Mucous secretion as observed after ultrarapid freezing and freeze substitution

Abstract

Most molecules synthesized for extracellular transport are believed to be secreted from the cell by exocytosis. Exocytosis is also generally accepted as the mechanism of mucous cell secretion. We are studying the control of mucous and fluid secretion in a gastropod mollusc, Agriolimax columbianus, and have shown that it is an excellent model for the study of mucous secretions because of the copiousness and precise control of these secretions and the fact that they are released onto an exposed body surface, rather than within body cavities. Our observations of chemically-fixed material (glutaraldehye followed by osmium) showed “omega figures” typical of mucous vesicle exocytosis. On the other hand, in vivo observations showed that membrane-bound vesicles were shed intact from mucous cells.2 As an alternative to chemical fixation, we used a physical method of fixation, ultrarapid freezing, to determine more accurately the ultrastructural basis of mucous cell secretion.Intact body wall of the terrestrial slug, A. columbianus, was frozen with a Cryopress™ (Med-Vac Inc., St. Louis, MO) by slamming the tissue against the face of a polished copper block cooled in liquid N2 The frozen tissue was then freeze-substituted in cold (-80°C) osmium-containing acetone for several days, then brought to room temperature and embedded in Epon. Sections for EM were observed in a JEOL-100S TEM.