Fig 1.
STIM1 is enriched in patches at the C. trachomatis inclusion membrane.
Confocal micrographs of HeLa cells infected with C. trachomatis for 24h and stained using antibodies against the C. trachomatis inclusion membrane protein IncA (A-B) (left panels, IncA, green (A) and red (B)). The cells were transfected with a mCherrySTIM1 construct 18h pre-infection (A) (middle panels, mChSTIM1, red) or stained using antibodies against STIM1 (B) (middle panels, STIM1, green). The merge is shown on the right. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusion. Scale bar: 10μm.
Fig 2.
The ER markers Rtn3C and Sec61ß are not enriched at the C. trachomatis inclusion membrane.
Confocal micrographs of HeLa cells co-expressing mCherrySTIM1 (A-B) (left panels, mChSTIM1, red) and GFP-Rtn3C (A) (middle panels, Rtn3C, green) or GFP-Sec61ß (B) (middle panels, Sec61ß, green) and infected with C. trachomatis for 24h. The merge is shown on the right. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusion. Scale bar: 10μm.
Fig 3.
STIM1 is enriched at ER-Inclusion MCSs.
Electron micrographs of HeLa cells expressing HRP-STIM1, infected with C. trachomatis for 24h and processed for conventional transmission electron microscopy coupled with peroxidase cytochemistry. White arrows and black arrowheads respectively indicate ER structures displaying high and low level of HRP-STIM1. The area outline by the box in A is shown at higher magnification in B. RB: Reticulate Body. Scale Bars: 1 μm (A), 500 nm (B).
Fig 4.
STIM1 colocalizes with CERT and VAPB at the C. trachomatis inclusion membrane.
Confocal micrographs of HeLa cells co-expressing YFP-CERT (yellow), CFP-VAPB (cyan) and mCherrySTIM1 (red) and infected with C. trachomatis for 24h. The merge is shown on the right. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusion. N: Nucleus. Scale bar: 10μm.
Fig 5.
STIM1 is associated with the C. trachomatis inclusion membrane throughout the developmental cycle.
A-B. Confocal micrographs of HeLa cells co-expressing YFP-CERT (yellow) and mCherrySTIM1 (mCh-STIM1, red) and infected with C. trachomatis expressing CFP (CtL2 CFP, cyan) for 4h (A), 8h (B). The extended focus view of 10 0.2 μm individual planes crossing the middle of the cell is shown. The boundary of the cell and the nucleus (N) are outlined and the area outlined by a box is shown at higher magnification below the whole cell micrograph. The merge is shown on the right. Scale bar: 10 μm. C-D. HeLa cells, as described in A and B, were infected for 14h (C) or 48h (D). Three small inclusions that have not yet fused are shown (C). The merge is shown on the right. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusions. N: Nucleus. Scale bars: 4μm (C), 20μm (D).
Fig 6.
STIM1 cellular localization in response to Ca2+ store depletion.
A. Confocal micrographs of HeLa cells co-expressing mCherry-STIM1 (left panels, mChSTIM1, red) and Orai1-GFP (middle panels, Orai1-GFP, green) and infected with C. trachomatis for 24h. The merge is shown on the right. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisks in the XY View panels indicate the position of the inclusions. Scale bar: 10μm. B. Confocal micrographs of live HeLa cells expressing mCherry-STIM1 (mCh-STIM1, red) and infected for 24h with a strain of C. trachomatis that expresses GFP (CtL2 GFP, green). The images were acquired before (left panels, Before Tg) and 5 min after addition of Thapsigargin (Tg) (right panels, 5min Tg). The top and bottom panels respectively correspond to the mCh-STIM1 signal alone and the merge. An extended focus view combining all the confocal planes is shown. The arrow indicates STIM1 enrichment at ER-Inclusion MCSs. The asterisk indicates the position of the inclusion. Scale bar: 10μm.
Fig 7.
The CAD domain of STIM1 is required for STIM1 localization to ER-Inclusion MCSs.
A. Schematic representation of the major domains of the STIM1 protein, their respective amino acid residue position and their respective cellular localization (ER lumen or Cytosol). Signal: signal peptide; EF: EF-hand; SAM: sterile alpha motif; TM: transmembrane domain; CC1: coiled-coil 1; CC2: coiled-coil 2; CAD: CRAC activation domain; S/P: Serine-proline-rich region; K: lysine-rich region. B. Confocal micrographs of HeLa cells expressing the indicated mCherry-STIM1 construct (red) and infected with C. trachomatis for 24h. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusion and N indicates the nucleus. Scale bar: 10μm. C. Quantification of inclusion association of the indicated mCh-STIM1 constructs compared to full-length mCh-STIM1. *** p value <0.001. D. Confocal micrographs of HeLa cells expressing YFP-CAD (CAD(342–448), yellow), and infected for 24h with a strain of C. trachomatis expressing CFP (cyan). The merge is shown on the right. Two representative examples of YFP-CAD pattern on the inclusion are shown. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusion. Scale bar: 10μm.
Fig 8.
STIM1 depletion does not affect C. trachomatis growth.
HeLa cells were transfected with control siRNA (GFPsi) or a pool of CERT siRNA (CERTsi) or a pool of siRNA against STIM1 (STIM1si) for 3 days and infected with a strain of C. trachomatis expressing mCherry. (A) For each condition, the surface area of the inclusions was determined (Arbitrary Units). Each circle, square or triangle represents data from a single inclusion. (B) The number of infectious bacteria (IFUs/ml) was determined 48h p.i. (A-B) Data show the mean and standard deviation of biological triplicate of a representative experiment. ***P<0.0001 (student’s t test).
Fig 9.
Schematic representation of ER-Inclusion MCSs.
CERT (green) localizes to ER-Inclusion MCSs. The PH domain of CERT binds to the C. trachomatis inclusion membrane protein IncD (red) and the FFAT motif of CERT allows CERT association with VAPA/B (Yellow) on the ER. In addition to CERT, VAPA/B and IncD, the ER Calcium sensor STIM1 (dark blue) also localizes to ER-Inclusion MCSs. The CAD domain of STIM1 is required for association with the inclusion. Future work will determine whether a Chlamydia factor X (light pink) is involved in this process.