RapB Regulates Cell Adhesion and Migration in Dictyostelium, Similar to RapA.

Ras small GTPases act as molecular switches in various cellular signaling pathways, including cell migration, proliferation, and differentiation. Three Rap proteins are present in Dictyostelium; RapA, RapB, and RapC. RapA and RapC have been reported to have opposing functions in the control of cell adhesion and migration. Here, we investigated the role of RapB, a member of the Ras GTPase subfamily in Dictyostelium, focusing on its involvement in cell adhesion, migration, and developmental processes. This study revealed that RapB, similar to RapA, played a crucial role in regulating cell morphology, adhesion, and migration. rapB null cells, which were generated by CRISPR/Cas9 gene editing, displayed altered cell size, reduced cell-substrate adhesion, and increased migration speed during chemotaxis. These phenotypes of rapB null cells were restored by the expression of RapB and RapA, but not RapC. Consistent with these results, RapB, similar to RapA, failed to rescue the phenotypes of rapC null cells, spread morphology, increased cell adhesion, and decreased migration speed during chemotaxis. Multicellular development of rapB null cells remained unaffected. These results suggest that RapB is involved in controlling cell morphology and cell adhesion. Importantly, RapB appears to play an inhibitory role in regulating the migration speed during chemotaxis, possibly by controlling cell-substrate adhesion, resembling the functions of RapA. These findings contribute to the understanding of the functional relationships among Ras subfamily proteins.

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.

Opposite functions of RapA and RapC in cell adhesion and migration in Dictyostelium.

There are three Rap proteins in Dictyostelium. RapA is a key regulator of cell adhesion and cytoskeletal rearrangement. Recently, RapC has been reported to be involved in cytokinesis, cell migration, and multicellular development. Here, we compare the functions of RapA and RapC using cells expressing or lacking Rap proteins, and confirm that RapA and RapC have opposite functions in cell spreading, adhesion, and migration. On the other hand, RapC has a unique function in cytokinesis and multicellular development. Activated RapA appears to stimulate spreading and adhesion of the cells to the substrate, possibly resulting in a decrease in the migration speed of the cells during chemotaxis without affecting the directionality, whereas RapC suppresses cell spreading and adhesion, thereby increasing the migration speed. Cells lacking RapC were defective in cytokinesis and multicellular development and showed multinucleation and formation of multiple tips from a mound during development. At the C-terminus, RapC has an additional stretch of amino acids, which is not found in RapA. The mechanism through which RapA and RapC perform their opposite functions in diverse cellular processes should be characterized further to understand the Rap signaling pathways in detail. ABBREVIATIONS: GAP; GTPase-activating proteins; GEF; guanine nucleotide exchanging factor; WT; wild type; CA; constitutively active; DN; dominantly negative.

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.

WasC, a WASP family protein, is involved in cell adhesion and migration through regulation of F-actin polymerization in Dictyostelium.

The actin cytoskeleton is involved in the regulation of cell morphology and migration. Wiskott-Aldrich Syndrome proteins (WASPs) play an important role in controlling actin polymerization by activating the Arp2/3 complex. The present study investigated the roles of WasC, one of the 3 WASPs in Dictyostelium, in cellular processes. Cells lacking WasC displayed strong cell adhesion and approximately 1.5-fold increase in F-actin levels as compared to the wild-type cells. Loss of wasC caused defects in phagocytosis and decreased the migration speed in chemoattractant-mediated cell migration but did not affect directionality. WasC was localized to the protruding region in migrating cells and, transiently and rapidly translocated to the cell cortex in response to chemoattractant stimulation, in an F-actin dependent manner. Our results suggest that WasC is involved in cell adhesion and migration by regulating F-actin polymerization at the leading edge of migrating cells, probably as a negative regulator. The increased strength of adhesion in wasC null cells is likely to decrease the migration speed but not the directionality.

Loss of RapC causes defects in cytokinesis, cell migration, and multicellular development of Dictyostelium.

The small GTPase Ras proteins are involved in diverse cellular processes. We investigated the functions of RapC, one of 15 Ras subfamily GTPases in Dictyostelium. Loss of RapC resulted in a spread shape of cells; severe defects in cytokinesis leading to multinucleation; decrease of migration speed in chemoattractant-mediated cell migration, likely through increased cell adhesion; and aberrations in multicellular development producing abnormal multiple tips from one mound and multi-branched developmental structures. Defects in cells lacking RapC were rescued by expressing GFP-RapC in rapC null cells. Our results demonstrate that RapC, despite its high sequence homology with Rap1, plays a negative role in cell spreading and cell adhesion, in contrast to Rap1, which is a key regulator of cell adhesion and cytoskeleton rearrangement. In addition, RapC appears to have a unique function in multicellular development and is involved in tip formation from mounds. This study contributes to the understanding of Ras-mediated cellular processes.

CBP7 Interferes with the Multicellular Development of Dictyostelium Cells by Inhibiting Chemoattractant-Mediated Cell Aggregation.

Calcium ions are involved in the regulation of diverse cellular processes. Fourteen genes encoding calcium binding proteins have been identified in Dictyostelium. CBP7, one of the 14 CBPs, is composed of 169 amino acids and contains four EF-hand motifs. Here, we investigated the roles of CBP7 in the development and cell migration of Dictyostelium cells and found that high levels of CBP7 exerted a negative effect on cells aggregation during development, possibly by inhibiting chemoattractant-directed cell migration. While cells lacking CBP7 exhibited normal development and chemotaxis similar that of wild-type cells, CBP7 overexpressing cells completely lost their chemotactic abilities to move toward increasing cAMP concentrations. This resulted in inhibition of cellular aggregation, a process required for forming multicellular organisms during development. Low levels of cytosolic free calcium were observed in CBP7 overexpressing cells, which was likely the underlying cause of their lack of chemotaxis. Our results demonstrate that CBP7 plays an important role in cell spreading and cell-substrate adhesion. cbp7 null cells showed decreased cell size and cell-substrate adhesion. The present study contributes to further understanding the role of calcium signaling in regulation of cell migration and development.

Loss of FrmB results in increased size of developmental structures during the multicellular development of Dictyostelium cells.

FERM domain-containing proteins are involved in diverse biological and pathological processes, including cell-substrate adhesion, cell-cell adhesion, multicellular development, and cancer metastasis. In this study, we determined the functions of FrmB, a FERM domain-containing protein, in the cell morphology, cell adhesion, and multicellular development of Dictyostelium cells. Our results show that FrmB appears to play an important role in regulating the size of developmental structures. frmB null cells showed prolonged aggregation during development, resulting in increased size of developmental structures, such as mounds and fruiting bodies, compared to those of wild-type cells, whereas FrmB overexpressing cells exhibited decreased size of developmental structures. These results suggest that FrmB may be necessary for limiting the sizes of developmental structures. Loss of FrmB also resulted in decreased cell-substrate adhesion and slightly increased cell area, suggesting that FrmB had important roles in the regulation of cell adhesion and cell morphology. These studies would contribute to our understanding of the intertwined and overlapped functions of FERM domain-containing proteins.

Minimal amino acids in the I/LWEQ domain required for anterior/posterior localization in Dictyostelium.

Establishment of cell polarity is mediated by a series of signaling molecules that are asymmetrically activated or localized in the cell upon extracellular stimulation. To understand the mechanism that mediates anterior/posterior asymmetric localization of RapGAP3 during migration, we determined the minimally required amino acids in the I/LWEQ domain that cause posterior localization and found that the minimal region of the F-actin binding domain for posterior localization could, with some additional deletion at the C-terminal, localize to the anterior. Analysis of the localization and translocation kinetics to the cell cortex of the truncated proteins suggests that the required regions for anterior/posterior localization might have a preferential binding affinity to preexisting F-actins at the rear and lateral sides of the cell or newly formed F-actins at the front of the cell, leading to distinct differential sites of the cell.