Figure 2. Elemental characterization of mitochondrial granules.

(A) EDX identifies calcium (Ca) and phosphorus (P) enrichment in mitochondria. Areas of a WI-38 fibroblast subjected to EDX (boxed) were imaged prior to spectroscopic analysis. Scale bar is 400 nm. (B) Schematic (not to scale) showing collection of BF and DF CSTET data. (C–E) Scale bar is 200 nm. Sections of BF (top) and DF (bottom) reconstructions display (C) mitochondrial granules (arrowheads), (D) a polyribosome (arrowheads), and (E) a lipid droplet (dr; arrowheads) and a vesicle (v). (F) Color-coded 3D volume rendering (red: high density; blue: low density) showing heterogeneity of density in granules of a WI-38 fibroblast. Granules are presented against backdrops of projected BF volume densities with inverted contrast. (G) Zoom in on the boxed region of F. Scale bar is 50 nm. Sections 10-nm thick from zlTEM tomographic reconstructions are shown for (H) a thin region of a WI-38 fibroblast cell displaying mitochondrial granules and (I) synthetic amorphous calcium phosphate. The synthetic particles shown here were obtained 1.5 min after mixing of calcium and phosphate solutions. Red arrows in panels H and I highlight particles for comparison. Scale bar in H is 50 nm and applies also to I. (J) EDX of synthetic amorphous calcium phosphate (top) compared to an adjacent region of vitrified solution (bottom).

Figure 2—figure supplement 1. Intensity thresholding for quantitative estimation of mitochondrial granule scattering.

The BF tomogram obtained from the HDMEC cell imaged in Figure 1—figure supplement 2B was used for analysis. A movie of the tilt series and reconstruction of this cell region are shown in Videos 7 and 8. Mean intensity in the selected slice is 9.7, with standard deviation 2.4. The aqueous medium filled in at a threshold of 16 (not shown). With a threshold of 3.0, dense parts of mitochondrial granules were selected (top). The densest peaks reached an intensity value of 0.3 (not shown). With a threshold of 5.3, most of the granule volumes and the densest regions of ribosomes were selected (middle). A threshold of 7.5 captures most of the ribosomal volumes (bottom). Scale bar is 400 nm. These data are used in Table 3 to estimate the material densities of granules.

Figure 2—figure supplement 2. Large granules are observed in mitochondria of murine embryonic fibroblasts (MEFs) and are independent of the presence of the mitochondrial calcium uniporter MCU.

(A) Mitochondria in wild-type MEFs. Scale bars are 400 nm. (B) PCR was used to confirm the MCU -/- and +/+genotypes (Pan et al., 2013). (C) Mitochondria from MCU -/- MEFs contain granules. Scale bars are 200 nm in the left panel and 400 nm in the right. (D) EDX confirms the presence of calcium and phosphorus in granules of MCU -/- MEFs. The gold (Au) peak partially overlapping the phosphorus peak is present in this spectrum but not that shown in main Figure 2A due to a change in the microscope aperture hardware that occurred between the two experiments.