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© The Rockefeller University Press, 0021-9525/1997//1271 $5.00
The Journal of Cell Biology, Volume 136, Number 6, , 1997 1271-1286

Article

Inactivation of Two 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



Greg Buczynski*, Bryon Grove§, Anson Nomura**, Maurice Kleve||,¶, John Bush||, Richard A. Firtel**, and James Cardelli*,{ddagger}

* Department of Microbiology and Immunology, {ddagger} 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

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 ({Delta}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, {Delta}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 {alpha}-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 {Delta}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, {Delta}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.

Abbreviations used in this paper: CHC, clathrin heavy chain; CV, contractile vacuole; FD, FITC-dextran; PI, phosphatidylinositide.

This research was supported by a National Institutes of Health (NIH) grant DK 39232-05 (J. Cardelli) and a National Science Foundation grant MCB-9507494 (J. Cardelli). Dr. Buczynski was supported by an NIH postdoctoral fellowship F32 GM17073. We also acknowledge support from the Louisiana State University MC Center for Excellence in Cancer Research and the Center for Excellence in Arthritis and Rheumatology. Dr. Bush was supported by a grant from the Arkansas Science and Technology Authority.

Please address all correspondence to J. Cardelli, Department of Microbiology and Immunology, Louisiana State University Medical Center, 1501 Kings Highway/P.O. Box 33932, Shreveport, LA 71130-3932. Tel.: (318) 675-5756. Fax: (318) 675-5764. E-Mail: jcarde{at}nomvs.lsumc.edu

This paper is dedicated to the memory of Jason A. Cardelli, Ph.D., Assistant Professor of Astronomy.


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