Autophagy of metabolically inert substances injected into fibroblasts in culture

We have examined the morphologic characteristics of fibroblasts cultured from the beige mouse, a genetic variant phenotypically similar to human Chediak-Higashi syndrome (CHS). These cultured fibroblasts are characterized by large, amorphous dense body inclusions in their cytoplasm, often as large as the cell nucleus. Using time-lapse video phase-contrast microscopy, we have observed the formation of these large dense bodies through fusion of relatively normal-appearing lysosomes in the beige mouse fibroblast. After formation of these large inclusions, cells occasionally extruded the contents of these structures through apparent fusion with the plasma membrane and rapid exocytosis. Fibroblasts cultured from normal black mice showed no evidence of fusion between lysosomes or exocytosis of lysosomes. However, the uptake of extracellular medium through macropinocytosis, subsequent actions of lysosomes on these macropinosomes through saltatory motion, cellular migration and ruffling activity appeared normal in beige mouse fibroblasts. Immunocytochemical localization of α₂-macroglobulin, a normal serum protein commonly incorporated into lysosomes in cultured fibroblasts by receptor-mediated endocytosis, showed that the large dense bodies contained α₂-macroglobulin, in keeping with their lysosomal origin. This suggested further that receptor-mediated endocytosis in these cells was relatively normal. In addition, light and electron microscopic cytochemistry showed these large inclusions to be acid-phosphatase positive, further characterizing them as lysosomal. The electron microscopic appearance of these dense inclusions was consistent with their origin through repeated fusion of lysosomes. It is suggested that a primary defect in this disease may be the ability of mature lysosomal membranes to fuse, unlike normal lysosomal membranes, indicating perhaps an alteration in some specific component of the lysosomal membranes in CHS.