Chemotaxis-competent cells respond to a variety of ligands by activating second messenger pathways leading to changes in the actin/myosin cytoskeleton and directed cell movement. We demonstrate that Dictyostelium Akt/PKB, a homologue of mammalian Akt/ PKB, is very rapidly and transiently activated by the chemoattractant cAMP. This activation takes place through G protein-coupled chemoattractant receptors via a pathway that requires homologues of mammalian p110 phosphoinositide-3 kinase. pkbA null cells exhibit aggregation-stage defects that include aberrant chemotaxis, a failure to polarize properly in a chemoattractant gradient and aggregation at low densities. Mechanistically, we demonstrate that the PH domain of Akt/ PKB fused to GFP transiently translocates to the plasma membrane in response to cAMP with kinetics similar to those of Akt/PKB kinase activation and is localized to the leading edge of chemotaxing cells in vivo. Our results indicate Akt/PKB is part of the regulatory network required for sensing and responding to the chemoattractant gradient that mediates chemotaxis and aggregation.

Our results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during Dictyostelium multicellular development. The results suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB functions predominantly during aggregation to control cell polarity and chemotaxis, whereas PKBR-1 is required for morphogenesis during multicellular development.

We have used the chemotactic ability of Dictyostelium cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B 61L or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B 61L and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B 17N do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis.C hemotaxis, directed cell movement toward a chemoattractant agent, is involved in diverse biological responses, including wound healing in vertebrates, migration of tumor cells, metastasis of cancer cells, and aggregation leading to the formation of the multicellular organism in Dictyostelium (1-5). This process is activated by a large and diverse number of extracellular ligands that bind to cell surface receptors and leads to the directed reorganization of the actin and myosin cytoskeletons, pseudopod extension in the direction of the chemoattractant source, and cell movement via pathways that are thought to be highly conserved between mammals and Dictyostelium (3, 6-8).In polymorphonuclear leukocytes, macrophage, and Dictyostelium cells, chemotaxis can be mediated through G proteincoupled cell surface receptors, and in mammalian cells, the response is thought to function through G i via release of G␤␥ subunits (9-11). In Dictyostelium, the chemoattractants folic acid and cAMP (the chemoattractant that mediates aggregation) function through the folate receptor and cAMP receptor cAR1 via heterotrimeric G proteins containing the coupled G␣ subunits G␣4 and G␣2, respectively (3, 12, 13). In Dictyostelium, the second messenger cGMP, produced by receptor activation of guanylyl cyclase, plays an essential role in regulating changes in the actin and myosin cytoskeletons (14). Other components such as the Dictyostelium Akt͞PKB, Ras protein RasG, the Ras exchange factor AleA, a MAP kinase cascade, a novel Rasinteracting protein RIP3, phosphatidyl inositol-3 kinase, and PAKa, a homologue of mammalian PAK1, are involved in different aspects of the signaling pathway that leads to cell movement and chemotaxis (2,(15)(16)(17)(18)(19)(20). Transduction of the chemotactic signal to second messengers induces rearrangement of cytoskeletal components, ...

We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin-enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell-cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.