Endoplasmic Reticulum-Golgi-Exocytic

Molecular Biology of the Cell Vol. 8,1501-1512, August 1997

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, and Fabio Grohovaz* Consiglio Nazionale delle Ricerche, Cellular and Molecular Pharmacology Center; B. Ceccarelli Center, Department of Pharmacology, University of Milan; and DIBIT, S. Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy Submitted February 26, 1997; Accepted May 10, 1997 Monitoring Editor: Suzanne R. Pfeffer

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. INTRODUCTION During last decades, significant progress of cell thethe During

niques such as high-resolution immunocytochemistry has not In contrast, thislopa fractionation. te opnnsta h aewt be subcellular been the case with other components that also play elements roles in cell life, i.e., inorganic fundamental extento calcium. with special reference Despite the

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ment has remained so far largely undefined. In fact, during cell preparation (fixation, homogenization, and centrifugation), artifactual redistributions (release and, in some cases, even accumulation) were shown to take place in various organelles, such as the endoplasmic reticulum (ER)1 and mitochondria (see Pozzan et al., 1994, and references therein). Even when calcium distribution was appropriately preserved by cell freezing, and microscopic samples thus prepared were analyzed by microanalytical techniques such as X-ray electron probe microanalysis or secondary ion mass spectrometry, the results were often unsatisfactory. Resolution was in fact low primarily due to problems with the identification of the various organelles and to the need to use thick (0.5-1 ,um) sections for appreciable signals to be generated (Andrews and Reese, 1986; Fiori et al., 1988; Hall, 1988; Lefurgey et al., 1988; Linton and Goldsmith, 1992; Chandra et al., 1994; Pozzan et al., 1994). Recently, a protocol for high-resolution revelation of the cellular distribution of calcium has been developed in our laboratory (Grohovaz et al., 1996) using electron energy loss spectroscopy (EELS) employed in its imaging mode (ESI; Ottensmeyer and Andrew, 1980; Colliex, 1986; Leapman and Ornberg, 1988). In this microanalytical approach, the electrons that are scattered inelastically by the specimen are collected, and the information about their energy distribution is converted into maps of the local elemental composition. Although available for many years, and known to offer distinct advantages in terms of both spatial resolution and sensitivity (the estimated detection threshold is three calcium atoms in a 10-nm diameter spot, Shuman and Somlyo, 1987), the technique has not been used extensively in biology. Most of the studies have in fact been carried out on samples first exposed to chemical treatments, with fixatives and/or dehydration agents, that are known to induce, or to be inadequate to prevent, artifactual changes of the cell native state (Echlin, 1992). In our previous work (Grohovaz et al. 1996), fibers of a thin frog muscle were first quick frozen and slowly freeze-dried at low temperature [quick freezing-freeze drying (qf-fd)], after which fixation with OSO4 vapors was applied. The subsequent direct resin embedding of the tissue permitted the preparation of ultrathin (