scholarly journals The Rac activator Tiam1 is required for α3β1-mediated laminin-5 deposition, cell spreading, and cell migration

2005 ◽  
Vol 171 (5) ◽  
pp. 871-881 ◽  
Author(s):  
Irene H.L. Hamelers ◽  
Cristina Olivo ◽  
Alexander E.E. Mertens ◽  
D. Michiel Pegtel ◽  
Rob A. van der Kammen ◽  
...  
Keyword(s):  
Mouse Skin ◽  
Nucleotide Exchange ◽  
Laminin 5 ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
T Lymphoma ◽  
Guanosine Triphosphatase ◽  
And Migration ◽  
Migration Of Cells

The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1−/−) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1−/− keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1−/− cells are unable to activate Rac upon α3β1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in α3β1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

scholarly journals Tuba, a Cdc42 GEF, is required for polarized spindle orientation during epithelial cyst formation

2010 ◽  
Vol 189 (4) ◽  
pp. 661-669 ◽  
Author(s):  
Yi Qin ◽  
Walter H. Meisen ◽  
Yi Hao ◽  
Ian G. Macara
Keyword(s):  
Cyst Formation ◽  
Cell Polarization ◽  
Spindle Orientation ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Lateral Plane ◽  
Similar Phenotype ◽  
Guanosine Triphosphatase

The Cdc42 guanosine triphosphatase is essential for cell polarization in several organisms and in vitro for the organization of polarized epithelial cysts. A long-standing question concerns the identity of the guanine nucleotide exchange factor (GEF) that controls this process. Using Madin–Darby canine kidney cells grown in Matrigel, we screened 70 GEFs by RNA interference. Of these, six positives were identified that caused a multilumen phenotype, including Tuba, a Cdc42-specific GEF localized below the apical cortex. Loss of Tuba abolishes Cdc42 enrichment at the apical cortex. Normal lumen formation is rescued by human Tuba or active Cdc42 but not by a GEF-negative Tuba mutant. Silencing Cdc42 causes a similar phenotype, including multilumen formation and reduced atypical protein kinase C (aPKC) activity. Lumen disorganization after depletion of Tuba or Cdc42 or inhibition of aPKC is caused by defective spindle orientation. Together, our findings implicate Tuba as a key activator of the Cdc42 GTPase during epithelial ductal morphogenesis, which in turn activates apical aPKC to ensure that spindles orient parallel to the lateral plane.

scholarly journals Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export

2014 ◽  
Vol 206 (6) ◽  
pp. 751-762 ◽  
Author(s):  
Kota Saito ◽  
Koh Yamashiro ◽  
Noriko Shimazu ◽  
Tomoya Tanabe ◽  
Kenji Kontani ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Secretory Proteins ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Transport Vesicles ◽  
Nucleotide Exchange Factor ◽  
Receptor Component ◽  
Er Exit Sites ◽  
Guanosine Triphosphatase ◽  
Trimeric Structure

Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60–90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate–bound Sar1 generated by Sec12 localized at ER exit sites.

scholarly journals Calcium-RasGRP2-Rap1 signaling mediates CD38-induced migration of chronic lymphocytic leukemia cells

2018 ◽  
Vol 2 (13) ◽  
pp. 1551-1561 ◽  
Author(s):  
Silvia Mele ◽  
Stephen Devereux ◽  
Andrea G. Pepper ◽  
Elvira Infante ◽  
Anne J. Ridley
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Cd38 Expression ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Key Points ◽  
And Migration

Key Points Basal intracellular Ca2+ levels and migration increase with higher CD38 expression in CLL cells. Rap1 and the Rap1 guanine-nucleotide exchange factor RasGRP2 are required for CLL migration and regulated by CD38 levels.

scholarly journals Myoblast Migration and Directional Persistence Affected by Syndecan-4-Mediated Tiam-1 Expression and Distribution

2020 ◽  
Vol 21 (3) ◽  
pp. 823 ◽  
Author(s):  
Daniel Becsky ◽  
Szuzina Gyulai-Nagy ◽  
Arpad Balind ◽  
Peter Horvath ◽  
Laszlo Dux ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Underlying Mechanism ◽  
Small Gtpase ◽  
Asymmetric Distribution ◽  
Nucleotide Exchange ◽  
Muscle Stem Cells ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
T Lymphoma ◽  
Migration Capacity

Skeletal muscle is constantly renewed in response to injury, exercise, or muscle diseases. Muscle stem cells, also known as satellite cells, are stimulated by local damage to proliferate extensively and form myoblasts that then migrate, differentiate, and fuse to form muscle fibers. The transmembrane heparan sulfate proteoglycan syndecan-4 plays multiple roles in signal transduction processes, such as regulating the activity of the small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) by binding and inhibiting the activity of Tiam1 (T-lymphoma invasion and metastasis-1), a guanine nucleotide exchange factor for Rac1. The Rac1-mediated actin remodeling is required for cell migration. Syndecan-4 knockout mice cannot regenerate injured muscle; however, the detailed underlying mechanism is unknown. Here, we demonstrate that shRNA-mediated knockdown of syndecan-4 decreases the random migration of mouse myoblasts during live-cell microscopy. Treatment with the Rac1 inhibitor NSC23766 did not restore the migration capacity of syndecan-4 silenced cells; in fact, it was further reduced. Syndecan-4 knockdown decreased the directional persistence of migration, abrogated the polarized, asymmetric distribution of Tiam1, and reduced the total Tiam1 level of the cells. Syndecan-4 affects myoblast migration via its role in expression and localization of Tiam1; this finding may facilitate greater understanding of the essential role of syndecan-4 in the development and regeneration of skeletal muscle.

scholarly journals In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

2004 ◽  
Vol 15 (11) ◽  
pp. 4990-5000 ◽  
Author(s):  
Adriana Pagano ◽  
Pascal Crottet ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Brefeldin A ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Vesicle Formation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and γ-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

scholarly journals Induction of cell retraction by the combined actions of Abl–CrkII and Rho–ROCK1 signaling

2008 ◽  
Vol 183 (4) ◽  
pp. 711-723 ◽  
Author(s):  
XiaoDong Huang ◽  
Diana Wu ◽  
Hua Jin ◽  
Dwayne Stupack ◽  
Jean Y.J. Wang
Keyword(s):  
Nucleotide Exchange ◽  
Downstream Target ◽  
Abl Tyrosine Kinase ◽  
Guanine Nucleotide Exchange ◽  
Cell Matrix ◽  
Nucleotide Exchange Factor ◽  
Wide Range ◽  
Important Regulator ◽  
Guanosine Triphosphatase ◽  
Cell Retraction

Dynamic modulation of cell adhesion is integral to a wide range of biological processes. The small guanosine triphosphatase (GTPase) Rap1 is an important regulator of cell–cell and cell–matrix adhesions. We show here that induced expression of activated Abl tyrosine kinase reduces Rap1-GTP levels through phosphorylation of Tyr221 of CrkII, which disrupts interaction of CrkII with C3G, a guanine nucleotide exchange factor for Rap1. Abl-dependent down-regulation of Rap1-GTP causes cell rounding and detachment only when the Rho–ROCK1 pathway is also activated, for example, by lysophosphatidic acid (LPA). During ephrin-A1–induced retraction of PC3 prostate cancer cells, we show that endogenous Abl is activated and disrupts the CrkII–C3G complex to reduce Rap1-GTP. Interestingly, ephrin-A1–induced PC3 cell retraction also requires LPA, which stimulates Rho to a much higher level than that is activated by ephrin-A1. Our results establish Rap1 as another downstream target of the Abl–CrkII signaling module and show that Abl–CrkII collaborates with Rho–ROCK1 to stimulate cell retraction.

scholarly journals ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment

2015 ◽  
Vol 26 (23) ◽  
pp. 4265-4279 ◽  
Author(s):  
Joseph C. Salem ◽  
Marta M. Reviriego-Mendoza ◽  
Lorraine C. Santy
Keyword(s):  
Adhesion Formation ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Recycling Endosomes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Migration ◽  
Regulated Trafficking ◽  
Migration Response

When expressed in epithelial cells, cytohesin-2/ARNO, a guanine nucleotide exchange factor (GEF) for ARF small GTPases, causes a robust migration response. Recent evidence suggests that cytohesin-2/ARNO acts downstream of small the GTPase R-Ras to promote spreading and migration. We hypothesized that cytohesin-2/ARNO could transmit R-Ras signals by regulating the recycling of R-Ras through ARF activation. We found that Eps15-homology domain 1 (EHD1), a protein that associates with the endocytic recycling compartment (ERC), colocalizes with active R-Ras in transiently expressed HeLa cells. In addition, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Knockdown or expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1 and inhibits cell spreading. E156K-ARNO also causes a reduction in focal adhesion size and number. Finally, we demonstrate that R-Ras/ARNO signaling is required for recycling of α5-integrin and R-Ras to the plasma membrane. These data establish a role for cytohesin-2/ARNO as a regulator of R-Ras and integrin recycling and suggest that ARF-regulated trafficking of R-Ras is required for R-Ras–dependent effects on spreading and adhesion formation.

scholarly journals Lte1 contributes to Bfa1 localization rather than stimulating nucleotide exchange by Tem1

2009 ◽  
Vol 187 (4) ◽  
pp. 497-511 ◽  
Author(s):  
Marco Geymonat ◽  
Adonis Spanos ◽  
Geoffroy de Bettignies ◽  
Steven G. Sedgwick
Keyword(s):  
Mutational Analysis ◽  
Spindle Pole ◽  
Cell Polarization ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Spindle Pole Bodies ◽  
Guanosine Triphosphatase ◽  
Spindle Position ◽  
Mitotic Regulation

Lte1 is a mitotic regulator long envisaged as a guanosine nucleotide exchange factor (GEF) for Tem1, the small guanosine triphosphatase governing activity of the Saccharomyces cerevisiae mitotic exit network. We demonstrate that this model requires reevaluation. No GEF activity was detectable in vitro, and mutational analysis of Lte1’s putative GEF domain indicated that Lte1 activity relies on interaction with Ras for localization at the bud cortex rather than providing nucleotide exchange. Instead, we found that Lte1 can determine the subcellular localization of Bfa1 at spindle pole bodies (SPBs). Under conditions in which Lte1 is essential, Lte1 promoted the loss of Bfa1 from the maternal SPB. Moreover, in cells with a misaligned spindle, mislocalization of Lte1 in the mother cell promoted loss of Bfa1 from one SPB and allowed bypass of the spindle position checkpoint. We observed that lte1 mutants display aberrant localization of the polarity cap, which is the organizer of the actin cytoskeleton. We propose that Lte1’s role in cell polarization underlies its contribution to mitotic regulation.

scholarly journals Gef1p, a New Guanine Nucleotide Exchange Factor for Cdc42p, Regulates Polarity inSchizosaccharomyces pombe

2003 ◽  
Vol 14 (1) ◽  
pp. 313-323 ◽  
Author(s):  
Pedro M. Coll ◽  
Yadira Trillo ◽  
Amagoia Ametzazurra ◽  
Pilar Perez
Keyword(s):  
Cell Morphology ◽  
Rho Gtpases ◽  
Genetic Interaction ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Schizosaccharomyces pombe cdc42+regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1+increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression ofgef1+causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1+deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1Δ scd1Δ is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1+orscd1+causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.

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