Ultrastructural studies on cellular autophagy : structure of limiting membranes and route of enzyme delivery

Abstract

In autophagy, a portion of cytoplasm is first segregated into a membrane-bound vacuole which then acquires lysosomal enzymes. The present study investigated: 1) the structure of autophagic-vacuole membranes in Ehrlich ascites cells and mouse hepatocytes; 2) the role of microtubules in autophagy in Ehrlich ascites cells; and 3) the route of enzyme delivery to autophagic vacuoles in cultured rat fibroblasts. The density of membrane particles in freeze-fracture replicas reflects the protein content of membranes. In hepatocytes, the membranes of early vacuoles were devoid of particles, whereas those in ascites cells contained particles. In both cell types, however, particle density was higher in later vacuoles. The membranes of nascent vacuoles were weakly labelled with filipin, which detects cholesterol, whereas those of the later vacuoles were heavily labelled. The increase in protein and cholesterol content may be associated with enzyme delivery, since the lysosomal membranes were rich in protein and cholesterol. The microtubule inhibitor vinblastine accumulated later autophagic vacuoles in Ehrlich ascites cells. Thus, microtubule disruption did not prevent the entry of lysosomal enzymes into nascent vacuoles. Since the rate of proteolysis did not increase, the accumulation must have been caused by retarded degradation in later vacuoles. In fibroblasts, mannose 6-phosphate receptor (MPR), which serves as a receptor for lysosomal enzymes, and cathepsin L, a lysosomal proteinase, were detected in early autophagic vacuoles. Inhibitors of MPR-mediated transport did not prevent cathepsin L delivery. Thus, the enzyme is not transported directly from the trans-Golgi. On the other hand, studies with endocytic markers showed that autophagic vacuoles fused with late endosomes. Acidification of the vacuoles occurred concomitantly with cathepsin L delivery, but the inhibition of acidification did not prevent the transport of cathepsin L. This suggests that in fibroblasts late endosomes are an important source of lysosomal enzymes and perhaps also proton pumps for autophagic vacuoles.