Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry.
Calpactin I complex, a calcium-dependent phospholipid-binding protein, promotes aggregation of chromaffin vesicles at physiological micromolar calcium ion levels. Calpactin I complex was found to be a globular molecule with a diameter of 10.7 +/- 1.7 (SD) nm on mica. When liposomes were aggregated by calpactin, quick-freeze, deep-etching revealed fine thin strands (6.5 +/- 1.9 [SD] nm long) cross-linking opposing membranes in addition to the globules on the surface of liposomes. Similar fine strands were also observed between aggregated chromaffin vesicles when they were mixed with calpactin in the presence of Ca2+ ion. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structures approximately 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy on frozen ultrathin sections showed that calpactin I was closely associated with the inner face of the plasma membranes and was especially conspicuous between plasma membranes and adjacent vesicles in chromaffin cells. These in vivo and in vitro data strongly suggest that calpactin I complex changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells.
Nakata
T
T
Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
Sobue
K
K
Hirokawa
N
N
D000313:Adrenal Medulla / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D017302:Annexins; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000648:ultrastructure; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure; D005089:Exocytosis; D005455:Fluorescent Antibody Technique; D005613:Freeze Etching; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D011487:Protein Conformation
D017302:Annexins; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins