Cellular functions & pharmacological manipulations of the small GTPase Rho & Rho effectors

e13011 Background: Glioblastoma is the grade 4 astrocytoma which is notorious for its highly invasive phenotype, very low survival rate and generally poor responses to conventional therapies. Glioblastoma stem-like cells (GSC), which are usually more resistant to therapeutic treatment, may account for the poor prognosis of this disease. Rac (Ras-related C3 botulinum toxin substrate) is a subfamily of Rho small GTPase which function is regulation of actin cytoskeleton rearrangement. While Rac1 is expressed ubiquitous in different tissues and cells, Rac2 is highly expressed in the mesenchymal subtype of glioblastoma according to the TCGA (the Cancer Genome Atlas) database, and Rac3 is mainly expressed in the brain. Methods: We used Rac proteins overexpressing-glioblastoma cellines derived GSC and Rac proteins specific siRNA harboring-GSC to perform colony formation assay and migration assay. Results: Here we report that Rac proteins overexpressing glioblastoma stem-like cells derived from glioblastoma cell lines have higher proliferation rate and stronger responses to LPA-induced cell migration. Knocking-down their expression by specific siRNA reduces the proliferation and migration of these cells. Instead of Rac1, Rac2 and Rac3 are more effective on promoting proliferation and migration of glioblastoma stem-like cells. Moreover, Rac proteins promote glioblastoma progression is associated with activation of JAK-STAT and ERK pathway. Conclusions: Although Rac1 is the most studied one in the Rac family, and has also been implicated in the progression of different cancers, however, it is homogeneously expressed in all different tissues, and plays important roles in normal cellular functions involving cell movement, such as wound healing, make it not a good candidate for specific drug targeting. According to our results, Rac2 or Rac3 serve as a better potential therapeutic targets for glioblastoma treatment.

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Rho and Anillin-dependent Control of mDia2 Localization and Function in Cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e10-04-0324 ◽

2010 ◽

Vol 21 (18) ◽

pp. 3193-3204 ◽

Cited By ~ 62

Author(s):

Sadanori Watanabe ◽

Katsuya Okawa ◽

Takashi Miki ◽

Satoko Sakamoto ◽

Tomoko Morinaga ◽

...

Keyword(s):

Rna Interference ◽

Rho Gtpase ◽

Small Gtpase ◽

Cleavage Furrow ◽

3T3 Cells ◽

Actin Nucleation ◽

Inhibitory Domain ◽

And Function ◽

Rho Effectors ◽

Nih 3T3

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.

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Rho Kinase Regulates the Intracellular Micromechanical Response of Adherent Cells to Rho Activation

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0218 ◽

2004 ◽

Vol 15 (7) ◽

pp. 3475-3484 ◽

Cited By ~ 66

Author(s):

Thomas P. Kole ◽

Yiider Tseng ◽

Lawrence Huang ◽

Joseph L. Katz ◽

Denis Wirtz

Keyword(s):

Mechanical Behavior ◽

Molecular Mechanisms ◽

Mechanical Response ◽

Sol Gel ◽

Critical Role ◽

Rho Kinase ◽

Small Gtpase ◽

Cytoskeletal Proteins ◽

Cellular Functions

Local sol-gel transitions of the cytoskeleton modulate cell shape changes, which are required for essential cellular functions, including motility and adhesion. In vitro studies using purified cytoskeletal proteins have suggested molecular mechanisms of regulation of cytoskeleton mechanics; however, the mechanical behavior of living cells and the signaling pathways by which it is regulated remains largely unknown. To address this issue, we used a nanoscale sensing method, intracellular microrheology, to examine the mechanical response of the cell to activation of the small GTPase Rho. We observe that the cytoplasmic stiffness and viscosity of serum-starved Swiss 3T3 cells transiently and locally enhances upon treatment with lysophosphatidic acid, and this mechanical behavior follows a trend similar to Rho activity. Furthermore, the time-dependent activation of Rho decreases the degree of microheterogeneity of the cytoplasm. Our results reveal fundamental differences between intracellular elasticity and cellular tension and suggest a critical role for Rho kinase in the regulation of intracellular mechanics.

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R-Ras Regulates Exocytosis by Rgl2/Rlf-mediated Activation of RalA on Endosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e06-08-0765 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1850-1860 ◽

Cited By ~ 39

Author(s):

Akiyuki Takaya ◽

Takahiro Kamio ◽

Michitaka Masuda ◽

Naoki Mochizuki ◽

Hirofumi Sawa ◽

...

Keyword(s):

Resonance Energy Transfer ◽

Resonance Energy ◽

Small Gtpase ◽

Nucleotide Exchange ◽

Cellular Functions ◽

Recycling Endosomes ◽

Nucleotide Exchange Factor ◽

Wide Range ◽

Ras Family

R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.

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Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00404.2010 ◽

2011 ◽

Vol 300 (6) ◽

pp. L840-L850 ◽

Cited By ~ 21

Author(s):

Peter V. Usatyuk ◽

Donghong He ◽

Vytas Bindokas ◽

Irina A. Gorshkova ◽

Evgeny V. Berdyshev ◽

...

Keyword(s):

Signal Transduction ◽

Signaling Pathways ◽

Adherens Junction ◽

Small Gtpase ◽

Cell Periphery ◽

Cellular Functions ◽

S1p Receptors ◽

Sphingosine 1 Phosphate ◽

Lung Endothelial Cells ◽

Barrier Regulation

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca2+ mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.

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Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1409667112 ◽

2015 ◽

Vol 112 (3) ◽

pp. E267-E276 ◽

Cited By ~ 51

Author(s):

Sulagna Das ◽

Taofei Yin ◽

Qingfen Yang ◽

Jingqiao Zhang ◽

Yi I. Wu ◽

...

Keyword(s):

Single Molecule ◽

Membrane Lipids ◽

Exchange Process ◽

Single Particle Tracking ◽

Small Gtpase ◽

Nucleotide Exchange ◽

Membrane Translocation ◽

Cellular Functions ◽

Single Molecule Tracking ◽

Spatial Regulation

Polarized Rac1 signaling is a hallmark of many cellular functions, including cell adhesion, motility, and cell division. The two steps of Rac1 activation are its translocation to the plasma membrane and the exchange of nucleotide from GDP to GTP. It is, however, unclear whether these two processes are regulated independent of each other and what their respective roles are in polarization of Rac1 signaling. We designed a single-particle tracking (SPT) method to quantitatively analyze the kinetics of Rac1 membrane translocation in living cells. We found that the rate of Rac1 translocation was significantly elevated in protrusions during cell spreading on collagen. Furthermore, combining FRET sensor imaging with SPT measurements in the same cell, the recruitment of Rac1 was found to be polarized to an extent similar to that of the nucleotide exchange process. Statistical analysis of single-molecule trajectories and optogenetic manipulation of membrane lipids revealed that Rac1 membrane translocation precedes nucleotide exchange, and is governed primarily by interactions with phospholipids, particularly PI(3,4,5)P3, instead of protein factors. Overall, the study highlights the significance of membrane translocation in spatial Rac1 signaling, which is in addition to the traditional view focusing primarily on GEF distribution and exchange reaction.

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Membrane Protrusion Formation Mediated by Rho/ROCK Signalling and Modulation of Chloride Flux

10.1101/600379 ◽

2019 ◽

Author(s):

Akiko Hori ◽

Kenji Nishide ◽

Yuki Yasukuni ◽

Kei Haga ◽

Wataru Kakuta ◽

...

Keyword(s):

Calcium Ion ◽

Chloride Ion ◽

Intersection Point ◽

Small Gtpase ◽

Membrane Protrusion ◽

Cellular Functions ◽

Membrane Morphology ◽

Amino Acid Stretch ◽

Underlying Mechanisms

ABSTRACTMembrane protrusion is an important structural property associated with various cellular functions. The pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to the protrusions and plays a pivotal role in migration and the determination of cellular morphology; however, the underlying mechanisms have been elusive. Here, we demonstrate that Prom1 is sufficient to trigger membrane protrusion formation. Overexpression of Prom1 in the RPE-1 cells triggers multiple long cholesterol-enriched protrusions, independently from actin and tubulin polymerisation. For this protrusion formation, the five amino acid stretch located at the carboxyl cytosolic region is essential. Moreover, the small GTPase Rho and its effector kinase ROCK are essential for this protrusion formation, and the intersection point of active Rho and Prom1 is where the protrusion formation initiates. Importantly, Prom1 causes the chloride ion efflux induced by calcium ion uptake, and protrusion formation is closely associated with the chloride efflux activity. Altogether, this study has elucidated that Prom1 plays critical roles for the membrane morphology and chloride ion flux.

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The polar Ras-like GTPase MglA activates type IV pilus via SgmX to enable twitching motility inMyxococcus xanthus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2002783117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28366-28373

Author(s):

Romain Mercier ◽

Sarah Bautista ◽

Maëlle Delannoy ◽

Margaux Gibert ◽

Annick Guiseppi ◽

...

Keyword(s):

Molecular Mechanisms ◽

Kin Recognition ◽

Small Gtpase ◽

Extracellular Dna ◽

Host Cells ◽

Twitching Motility ◽

Cellular Functions ◽

Spatial Regulation ◽

Type Iv ◽

The Social

Type IV pili (Tfp) are highly conserved macromolecular structures that fulfill diverse cellular functions, such as adhesion to host cells, the import of extracellular DNA, kin recognition, and cell motility (twitching). Outstandingly, twitching motility enables a poorly understood process by which highly coordinated groups of hundreds of cells move in cooperative manner, providing a basis for multicellular behaviors, such as biofilm formation. In the social bacteriaMyxococcus xanthus, we know that twitching motility is under the dependence of the small GTPase MglA, but the underlying molecular mechanisms remain elusive. Here we show that MglA complexed to GTP recruits a newly characterized Tfp regulator, termed SgmX, to activate Tfp machines at the bacterial cell pole. This mechanism also ensures spatial regulation of Tfp, explaining how MglA switching provokes directional reversals. This discovery paves the way to elucidate how polar Tfp machines are regulated to coordinate multicellular movements, a conserved feature in twitching bacteria.

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Nir2, a Human Homolog of Drosophila melanogaster Retinal Degeneration B Protein, Is Essential for Cytokinesis

Molecular and Cellular Biology ◽

10.1128/mcb.22.14.5064-5075.2002 ◽

2002 ◽

Vol 22 (14) ◽

pp. 5064-5075 ◽

Cited By ~ 30

Author(s):

Vladimir Litvak ◽

Donguha Tian ◽

Shari Carmon ◽

Sima Lev

Keyword(s):

Time Lapse ◽

Eukaryotic Cell ◽

Small Gtpase ◽

Cleavage Furrow ◽

Cleavage Sites ◽

Cellular Functions ◽

Daughter Cells ◽

Interphase Cells ◽

Phosphatidylinositol Transfer ◽

Cytoskeletal Networks

ABSTRACT Cytokinesis, the final stage of eukaryotic cell division, ensures the production of two daughter cells. It requires fine coordination between the plasma membrane and cytoskeletal networks, and it is known to be regulated by several intracellular proteins, including the small GTPase Rho and its effectors. In this study we provide evidence that the protein Nir2 is essential for cytokinesis. Microinjection of anti-Nir2 antibodies into interphase cells blocks cytokinesis, as it results in the production of multinucleate cells. Immunolocalization studies revealed that Nir2 is mainly localized in the Golgi apparatus in interphase cells, but it is recruited to the cleavage furrow and the midbody during cytokinesis. Nir2 colocalizes with the small GTPase RhoA in the cleavage furrow and the midbody, and it associates with RhoA in mitotic cells. Its N-terminal region, which contains a phosphatidylinositol transfer domain and a novel Rho-inhibitory domain (Rid), is required for normal cytokinesis, as overexpression of an N-terminal-truncated mutant blocks cytokinesis completion. Time-lapse videomicroscopy revealed that this mutant normally initiates cytokinesis but fails to complete it, due to cleavage furrow regression, while Rid markedly affects cytokinesis due to abnormal contractility. Rid-expressing cells exhibit aberrant ingression and ectopic cleavage sites; the cells fail to segregate into daughter cells and they form a long unseparated bridge-like cytoplasmic structure. These results provide new insight into the cellular functions of Nir2 and introduce it as a novel regulator of cytokinesis.

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