Activated Rap1A Induces Osteoblastic Differentiation and Cell Adhesion

김혜선,전택중 조선대학교 기초과학연구원 2016 조선자연과학논문집 Vol.9 No.3

Rap1 is a key regulator of cell adhesion and migration. Although increasing evidence indicates that the Rap1 signaling pathway is involved in the process of bone remodeling, the mechanism by which Rap1 regulates osteoblastic differentiation and cell adhesion remains unknown. Here, we investigated the morphological characteristics and osteoblastic differentiation of cells expressing constitutively activated form of Rap1A (Rap1ACA) or Rap1 GTPase activating protein Rap1GAP and found that activated Rap1 induces osteoblastic differentiation and cell adhesion as well as cell spreading. When osteoblastic differentiation was induced, Rap1ACA cells showed considerably higher levels of calcium deposits than the wild-type and Rap1GAP-overexpressing cells did. Rap1ACA cells showed increased spreading and size, as well as strong cell adhesion and significantly decreased growth rates. F-actin staining using phalloidin revealed several thin thread-like filopodia around the protrusions in Rap1ACA cells, which possibly contribute to the increased cell adhesion.

Cell Migration: Regulation of Cytoskeleton by Rap1 in Dictyostelium discoideum

이미래,전택중 한국미생물학회 2012 The journal of microbiology Vol.50 No.4

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.

세포 이동에서 PI3K 억제제인 LY294002의 효과

김원범,전택중 조선대학교 기초과학연구원 2022 조선자연과학논문집 Vol.15 No.3

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

전지현,김동주,전택중 한국통합생물학회 2021 Animal cells and systems Vol.25 No.4

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 Cterminus, 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.

The I/LWEQ Domain in RapGAP3 Required for Posterior Localization in Migrating Cells

이미래,김혜선,전택중 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.4

Cell migration requires a defined cell polarity which is formed by diverse cytoskeletal components differentially localized to the poles of cells to extracellular signals. RapGAP3 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 RapGAP3. 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 post-erior regions of the cell.

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

Injun Cha,전택중 한국분자세포생물학회 2011 Molecules and cells Vol.32 No.3

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 chemo-taxing Dictyostelium cells. Most of the cortexillin I was enriched on the lateral sides of moving cells. Upon che-moattractant stimulation, cortexillin I was rapidly re-leased from the cortex followed by a transient transloca-tion 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-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.

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

박병규,신동엽,전택중 한국분자세포생물학회 2018 Molecules and cells Vol.41 No.2

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 lack-ing 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.

Regulation of Actin Cytoskeleton by Rap1 Binding to RacGEF1

Hyemin Mun,전택중 한국분자세포생물학회 2012 Molecules and cells Vol.34 No.1

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.

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

김혜선,이미래,전택중 한국미생물학회 2017 The journal of microbiology Vol.55 No.9

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

김혜선,신동엽,전택중 한국미생물학회 2017 The journal of microbiology Vol.55 No.5

Establishment of cell polarity is mediated by a series of signalingmolecules that are asymmetrically activated or localizedin the cell upon extracellular stimulation. To understand themechanism that mediates anterior/posterior asymmetriclocalization of RapGAP3 during migration, we determinedthe minimally required amino acids in the I/LWEQ domainthat cause posterior localization and found that the minimalregion of the F-actin binding domain for posterior localizationcould, with some additional deletion at the C-terminal,localize to the anterior. Analysis of the localization and translocationkinetics to the cell cortex of the truncated proteinssuggests that the required regions for anterior/posterior localizationmight have a preferential binding affinity to preexistingF-actins at the rear and lateral sides of the cell ornewly formed F-actins at the front of the cell, leading to distinctdifferential sites of the cell.