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1.
Nat Rev Mol Cell Biol ; 9(9): 690-701, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18719708

RESUMEN

Rho GTPases are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking and cytokinesis. These proteins are conserved from plants and yeast to mammals, and function by interacting with and stimulating various downstream targets, including actin nucleators, protein kinases and phospholipases. The roles of Rho GTPases have been extensively studied in different mammalian cell types using mainly dominant negative and constitutively active mutants. The recent availability of knockout mice for several members of the Rho family reveals new information about their roles in signalling to the cytoskeleton and in development.


Asunto(s)
Mamíferos/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Neuronas/enzimología , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo
2.
J Leukoc Biol ; 89(4): 577-86, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21233409

RESUMEN

Statins are known to inhibit signaling of Ras superfamily GTPases and reduce T cell adhesion to ICAM-1. Here, we address the hypothesis that statins affect T cell adhesion and migration by modulating the function of specific GTPases. Statins inhibit the synthesis of mevalonic acid, which is required for farnesyl and geranylgeranyl isoprenoid synthesis. Ras superfamily GTPases are post-translationally isoprenylated to facilitate their anchorage to membranes, where they function to stimulate signal transduction processes. We demonstrate that 1 µM statin inhibits the adhesion, migration, and chemotaxis of the T-ALL cell line CCRF-CEM and TEM of CCRF-CEM and PEER T-ALL cells, but higher statin concentrations are needed to inhibit adhesion of primary T cells. Similar effects are observed following treatment with GGTI-298 or RNA interference-mediated knockdown of Rap1b but not Rap1a, Rac1, Rac2, RhoA, or Cdc42. Statins also alter Rap1 activity and Rap1b localization. Rap1 levels are higher in primary T cells than T-ALL cells, which could explain their reduced sensitivity to statins. These results demonstrate for the first time that the closely related Rap1a and Rap1b isoforms have different functions and suggest that statins or Rap1b depletion could be used to reduce tissue invasion in T-ALL.


Asunto(s)
Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Lovastatina/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Simvastatina/farmacología , Proteínas de Unión al GTP rap/metabolismo , Western Blotting , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Técnicas para Inmunoenzimas , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismo
3.
Small GTPases ; 1(3): 174-179, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21686273

RESUMEN

T cells need to cross endothelial barriers during immune surveillance and inflammation. This involves T-cell adhesion to the endothelium followed by polarization and crawling with a lamellipodium at the front and contractile uropod at the back. T cells subsequently extend lamellipodia and filopodia under the endothelium in order to transmigrate. Rho GTPases play key roles in cell migration by regulating cytoskeletal dynamics and cell adhesion. We have found that the Rho GTPase RhoA is required for efficient T-cell polarization and migration on endothelial cells as well as transendothelial migration. RhoA-depleted cells lack both lamellipodia and uropods, and instead have narrow protrusions extending from a rounded cell body. Using a RhoA activity biosensor, we have shown that RhoA is active at the leading edge in lamellipodia and filopodia of crawling and transmigrating T cells, as well as in the uropod. In lamellipodia, its activity correlates with both protrusion and retraction. We predict that RhoA signals via the formin mDIA 1 during lamellipodial protrusion whereas it induces lamellipodial retraction via the kinase ROCK and actomyosin contractility. We propose that different guanine-nucleotide exchange factors (GEFs) are responsible for coordinating RhoA activation and signaling in different regions of transmigrating T cells.

4.
PLoS One ; 5(1): e8774, 2010 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-20098744

RESUMEN

BACKGROUND: Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs. CONCLUSIONS/SIGNIFICANCE: Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning.


Asunto(s)
Microtúbulos/fisiología , Transducción de Señal , Linfocitos T/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Línea Celular Tumoral , Polaridad Celular/efectos de los fármacos , Humanos , Nocodazol/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología
5.
J Cell Biol ; 190(4): 553-63, 2010 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-20733052

RESUMEN

Transendothelial migration (TEM) is a tightly regulated process whereby leukocytes migrate from the vasculature into tissues. Rho guanosine triphosphatases (GTPases) are implicated in TEM, but the contributions of individual Rho family members are not known. In this study, we use an RNA interference screen to identify which Rho GTPases affect T cell TEM and demonstrate that RhoA is critical for this process. RhoA depletion leads to loss of migratory polarity; cells lack both leading edge and uropod structures and, instead, have stable narrow protrusions with delocalized protrusions and contractions. By imaging a RhoA activity biosensor in transmigrating T cells, we find that RhoA is locally and dynamically activated at the leading edge, where its activation precedes both extension and retraction events, and in the uropod, where it is associated with ROCK-mediated contraction. The Rho guanine nucleotide exchange factor (GEF) GEF-H1 contributes to uropod contraction but does not affect the leading edge. Our data indicate that RhoA activity is dynamically regulated at the front and back of T cells to coordinate TEM.


Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Movimiento Celular/fisiología , Transducción de Señal/fisiología , Linfocitos T , Proteína de Unión al GTP rhoA/metabolismo , Animales , Línea Celular , Estructuras de la Membrana Celular/ultraestructura , Polaridad Celular , Endotelio/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Linfocitos T/citología , Linfocitos T/fisiología , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/genética
6.
Eur J Immunol ; 34(6): 1752-61, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15162446

RESUMEN

One of beneficial effects of glucocorticoids (GC) in inflammation may be the augmentation of macrophages' capacity for phagocytosis of apoptotic cells, a process that has a central role in resolution of inflammation. Here we define the phenotype of GC-treated monocyte-derived macrophages, comparing to IFN-gamma-treated and IL-4-treated monocyte-derived macrophages and combinatorial treatment. Our data indicate that the cytokine microenvironment at an inflammatory site will critically determine monocyte functional capacity following treatment with GC. In particular, whilst GC exert dominant regulatory effects over IFN-gamma in terms of cell surface receptor repertoire and morphology, the acquisition of a macrophage capacity for clearance of apoptotic cells is prevented by combined treatment. In terms of mechanism, GC augmentation of phagocytosis was reversed even when monocytes were pre-incubated with GC for the first 24 h of culture, a period that is critical for induction of a highly phagocytic macrophage phenotype. These findings have important implications for the effectiveness of GC in promoting acquisition of a pro-phagocytic macrophage phenotype in inflammatory diseases associated with high levels of IFN-gamma


Asunto(s)
Glucocorticoides/antagonistas & inhibidores , Interferón gamma/farmacología , Macrófagos/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Dexametasona/antagonistas & inhibidores , Dexametasona/inmunología , Dexametasona/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Glucocorticoides/inmunología , Glucocorticoides/farmacología , Humanos , Inmunofenotipificación , Interleucina-4/farmacología , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología
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