A ROLE FOR PHOSPHOINOSITIDE 3-KINASE IN THE COMPLETION OF MACROPINOCYTOSIS AND PHAGOCYTOSIS BY MACROPHAGES

Phosphoinositide 3-kinase (PI3-kinase) has been implicated in growth f actor signal transduction and vesicular membrane traffic. It is though t to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibi tors of PI3-kinase inhibit endocytosis in macrophages, not by interfer ing with the initiation of the process but rather by preventing its co mpletion. Consistent with earlier studies, the inhibitors wortmannin a nd LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had Little effect on the receptor-mediated end ocytosis of diI-labeled, acetylated, low density lipoprotein. Large so lute probes of endocytosis reported greater inhibition by wortmannin t han smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis ar e actin-mediated processes, we expected that their inhibition by wortm annin resulted from deficient signaling from macrophage colony-stimula ting factor (M-CSF) receptors or Fc receptors to the actin cytoskeleto n. However, video microscopy showed cell surface ruffling in wortmanni n-treated cells, and increased ruffling after addition of M-CSF or pho rbol myristate acetate. Quantitative measurements of video data report ed slightly diminished ruffling in wortmannin-treated cells. Remarkabl y, the ruffles that formed in wortmannin-treated macrophages all reced ed into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich ps eudopodia along IgG-opsonized sheep erythrocytes, but instead prevente d them from closing into phagosomes. These findings indicate that PI3- kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.