High-resolution calcium mapping of the endoplasmic reticulum-Golgi- exocytic membrane system Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells

Roberta Pezzati, Mario Bossi, Paola Podini, Jacopo Meldolesi, Fabio Grohovaz

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100 Citations (Scopus)

Abstract

The calcium pools segregated within the endoplasmic reticulum, Golgi complex, 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 established. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the electron energy loss spectroscopy technique. The preparation procedure used included quick freezing of cell monolayers, followed by freeze-drying, fixation with OsO4 vapors, resin embedding, and cutting of very thin sections. Conventional electron microscopy and high-resolution immunocytochemistry revealed a high degree of structural preservation, a condition in which inorganic elements are expected to maintain their native distribution. Within these cells, calcium signals of nucleus, cytosol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic reticulum, the distribution was heterogeneous with strongly positive cisternae (more often the nuclear envelope and stacks of parallel elements that are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cisternae. The Golgi complexes were labeled strongly and uniformly in all cisternae and part of their vesicles, with no appreciable differences along the cis-trans axis. Weaker or negative signals were recorded from the trans- Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. These results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the various organelles, and about the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.

Original languageEnglish
Pages (from-to)1501-1512
Number of pages12
JournalMolecular Biology of the Cell
Volume8
Issue number8
Publication statusPublished - 1997

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PC12 Cells
Endoplasmic Reticulum
Electrons
Calcium
Organelles
Membranes
Golgi Apparatus
Electron Energy-Loss Spectroscopy
trans-Golgi Network
Freeze Drying
Nuclear Envelope
Eukaryotic Cells
Cytosol
Freezing
Electron Microscopy
Mitochondria
Immunohistochemistry

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

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High-resolution calcium mapping of the endoplasmic reticulum-Golgi- exocytic membrane system Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells. / Pezzati, Roberta; Bossi, Mario; Podini, Paola; Meldolesi, Jacopo; Grohovaz, Fabio.

In: Molecular Biology of the Cell, Vol. 8, No. 8, 1997, p. 1501-1512.

Research output: Contribution to journalArticle

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