Programmed cell death. Cytochemical evidence for accumulation of calcium in mitochondria and its translocation into lysosomes: X-ray microanalysis in metamorphosing insect muscles.

The intersegmental muscles in the metamorphosing silkmoth Antheraea polyphemus were examined by two electron cytochemical procedures for demonstration of calcium compartmentation during the two-day period of degeneration after emergence. Muscle fibres were treated with either oxalate-pyroantimonate, or phosphate-pyroantimonate procedures. The elemental composition of the reaction product arising from the oxalate procedure was determined with electron probe X-ray microanalysis of unstained thin sections by energy dispersive spectrometry and wavelength dispersive spectrometry. The wavelength dispersive data revealed high peaks of calcium and antimony in the electron-dense precipitates. No reaction was obtained in muscles after treatment with the phosphate-pyroantimonate method. Shortly after the emergence of the moth, very few calcium deposits were found in the mitochondria, which also contained amorphous matrix densities. During the rapid lytic phase (17 and 30 h after ecdysis), the mitochondria, autophagic vacuoles sequestering mitochondria, and lysosomal dense bodies issuing from the latter were highly reactive in each muscle fibre. These results demonstrate that the collapse of tracheae (hypoxic conditions) is correlated with the calcium overload of mitochondria when the cell calcium homeostasis is apparently lost. Such calcium overload of the mitochondria appears to cause irreversible damage to these organelles which are then sequestered in autophagic vacuoles. This mitochondrial autophagic process leads to calcium translocation into a lysosomal compartment. We suggest that the calcium lysosomal stores may have a transient function of cell detoxification and stimulation of calcium-dependent degradative processes prior to the final muscle collapse.