3D visualization of mitochondrial solid-phase calcium stores in whole cells

Abstract

The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results 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.

Inorganic polyphosphate (polyP) is required for sustained free mitochondrial calcium elevation, following stimulated calcium uptake.

“…It has been previously demonstrated that the calcium present in the mitochondrial matrix is buffered in dense calcium-phosphate granules, (Nicholls and Chalmers, 2004). These granules have been found in live cells, after pathological calcium overload, as well as in cells under normal culture conditions, (Wolf et al, 2017). While these granules contain precipitated (non-active) calcium, they can be easily dissolved by mitochondria, when buffering is needed.…”

The Mitochondrial Permeability Transition Phenomenon Elucidated by Cryo-EM Reveals the Genuine Impact of Calcium Overload on Mitochondrial Structure and Function

“…At this point, we believe the calcium buffering in the form of 434 calcium phosphate granules system becomes relevant. In a separate study, calcium phosphate granule 435 deposits were observed in the matrix near cristae junctions in a variety of different eukaryotic cells under 436 physiological conditions(Wolf et al, 2017). Given the relevance of calcium in bioenergetics, the presence of 437 these calcium deposits may exert some degree of control over mitochondrial signaling and metabolism.…”

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

“…As such there is a need to optimize imaging conditions such that SNR, contrast, and resolution are maximized for images collected from thick samples with low electron fluxes. While this has been demonstrated experimentally for resin embedded cells or cryogenic thin sections (16,18), and discussed in theory for liquid and cryogenic samples (19)(20)(21)(22), at the time of this writing the optimal imaging modalities have not been demonstrated experimentally for LC-TEM across equivalent samples on the same instrument. Furthermore, despite demonstrations of hydrated cells imaged with LC-TEM in the literature, images collected with LC-TEM do not consistently show the same level of detail as those imaged with cryo-EM.…”