Earlier work (2002–2012) on RhoGDI1, Rho GTPase crosstalk and integrin-linked kinase.
Between 2002 and 2012, during my PhD and postdoctoral work (including a postdoctoral fellowship at the University of North Carolina at Chapel Hill), I worked on the regulation of Rho GTPases, small molecular switches that control cytoskeletal organization and cell adhesion.
At UNC, this work centered on RhoGDI1, a regulator of the Rho GTPase cycle. We showed that RhoGDI1 controls the crosstalk, degradation and activity of Rho GTPases (Boulter et al., 2010), with a companion review discussing this regulatory mechanism (Garcia-Mata et al., 2011) and a commentary putting it in the context of the broader Rho GTPase field (Boulter & Garcia-Mata, 2010). We also reviewed alternative, non-canonical mechanisms of Rho GTPase regulation (Boulter et al., 2012) and described experimental approaches to study RhoGDI1-dependent degradation of Rho GTPases (Boulter & Garcia-Mata, 2012). Related work from the same lab addressed the nuclear localization and activation of RhoA (Dubash et al., 2011), the role of RhoG in endothelial cells (van Buul et al., 2007), and the structure and function of focal adhesions (Dubash et al., 2009).
Earlier, during my PhD, I worked on integrin-linked kinase (ILK) and its role in cell-matrix adhesion dynamics, including its regulation of Rac-1 (Boulter et al., 2006), its function as part of a modular signaling platform (Boulter & Van Obberghen-Schilling, 2006), and its requirement for the assembly of matrix-forming adhesions in endothelial cells (Vouret-Craviari et al., 2004). This period also included work on Rho GTPase and Src signalling downstream of thrombin and sphingosine-1-phosphate in endothelial cells (Vouret-Craviari et al., 2002).
This earlier work laid the foundations of my current interest in RhoA regulation.
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.
@article{boulter2012off,title={Off the beaten paths: alternative and crosstalk regulation of Rho GTPases},author={Boulter, Etienne and Estrach, Soline and Garcia-Mata, Rafael and Féral, Chloé C},journal={The FASEB Journal},volume={26},number={2},pages={469–479},year={2012},month=feb,doi={10.1096/fj.11-192252},publisher={Wiley},}
Methods Mol Biol
Analysis of the Role of RhoGDI1 and Isoprenylation in the Degradation of RhoGTPases
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.
@incollection{boulter2012analysis,title={Analysis of the Role of RhoGDI1 and Isoprenylation in the Degradation of RhoGTPases},author={Boulter, Etienne and Garcia-Mata, Rafael},booktitle={Rho GTPases},pages={97–105},year={2012},doi={10.1007/978-1-61779-442-1_7},publisher={Springer New York},}
2011
Nat Rev Mol Cell Biol
The ‘invisible hand’: regulation of RHO GTPases by RHOGDIs
Rafael Garcia-Mata, Etienne Boulter, and Keith Burridge
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.
@article{garciamata2011invisible,title={The ‘invisible hand’: regulation of RHO GTPases by RHOGDIs},author={Garcia-Mata, Rafael and Boulter, Etienne and Burridge, Keith},journal={Nature Reviews Molecular Cell Biology},volume={12},number={8},pages={493–504},year={2011},month=jul,doi={10.1038/nrm3153},publisher={Springer Science and Business Media LLC},}
BackgroundRho 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 findingsUsing 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/significanceThese 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.
@article{dubash2011small,title={The Small GTPase RhoA Localizes to the Nucleus and Is Activated by Net1 and DNA Damage Signals},author={Dubash, Adi D and Guilluy, Christophe and Srougi, Melissa C and Boulter, Etienne and Burridge, Keith and García-Mata, Rafael},journal={PLoS ONE},volume={6},number={2},pages={e17380},year={2011},month=feb,doi={10.1371/journal.pone.0017380},publisher={Public Library of Science (PLoS)},}
2010
Nat Cell Biol
Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1
Etienne Boulter, Rafael Garcia-Mata, Christophe Guilluy, and 4 more authors
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.
@article{boulter2010regulation,title={Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1},author={Boulter, Etienne and Garcia-Mata, Rafael and Guilluy, Christophe and Dubash, Adi and Rossi, Guendalina and Brennwald, Patrick J and Burridge, Keith},journal={Nature Cell Biology},volume={12},number={5},pages={477–483},year={2010},month=apr,doi={10.1038/ncb2049},publisher={Springer Science and Business Media LLC},}
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.
@article{boulter2010rhogdi,title={RhoGDI: A rheostat for the Rho switch},author={Boulter, Etienne and Garcia-Mata, Rafael},journal={Small GTPases},volume={1},number={1},pages={65–68},year={2010},month=jul,doi={10.4161/sgtp.1.1.12990},publisher={Informa UK Limited},}
2009
Int Rev Cell Mol Biol
Chapter 1. Focal adhesions: new angles on an old structure
Adi D Dubash, Marisa M Menold, Thomas Samson, and 4 more authors
In International Review of Cell and Molecular Biology, 2009
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.
@incollection{dubash2009chapter,title={Chapter 1. Focal adhesions: new angles on an old structure},author={Dubash, Adi D and Menold, Marisa M and Samson, Thomas and Boulter, Etienne and García-Mata, Rafael and Doughman, Renee and Burridge, Keith},booktitle={International Review of Cell and Molecular Biology},pages={1–65},year={2009},doi={10.1016/s1937-6448(09)77001-7},publisher={Elsevier},}
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.
@article{vanbuul2007rhog,title={RhoG regulates endothelial apical cup assembly downstream from ICAM1 engagement and is involved in leukocyte trans-endothelial migration},author={van Buul, Jaap D and Allingham, Michael J and Samson, Thomas and Meller, Julia and Boulter, Etienne and García-Mata, Rafael and Burridge, Keith},journal={The Journal of Cell Biology},volume={178},number={7},pages={1279–1293},year={2007},month=sep,doi={10.1083/jcb.200612053},publisher={Rockefeller University Press},}
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.
@article{boulter2006regulation,title={Regulation of cell‐matrix adhesion dynamics and Rac‐1 by integrin linked kinase},author={Boulter, Etienne and Grall, Dominique and Cagnol, Sébastien and Van Obberghen-Schilling, Ellen},journal={The FASEB Journal},volume={20},number={9},pages={1489–1491},year={2006},month=may,doi={10.1096/fj.05-4579fje},publisher={Wiley},}
Eur J Cell Biol
Integrin-linked kinase and its partners: A modular platform regulating cell–matrix adhesion dynamics and cytoskeletal organization
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.
@article{boulter2006integrin,title={Integrin-linked kinase and its partners: A modular platform regulating cell–matrix adhesion dynamics and cytoskeletal organization},author={Boulter, Etienne and Van Obberghen-Schilling, Ellen},journal={European Journal of Cell Biology},volume={85},number={3-4},pages={255–263},year={2006},month=apr,doi={10.1016/j.ejcb.2005.09.002},publisher={Elsevier BV},}
Integrins play a key role in regulating endothelial cell survival, migration and differentiated function during angiogenic blood-vessel remodeling. Integrin-linked kinase (ILK) is a multidomain protein that interacts with the cytoplasmic tail of integrin beta subunits and is thought to participate in integrin-mediated signal transduction. We report here that attenuation of ILK expression in cultured bovine aortic endothelial cells by RNA interference had marked effects on surface distribution of alpha5beta1 integrin and the organization of cell-matrix adhesions characterized by the disappearance of fibrillar (3D-like) adhesions that are rich in alpha5beta1 and paxillin, and associated fibrillar fibronectin matrix. This defect was not caused by a decrease in fibronectin mRNA levels or by intracellular retention of the protein. Adhesion to surface-adsorbed matrix proteins based on beta1 and beta3 integrin was enhanced following ILK depletion, whereas cell spreading, migration and multilayer alignment into capillary-like structures on Matrigel were impaired. We conclude that ILK is an important regulator of the endothelial phenotype and vascular network formation by directing the assembly and/or maturation of alpha5beta1-competent matrix-forming adhesions.
@article{vouretcraviari2004ilk,title={ILK is required for the assembly of matrix-forming adhesions and capillary morphogenesis in endothelial cells},author={Vouret-Craviari, Valérie and Boulter, Etienne and Grall, Dominique and Matthews, Cédric and Van Obberghen-Schilling, Ellen},journal={Journal of Cell Science},volume={117},number={19},pages={4559–4569},year={2004},month=sep,doi={10.1242/jcs.01331},publisher={The Company of Biologists},}
Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.
@article{vouretcraviari2002distinct,title={Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells},author={Vouret-Craviari, Valérie and Bourcier, Christine and Boulter, Etienne and van Obberghen-Schilling, Ellen},journal={Journal of Cell Science},volume={115},number={12},pages={2475–2484},year={2002},month=jun,doi={10.1242/jcs.115.12.2475},publisher={The Company of Biologists},}