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

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.

Inactivation of the KlPMR1 gene of Kluyveromyces lactis results in defective cell-wall morphogenesis

“…Protein folding (Gething & Sambrook, 1992), interaction of misfolded proteins with the BiP chaperone (Suzuki et af., 1991) and protein degradation are also affected by the Ca2+ content in the ER (Wileman et af., 1991). A high concentration of calcium has also been reported in the Golgi apparatus (Pezzati et al, 1997). In this compartment, calcium ions are involved in the glycosylation of secreted proteins (Rudolph et af., 1989), the control of protein trafficking and sorting (Carnell & Moore, 1994), the selective aggregation of cargo proteins (Chanat & Huttner, 1991), and precursor processing in yeast (Fuller et af., 1989) and higher eukaryotes (Oda, 1992).…”

Calcium Mobilization via Intracellular Ion Channels, Store Organization and Mitochondria in Smooth Muscle

“…In some cells, multiple stores do exist unequivocally. Different Ca 2+ concentrations have been measured in various regions of the store using recombinant aequorin [47], electron microscopic determination of Ca 2+ content [50] or fluorescent indicators loaded into the cell [34], suggesting that discontinuities exist within the structures surrounding the lumen itself. The store [34] may adopt different configurations within the cell and components may even detach and reattach, so influencing the pattern and distribution of Ca 2+ release channel [51].…”

Calcium dynamics in the secretory granules of neuroendocrine cells

“…This high calcium content is not exclusive of chromaffin granules but instead it is also found at similar or even higher values in most types of secretory granules, e.g. zymogen granules of pancreatic acinar cells [15], small synaptic vesicles of the neuromuscular junction [13], insulin granules [16] and many others (see [17] for a review).…”

“…Winkler and Westhead [9] calculated a total of 90,000 Ca 2+ ions for a vesicle with 270 nm internal diameter, which means a concentration of 15 mM. Studies of total calcium made by electron energy loss imaging spectroscopy in PC12 cells show also clearly that dense granules have a high calcium content (>10 mM), with a signal comparable to that obtained in the Golgi complex and some regions of the ER [13]. Accordingly, in PC12 cells, the acidic calcium pool accounted for 170 mol/l cell water, which corresponds to an internal total concentration of about 30 mM [14].…”

“…The free [Ca 2+ ] in the ER has been also measured with low-Ca 2+ -affinity aequorin and is in the 500-800 M range [7], so that the Ca 2+ -bound/Ca 2+ -free relationship in the ER is about 10. In addition, the total calcium distribution in the ER is heterogeneous, with strongly positive cisternae (including the nuclear envelope) lying in the proximity of or even in direct continuity with other, apparently negative cisternae [13]. This heterogeneity must surely reflect the distribution of the Ca 2+ -binding proteins in the lumen of the ER, as most of the calcium content of the ER is bound with low affinity (K d = 1-4 mM) to several Ca 2+ -binding proteins, mainly calreticulin in the ER and calsequestrin in the SR [2,18].…”