INACTIVATION OF 2 DICTYOSTELIUM-DISCOIDEUM GENES, DDPIK1 AND DDPIK2, ENCODING PROTEINS RELATED TO MAMMALIAN PHOSPHATIDYLINOSITIDE 3-KINASES, RESULTS IN DEFECTS IN ENDOCYTOSIS, LYSOSOME TO POSTLYSOSOME TRANSPORT, AND ACTIN CYTOSKELETON ORGANIZATION

Phosphatidylinositide 3-kinases (PI 3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles , There are at least three genes in Dictyostelium discoideum, DnPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mamm alian 110-kD PI-3 kinase in amino acid sequence within the kinase doma in. A mutant disrupted in DnPIK1 and DdPIK2 (Delta ddpik1/ddpik2) grow s slowly in liquid medium, Using FITC-dextran (FD) as a fluid phase ma rker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and bioc hemical approaches indicated that the transport rate of fluid-phase fr om acidic lysosomes to non-acidic postlysosomal vacuoles was reduced i n mutant cells resulting in a reduction in efflux of fluid phase, Muta nt cells were also almost completely devoid of large postlysosomal vac uoles as determined by transmission EM, However, Delta ddpik1/ddpik2 c ells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the tr ansport rates along the secretory pathway and the sorting efficiency o f the lysosomal enzyme alpha-mannosidase were normal in the mutant str ain. Furthermore, the contractile vacuole network of membranes (probab ly connected to the endosomal pathway by membrane traffic) was functio nally and morphologically normal in mutant cells. Light microscopy rev ealed that Delta ddpik1/ddpik2 cells appeared smaller and more irregul arly shaped than wild-type cells; 1-3 % of the mutant cells were also connected by a thin cytoplasmic bridge, Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and ve rtically from the cell surface, and fluorescent microscopy indicated t hat these filopodia were enriched in F-actin which accumulated in a co rtical pattern in control cells. Finally, Delta ddpik1/ddpik2 cells re sponded and moved more rapidly towards cAMP. Together, these results s uggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate mul tiple steps in the endosomal pathway, and function in the regulation o f cell shape and movement perhaps through changes in actin organizatio n.