Dynamic subcellular localization of DydA in Dictyostelium cells.

DydA plays an important role in chemotaxis, development, and cell growth as an adaptor protein that connects Ras signaling and cytoskeletal rearrangement. DydA is a downstream effector of RasG and is involved in controlling cell polarity and pseudopodia formation during chemoattractant-directed cell migration. To understand the mechanism by which DydA functions on the cell migration, we investigated the dynamic subcellular localization of DydA in response to chemoattractant stimulation and found that DydA rapidly and transiently translocated to the cell cortex through the RA domain and the PRM region in DydA in response to chemoattractant stimulation. The PRM region appears to play a primary role in the translocation of DydA to the cell cortex and in its localization to the actin foci at the bottom of cells. Colocalization experiments of GFP-PRM with RFP-coronin indicated that GFP-PRM preceded GFP-coronin by 2-3 s in response to chemoattractant stimulation. These results suggest that the PRM region plays an indispensable role in relaying upstream regulators, such as RasG, to downstream effectors by mediating the localization of DydA to the cell cortex upon chemoattractant stimulation.

Phosphatidylinositol 3-kinases play a suppressive role in cell motility of vegetative Dictyostelium cells.

Phosphatidylinositol 3-Kinase (PI3K) is a key regulator of cell motility during chemotaxis and plays an important role in relaying and amplifying the shallow gradient of chemoattractant signals to ultimately mediate rearrangements of the actin cytoskeleton. To determine whether PI3K plays a similar role in electrotaxis as in chemotaxis, we examined directional cell migration in response to an electric field (EF) and unexpectedly found that the role of PI3K in regulating cell motility differs depending on the state of Dictyostelium cells. Contrary to chemotaxis experiments using aggregation-competent cells, in the cell migration assay, we used a recently developed method for electrotaxis using 3-h starved cells. Wild-type cells starved for 3 h showed increased motility in the presence of LY294002, a PI3K inhibitor, whereas aggregation-competent cells showed slightly decreased motility, indicating the effect of LY294002 on cell motility differ depending on the state of the cells. Consistent with these results, pi3k null cells in the vegetative state exhibited increased motility similar to that in the presence of LY294002, compared to wild-type cells. These findings were confirmed through random migration experiments. These results suggest that PI3Ks play a suppressive role in regulating cell motility of vegetative Dictyostelium cells and that the suppressive effect is reversed on inhibition or lack of PI3Ks, leading to high motility.

Reversible function of RapA with the C-terminus of RapC in Dictyostelium.

Rap small GTPases are involved in diverse signaling pathways associated with cell growth, proliferation, and cell migration. There are three Rap proteins in Dictyostelium, RapA, RapB, and RapC. RapA is a key regulator in the control of cell adhesion and migration. Recently RapA and RapC have been reported to have opposite functions in the regulation of cellular processes. In this study, we demonstrate that the C-terminus of RapC, which is not found in RapA, is essential for the opposite functions of RapC and is able to reverse the functions of RapA when fused to the tail of RapA. Cells lacking RapC displayed several defective phenotypes, including spread morphology, strong adhesion, and decreased cell migration compared to wild-type cells. These phenotypes were rescued by full-length RapC, but not by RapC missing the C-terminus. Furthermore, recombinant RapA fused with the C-terminus of RapC completely recovered the phenotypes of rapC null cells, indicating that the functions of RapA were modified to become similar to those of RapC by the C-terminus of RapC with respect to cell morphology, cell adhesion and migration, cytokinesis, and development. These results suggest that the C-terminal residues of RapC are able to suppress and change the functions of other Ras proteins in Ras oncogenic signaling pathways.