Autophagy in the epithelial cells of murine seminal vesicle in vitro - Formation of large sheets of nascent isolation membranes, sequestration of thenucleus and inhibition by wortmannin and 3-methyladenine

The spontaneous autophagic activity in epithelial cells of isolated tissue slices of murine seminal vesicle is strongly enhanced by short (5 min) pret reatment in a medium containing 0.03% Triton X-100. In addition to the sign ificant increase in the cytoplasmic volume fraction and the mean size of au tophagic vacuoles, the appearance of shorter or longer smooth membrane pair s located between cisterns of rough endoplasmic reticulum (RER) and in the vicinity of nucleus is also greatly stimulated. Their morphological feature s observed after application of various fixation methods, freeze-substituti on and freeze-fracture techniques show that they are unclosed nascent isola tion membranes, representing a unique class of intracellular membranes. The y may grow around the nucleus, leading to its complete autophagic sequestra tion and degradation, which is observed here for the first time. Treatment with 3-methyladenine or wortmannin inhibits the formation of autophagosomes , leading to their regression with a halving time of 7 min. In contrast, th ese inhibitors cause extremely fast shrinking of nascent isolating membrane s, leading to their complete disappearance within 7 min. We propose chat th e early events of autophagy involve three main steps: initiation, growth an d closure, and suggest that the growth of nascent isolation membranes is re versible i.e. the membranes may be subject to disassembly before their clos ure is completed. Bending and closure of the isolation membrane and the sta bility of neighbouring cellular structures appear as important determinants of size regulation. These early steps of autophagy are good candidates for very fast accommodation to changing conditions and subtle regulation by ph osphoinositide kinases as indicated by wortmannin sensitivity.