Taeck J. Jeon

Cell migration: regulation of cytoskeleton by Rap1 in Dictyostelium discoideum.

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.

Regulation of Actin Cytoskeleton by Rap1 Binding to RacGEF1

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.

Chemoattractant-mediated Rap1 activation requires GPCR/G proteins

Rap1 is rapidly activated upon chemoattractant stimulation and plays an important role in cell adhesion and cytoskeletal reorganization during chemotaxis. Here, we demonstrate that G-protein coupled receptors and G-proteins are essential for chemoattractant-mediated Rap1 activation in Dictyostelium. The rapid Rap1 activation upon cAMP chemoattractant stimulation was absent in cells lacking chemoattractant cAMP receptors cAR1/cAR3 or a subunit of the heterotrimeric G-protein complex Gα2. Loss of guanylyl cyclases GCA/SGC or a cGMP-binding protein GbpC exhibited no effect on Rap1 activation kinetics. These results suggest that Rap1, a key regulator for the regulation of cytoskeletal reorganization during cell movement, is activated through the G-protein coupled receptors cAR1/cAR3 and Gα2 proteins in a way independent of the cGMP signaling pathway.

Dynamic Localization of the Actin-Bundling Protein Cortexillin I during Cell Migration

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 in-creased 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-tantmediated 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.

Effects of fucoidan on cell morphology and migration in osteoblasts

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.

DNA adenine methylation of sams1 gene in symbiont-bearing Amoeba proteus

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.

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.