Fig 2: Mutations in BH2 domain of Bcl-xL disrupt physical and functional interaction with Drp1a. CED-9 and Bcl-xL protein sequences. Green and yellow boxes specify BH1 and BH2 domains, respectively. Red boxes denote conserved mutated amino acids within each domain.b. Immunoprecipitation by an anti-FLAG antibody of the wild type and mutant (M1 and M2) FLAG-tagged Bcl-xL proteins. Samples were immunoblotted for Drp1 and Bcl-xL. Lysate studies are shown in the right panel.c. Normalized (to starting value) fluorescence change of synaptopHluorin puncta in hippocampal neurons expressing indicated constructs before, during and after electrical stimulation (with 100 action potentials, 10 Hz), n=10 puncta each from 3 cells each from one culture.d. Group data for all experiments shown in c (*p=0.0169; ***p=0.0004).e. Model showing order of events: 1) Neuronal stimulation leads to calcium influx. 2) Calcium binds to and activates Calmodulin (CaM), which is inhibited by calmidazolium (CaMi). 3) CaM causes the translocation of Bcl-xL and Drp1 to clathrin-coated pits where Bcl-xL binds to and activates Drp1. 4) Drp1 is in part responsible for the formation of normally curved endocytotic vesicles. Knock down or functional inhibition of Bcl-xL, Drp1 or CaM disrupts this process, slowing endocytosis.

Fig 3: Bcl-xL over-expression enhances the rate of release of styryl dyes in hippocampal neuronsa. Diagram of protocols for experiments (1-3) described in text.b. Examples of exponential fits to normalized fluorescence change of FM 5-95 puncta during stimulation with 90 mM KCl. Left panel: Bi-exponential fit to normalized fluorescence change from cells over-expressing Bcl-xL (ExpDec2 of Bcl-xL=bi-exponential decay of fluorescence). Center panel: Dotted lines model two different exponents of left panel (ExpDec of Bcl-xL=bi-exponential fit to fluorescence decay shown as one dotted line for each exponent). Right panel: Single exponential fit to the normalized fluorescence change from a GFP control neuron (ExpDec1 of control=single exponential decay of fluorescence).c. Time course of normalized fluorescence of 5 µM Fluo-4 measured in synaptic boutons during electrical stimulation (100 action potentials at 10 Hz; n=15 DsRed expressing cells, n=15 DsRed-Bcl-xL expressing cells).d. Peak fluorescence after loading with Exp 1 protocol (prior to unloading) of individual FM 5-95 puncta (n=10 GFP CTL puncta, 14 GFP- Bcl-xL puncta; **p < 0.0013; three independent cultures). In all experiments, Bcl-xL expressing and GFP-expressing controls are pairs from the same culture.e. Normalized fluorescence change in 90 mM KCl (n=10 GFP CTL puncta, 14 GFP- Bcl-xL puncta).f. Time constants for exponential fits to all experiments in e.g. Relative weights of exponential pools for experiments in e.h. Peak fluorescence of FM 5-95 puncta (protocol for Exp 2); n=14 GFP CTL puncta, 15 GFP-Bcl-xL puncta; three independent cultures; ***p < 0.0001).i. Normalized fluorescence change in 90 mM KCl (Exp 2 protocol; n=14 GFP CTL puncta, 15 GFP-Bcl-xL puncta).j. Time constants for exponential fits to experiments in i.k. Relative weights of exponential pools for experiments in i.l. Peak fluorescence of FM 5-95 puncta (Exp 3 protocol; n=11 GFP CTL puncta, 20 GFP-Bcl-xL puncta; three independent cultures).m. Normalized fluorescence change in 90 mM KCl (Exp 3 protocol; n=5 GFP CTL puncta, 10 GFP-Bcl-xL puncta). The initial 10 s of fluorescence decline is shown. 5 CTL puncta are represented to emphasize the bi-exponential effect found in a subgroup of CTL puncta. No punctal fluorescence changes from GFP-Bcl-xL expressing cells required bi-exponential fits.n. Time constants for exponential fits to experiments such as in m (n=9 GFP CTL puncta, n=10 GFP-Bcl-xL puncta).o. Relative weights of exponential pools for experiments in m. Statistics are represented as mean +/- S.E.M.

Fig 4: Colocalization of BAX and BCL2L1. Oocytes that underwent either TN/TN IVM or HS/TN IVM were fixed and used for IHC to determine colocalization. Neither the time point during IVM nor the temperature treatment affected colocalization of antibody-labeled BCL2L1 and BAX in the oocyte. Based on level of fluorescence, there appeared to be a qualitative increase in BAX (red) at 21 h of IVM under HS compared to 21 h of IVM at TN conditions, though there appeared to be no effect on the abundance of BCL2L1 (green; A). The comparison between 21-h TN (B), 21-h HS (C), or negative control (D) shows that there was punctate yellow signal representing colocalization in both TN and HS oocytes. For A, the white bar in images in the left and middle columns represent 50 µm, and the white bar in images in the right column represents 20 µm. The white arrows point to areas of colocalization. The images in the right column are magnified 5× images from the same oocyte from the middle column

Fig 5: Bcl-xl reconstructs the mitochondrial network. (A) Fluorescence microscopic images of living cells transfected with the indicated amounts of YFP (empty vector) or YFP-Bcl-xl plasmids, and also stained with MitoTracker. Scale bars: 2 µm. Line scans show the fluorescence intensities of YFP signals (yellow) and mitochondria stained by MitoTracker (red) along the selected white arrows in the merge panels. (B) Percentages of cells with indicated mitochondrial morphologies in YFP-positive cells (n = 150) transfected with the indicated amounts of plasmids. Data were collected from three independent experiments. The error bars represent SD. (C) Expression level of Bcl-xl in cells transfected with the indicated amounts of YFP (empty vector) or YFP-Bcl-xl assessed by western blotting.