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* Department of Microbiology and Immunology, 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, DdPIK1, DdPIK2, and DdPIK3, encoding
proteins most closely related to the mammalian 110-kD
PI-3 kinase in amino acid sequence within the kinase
domain. A mutant disrupted in DdPIK1 and DdPIK2
( Center for Excellence in Cancer Research, and § Department of Cell Biology
and Anatomy, Louisiana State University Medical Center, Shreveport, Louisiana 71130; Department of Biology and ¶ Altheimer
Microscopy Laboratory, University of Arkansas at Little Rock, Little Rock, Arkansas 72204; ** Center for Molecular Genetics,
University of California, La Jolla, California 92093-0634
ddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria.
Microscopic and biochemical approaches indicated that
the transport rate of fluid-phase from acidic lysosomes
to non-acidic postlysosomal vacuoles was reduced in
mutant cells resulting in a reduction in efflux of fluid
phase. Mutant cells were also almost completely devoid
of large postlysosomal vacuoles as determined by transmission EM. However, ddpik1/ddpik2 cells functioned
normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme
-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway
by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed
that ddpik1/ddpik2 cells appeared smaller and more
irregularly 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
vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in
F-actin which accumulated in a cortical pattern in control cells. Finally, ddpik1/ddpik2 cells responded and
moved more rapidly towards cAMP. Together, these
results suggest that Dictyostelium DdPIK1 and DdPIK2
gene products regulate multiple steps in the endosomal
pathway, and function in the regulation of cell shape
and movement perhaps through changes in actin organization.
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