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Journal of Neuroscience, Vol 12, 2186-2197, Copyright © 1992 by Society for Neuroscience
Organization of cortical cytoskeleton of cultured chromaffin cells and involvement in secretion as revealed by quick-freeze, deep-etching, and double-label immunoelectron microscopy
T Nakata and N Hirokawa
Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
We have studied the organization of the cytoskeleton in both unstimulated
and stimulated cultured chromaffin cells, as well as its relationship with
their secretory process by exocytosis. We found the spatial heterogeneity
in the intensity of cortical rhodamine-phalloidin staining within a cell.
The overall staining pattern or intensity was minimally altered after
stimulation, although dopamine-beta-hydroxylase (DBH) antigen, a marker for
the chromaffin granule membrane, was exposed preferentially on the plasma
membrane areas with lower intensity of rhodamine-phalloidin staining. Using
the quick-freeze, deep-etch technique, we found the heterogeneity in the
organization of cortical cytoskeletal networks--some regions have actin
filament bundles running parallel to the plasma membrane interspersed
between granules and the plasma membrane, while others have few actin
filaments beneath the plasma membrane before stimulation. Actin filaments
were rarely observed in the inner cytoplasm. We did not observe the overall
change in its organization after stimulation. Double-label immunogold EM
using anti-DBH antibody and anti-actin antibody combined with statistical
analysis showed that (1) DBH was exposed on the plasma membrane
preferentially where actin was sparse after stimulation (significant at
less than 0.1%), although (2) regions having sparse actin were not always
the sites for DBH exposure, and (3) the cortical actin zone was sometimes
disrupted at the DBH-exposed sites after stimulation. The present data
suggested that (1) secretion is related to heterogeneous organization of
cortical cytoskeleton after stimulation and (2) massive synchronized
reorganization of the cytoskeleton in the whole cell is not necessary for
secretion, although small changes of the cytoskeleton might occur under
local regulation at each exocytotic site at the moment of the release.
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