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1.
Nat Rev Mol Cell Biol ; 12(8): 493-504, 2011 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-21779026

RESUMEN

The 'invisible hand' is a term originally coined by Adam Smith in The Theory of Moral Sentiments to describe the forces of self-interest, competition and supply and demand that regulate the resources in society. This metaphor continues to be used by economists to describe the self-regulating nature of a market economy. The same metaphor can be used to describe the RHO-specific guanine nucleotide dissociation inhibitor (RHOGDI) family, which operates in the background, as an invisible hand, using similar forces to regulate the RHO GTPase cycle.


Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Evolución Molecular , Inhibidores de Disociación de Guanina Nucleótido/química , Inhibidores de Disociación de Guanina Nucleótido/genética , Humanos , Microdominios de Membrana/metabolismo , Redes y Vías Metabólicas , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos , Neoplasias/metabolismo , Fosforilación , Filogenia , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión al GTP rho/química , Inhibidor alfa de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico
2.
J Cell Sci ; 133(1)2020 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-31822630

RESUMEN

Mechanical cues are essential for the regulation of cell and tissue physiology. Hence, it has become an utmost necessity for cell biologists to account for those mechanical parameters when investigating biological processes and they need devices to manipulate cells accordingly. Here, we report a simple mechanical cell-stretching system that can generate uniaxial cyclic mechanical stretch on cells in tissue culture. This system is based upon a low-cost battery-powered uniaxial cyclic mechanical stretcher exclusively built out of LEGO® parts combined with a stretchable poly(dimethylsiloxane) tissue culture plate in order to grow and stretch cells. We characterize the system and show that it can be used in a wide variety of downstream applications, including immunofluorescence, western blotting and biochemical assays. We also illustrate how this system can be useful in a study as we investigated the behavior of integrin adhesion complexes upon cell stretching. We therefore present a cost-effective, multipurpose cell-stretching system that should help to increase understanding of mechanical signaling.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Células Cultivadas/metabolismo , Estrés Mecánico , Células Cultivadas/citología , Humanos
3.
FASEB J ; 26(2): 469-79, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22038046

RESUMEN

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.


Asunto(s)
Proteínas de Unión al GTP rho/metabolismo , Animales , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Modelos Biológicos , Fosforilación , Conformación Proteica , Procesamiento Proteico-Postraduccional , Proteolisis , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/genética
4.
J Cell Biol ; 178(7): 1279-93, 2007 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-17875742

RESUMEN

During trans-endothelial migration (TEM), leukocytes use adhesion receptors such as intercellular adhesion molecule-1 (ICAM1) to adhere to the endothelium. In response to this interaction, the endothelium throws up dynamic membrane protrusions, forming a cup that partially surrounds the adherent leukocyte. Little is known about the signaling pathways that regulate cup formation. In this study, we show that RhoG is activated downstream from ICAM1 engagement. This activation requires the intracellular domain of ICAM1. ICAM1 colocalizes with RhoG and binds to the RhoG-specific SH3-containing guanine-nucleotide exchange factor (SGEF). The SH3 domain of SGEF mediates this interaction. Depletion of endothelial RhoG by small interfering RNA does not affect leukocyte adhesion but decreases cup formation and inhibits leukocyte TEM. Silencing SGEF also results in a substantial reduction in RhoG activity, cup formation, and TEM. Together, these results identify a new signaling pathway involving RhoG and its exchange factor SGEF downstream from ICAM1 that is critical for leukocyte TEM.


Asunto(s)
Movimiento Celular , Polaridad Celular , Células Endoteliales/citología , Células Endoteliales/enzimología , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/citología , Proteínas de Unión al GTP rho/metabolismo , Animales , Células COS , Adhesión Celular , Extensiones de la Superficie Celular/metabolismo , Chlorocebus aethiops , Activación Enzimática , Proteínas Fluorescentes Verdes/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HL-60 , Humanos , Molécula 1 de Adhesión Intercelular/química , Leucocitos/enzimología , Leucocitos/ultraestructura , Microesferas , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Dominios Homologos src
5.
Trends Biotechnol ; 40(9): 1073-1087, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35314074

RESUMEN

LEGO® is a brand of toys that have entertained generations of children. Beyond amusement, LEGO® bricks also constitute a building ecosystem of their own that creators from the general public, as well as scientists and engineers, can use to design and assemble devices for all purposes, including scientific research and biotechnology. We describe several of these constructions to highlight the construction properties of LEGO® and their advantages, caveats, and impact in biotechnology. We also discuss how this emerging trend in LEGO® building pairs with a growing interest in open-access and frugal science which aims to provide access to technology to all scientists regardless of financial wealth and technological prowess.


Asunto(s)
Biotecnología , Ecosistema , Niño , Humanos , Juego e Implementos de Juego
6.
STAR Protoc ; 2(2): 100437, 2021 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-33899018

RESUMEN

Mechanical signals are essential for the regulation of many biological processes. Therefore, it has become paramount to account for these mechanical parameters when exploring biological processes. Here, we describe a protocol to apply cyclic uniaxial stretch on cells in culture using a LEGO®-based mechanical stretcher and a flexible custom-made polydimethylsiloxane culture vessel as well as validated downstream applications. While this system offers an out-of-the-box limited type of simulation, it provides a reliable and low-cost opportunity to perform cell stretching. For complete details on the use and execution of this protocol, please refer to Boulter et al. (2020).


Asunto(s)
Fenómenos Biomecánicos/fisiología , Técnicas de Cultivo de Célula , Estrés Mecánico , Técnicas de Cultivo de Tejidos , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Células Cultivadas/citología , Células Cultivadas/fisiología , Dimetilpolisiloxanos/química , Diseño de Equipo , Fibroblastos/citología , Fibroblastos/fisiología , Células HeLa , Humanos , Técnicas de Cultivo de Tejidos/instrumentación , Técnicas de Cultivo de Tejidos/métodos
7.
Cell Metab ; 29(1): 124-140.e10, 2019 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-30293773

RESUMEN

Dysregulation of extracellular matrix (ECM) deposition and cellular metabolism promotes tumor aggressiveness by sustaining the activity of key growth, invasion, and survival pathways. Yet mechanisms by which biophysical properties of ECM relate to metabolic processes and tumor progression remain undefined. In both cancer cells and carcinoma-associated fibroblasts (CAFs), we found that ECM stiffening mechanoactivates glycolysis and glutamine metabolism and thus coordinates non-essential amino acid flux within the tumor niche. Specifically, we demonstrate a metabolic crosstalk between CAF and cancer cells in which CAF-derived aspartate sustains cancer cell proliferation, while cancer cell-derived glutamate balances the redox state of CAFs to promote ECM remodeling. Collectively, our findings link mechanical stimuli to dysregulated tumor metabolism and thereby highlight a new metabolic network within tumors in which diverse fuel sources are used to promote growth and aggressiveness. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in cancer.


Asunto(s)
Ácido Aspártico/metabolismo , Neoplasias de la Mama/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma/metabolismo , Ácido Glutámico/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Fibroblastos Asociados al Cáncer/patología , Línea Celular , Matriz Extracelular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAP
8.
Nat Commun ; 9(1): 4862, 2018 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-30451822

RESUMEN

Mechanical and metabolic cues independently contribute to the regulation of cell and tissue homeostasis. However, how they cross-regulate each other during this process remains largely unknown. Here, we show that cellular metabolism can regulate integrin rigidity-sensing via the sphingolipid metabolic pathway controlled by the amino acid transporter and integrin coreceptor CD98hc (SLC3A2). Genetic invalidation of CD98hc in dermal cells and tissue impairs rigidity sensing and mechanical signaling downstream of integrins, including RhoA activation, resulting in aberrant tissue mechanical homeostasis. Unexpectedly, we found that this regulation does not occur directly through regulation of integrins by CD98hc but indirectly, via the regulation of sphingolipid synthesis and the delta-4-desaturase DES2. Loss of CD98hc decreases sphingolipid availability preventing proper membrane recruitment, shuttling and activation of upstream regulators of RhoA including Src kinases and GEF-H1. Altogether, our results unravel a novel cross-talk regulation between integrin mechanosensing and cellular metabolism which may constitute an important new regulatory framework contributing to mechanical homeostasis.


Asunto(s)
Fibroblastos/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Mecanotransducción Celular , Complejos Multienzimáticos/genética , Oxidorreductasas/genética , Esfingolípidos/biosíntesis , Animales , Dermis/citología , Dermis/metabolismo , Fibroblastos/citología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/deficiencia , Regulación de la Expresión Génica , Homeostasis , Lipogénesis , Ratones , Ratones Transgénicos , Complejos Multienzimáticos/metabolismo , Oxidorreductasas/metabolismo , Cultivo Primario de Células , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo
9.
J Invest Dermatol ; 138(12): 2511-2521, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-29906411

RESUMEN

Skin homeostasis relies on fine-tuning of epidermis-dermis interactions and is affected by aging. While extracellular matrix (ECM) proteins, such as integrins, are involved in aging, the molecular basis of the skin changes needs to be investigated further. Here, we showed that integrin co-receptor, SLC3A2, required for cell proliferation, is expressed at the surface of resting dermal fibroblasts in young patients and is reduced drastically with aging. In vivo SLC3A2 dermal fibroblast deletion induced major skin phenotypes resembling premature aging. Knockout mice (3 months old) presented strong defects in skin elasticity due to altered ECM assembly, which impairs epidermal homeostasis. SLC3A2 dermal fibroblast loss led to an age-associated secretome profile, with 77% of identified proteins belonging to ECM and ECM-associated proteins. ECM not only contributes to skin mechanical properties, but it is also a reservoir of growth factors and bioactive molecules. We demonstrate that dermal fibroblast SLC3A2 is required for ECM to fully exert its structural and reservoir role allowing proper and efficient TGF-ß localization and activation. We identified SLC3A2 as a protective controller of dermal ECM stiffness and quality required to maintain the epidermis to dermis interface as functional and dynamic.


Asunto(s)
Envejecimiento Prematuro/genética , Dermis/patología , Epitelio/fisiología , Fibroblastos/fisiología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Animales , Proliferación Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Homeostasis , Humanos , Ratones , Ratones Noqueados , Transporte de Proteínas , Factor de Crecimiento Transformador beta/metabolismo
10.
FASEB J ; 20(9): 1489-91, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16723384

RESUMEN

Extracellular matrix (ECM) receptors of the integrin family initiate changes in cell shape and motility by recruiting signaling components that coordinate these events. Integrin-linked kinase (ILK) is one such partner of beta1 integrins that participates in dynamic rearrangement of cell-matrix adhesions and cell spreading by mechanisms that are not well understood. To further elucidate the role of ILK in these events, we engineered a chimeric molecule comprising ILK fused to a membrane-targeted green fluorescent protein (ILK-GFP-F). ILK-GFP-F is highly enriched in cell-matrix adhesions, and its expression in fibroblasts leads to an accumulation of focal adhesions (2-5 microm) and elongated adhesions (>5 microm). ILK-GFP-F enhances cell spreading on fibronectin and induces a constitutive increase in the levels of GTP-bound Rac-1. Conversely, ILK knock-down by siRNA transfection decreases active Rac-1. Endogenous ILK was found to associate with PKL (paxillin kinase linker) and the Rac/Cdc42 guanine nucleotide exchange factor betaPIX. Further, expression of a dominant negative betaPIX mutant reversed the increase in active Rac-1 levels of ILK-GFP-F-expressing cells, thus placing betaPIX in the pathway leading from ILK to Rac-1 activation. However, expression of constitutively active Rac only partially restores the spreading defects of ILK-depleted cells, suggesting that an additional ILK-dependent signal is required for cell spreading.


Asunto(s)
Endotelio Vascular/fisiología , Matriz Extracelular/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteína de Unión al GTP rac1/metabolismo , Animales , Aorta , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Vectores Genéticos , Cinética , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Transfección , Proteína de Unión al GTP rac1/genética
11.
Eur J Cell Biol ; 85(3-4): 255-63, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16546570

RESUMEN

Integrin-linked kinase (ILK) represents a key component of integrin signaling complexes that functions in concert with multiple binding partners to transmit cues from the extracellular matrix environment to the actin cytoskeleton. Both gain- and loss-of-function approaches to study ILK have confirmed the essential role of this protein in regulating cell-matrix adhesion dynamics and cytoskeletal organization.


Asunto(s)
Citoesqueleto de Actina/fisiología , Uniones Célula-Matriz/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Citoesqueleto de Actina/ultraestructura , Animales , Adhesión Celular , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Humanos , Modelos Biológicos , Modelos Genéticos , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo
12.
Eur J Cell Biol ; 95(11): 475-482, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27461124

RESUMEN

Skin, by nature, is very similar to the Rouquayrol-Denayrouze suit mentioned by Jules Verne in Twenty Thousand Leagues Under the Sea: it allows "to risk (…) new physiological conditions without suffering any organic disorder". Mechanical cues, to the same extent as other environmental parameters, are such "new physiological conditions". Indeed, skin's primary function is to form a protective barrier to shield inner tissues from the external environment. This requires unique mechanical properties as well as the ability to sense mechanical cues from the environment in order to prevent or repair mechanical damages as well as to function as the primary mechanosensory interface of the whole body.


Asunto(s)
Dermis/fisiología , Mecanotransducción Celular/fisiología , Fenómenos Fisiológicos de la Piel , Animales , Humanos
13.
Cancer Metab ; 3: 8, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26322231

RESUMEN

BACKGROUND: Mitochondria are more than just the powerhouse of cells; they dictate if a cell dies or survives. Mitochondria are dynamic organelles that constantly undergo fusion and fission in response to environmental conditions. We showed previously that mitochondria of cells in a low oxygen environment (hypoxia) hyperfuse to form enlarged or highly interconnected networks with enhanced metabolic efficacy and resistance to apoptosis. Modifications to the appearance and metabolic capacity of mitochondria have been reported in cancer. However, the precise mechanisms regulating mitochondrial dynamics and metabolism in cancer are unknown. Since hypoxia plays a role in the generation of these abnormal mitochondria, we questioned if it modulates mitochondrial function. The mitochondrial outer-membrane voltage-dependent anion channel 1 (VDAC1) is at center stage in regulating metabolism and apoptosis. We demonstrated previously that VDAC1 was post-translationally C-terminal cleaved not only in various hypoxic cancer cells but also in tumor tissues of patients with lung adenocarcinomas. Cells with enlarged mitochondria and cleaved VDAC1 were also more resistant to chemotherapy-stimulated cell death than normoxic cancer cells. RESULTS: Transcriptome analysis of mouse embryonic fibroblasts (MEF) knocked out for Vdac1 highlighted alterations in not only cancer and inflammatory pathways but also in the activation of the hypoxia-inducible factor-1 (HIF-1) signaling pathway in normoxia. HIF-1α was stable in normoxia due to accumulation of reactive oxygen species (ROS), which decreased respiration and glycolysis and maintained basal apoptosis. However, in hypoxia, activation of extracellular signal-regulated kinase (ERK) in combination with maintenance of respiration and increased glycolysis counterbalanced the deleterious effects of enhanced ROS, thereby allowing Vdac1 (-/-) MEF to proliferate better than wild-type MEF in hypoxia. Allografts of RAS-transformed Vdac1 (-/-) MEF exhibited stabilization of both HIF-1α and HIF-2α, blood vessel destabilization, and a strong inflammatory response. Moreover, expression of Cdkn2a, a HIF-1-target and tumor suppressor gene, was markedly decreased. Consequently, RAS-transformed Vdac1 (-/-) MEF tumors grew faster than wild-type MEF tumors. CONCLUSIONS: Metabolic reprogramming in cancer cells may be regulated by VDAC1 through vascular destabilization and inflammation. These findings provide new perspectives into the understanding of VDAC1 in the function of mitochondria not only in cancer but also in inflammatory diseases.

14.
Cancer Res ; 74(23): 6878-89, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25267066

RESUMEN

CD98hc (SLC3A2) is the heavy chain component of the dimeric transmembrane glycoprotein CD98, which comprises the large neutral amino acid transporter LAT1 (SLC7A5) in cells. Overexpression of CD98hc occurs widely in cancer cells and is associated with poor prognosis clinically, but its exact contributions to tumorigenesis are uncertain. In this study, we showed that genetic deficiency of CD98hc protects against Ras-driven skin carcinogenesis. Deleting CD98hc after tumor induction was also sufficient to cause regression of existing tumors. Investigations into the basis for these effects defined two new functions of CD98hc that contribute to epithelial cancer beyond an intrinsic effect of CD98hc on tumor cell proliferation. First, CD98hc increased the stiffness of the tumor microenvironment. Second, CD98hc amplified the capacity of cells to respond to matrix rigidity, an essential factor in tumor development. Mechanistically, CD98hc mediated this stiffness sensing by increasing Rho kinase (ROCK) activity, resulting in increased transcription mediated by YAP/TAZ, a nuclear relay for mechanical signals. Our results suggest that CD98hc contributes to carcinogenesis by amplifying a positive feedback loop, which increases both extracellular matrix stiffness and resulting cellular responses. This work supports a rationale to explore the use of CD98hc inhibitors as cancer therapeutics.


Asunto(s)
Carcinogénesis/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Integrinas/metabolismo , Proteínas ras/metabolismo , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinogénesis/patología , Proteínas de Ciclo Celular , Proliferación Celular/fisiología , Células Cultivadas , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Mecanotransducción Celular , Ratones , Fosfoproteínas/metabolismo , Transducción de Señal/fisiología , Piel/metabolismo , Piel/patología , Factores de Transcripción/metabolismo , Microambiente Tumoral/fisiología , Proteínas Señalizadoras YAP , Quinasas Asociadas a rho/metabolismo
15.
J Exp Med ; 210(1): 173-90, 2013 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-23296466

RESUMEN

Skin aging is linked to reduced epidermal proliferation and general extracellular matrix atrophy. This involves factors such as the cell adhesion receptors integrins and amino acid transporters. CD98hc (SLC3A2), a heterodimeric amino acid transporter, modulates integrin signaling in vitro. We unravel CD98hc functions in vivo in skin. We report that CD98hc invalidation has no appreciable effect on cell adhesion, clearly showing that CD98hc disruption phenocopies neither CD98hc knockdown in cultured keratinocytes nor epidermal ß1 integrin loss in vivo. Instead, we show that CD98hc deletion in murine epidermis results in improper skin homeostasis and epidermal wound healing. These defects resemble aged skin alterations and correlate with reduction of CD98hc expression observed in elderly mice. We also demonstrate that CD98hc absence in vivo induces defects as early as integrin-dependent Src activation. We decipher the molecular mechanisms involved in vivo by revealing a crucial role of the CD98hc/integrins/Rho guanine nucleotide exchange factor (GEF) leukemia-associated RhoGEF (LARG)/RhoA pathway in skin homeostasis. Finally, we demonstrate that the deregulation of RhoA activation in the absence of CD98hc is also a result of impaired CD98hc-dependent amino acid transports.


Asunto(s)
Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Queratinocitos/metabolismo , Piel/metabolismo , Cicatrización de Heridas/fisiología , Factores de Edad , Animales , Proteína Tirosina Quinasa CSK , Adhesión Celular/genética , Movimiento Celular/genética , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Epidermis/metabolismo , Epidermis/patología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Folículo Piloso/metabolismo , Homeostasis , Integrinas/metabolismo , Queratinocitos/patología , Ratones , Ratones Transgénicos , Especies Reactivas de Oxígeno/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Fenómenos Fisiológicos de la Piel , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA , Familia-src Quinasas/metabolismo
16.
Methods Mol Biol ; 827: 97-105, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22144270

RESUMEN

RhoGDI1 is one of the three major regulators of the Rho switch along with RhoGEFs and RhoGAPs. RhoGDI1 extracts prenylated Rho proteins from lipid membranes, sequesters them in the cytosol, and prevents nucleotide exchange or hydrolysis. In addition, RhoGDI1 protects prenylated Rho proteins from degradation. Here, we describe techniques to monitor Rho proteins degradation upon depletion of RhoGDI1 and their dependence upon prenylation for degradation.


Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Prenilación de Proteína/fisiología , Proteínas de Unión al GTP rho/metabolismo , Western Blotting , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Lovastatina/farmacología , Prenilación de Proteína/efectos de los fármacos , ARN Interferente Pequeño , Transfección , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico
17.
PLoS One ; 6(2): e17380, 2011 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-21390328

RESUMEN

BACKGROUND: Rho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells. PRINCIPAL FINDINGS: Using an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus. CONCLUSIONS/SIGNIFICANCE: These results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response.


Asunto(s)
Núcleo Celular/metabolismo , Daño del ADN/fisiología , Proteínas Oncogénicas/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Distribución Tisular/efectos de los fármacos
18.
Small GTPases ; 1(1): 65-68, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21686121

RESUMEN

Regulation of the Rho switch has been typically centered on their main regulators, RhoGEFs and RhoGAPs. On the side, RhoGDI proteins have been considered mostly as passive regulators devoid of catalytic activity simply holding Rho proteins in the cytosol. In the May issue of Nature Cell Biology,1 we describe a novel evolutionary conserved function for RhoGDI1 as a chaperoning protein which prevents degradation of prenylated Rho GTPases. The limited amount of RhoGDI1 in cells generates a competitive balance between GTPases in order to prevent their degradation. Therefore, this creates a crosstalk regulatory mechanism of Rho proteins, whereby the level of one Rho protein can affect both the level and activity of the others. For example, overexpression of a single GTPase will promote the degradation and inactivation of all endogenous Rho proteins bound to GDI. These results suggest that some of the conclusions drawn from studies that manipulate Rho protein levels may need to be reevaluated. Here, we discuss some of the consequences of this mechanism on the regulation of Rho proteins, the dissociation of Rho-RhoGDI complexes by GDF and whether this regulation might be extended to other GTPases of the Ras superfamily.

19.
Nat Cell Biol ; 12(5): 477-83, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20400958

RESUMEN

At steady state, most Rho GTPases are bound in the cytosol to Rho guanine nucleotide dissociation inhibitors (RhoGDIs). RhoGDIs have generally been considered to hold Rho proteins passively in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while activating the remaining membrane-bound fraction. Because RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression itself but from additional effects on the levels and activity of other Rho GTPases as a result of competition for binding to RhoGDI1; this may require a re-evaluation of previously published studies that rely exclusively on these techniques.


Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/fisiología , Proteínas de Unión al GTP rho/metabolismo , Línea Celular , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Homeostasis , Humanos , Unión Proteica , Pliegue de Proteína , Prenilación de Proteína , Estabilidad Proteica , Receptor Cross-Talk , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico
20.
Int Rev Cell Mol Biol ; 277: 1-65, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19766966

RESUMEN

Focal adhesions have been intensely studied ever since their discovery in 1971. The last three decades have seen major advances in understanding the structure of focal adhesions and the functions they serve in cellular adhesion, migration, and other biological processes. In this chapter, we begin with a historical perspective of focal adhesions, provide an overview of focal adhesion biology, and highlight recent major advances in the field. Specifically, we review the different types of matrix adhesions and the role different Rho GTPases play in their formation. We discuss the relative contributions of integrin and syndecan adhesion receptors to the formation of focal adhesions. We also focus on new insights gained from studying focal adhesions on biomaterial surfaces and from the growing field of mechanotransduction. Throughout this chapter, we have highlighted areas of focal adhesion biology where major questions still remain to be answered.


Asunto(s)
Adhesiones Focales/metabolismo , Animales , Humanos , Receptores de Superficie Celular/metabolismo , Proteínas de Unión al GTP rho/metabolismo
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