scholarly journals Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

2013 ◽  
Vol 204 (1) ◽  
pp. 111-127 ◽  
Author(s):  
Ceniz Zihni ◽  
Peter M.G. Munro ◽  
Ahmed Elbediwy ◽  
Nicholas H. Keep ◽  
Stephen J. Terry ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Apical Membrane ◽  
Apical Margin ◽  
Nucleotide Exchange ◽  
Lateral Border ◽  
Guanine Nucleotide Exchange ◽  
Kinase C ◽  
Nucleotide Exchange Factor ◽  
Junction Formation ◽  
Interfering Rna

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation.

scholarly journals Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity

2017 ◽  
Vol 28 (2) ◽  
pp. 252-260 ◽  
Author(s):  
Travis R. Ruch ◽  
David M. Bryant ◽  
Keith E. Mostov ◽  
Joanne N. Engel
Keyword(s):  
Apical Membrane ◽  
Protein Localization ◽  
Apical Surface ◽  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Signaling Mechanism ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Chemically Induced Dimerization ◽  
Phosphatidylinositol 3

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens.

scholarly journals Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6

2008 ◽  
Vol 183 (3) ◽  
pp. 499-511 ◽  
Author(s):  
Sophia Semerdjieva ◽  
Barry Shortt ◽  
Emma Maxwell ◽  
Sukhdeep Singh ◽  
Paul Fonarev ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Coated Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Steady State Levels ◽  
Endocytic Vesicles ◽  
Interfering Rna

Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5S34N, or small interfering RNA (siRNA)–mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to α-adaptin ear, which displaces the ear-associated μ2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-μ2 levels, and expression of a phosphomimetic μ2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5S35N expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P2) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating μ2 dephosphorylation and PtdIns(4,5)P2 levels in CCVs.

scholarly journals Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

2010 ◽  
Vol 30 (22) ◽  
pp. 5421-5431 ◽  
Author(s):  
Martijn Gloerich ◽  
Bas Ponsioen ◽  
Marjolein J. Vliem ◽  
Zhongchun Zhang ◽  
Jun Zhao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Cyclic Amp ◽  
Cell Junction ◽  
Nucleotide Exchange ◽  
Erm Proteins ◽  
Spatial Regulation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Interfering Rna

ABSTRACT Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.

scholarly journals Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes

2009 ◽  
Vol 20 (12) ◽  
pp. 2900-2908 ◽  
Author(s):  
Kanako Tamura ◽  
Norihiko Ohbayashi ◽  
Yuto Maruta ◽  
Eiko Kanno ◽  
Takashi Itoh ◽  
...  
Keyword(s):  
Binding Protein ◽  
Small Gtpase ◽  
Ankyrin Repeat ◽  
Dramatic Reduction ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
A Cells ◽  
Nucleotide Exchange Factor ◽  
Specific Effector ◽  
Interfering Rna

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.

scholarly journals Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO

2016 ◽  
Vol 216 (1) ◽  
pp. 181-197 ◽  
Author(s):  
Nisha Bte Mohd Rafiq ◽  
Zi Zhao Lieu ◽  
Tingting Jiang ◽  
Cheng-han Yu ◽  
Paul Matsudaira ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Myosin Ii ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Cell Matrix ◽  
Nucleotide Exchange Factor ◽  
Myosin Iia ◽  
Interfering Rna

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)–bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

scholarly journals Grb2 Is a Negative Modulator of the Intrinsic Ras-GEF Activity of hSos1

2006 ◽  
Vol 17 (8) ◽  
pp. 3591-3597 ◽  
Author(s):  
Natasha Zarich ◽  
José Luis Oliva ◽  
Natalia Martínez ◽  
Rocío Jorge ◽  
Alicia Ballester ◽  
...  
Keyword(s):  
Terminal Region ◽  
Full Length ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Before And After ◽  
Carboxyl Terminal ◽  
Interfering Rna ◽  
Exchange Activity

hSos1 is a Ras guanine-nucleotide exchange factor. It was suggested that the carboxyl-terminal region of hSos1 down-regulates hSos1 functionality and that the intrinsic guanine-nucleotide exchange activity of this protein may be different before and after stimulation of tyrosine kinase receptors. Using different myristoylated hSos1 full-length and carboxyl-terminal truncated mutants, we show that Grb2 function accounts not only for recruitment of hSos1 to the plasma membrane but also for modulation of hSos1 activity. Our results demonstrate that the first two canonical Grb2 binding sites, inside the carboxyl-terminal region of hSos1, are responsible for this regulation. Following different approaches, such as displacement of Grb2 from the hSos1-Grb2 complex or depletion of Grb2 levels by small interfering RNA, we found that the full-length Grb2 proteins mediate negative regulation of the intrinsic Ras guanine-nucleotide exchange activity of hSos1.

scholarly journals Rab1b Interacts with GBF1 and Modulates both ARF1 Dynamics and COPI Association

2007 ◽  
Vol 18 (7) ◽  
pp. 2400-2410 ◽  
Author(s):  
Pablo Monetta ◽  
Ileana Slavin ◽  
Nahuel Romero ◽  
Cecilia Alvarez
Keyword(s):  
Time Lapse ◽  
Limited Range ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Golgi Membranes ◽  
Golgi Transport ◽  
Nucleotide Exchange Factor ◽  
Er Exit Sites ◽  
Interfering Rna

Assembly of the cytosolic coat protein I (COPI) complex at the ER–Golgi interface is directed by the ADP ribosylation factor1 (Arf1) and its guanine nucleotide exchange factor (GBF1). Rab1b GTPase modulates COPI recruitment, but the molecular mechanism underlying this action remains unclear. Our data reveal that in vivo expression of the GTP-restricted Rab1b mutant (Rab1Q67L) increased the association of GBF1 and COPI to peripheral structures localized at the ER exit sites (ERES) interface. Active Rab1b also stabilized Arf1 on Golgi membranes. Furthermore, we characterized GBF1 as a new Rab1b effector, and showed that its N-terminal domain was involved in this interaction. Rab1b small interfering RNA oligonucleotide assays suggested that Rab1b was required for GBF1 membrane association. To further understand how Rab1b functions in ER-to-Golgi transport, we analyzed GFP-Rab1b dynamics in HeLa cells. Time-lapse microscopy indicated that the majority of the Rab1b-labeled punctuated structures are relatively short-lived with limited-range movements. FRAP of Golgi GFP-Rab1bwt showed rapid recovery (t1/2 120 s) with minimal dependence on microtubules. Our data support a model where Rab1b-GTP induces GBF1 recruitment at the ERES interface and at the Golgi complex where it is required for COPII/COPI exchange or COPI vesicle formation, respectively.

scholarly journals The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

2012 ◽  
Vol 23 (15) ◽  
pp. 2831-2844 ◽  
Author(s):  
Jos van Rijssel ◽  
Jeffrey Kroon ◽  
Mark Hoogenboezem ◽  
Floris P. J. van Alphen ◽  
Renske J. de Jong ◽  
...  
Keyword(s):  
Structure Formation ◽  
Apical Membrane ◽  
Transendothelial Migration ◽  
Dependent Manner ◽  
Membrane Protrusion ◽  
Nucleotide Exchange ◽  
Leukocyte Transmigration ◽  
Guanine Nucleotide Exchange ◽  
Leukocyte Transendothelial Migration ◽  
Nucleotide Exchange Factor

Leukocyte transendothelial migration involves the active participation of the endothelium through the formation of apical membrane protrusions that embrace adherent leukocytes, termed docking structures. Using live-cell imaging, we find that prior to transmigration, endothelial docking structures form around 80% of all neutrophils. Previously we showed that endothelial RhoG and SGEF control leukocyte transmigration. In this study, our data reveal that both full-length Trio and the first DH-PH (TrioD1) domain of Trio, which can activate Rac1 and RhoG, interact with ICAM-1 and are recruited to leukocyte adhesion sites. Moreover, upon clustering of ICAM-1, the Rho-guanine nucleotide exchange factor Trio activates Rac1, prior to activating RhoG, in a filamin-dependent manner. We further show that docking structure formation is initiated by ICAM-1 clustering into ring-like structures, which is followed by apical membrane protrusion. Interestingly, we find that Rac1 is required for ICAM-1 clustering, whereas RhoG controls membrane protrusion formation. Finally, silencing endothelial Trio expression or reducing TrioD1 activity without affecting SGEF impairs both docking structure formation and leukocyte transmigration. We conclude that Trio promotes leukocyte transendothelial migration by inducing endothelial docking structure formation in a filamin-dependent manner through the activation of Rac1 and RhoG.

scholarly journals A Two-Tiered Mechanism Enables Localized Cdc42 Signaling during Enterocyte Polarization

2017 ◽  
Vol 37 (7) ◽  
Author(s):  
Lucas J. M. Bruurs ◽  
Susan Zwakenberg ◽  
Mirjam C. van der Net ◽  
Fried J. Zwartkruis ◽  
Johannes L. Bos
Keyword(s):  
Molecular Mechanisms ◽  
Apical Membrane ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Differential Diffusion ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Dual Mechanism

ABSTRACT Signaling by the small GTPase Cdc42 governs a diverse set of cellular processes that contribute to tissue morphogenesis. Since these processes often require highly localized signaling, Cdc42 activity must be clustered in order to prevent ectopic signaling. During cell polarization, apical Cdc42 signaling directs the positioning of the nascent apical membrane. However, the molecular mechanisms that drive Cdc42 clustering during polarity establishment are largely unknown. Here, we demonstrate that during cell polarization localized Cdc42 signaling is enabled via activity-dependent control of Cdc42 mobility. By performing photoconversion experiments, we show that inactive Cdc42-GDP is 30-fold more mobile than active Cdc42-GTP. This switch in apical mobility originates from a dual mechanism involving RhoGDI-mediated membrane dissociation of Cdc42-GDP and Tuba-mediated immobilization of Cdc42-GTP. Interference with either mechanism affects Cdc42 clustering and as a consequence impairs Cdc42-mediated apical membrane clustering. We therefore identify a molecular network, comprised of Cdc42, the guanine nucleotide exchange factor (GEF) Tuba, and RhoGDI, that enables differential diffusion of inactive and active Cdc42 and is required to establish localized Cdc42 signaling during enterocyte polarization.

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