HIGH-RESOLUTION CALCIUM MAPPING OF THE ENDOPLASMIC-RETICULUM GOLGI EXOCYTIC MEMBRANE SYSTEM - ELECTRON-ENERGY-LOSS IMAGING ANALYSIS OF QUICK FROZEN FREEZE-DRIED PC12 CELLS

The calcium pools segregated within the endoplasmic reticulum, Golgi c omplex, exocytic, and other organelles are believed to participate in the regulation of a variety of cell functions. Until now, however, the precise intracellular distribution of the element had not been establ ished. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the elec tron energy loss spectroscopy technique. The preparation procedure use d included quick freezing of cell monolayers, followed by freeze-dryin g, fixation with OsO4 vapors, resin embedding, and cutting of very thi n sections. Conventional electron microscopy and high-resolution immun ocytochemistry revealed a high degree of structural preservation, a co ndition in which inorganic elements are expected to maintain their nat ive distribution. Within these cells, calcium signals of nucleus, cyto sol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic retic ulum, the distribution was heterogeneous with strongly positive cister nae (more often the nuclear envelope and stacks of parallel elements t hat are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cister nae. The Golgi complexes were labeled strongly and uniformly in all ci sternae and part of their vesicles, with no appreciable differences al ong the cis-trans axis. Weaker or negative signals were recorded from the tl ans-Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. Th ese results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the various organelles, and abo ut the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.