MONODANSYLCADAVERINE (MDC) IS A SPECIFIC IN-VIVO MARKER FOR AUTOPHAGIC VACUOLES

We report the use of the autofluorescent compound monodansylcadaverine (MDC) for in vivo labeling of autophagic vacuoles. When applied to va rious cell types (PaTu 8902, MDCK I, PC12, AR4-2J, WI-38) in culture, spherical structures were observed by fluorescence microscopy, predomi nantly located in the perinuclear region. Only PC12 and WI-38 cells ha d some of these labeled structures in their filopodiae. Dose-response experiments with PaTu 8902 showed that the optimal concentration for i n vivo labeling was 0.05 to 0.1 mM, while cells detached and disintegr ated, when MDC concentration exceeded 0.1 mM. After incubation with MD C and subcellular fractionations of PaTu 8902 cells on sucrose density gradients, a narrow fluorescence peak at 20 to 26% sucrose concentrat ion equal to densities of about 1.081 to 1.108 g/cm(3) was observed. U ltrastructural analysis of these fractions revealed autophagic vacuole s in different stages of their development. To investigate whether end osomal compartments were also labeled by MDC, we coincubated PaTu 8902 cells with MDC and the fluid-phase markers, RITC-dextran and ferritin , respectively. Fluorescence measurements after subcellular fractionat ions as well as fine structural analysis indicated that MDC-labeled au tophagic vacuoles did not contain fluid-phase markers and were spatial ly separated from endosomal compartments. We further could demonstrate , after subcellular fractionation procedures, that MDC-labeled organel les contained the lysosomal enzymes acid phosphatase and the mature fo rm of cathepsin D. Membrane markers of rough endoplasmic reticulum (TR AM and sec61 beta), and for smooth endoplasmic reticulum (cytochrome P -450) were not detected in the same fractions. These results indicate that MDC accumulates as a selective fluorescent marker for autophagic vacuoles under in vivo conditions and is not present in the early and late endosome.