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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.

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.

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.

Chemotaxis, which is G protein-coupled receptor (GPCR)-mediated directional cell migration, plays pivotal roles in diverse human diseases, including recruitment of leukocytes to inflammation sites and metastasis of cancer. It is still not fully understood how eukaryotes sense and chemotax in response to chemoattractants with an enormous concentration range. A genetically traceable model organism, Dictyostelium discoideum, is the best-studied organism for GPCR-mediated chemotaxis. Recently, we have shown that C2GAP1 controls G protein coupled receptor-mediated Ras adaptation and chemotaxis. Here, we investigated the molecular mechanism and the biological function of C2GAP1 membrane targeting for chemotaxis. We show that calcium and phospholipids on the plasma membrane play critical roles in membrane targeting of C2GAP1. Cells lacking C2GAP1 (c2gapA–) displayed an improved chemotaxis in response to chemoattractant gradients at subsensitive or low concentrations (1 μM). Taken together, our results demonstrate that the membrane targeting of C2GAP1 enables Dictyostelium to sense chemoattractant gradients at a higher concentration range. This mechanism is likely an evolutionarily conserved molecular mechanism of Ras regulation in the adaptation and chemotaxis of eukaryotes.

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.

Motile cells manifest increased migration speed and directionality in gradients of stimuli, including chemoattractants, electrical potential, and substratum stiffness. Here, we demonstrate that Dictyostelium cells move directionally in response to an electric field with specific acceleration/deceleration kinetics of directionality and migration speed. Detailed analyses of the migration kinetics suggest that migration speed and directionality are separately regulated by Gβ and RasG, respectively, in EF-directed cell migration. Cells lacking Gβ, which is essential for all chemotactic responses in Dictyostelium, showed EF-directed cell migration with the same increase in directionality in an EF as wild-type cells. However, these cells failed to show induction of the migration speed upon EF stimulation as much as wild-type cells. Loss of RasG, a key regulator of chemoattractant-directed cell migration, resulted in almost complete loss of directionality, but similar acceleration/deceleration kinetics of migration speed as wild-type cells. These results indicate that Gβ and RasG are required for the induction of migration speed and directionality, respectively, in response to an EF, suggesting separation of migration speed and directionality even with intact feedback loops between mechanical and signaling networks.

Fucoidan, a sulfated polysaccharide found in brown algae, possesses various biological activities including anti-inflammatory and anti-cancer effects. In this study, we investigated the effects of fucoidan on the cell growth and morphology of human osteosarcoma MG-63 cells and found that fucoidan induces cell aggregation and apoptosis in osteosarcoma cells when the cells are treated with fucoidan upon seeding before becoming stably attached to the plates. Typical characteristics of apoptotic cells such as chromatin condensation and nuclear fragmentation were observed in fucoidan-treated cells. The number of annexin V-stained cells was increased by fucoidan treatment in a dose-dependent manner. Consistent with these results, cell viability and growth rate were decreased and cell spreading was inhibited in the presence of fucoidan, leading to rounded morphological changes of the cells. Our results demonstrate that fucoidan treatment results in cell aggregation through F-actin accumulation at the rou...

Fucoidan has been reported to induce the differentiation and mineralization of osteoblastic cells. Here we investigated the effects of fucoidan on the morphology and migration of osteoblasts and found that the effects of fucoidan on the migration of osteoblasts vary depending on the conditions under which the cells are treated. A migration assay using cells treated with fucoidan before stable attachment on plates showed slightly increased migration of fucoidan-treated cells compared to control cells, whereas wound healing experiments using stably attached cells exhibited a slightly decreased migration. The fucoidan-treated cells became shrunk and rounded and showed significantly decreased spreading and adhesion compared to control cells. F-actin was highly accumulated at the rounded cell cortex by fucoidan treatment. These studies suggest that the slightly increased migration of cells in the presence of fucoidan might be correlated to the morphological changes and decreased adhesion.

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.

Recent reports have demonstrated that the importance of Rap1-specific GTPase-activating proteins (GAPs) in the spatial and temporal regulation of Rap1 activity during cell migration and development in Dictyostelium. Here, we identified another putative Rap1 GAP-domain containing protein, showing high sequence homologies with those of human Rap1GAP and Dictyotelium RapGAP3, by bioinformatic search. Loss of RapGAP9 resulted in some defects in morphogenesis and development in Dicytostelium. rapGAP9 null cells were more flattened and spread, and highly multinucleated. Compared to wild-type cells, cells lacking RapGAP9 exhibited increased levels of F-actin and more filopodia. These results suggest that RapGAP9 is involved in the regulation of cytoskeleton reorganization and cytokinesis. rapGAP9 null cells showed a small increase of cell-substratum attachment and slightly lower moving speed and directionality compared to wild-type cells. In addition, the loss of RapGAP9 resulted in an altered morphology of fruiting body with a shorter length of stalk and spore. Identification and characterization of RapGAP9 in this study will provide further insights into the molecular mechanism by which Rap1 regulates cytoskeleton reorganization and morphogenesis 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.

Rap1 is rapidly and transiently activated in response to chemoattractant stimulation and helps establish cell polarity by locally modulating cytoskeletons. Here, we investigated the mechanisms by which Rap1 controls actin cytoskeletal reorganization in Dictyostelium and found that Rap1 interacts with RacGEF1 in vitro and stimulates F-actin polymerization at the sites where Rap1 is activated upon chemoattractant stimulation. Live cell imaging using GFP-coronin, a reporter for F-actin, demonstrates that cells expressing constitutively active Rap1 (Rap1CA) exhibit a high level of F-actin uniformly distributed at the cortex including the posterior and lateral sides of the chemotaxing cell. Examination of the localization of a PH-domain containing PIP3 reporter, PhdA-GFP, and the activation of Akt/Pkb and other Ras proteins in Rap1CA cells reveals that activated Rap1 has no effect on the production of PIP3 or the activation of Akt/Pkb and Ras proteins in response to chemoattractant stimulation. Rac family proteins are crucial regulators in actin cytoskeletal reorganization. In vitro binding assay using truncated RacGEF1 proteins shows that Rap1 interacts with the DH domain of RacGEF1. Taken together, these results suggest that Rap1-mediated F-actin polymerization probably occurs through the Rac signaling pathway by directly binding to RacGEF1.

Cortexillins are actin-bundling proteins that play a critical role in regulating cell morphology and actin cytoskeleton reorganization in Dictyostelium. Here, we investigated dynamic subcellular localization of cortexillin I in chemotaxing Dictyostelium cells. Most of the cortexillin I was enriched on the lateral sides of moving cells. Upon chemoattractant stimulation, cortexillin I was rapidly released from the cortex followed by a transient translocation to the cell cortex with a peak at ~5 s and a subsequent decrease to basal levels, indicating that localization of cor-texillin I at the cortex in chemotaxing cells is controlled by two more signaling components, one for the initial delocalization from the cortex and another for the translocation to the cortex ~5 s after chemoattractant stimulation. Loss of cortexillins leads to reduced cell polarity and an increased number of lateral pseudopodia during chemotaxis, suggesting that cortexillins play an inhibitory role in producing pseudopodia along the lateral sides of the cell. Cells lacking cortexillins displayed extended chemoattrac-tant-mediated Arp2/3 complex translocation kinetics to the cortex. Our present study provides a new insight into the function of cortexillins during reorganization of the actin cytoskeleton and cell migration.

The expression of amoeba sams genes is switched from sams1 to sams2 when amoebae are infected with Legionella jeonii. To elucidate the mechanism for the inactivation of host sams1 gene by endosymbiotic bacteria, methylation states of the sams1 gene of D and xD amoebae was compared in this study. The sams1 gene of amoebae was methylated at an internal adenine residue of GATC site in symbiont-bearing xD amoebae but not in symbiont-free D amoebae, suggesting that the modification might have caused the inactivation of sams1 in xD amoebae. The sams1 gene of xD amoebae was inactivated at the transcriptional level. Analysis of DNA showed that adenine residues in L. jeonii sams were also methylated, implying that L. jeonii bacteria belong to a Dam methylase-positive strain. In addition, both SAM and Met appeared to act as negative regulators for the expression of sams1 whereas the expression of sams2 was not affected in amoebae.

Spatial and temporal regulation of Rap1 is required for proper myosin assembly and cell adhesion during cell migration in Dictyostelium discoideum. Here, we identify a Rap1 guanosine triphosphatase–activating protein (GAP; RapGAP1) that helps mediate cell adhesion by negatively regulating Rap1 at the leading edge. Defects in spatial regulation of the cell attachment at the leading edge in rapGAP1− (null) cells or cells overexpressing RapGAP1 (RapGAP1OE) lead to defective chemotaxis. rapGAP1− cells have extended chemoattractant-mediated Rap1 activation kinetics and decreased MyoII assembly, whereas RapGAP1OE cells show reciprocal phenotypes. We see that RapGAP1 translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of chemotaxing cells via an F-actin–dependent pathway. RapGAP1 localization is negatively regulated by Ctx, an F-actin bundling protein that functions during cytokinesis. Loss of Ctx leads to constitutive and uniform RapGAP1 cortical localization. We suggest that RapGAP1 functions in the spatial and temporal regulation of attachment sites through MyoII assembly via regulation of Rap1–guanosine triphosphate.

We have investigated the role of Rap1 in controlling chemotaxis and cell adhesion in Dictyostelium discoideum. Rap1 is activated rapidly in response to chemoattractant stimulation, and activated Rap1 is preferentially found at the leading edge of chemotaxing cells. Cells expressing constitutively active Rap1 are highly adhesive and exhibit strong chemotaxis defects, which are partially caused by an inability to spatially and temporally regulate myosin assembly and disassembly. We demonstrate that the kinase Phg2, a putative Rap1 effector, colocalizes with Rap1–guanosine triphosphate at the leading edge and is required in an in vitro assay for myosin II phosphorylation, which disassembles myosin II and facilitates filamentous actin–mediated leading edge protrusion. We suggest that Rap1/Phg2 plays a role in controlling leading edge myosin II disassembly while passively allowing myosin II assembly along the lateral sides and posterior of the cell.

The expression of genes for S-adenosylmethionine synthetase (SAMS), which catalyzes the synthesis of S-adenosylmethionine (AdoMet), a major methyl donor in cells, was studied in symbiont-free (D) and symbiont-bearing (xD) amoeba strains to determine the effect of bacterial endosymbionts. The symbionts suppressed the expression of the gene in host xD amoebae, but amoebae still exhibited about half the enzyme activity found in symbiont-free D amoebae. The study was aimed at elucidating mechanisms of the suppression of the amoeba's gene and determining the alternative source for the gene product. Unexpectedly, we found a second sams (sams2) gene in amoebae, which encoded 390 amino acids. Results of experiments measuring SAMS activities and amounts of AdoMet in D and xD amoebae showed that the half SAMS activity found in xD amoebae came from the amoeba's SAMS2 and not from their endosymbionts. The expression of amoeba sams genes was switched from sams1 to sams2 as a result of infection with X-bacteria, raising the possibility that the switch in the expression of sams genes by bacteria plays a role in the development of symbiosis and the host-pathogen interactions. This is the first report showing such a switch in the expression of host sams genes by infecting bacteria.

Cell migration requires a defined cell polarity which is formed by diverse cytoskeletal components differentially localized to the poles of cells to extracellular signals. Rap- GAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of migrating cells. Here, we examined localization of truncated RapGAP3 proteins and found that the I/LWEQ domain in the central region of RapGAP3 was sufficient for posterior localization in migrating cells, as opposed to leading-edge localization of full-length Rap- GAP3. All truncated proteins accumulated at the leading edge of migrating cells exhibited clear translocation to the cell cortex in response to stimulation, whereas proteins localized to the posterior in migrating cells displayed no translocation to the cortex. The I/LWEQ domain appears to passively accumulate at the posterior region in migrating cells due to exclusion from the extended front region in response to chemoattractant stimulation rather than actively being localized to the back of cells. Our results suggest that posterior localization of the I/LWEQ domain of RapGAP3 is likely related to F-actin, which has probably different properties compared to newly formed F-actin at the leading edge of migrating cells, at the lateral and posterior regions of the cell.

Daydreamer (DydA), a new Mig10/RIAM/lamellipodin family adaptor protein, is a Ras effector required for cell polarization and directional movement during chemotaxis. DydA is phosphorylated by glycogen synthase kinase-3, which is required for some, but not all, of DydA's functions. gskA− cells exhibit very strong chemotactic phenotypes, a subset of which are exhibited by dydA− cells., How independent signaling pathways are integrated to holistically control a biological process is not well understood. We have identified Daydreamer (DydA), a new member of the Mig10/RIAM/lamellipodin (MRL) family of adaptor proteins that localizes to the leading edge of the cell. DydA is a putative Ras effector that is required for cell polarization and directional movement during chemotaxis. dydA− cells exhibit elevated F-actin and assembled myosin II (MyoII), increased and extended phosphoinositide-3-kinase (PI3K) activity, and extended phosphorylation of the activation loop of PKB and PKBR1, suggesting that DydA is involved in the negative regulation of these pathways. DydA is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is required for some, but not all, of DydA's functions, including the proper regulation of PKB and PKBR1 and MyoII assembly. gskA− cells exhibit very strong chemotactic phenotypes, as previously described, but exhibit an increased rate of random motility. gskA− cells have a reduced MyoII response and a reduced level of phosphatidylinositol (3,4,5)-triphosphate production, but a highly extended recruitment of PI3K to the plasma membrane and highly extended kinetics of PKB and PKBR1 activation. Our results demonstrate that GSK-3 function is essential for chemotaxis, regulating multiple substrates, and that one of these effectors, DydA, plays a key function in the dynamic regulation of chemotaxis.

Cell movement involves a coordinated regulation of the cytoskeleton, F-actin-mediated protrusions at the front and myosin-mediated contraction of the posterior of a cell. The small GTPase Rap1 functions as a key regulator in the spatial and temporal control of cytoskeleton reorganization for cell migration. This review outlines the establishment of cell polarity by differential localizations of the cytoskeleton and discusses the spatial and temporal regulation of cytoskeleton reorganization via the Rap1 signaling pathway during chemotaxis with a focus on recent advances in the study of chemotaxis using a simple eukaryotic model organism, Dictyostelium discoideum.

As a result of harboring obligatory bacterial endosymbionts, the xD strain of Amoeba proteus no longer produces its own S-adenosylmethionine synthetase (SAMS). When symbiont-free D amoebae are infected with symbionts (X-bacteria), the amount of amoeba SAMS decreases to a negligible level within four weeks, but about 47% of the SAMS activity, which apparently comes from another source, is still detected. Complete nucleotide sequences of sams genes of D and xD amoebae are presented and show that there are no differences between the two. Long-established xD amoebae contain an intact sams gene and thus the loss of xD amoeba's SAMS is not due to the loss of the gene itself. The open reading frame of the amoeba's sams gene has 1,281 nucleotides, encoding SAMS of 426 amino acids with a mass of 48 kDa and pI of 6.5. The amino acid sequence of amoeba SAMS is longer than the SAMS of other organisms by having an extra internal stretch of 28 amino acids. The 5'-flanking region of amoeba sams contains consensus-binding sites for several transcription factors that are related to the regulation of sams genes in E. coli and yeast. The complete nucleotide sequence of the symbiont's sams gene is also presented. The open reading frame of X-bacteria sams is 1,146 nucleotides long, encoding SAMS of 381 amino acids with a mass of 41 kDa and pI of 6.0. The X-bacteria SAMS has 45% sequence identity with that of A. proteus.

Rap1 is a key regulator of cell adhesion and cell motility in Dictyostelium. Here, we identify a Rap1-specific GAP protein (RapGAP3) and provide evidence that Rap1 signaling regulates cell–cell adhesion and cell migration within the multicellular organism. RapGAP3 mediates the deactivation of Rap1 at the late mound stage of development and plays an important role in regulating cell sorting during apical tip formation, when the anterior–posterior axis of the organism is formed, by controlling cell–cell adhesion and cell migration. The loss of RapGAP3 results in a severely altered morphogenesis of the multicellular organism at the late mound stage. Direct measurement of cell motility within the mound shows that rapGAP3− cells have a reduced speed of movement and, compared to wild-type cells, have a reduced motility towards the apex. rapGAP3− cells exhibit some increased EDTA/EGTA sensitive cell–cell adhesion at the late mound stage. RapGAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation, which is dependent on F-actin polymerization. We suggest that the altered morphogenesis and the cell-sorting defect of rapGAP3− cells may result in reduced directional movement of the mutant cells to the apex of the mound.