Many biologically important molecules have been shown to enter cells by receptor-mediated endocytosis. Among these are molecules which stimulate cell growth in vitro, such as insulin and epidermal growth factor. Acidification of endocytic vesicles has been shown to play a crucial role in the entry into the cytoplasm of many toxins and viruses, and since many internal binding sites for growth factors have been demonstrated, it is possible that a similar process is involved in growth factor action. This possibility will be studied by a combination of flow cytometry and cell sorting. The pathways followed after receptor-mediated endocytosis of growth factors by cultured mouse fibroblasts will be investigated using fluorescent analogues which are sensitive to their environment. The kinetics of acidification of endocytic vesicles containing molecules endocytosed specifically or non-specifically will be determined by dual fluorescence flow cytometry with a mixture of fluorescein (pH sensitive) and rhodamine (pH insensitive) analogues. The frequency with which different molecules are contained in the same endocytic vesicle will be determined by fluorescence energy transfer. These experiments will help resolve current controversies regarding differential processing of endocytosed molecules. The ability of a variety of lysomotropic agents to affect growth stimulation by insulin and epidermal growth factor will be determined. An important goal will be to detemine which effects of these agents (neutralization, vacuolization, inhibition of vesicle fusion) cause inhibition of growth stimulation. The point of action of these agents in the cell cycle will be determined by simultaneous flow cytometric measurement of DNA, RNA and protein content. To directly examine the role o acidification in growth factor action, spontaneous and temperature-sensitive mutants which are unable to acidify endocytic vesicles containing specific probes will be isolated from serum-requiring cells by flow sorting. These cell lines will be tested fo their ability to be stimulated by a variety of growth factors. In addition to their usefulness for studies of growth factor action, those cell lines which have defects in lysosomal acidification should be particularly valuable as recipients for nucleic acids and proteins.
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