Mouse lines with photo-activatable mitochondria to study mitochondrial dynamics

Abstract

Many pathological states involve dysregulation of mitochondrial fusion, fission, or transport. These dynamic events are usually studied in cells lines because of the challenges in tracking mitochondria in tissues. To investigate mitochondrial dynamics in tissues and disease models, we generated two mouse lines with photo-activatable mitochondria (PhAM). In the PhAM(floxed) line, a mitochondrially localized version of the photo-convertible fluorescent protein Dendra2 (mito-Dendra2) is targeted to the ubiquitously expressed Rosa26 locus, along with an upstream loxP-flanked termination signal. Expression of Cre in PhAM( floxed) cells results in bright mito-Dendra2 fluorescence without adverse effects on mitochondrial morphology. When crossed with Cre drivers, the PhAM(floxed) line expresses mito-Dendra2 in specific cell types, allowing mitochondria to be tracked even in tissues that have high cell density. In a second line (PhAM(excised) ), the expression of mito-Dendra2 is ubiquitous, allowing mitochondria to be analyzed in a wide range of live and fixed tissues. By using photo-conversion techniques, we directly measured mitochondrial fusion events in cultured cells as well as tissues such as skeletal muscle. These mouse lines facilitate analysis of mitochondrial dynamics in a wide spectrum of primary cells and tissues, and can be used to examine mitochondria in developmental transitions and disease states.

Figure 1. Construction of PhAM^floxed and PhAM^excised mouse lines

(A) Targeting of mito-Dendra2 into the Rosa26 locus. Homologous recombination of the targeting construct (schematic 2) in embryonic stem cells results in insertion of the Cre-dependent mito-Dendra2 cassette into the Rosa26 locus (schematic 3). In mice, removal of the neomycin selection marker by Flp recombinase results in the PhAM^floxed line (schematic 4), which can be mated to a Cre driver line to obtain cell-specific labeling of mitochondria. Germline excision of the termination signal produces the PhAM^excised line (schematic 5). Black arrowheads, loxP sites; stop symbol, termination cassette; gray diamonds, frt sites; half arrows, PCR primers for genotyping; short horizontal line, probe for Southern blot. (B) Representative Southern blot analysis of ES cell clones. Genomic DNA was digested with HindIII and hybridized with the Rosa26 probe indicated in schematic 1 of (A). (C) PCR genotyping of the PhAM^floxed strain for the wild-type or knock-in allele using the set of three primers in schematic 3 of (A). (D) PCR genotyping of the PhAM^excised strain using the three primers in schematic 4 of (A).

Figure 2. Tracking of mitochondria in PhAM^floxed tail fibroblasts

(A) Representative images of mitochondria in tail fibroblasts cultured from the PhAM^floxed mice. Tail fibroblasts were cultured in the absence (top) or presence of Cre-expressing retrovirus (bottom). Mitochondria are identified by immunostaining for HSP60 (red). The mito-Dendra2 fluorescence (green) was found only after expression of Cre. Mitochondrial morphology remains tubular (inset). Scale bar is 10 μm. (B) Quantification of mitochondrial morphology in wildtype and PhAM^floxed fibroblasts. The table shows the percentage of cells with the indicated morphology ± SEM (n=4). (C) Monitoring mitochondrial fusion in PhAM^floxed fibroblasts. A subset of mitochondria was photo-converted (red) and tracked by time-lapse imaging. Three still images from the resulting movie highlight a mitochondrial fusion event (arrowhead) and exchange of matrix contents. Scale bar is 5 μm. (D) Fluorescence line analysis of the two mitochondria undergoing fusion in the frames from (C). Each plot measures the red and green signals along the drawn line. The line analysis demonstrates that mitochondrial fusion results in the transfer of red fluorescence to the adjoining mitochondrion.

Figure 3. Ubiquitous expression of mito-Dendra2 in PhAM^excised tissues

Frozen tissue sections from the PhAM^excised mice. (A) pyramidal neurons in the cortex; (B) pyramidal neurons in the hippocampus; (C) Purkinje neurons of the cerebellum; (D) myocardium; (E) testis; (F) lung; (G) liver cannula, inset shows magnified image of the boxed region; (H) kidney cortex; (I) thymus. Cell counter stains are shown in red or purple. In (A–B), anti-Map2 (red) stains the dendritic processes of neurons; in (A–B), a fluorescent Nissl stain (purple) marks neurons; in (C), anti-calbindin (red) highlights Purkinje neurons; in (D–I), wheat germ agglutinin (WGA) labels cell borders. Scale bars, 10 μm.

Figure 4. Imaging of mito-Dendra2 in live isolated cells

The fluorescence of mito-Dendra2 (green) was imaged in a (A) spermatocyte, (B) myofiber, and (C) cardiomyocyte. In each case, a subset of mitochondria was irradiated with a 405 nm laser to photo-switch mito-Dendra2 (red). (D) Comparison of mito-Dendra2 (green) in a fixed myofiber with the Z-disc marker α-actinin (red). Since the myofiber in (D) was processed for immunostaining, the resolution of mitochondrial doublets is lower than (B). The far right panel is a higher magnification image of the boxed region. (E) Detection of mitochondrial fusion in isolated EDL muscle from a 2-month old animal. A subset of mitochondria was photo-converted and tracked. Intensity maps of the photo-converted signal show two mitochondrial fusion events (marked by arrowheads) over a 12-minute period. In the top fusion event, the transfer of red signal into an unconverted mitochondrion was detected. In the bottom event, fusion occurs between two photo-converted mitochondria and results in equalization of the intensity. Intensity values of the heat maps are indicated in the legend. Scale bars: 10 μm for sperm and 5 μm for myofibers and cardiomyocyte. (F, H) Changes in mitochondrial structure during postnatal muscle development. Whole EDL muscles were isolated and imaged by mito-Dendra2 fluorescence at indicated ages. Scale bars: 5 μm. (G, I) Ultrastructural analysis of fixed EDL sections. Mitochondria are indicated by arrowheads. Scale bars: 10 μm.

Figure 5. Purkinje-specific labeling of mitochondria

PhAM^floxed mice were crossed with a Purkinje-specific driver, Pcp2 Cre, and organotypic slice cultures were prepared from the offspring. (A) Merged image of mito-Dendra2 (green) and anti-calbindin (red). Two Purkinje cells express mito-Dendra2. (B) Single-channel image of anti-calbindin highlighting the borders of Purkinje neurons. (C) Single-channel image of mito-Dendra2 signal. (D) High magnification image of the boxed region in (C). Note the tight clusters of mitochondria in the distal dendritic branches (arrowheads). Scale bars: 10 μm.

Figure 6. Visualization of mitochondrial defects in Purkinje neurons lacking Mfn2

Frozen sections of cerebellum with stained for calbindin (red) and Dendra2 (green). The top panel is from a control animal with normal Purkinje neurons. The bottom panel is from a littermate lacking Mfn2 in Purkinje neurons due to the Pcp2 Cre driver. The last column shows high magnification images of the boxed regions. Scale bar: 10 μm in the merged image and 5 μm in the magnified image.