Fluorescence Visualization of Receptor-Bound Low Density Lipoprotein in Human Fibroblasts

Abstract

Previous electron microscopic studies have shown that receptors for plasma low density lipoprotein (LDL) in human fibroblasts are located predominantly in coated pits on the cell surface. In the current studies, LDL receptors were visualized at the light microscopic level by two methods: 1) indirect immuno-fluorescent staining of receptor-bound LDL, and 2) direct visualization of LDL particles that were reconstituted with a fluorescent cholesteryl ester derivative. By both techniques, LDL receptors were seen to be clustered in minute foci that were aligned in linear arrays on the cell surface. These arrays corresponded to linear arrays of coated pits that were visualized by immunofluorescent staining of the coat protein, clathrin. During the internalization of receptor-bound LDL, each fluorescent focus of LDL particles seemed to enter the cell as an individual unit without the formation of large patches. Immediately after the reconstituted fluorescent LDL entered the cell, the fluorescent dots became larger in size and fewer in number, suggesting rapid fusion of endocytic vesicles. After internalization of LDL, new unoccupied receptors rapidly appeared on the cell surface where they were again distributed in linear arrays. In fibroblasts from a patient with the LDL internalization defect (J.D.), LDL receptors fail to be incorporated in coated pits. By immuno-fluorescence, LDL that was bound to these mutant receptors produced fluorescent foci that tended to be smaller and more numerous than those of normal fibroblasts. Moreover, the fluorescent foci of LDL