the juxtanuclear area. (F) At these time points, Shiga toxin B-subunit colocalised extensively with TGN46 (Alexa594, red). Bars, 10 µm.

 

Figure 2
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  		Fig. 2. In SNX1-suppressed cells, retrograde endosome-to-TGN transport of FITC-STxB is markedly perturbed. HeLa cells were treated with either control or SNX1-specific siRNA. After 72 hours, toxin uptake assays using FITX-STxB (green channel) were performed. Cells were fixed at indicated times and immunolabeled for SNX1 (Alexa594, red channel). In cells treated with SNX1-specific siRNA, the immunofluorescence signal was markedly reduced. (A) When cells were fixed without prior incubation at 37°C, the toxin was found to label the plasma membrane to comparable levels in control and SNX1-suppressed conditions. (B) After 30 minutes of internalization, the toxin showed juxtanuclear enrichment in control siRNA-treated cells, but remained associated with more peripheral structures in SNX1-suppressed cells. Even after 60 minutes (C) and 120 minutes (D), only moderate amounts of the toxin had reached the TGN area in SNX1-suppressed cells with most cells still displaying vesicular enrichment of the toxin (asterisks). Bars, 20 µm.

 

Figure 3
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  		Fig. 3. Quantification of the defect in endosome-to-TGN transport in SNX1-suppressed cells. Toxin-uptake assays using control and SNX1-suppressed HeLa cells were performed as described. After fixation and immunolabeling with anti-SNX1 (Alexa594, red channel), samples were inspected by epifluorescence microscopy and scored blindly for either a TGN-like enrichment (x) or a vesicular appearance (*). Cells marked `o' were not scored because of low toxin levels. (A,B) Scoring phenotypes; FITC-STxB in green channel and TGN46 (Alexa594) in red channel for control and SNX1-suppressed cells after 30 minutes of uptake. (C) Representative data for a set of control and SNX1-suppressed cells (n>600) after 30 minutes of toxin uptake. (D) TGN-like accumulation of FITC-STxB over time (n>600 for each sample; scored for 30, 60 and 120 minutes) for SNX1-suppressed and control cells. (E) Colocalization of FITC-STxB and TGN46, digitally quantified from confocal images using MetaMorph software (see Materials and Methods for details). In control cells, 37±0.4% of FITC-STxB colocalized with TGN46 after 30 minutes of internalization, whereas SNX1-suppressed cells showed 12±1% colocalization. Values given are ± the standard deviation and were determined from the mean of individual visual fields for a total of n>50 cells. Bar, 20 µm.

 

Figure 4
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  		Fig. 4. SNX1-suppression does not grossly perturb Golgi complex and/or TGN appearance. (A,B) Control and SNX1-suppressed cells were subjected to toxin-uptake assay with FITC-STxB (green channel) for 30 minutes, and were then fixed and immunolabeled for either (A) SNX1 (Alexa594, red channel) and giantin (Alexa633, cyan channel) or (B) TGN46 (Alexa594, red channel). Bars, 20 µm.

 

Figure 5
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  		Fig. 5. Analysis of STxB sulfation in SNX1- and SNX2-suppressed cells. The levels of TGN-localized STxB in control and SNX1- and SNX2-suppressed cells (individually and jointly) was quantified using a biochemical sulfation assay (see Materials and Methods and text for details). (A) Representative western blot showing levels of siRNA-mediated protein suppression. The table summarizes quantified levels of suppression achieved (ImageQuant) from n>=4 assays (n=6, for SNX1 and SNX2 individually). (B) The autoradiograph shows a typical result of a sulfation experiment using the B-(Sulf2) construct. The graph summarizes results from six independent sets (each in duplicates) as percent of control. Autoradiographs were quantified by densitometry and normalized to overall levels of protein sulfation. In SNX1-suppressed cells, sulfation was reduced by 43±24.4% compared with control cells, whereas in SNX2-suppressed cells sulfation was reduced by only 22±26.7%; values are given as the mean ± standard deviation (s.d.). (C) Levels of siRNA-mediated suppression as determined by western blotting and quantified by densitometry were plotted against the levels of STxB sulfation (each data point represents mean from duplicates, normalized to global sulfation) for six experimental sets (pink triangles, SNX1 siRNA; blue triangles, SNX2 siRNA). Lower levels of SNX1-suppression (<90%) do not result in reduced sulfation (asterisks). The regions were determined using the lowest suppression level for SNX1 (x-axis) not inducing a reduction in STxB sulfation (100%, y-axis). (D) Graph shows mean levels (± s.d.) of overall protein sulfation for the different siRNA conditions from at least five different experiments (in duplicates).

 

Figure 6
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  		Fig. 6. Immunofluorescence analysis confirms that SNX2-suppression does not grossly perturb retrograde endosome-to-TGN transport of FITC-STxB. (A) FITC-STxB-uptake-assay (performed as described in Materials and Methods). Cells were fixed after 30 minutes and immunolabeled for SNX2 (Alexa633, red channel) and SNX1 (Alexa594, magenta channel). (B) After FITC-STxB internalization for 30 minutes in SNX2-suppressed cells, the toxin colocalized intensively with TGN46 (Alexa594, red channel); yellow in the merged image. (C) Colocalization analysis of STxB and TGN46 of confocal image sections using MetaMorph (see Materials and Methods for further details) in control cells (37±0.4%) and SNX2-suppressed cells (40±8%); s.d. determined from mean for individual visual fields for n>50 cells. Values are given as the mean ± standard deviation (s.d.); bars, 20 µm.

 

Figure 7
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  		Fig. 7. Quantification of STxB localization in SNX2-suppressed cells and cells that were jointly suppressed for SNX1 and SNX2. (A) The assay was performed as described for Fig. 3 and the localization of FITC-STxB in >600 cells was scored after 30 minutes of toxin uptake, as described for Fig. 3. (B) Representative data set for TGN-like accumulation of FITC-STxB over time (n>600 for each sample; scored for 30, 60 and 120 minutes) for control cells and, SNX2-suppressed cells and cells that were jointly suppressed for SNX1 and SNX2. (C) SNX2 clustering in the perinuclear area is dependent on SNX1. Control and SNX1-suppressed cell were subjected to a FITC-STxB-uptake assay (green channel). The toxin was surface-bound at 4°C and internalized at 37°C for 30 minutes. Fixed cells were immunolabeled with anti-SNX2 antibody (Alexa633, red cannel). Bar, 10 µm.

 

Figure 8
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  		Fig. 8. Retrograde transport of CTxB appears to be unaffected in SNX1-suppressed cells. Control and SNX1-suppressed cell were subjected to a CTxB-toxin-uptake assay using Alexa555-conjugated CTxB (red channel). The toxin was surface-bound at 4°C and then either fixed directly (A) or allowed to internalize at 37°C for the indicated times. Fixed cells were immunolabeled with anti-SNX1 (Alexa488, green channel). (B) After 15 minutes, CTxB was found on vesicular structures where it extensively colocalized with SNX1. The toxin displayed a similar vesicular appearance in SNX1-suppressed cells. (C,D) At later time points the toxin was found accumulated in the juxtanuclear area to similar extents in both control and SNX1-suppressed cells, where it colocalized with giantin (E). (F) Colocalization analysis of giantin and STxB in control cells (13±6% colocalization; 94 of 200 evaluated cells with detectable Alexa555-CTxB label), SNX1-suppressed cells (26±13%; 71 of 149 cells with label) and SNX2-suppressed cells (15±8%; 21 of 43 cells with label) using MetaMorph. Values are given as the mean ± standard deviation (s.d.); bars, 20 µm.

 

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© The Company of Biologists Ltd 2007