Nearly all eukaryotic cells exhibit fluid phase pinocytosis, the cellular internalization of extracellular fluid in plasma membrane derived vesicles. While there is increasing evidence that this internalization, and the subsequent intracellular traffic of vesicles through cytoplasm, is important in the regulation of cellular differentiation and growth, the molecular and cellular mechanisms which control this process are not known. Earlier work has shown that in thioglycollate-elicited mouse peritoneal macrophages (TMO), only a small percentage of the great quantity of fluid entering by pinocytosis is sent to lysosomes. Treatment of cells with the tumor promoter phorbol myristate acetate (PMA) increases both pinocytic influx and flow of pinocytosed fluid to lysosomes. The induced pinocytosis is distinct from constitutive pinocytosis in that only the former can be inhibited by colchicine or by expansion of the lysosomal compartment. Pinocytosis in the transformed mouse macrophage cell line J774.2 is similar to the PMA-induced process of TMO. This suggests that a pinocytosis like the PMA-inducible process of TMO is part of constitutive pinocytosis in growing or transformed cells. The work proposed here will further dissect the PMA-inducible pinocytosis of TMO, and determine whether that distinct type of pinocytic flow is characteristic of growth or transformation. First, pinosomes of unstimulated and PMA-stimulated cells will be compared morphologically, then purified and compared biochemically. Second, the role of phosphorylation in the PMA-inducible pinocytosis will be determined. Plasma membrane phosphoproteins will be labeled in situ with 32PO4, enriched by precipitating with antibodies against macrophage plasma membrane, then compared after various treatments which alter pinocytosis (plus, minus PMA, plus, minus colchicine, plus, minus expansion of the lysosomal compartment). Third, the cytoplasmic factors governing vesicle traffic in macrophages will be determined by the development and characterization of an in vitro pinosome-lysosome fusion system. Fourth, the route of pinocytosed fluid out of lysosomes will be traced using percoll and the fluorescent dye lucifer yellow in density shift experiments. Finally, pinocytosis by growing and transformed chick embryo fibroblasts will be analyzed to determine whether the type of pinocytosis induced in TMO by PMA, that is, a pinocytosis inhibitable by colchicine by expansion of the lysosomal compartment, is characteristic of growth or transformation.
