Mutant Rac1B expression in Dictyostelium: Effects on morphology, growth, endocytosis, development, and the actin cytoskeleton

Rad is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rad during motile processes, we have established D ictyostelium cell lines that conditionally overexpress epitope-tagged Dicty ostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expres sion of a constitutively active mutant (G12-->V or Q61-->L) or a dominant n egative mutant (T17-->N) generated amoebae with characteristic cellular def ects. The morphological appearance of actin-containing structures, intracel lular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhib ited cell growth and cytokinesis, and delayed multicellular development, su ggesting that DdRac1B plays important regulatory role(s) during these proce sses. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and th e longest developmental delays; DdRac1B-Q61L produced the strongest cytokin esis defect; and DdRac1B-T17N generated intermediate phenotypes. These cond itionally expressed DdRac1B proteins should facilitate the identification a nd characterization of the Rad signaling pathway in an organism that is ame nable to both biochemical and molecular genetic manipulations. Cell Motil. Cytoskeleton 46:285-304, 2000. (C) 2000 WiIey-Liss, Inc.