The C-terminal transmembrane domain of Bcl-xL mediates changes in mitochondrial morphology

Abstract

We investigate the effect of mitochondrial localization and the Bcl-x(L) C-terminal transmembrane (TM) domain on mitochondrial morphology and subcellular light scattering. CSM 14.1 cell lines stably expressed yellow fluorescent protein (YFP), YFP-Bcl-x(L,) YFP-Bcl-x(L)-DeltaTM, containing the remainder of Bcl-x(L) after deletion of the last 21 amino acids corresponding to the TM domain, or YFP-TM, consisting of YFP fused at its C-terminal to the last 21 amino acids of Bcl-x(L). YFP-Bcl-x(L) and YFP-TM localized to the mitochondria. Their expression decreased the intensity ratio of wide-to-narrow angle forward scatter by subcellular organelles, and correlated with an increase in the proportion of mitochondria with an expanded matrix having greatly reduced intracristal spaces as observed by electron microscopy. Cells expressing YFP-TM also exhibited significant autophagy. In contrast, YFP-Bcl-x(L)-DeltaTM was diffusely distributed in the cells, and its expression did not alter light scattering or mitochondrial morphology compared with parental cells. Expression of YFP-Bcl-x(L) or YFP-Bcl-x(L)-DeltaTM provided significant resistance to staurosporine-induced apoptosis. Surprisingly however, YFP-TM expression also conferred a moderate level of cell death resistance in response to staurosporine. Taken together, our results suggest the existence of a secondary Bcl-x(L) function that is mediated by the transmembrane domain, alters mitochondrial morphology, and is distinct from BH3 domain sequestration.

FIGURE 1

YFP constructs, YFP expression, and YFP localization in CSM 14.1 cell lines. (A) Bcl-x_L, Bcl-x_L-ΔTM, which lacks the last 21 amino acids of wild-type Bcl-x_L, or TM, which consists of the last 21 amino acids of Bcl-x_L, was fused to YFP. (B) YFP expression in CSM 14.1 cells was confirmed by Western blotting. As expected, a band between 29 and 37 kDa was found for cells transfected with YFP or YFP-TM. A band just above 50 kDa corresponds to the presence of YFP-Bcl-x_L and YFP-Bcl-x_L-ΔTM (Bcl-x_L is ∼26 kDa). (C) Differential interference contrast (upper left panel) and fluorescent images of representative cells transfected with YFP, YFP-BCL-x_L, YFP-BCL-x_L-ΔTM, and YFP-TM. The distribution of YFP-Bcl-x_L and YFP-TM (third and last panel pairs) coincides with the distribution of mitochondria labeled with anti-OxPhos Complex V antibody. Very bright and punctate mitochondria were also found in >50% of the YFP-TM transfected cells (arrows in last panel pair). YFP distribution is diffuse in cells transfected with YFP and YFP-BCL-x_L-ΔTM.

FIGURE 2

Optical scatter images (OSI) and optical scatter image ratio (OSIR) values in the CSM 14.1 cell variants. (A) Differential interference contrast (DIC, top panels), and OSI images of untransfected (CSM, leftmost panels) and transfected CSM 14.1 cells. OSI pixel values (color bar) encode local measurements of OSIR, which can be theoretically described by Eq. 1. Note that the image pixels encode OSIR × 100. (B) Mie theory prediction of the average OSIR per pixel expected from images of homogeneous suspensions of spheres with a given diameter. For spheres, the OSIR is expected to decrease monotonically with increasing sphere diameter D, for 0.015 μm < D < 2.75 μm. (C) Mean OSIR per cell for each of the five CSM 14.1 cell variants. Error bars represent the 95% confidence interval of the mean, and the number in parenthesis is the number of cells tested.

FIGURE 3

Pixel analysis of OSI images. (A) Pixel histograms normalized to the number of pixels with OSIR = 1.15 for the untransfected CSM 14.1 cells (black) and transfected CSM 14.1 variants (dark gray, YFP; light gray, YFP-Bcl-x_L; triangles, YFP-Bcl-x_L-ΔTM; circles, YFP-TM). Each histogram includes pixels from all the segmented cell images considered within a given variant. The line with connected small squares corresponds to the pixel histogram of the bright and punctate mitochondria, which were found by fluorescence in YFP-TM cells (see also last panel pair in Fig. 1 C) and segmented via a local intensity peak detection algorithm. (B) Mean of the unnormalized pixel histograms for each of the CSM 14.1 cell variants, and punctate mitochondria found in the YFP-TM cells. The error bar corresponds to the 95% confidence interval of the histogram mean.

FIGURE 4

Representative electron micrographs of mitochondria with a condensed matrix (black arrows) or an expanded matrix (white arrows) found in the transfected CSM 14.1 cell variants. Mitochondria with a condensed matrix were ones in which at least one crista could be discerned, while mitochondria with an expanded matrix had very reduced intracristal spaces, such that no cristae could be discerned under up to 50,000× magnification.

FIGURE 5

Proportion of observed mitochondria with expanded or condensed matrix within each cell variant.

FIGURE 6

Low magnification electron micrographs showing a large number of autophagocytic vesicles (arrows) in a representative YFP-TM cell, and comparison with a typical YFP-Bcl-x_L-expressing cell. (Inset) High magnification view of a few autophagocytic vesicles in a YFP-TM cell.

FIGURE 7

Percentage of dead CSM 14.1 and iBMK cells assessed by propidium iodide exclusion after 24 h of treatment with 1 μM staurosporine. Both cell types were stably transfected with YFP, YFP-BCL-x_L, YFP-BCL-x_L-ΔTM, or YFP-TM. Mean ± SD of at least three cell plates.