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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).

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