FIGURE 1.
Bcl-xL promotes ER to mitochondrial Ca2+ transfer without affecting ER or mitochondrial morphology. A, representative images showing intact WT and Bcl-xL-KO cells co-loaded with Rhod-2 and Fluo-2 during stimulation with 1 mm ATP to evoke InsP3-dependent ER Ca2+ release. B, representative time course plots of [Ca2+]cyto and [Ca2+]mito are shown. The peak amplitude (mean ± S.E.) of the [Ca2+]cyto response was significantly larger (p < 0.05; Student's t test) in Bcl-xL-KO (3.20 ± 0.36) compared with WT (1.93 ± 0.41), whereas the peak amplitude of the [Ca2+]mito transient was smaller in Bcl-xL-KO (1.83 ± 0.20) compared with WT (2.84 ± 0.38). C, WT and Bcl-xL-KO MEFs were labeled with enhanced YFP and RFP targeted to the ER and mitochondria, respectively. Representative confocal sections show mitochondria (red), ER (green), and regions of colocalization (white). The degree of ER-mitochondrial colocalization was assessed using Manders' coefficient, and no significant difference was observed between the coefficients calculated for WT (0.49 ± 0.01) and Bcl-xL-KO (0.48 ± 0.01) cells (p > 0.05; Student's t test). D, electron micrographs of WT and Bcl-xL-KO cells (19,500×). Arrows indicate points of close ER-mitochondrial apposition. E, the ER-mitochondrial contacts identified that were less than or equal to 100 nm were sorted into five groups (0–20, 21–40, 41–60, 61–80, and 81–100 nm) and graphed based on the number of contacts in each group normalized to the total number of contacts less than or equal to 100 nm (wild type, 88 contacts; Bcl-xL-KO, 92 contacts). F, the expression levels of several mitochondrial proteins were detected by Western blot. COX-IV, cytochrome c oxidase subunit IV.