3D Visualization of Mitochondrial Solid-Phase Calcium Stores in Intact Cells
Sharon G. Wolf1, Yael Mutsafi2, Tali Dadosh1, Tal Ilani2, Zipora Lansky2, Ben Horowitz2, Sarah Rubin3, Michael Elbaum3, and Deborah Fass2,
1Department of Chemical Research Support, 2Department of Structural Biology, and 3Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, 7610001 Israel
The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Over the past decade, the molecular identities of transporters involved in mitochondrial calcium influx and efflux have been determined. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. Using cryo-scanning transmission electron tomography (CSTET) to visualize mitochondria engaged in native interactions with other organelles in thick regions of vitrified intact mammalian cells, we found amorphous solid granules containing calcium and phosphorus to be pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering showed that these repositories can reach the equivalent of molar concentrations of dissolved ions. Granule formation or maintenance required mitochondrial polarization but not on the mitochondrial calcium uniporter. Mitochondrial granules appeared to be depleted in highly metabolically active cells, as fibroblasts engaged in intense extracellular matrix production and secretion upon reaching confluence tended to have many granule-free mitochondria, whereas slowly proliferating, sub-confluent fibroblasts and other cell types consistently showed granules. Our observations demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling. CSTET visualization of calcium sequestration in a variety of unfixed and unstained intact cells strengthens the notion that solid-state storage must be broadly considered in a general accounting of cellular calcium and is not restricted to mineralizing cell types.