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First published online 25 September 2007
doi: 10.1242/jcs.03486

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Reversible interactions between smooth domains of the endoplasmic reticulum and mitochondria are regulated by physiological cytosolic Ca²⁺ levels

Jacky G. Goetz^1,2,*, Hélène Genty^2,*, Pascal St-Pierre^1,*, Thao Dang^1,2, Bharat Joshi¹, Rémy Sauvé³, Wayne Vogl¹ and Ivan R. Nabi^1,

¹ Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver V6T 1Z3, Canada
² Department of Pathology and Cell Biology, Université de Montréal, Montreal H3C 3J7, Canada
³ Department of Physiology, Université de Montréal, Montreal H3C 3J7, Canada

View larger version (52K): [in this window] [in a new window]   Fig. 1. siRNA knockdown of expression of AMFR. (A) NIH-3T3 cells untransfected (UT) or transfected with Dicer AMFR siRNA (d-siRNA AMFR) or control siRNA (ctl siRNA) were western blotted with the 3F3A antibody against AMFR and antibody against -actin, as indicated. (B) NIH-3T3 cells untransfected (UT) or transfected with d-siRNA AMFR or control siRNA were fixed and immunofluorescently labelled with the 3F3A mAb and antibody against mtHSP70. Images were acquired with the same confocal settings (using a wide-open pinhole), and the bottom panels show digitally zoomed images of the inset (white box, first row), with the merge presenting 3F3A labelling in red and mitochondrial mtHSP70 labelling in green. (C) 3F3A labelling intensity was quantified from confocal images acquired with an open pinhole (see top row, panel B) of NIH-3T3 cells untransfected (UT) or transfected with d-siRNA AMFR or control siRNA. Total intensity per field was divided by the number of cells in the field (excluding cells touching the edges), determined by Hoechst nuclear labelling (data not shown). **P<0.001. Bar, 50 µm (top row); 10 µm (zoom, bottom row).

View larger version (111K): [in this window] [in a new window]   Fig. 2. Characterisation of the 3F3A-labelled ER domain. (A) Cos-7 cells were co-transfected with GFP-Sec61 and Rtn4a-MYC and visualised using the GFP tag and anti-MYC and 3F3A antibodies. Inset shows that 3F3A labelling does not overlap with the Rtn4a-labelled ER tubules or the perinuclear ER labelled for GFP-Sec61. (B) Cos-7 cells transfected with Rtn4a-MYC were labelled with anti-MYC, 3F3A and anti-Sec61 antibodies. Rtn4a distribution is distinct from both 3F3A and Sec61, and 3F3A shows only partial colocalisation with Sec61. Alternatively, FLAG-AMFR-transfected cells were immunofluorescently labelled for FLAG, 3F3A and either Sec61 (C) or mtHSP70 (E) or co-transfected with vector encoding Rtn4a-MYC and labelled for FLAG, 3F3A and MYC (D). Insets highlight colocalisation of 3F3A labelling with anti-FLAG (C), Rtn4a (D) and mtHSP70 (E). Cells labelled for Sec61 were pre-treated with RNAse. Bars, 20 µm; inset bars, 5 µm.

View larger version (91K): [in this window] [in a new window]   Fig. 3. Ca2+ sensitivity of the mitochondria-interaction of 3F3A-labelled SER tubules in ionomycin-treated cells. MDCK cells were treated with 1 µM ionomycin and either 200 µM EGTA (A,B,C,C'), 1 mM [Ca2+]ex (D,E,F,F') or 10 mM [Ca2+]ex (G,H,I,I') for 20 minutes and double immunofluorescently labelled with the 3F3A mAb and antibody against mtHSP70. Merged images (C,C',F,F',I,I') show 3F3A labelling in red and mtHSP70 in green and overlap in yellow. C',F' and I' show details of a zoomed region from images C,F and I, respectively. (J) The extent of dissociation of 3F3A-labelled SER tubules from mitochondria was quantified by mask overlay (black bars, left Y-axis) for cells treated with 1 µM ionomycin and 0, 1 and 10 mM [Ca2+]ex, as indicated. For the same conditions, the corresponding average [Ca2+]cyt determined using Fura-2-AM ratiometric labelling is shown (white bars, right Y-axis). (K) MDCK cells were treated with 10 µM thapsigargin for 20 minutes (TG 20 min) or with 1 µM ionomycin and 10 µM thapsigargin for 20 (TG+Iono 20 min) or 60 (TG+Iono 60 min) minutes. Cells were double immunofluorescently labelled for 3F3A and mtHSP70 and dissociation of 3F3A-labelled SER tubules from mitochondria quantified (black bars, left Y-axis). The corresponding average [Ca2+]cyt (white bars, right Y-axis) is shown. The mask overlay data represent the average (±s.e.m.) of three independent experiments and the [Ca2+]cyt values the average of at least ten Fura-2-AM experiments. Bars, 20 µm (I); 2 µm (I').

View larger version (77K): [in this window] [in a new window]   Fig. 4. Electron microscopy analysis of the Ca2+ sensitivity of the ER-mitochondria interaction. MDCK cells grown on filters were treated with 1 µM ionomycin and either 200 µM EGTA (A,D,E), 1 mM [Ca2+]ex (B,F) or 10 mM [Ca2+]ex (C,G) for 20 minutes and then processed for electron microscopy. D shows details of a zoomed region from A, with examples of smooth ER tubules and rough ER tubules indicated by arrows (rough arrays point to linear arrays of membrane-bound ribosomes). E, F and G represent masks of images A, B and C, respectively, outlining mitochondria (green), smooth ER tubules (red) and rough ER tubules (blue). The shortest distance of ER tubules from the nearest mitochondria was measured from cells plated on plastic, fixed, scraped and embedded as a cell pellet (H) and from cells grown on filters and fixed and embedded in situ (I) (*P<0.001 for smooth tubules, 1 mM [Ca2+]ex relative to EGTA and 10 mM [Ca2+]ex). J presents the average number of ER tubules found within 50 nm of individual mitochondria (n=5; *P<0.01 for smooth tubules, 1 mM [Ca2+]ex relative to EGTA and 10 mM [Ca2+]ex; **P<0.01 smooth tubules relative to rough tubules). Bars, 200 nm (A-C); 100 nm (D).

View larger version (93K): [in this window] [in a new window]   Fig. 5. IP3R-mediated ER Ca2+ release induces transient SER tubule-mitochondria dissociation. MDCK cells were treated with 10 µM ATP for up to 5 minutes. Treated MDCK cells were fixed at time 0 (A-C'), 2 (D-F') or 5 (G-I') minutes and then double immunofluorescently labelled for 3F3A and mtHSP70. Merged images (C,C',F,F',I,I') show 3F3A (red) and mitochondria (green), co-distribution in yellow. C', F' and I' show details of the boxed regions from images C, F and I, respectively. (J) At various times of ATP treatment without (solid lines) or with xestospongin C (XeC) pre-treatment (dashed lines), the extent of dissociation of 3F3A-labelled SER tubules from mitochondria was quantified (blue, right Y-axis) and the corresponding average [Ca2+]cyt determined (red, left Y-axis). The data represent the average (±s.e.m.) of three independent experiments and [Ca2+]cyt values the average of five Fura-2-AM experiments. Bars, 20 µm (H); 2 µm (I).

View larger version (166K): [in this window] [in a new window]   Fig. 6. Relationship of 3F3A-labelled SER tubules to other ER markers upon ATP-induced mitochondrial dissociation. MDCK cells untreated (A,B,E,F) or treated for 2 minutes with 10 µM ATP (C,D,G,H) were fixed and double immunofluorescently labelled for 3F3A and calnexin (A-D) or for 3F3A and SERCA (E-H). Merged images show 3F3A (red) and calnexin (green) co-distribution (A'-C') or 3F3A (red) and SERCA (green) co-distribution (E'-G') in yellow. A'', C'', E'' and G'' show details of the boxed regions from images A', C', E' and G', respectively. Bars, 20 µm (G'); 4 µm (G'').

View larger version (122K): [in this window] [in a new window]   Fig. 7. IP3R and 3F3A colocalise to mitochondria-associated ER but segregate upon ATP treatment. (A) MDCK cells either untreated or treated with 10 µM ATP for 2 or 5 minutes were fixed and immunofluorescently labelled for IP3R, 3F3A and mitochondrial ATP-synthase (subunit ). (B) Details of zoomed regions from merges of 3F3A (red) and IP3R (green). (C) The extent of colocalisation of 3F3A and IP3R-labelled ER domains with mitochondria and between the 3F3A and IP3R-labelled ER domains at various times of ATP treatment was quantified using the Pearson coefficient. *P<0.01; **P<0.005 relative to 0 min. Bars, 20 µm (A); 2 µm (B).

View larger version (63K): [in this window] [in a new window]   Fig. 8. Distribution of 3F3A-labelled SER tubules in RAS-transformed NIH-3T3 cells is regulated by [Ca2+]cyt. (A) Expression of AMFR and AMF was assessed in NIH-3T3 and RAS-transformed NIH-3T3 (NIH-Ras) cells by western blot using -actin as a loading control. The bar graph shows quantitative immunofluorescence analysis of 3F3A labelling intensity from confocal images of NIH-3T3 and NIH-Ras cells acquired with an open pinhole (*P<0.005). (B) NIH-3T3 and NIH-Ras cells were immunofluorescently labelled for 3F3A (red) and mtHSP70 (mito; green) and zooms of boxed regions of the merged images presented in the bottom row. Bars, 20 µm; 2 µm (zoom). (C) Average [Ca2+]cyt using Fura-2-AM ratiometric labelling and 3F3A-labelled SER tubule dissociation from mitochondria quantified by the mask overlay approach for NIH-3T3 and NIH-Ras cells. (D) Dissociation of 3F3A-labelled SER tubules from mitochondria was quantified by mask overlay in NIH-3T3 and NIH-Ras cells either left untreated (Control) or treated with 1 µM ionomycin in the presence of 200 µM EGTA or 1 mM [Ca2+]ex, as indicated.

View larger version (66K): [in this window] [in a new window]   Fig. 9. Segregation of 3F3A and IP3R-labelled ER domains in RAS-transformed NIH-3T3 cells. NIH-3T3 (A-G) and RAS-transformed NIH-3T3 (NIH-Ras; H-N) cells were immunofluorescently labelled for mtHSP70 (mito; A,H), 3F3A (B,I) and IP3R (C,J). Merged images show 3F3A (red) and mtHSP70 (mito; green) (D,K), IP3R (red) and mtHSP70 (mito; green) (E,L), or 3F3A (red) and IP3R (green) (F,M). G and N show details of the boxed regions from images F and M, respectively. Bars, 20 µm (M); 2 µm (N).