Spatially Distinct Binding of Cdc42 to PAK1 and N-WASP in Breast Carcinoma Cells

Parsons, Maddy; Monypenny, James; Ameer-Beg, Simon; Millard, Thomas H.; Machesky, Laura M.; Peter, Matthias; Keppler, Melanie D.; Schiavo, Giampietro; Watson, Rose; Chernoff, Jonathan; Zicha, Daniel; Vojnovic, B.; Ng, Tony

doi:10.1128/mcb.25.5.1680-1695.2005

Cited by 86 publications

(83 citation statements)

References 58 publications

(47 reference statements)

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“…Acquisition times on the order of 300 s at low excitation power were used to achieve sufficient photon statistics for fitting, avoiding either pulse pile-up or significant photobleaching. Data were analyzed as previously described (50,51). The FRET efficiency is related to the molecular separation of donor and acceptor and the fluorescence lifetime of the interacting fraction by…”

Section: Methodsmentioning

confidence: 99%

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

Rzeniewicz

Newe

Gallardo

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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103

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L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/ CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.

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Section: Methodsmentioning

confidence: 99%

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

Rzeniewicz

Newe

Gallardo

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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103

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“…28 A similar approach could be used to examine the spatio-temporal binding of RhoA to its effectors mDIA1 and ROCK at the leading edge of migrating T cells, in order to determine their contributions to extension and retraction.…”

Section: A Possible Role For Rhoa In Integrin Clustersmentioning

confidence: 99%

Multiple roles for RhoA during T cell transendothelial migration

Heasman

Ridley

2010

Small GTPases

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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 mdia1 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. IntroductionDuring inflammation leukocytes are recruited from the vasculature into tissues using a specialized migratory pathway. Rho GTPase family proteins are key regulators of cytoskeletal dynamics and cell migration, 3 and thus would be expected to contribute to the process of TEM. Most Rho GTPases cycle between an inactive, GDP-bound form and an active, GTP-bound form, which interacts with and activates downstream targets. Rho GTPases are stimulated by guanine nucleotide exchange factors (GEFs), which stimulate exchange of GDP for GTP and inactivated by GTPaseactivating proteins (GAPs), which stimulate GTP hydrolysis.In our recent paper we used an RNAi screen to identify which Rho GTPases are important for T cell TEM. 4 We found that knockdown of RhoA induced the strongest inhibition of this process and so subsequently examined the role of RhoA in detail during both T cell crawling on EC and subsequent TEM. Significantly, active RhoA was observed at both the front and back of polarized T cells as they crawled on EC and transmigrated, as well as in dynamic puncta in the basal membrane and filopodia extending beneath the endothelium of transmigrating cells ( fig. 1). Here we discuss the implications of these findings in the context of current understanding of RhoA signaling and the potential function of these cellular processes in T-cell migration. Extra View to: Heasman SJ, Carlin LM, Cox S, Ng T, Ridley AJ. Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration.

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“…GST-Dbl(DH/PH) was subcloned into pGEX-KG from pRK5-myc-Dbl using BamHI. The PAK1-green fluorescent protein (GFP) construct was previously described (Parsons et al, 2005). Onco-Dbl (pRK5-myc-Dbl) and FGD1 (pRK5-myc-FGD1) were generous gifts from Professor Alan Hall (MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, London, United Kingdom).…”

Section: Plasmid Constructsmentioning

confidence: 99%

“…After extensive washes in lysis buffer, samples were resuspended in Laemmli sample buffer containing SDS. Cdc42 and Rac1 GTPase activity assays were performed as previously described (Parsons et al, 2005) using GST-PBD-PAK1. Rho GTPase activity assays were performed using GST-Rhotekin agarose (Upstate).…”

Section: Protein Purification and In Vitro Pulldown Assaysmentioning

confidence: 99%

“…We therefore examined the functional behavior of Cdc42 and Rac in MDA-MB-231 cells in the presence of full-length ezrin or N-ERMAD(E244K). Cells expressing PAK1-GFP and Cdc42-myc in the absence or presence of either full-length ezrin or N-ERMAD(E244K) were left unstained or were labeled after fixation with an anti-myc-Cy3 IgG and then subjected to multiphoton FLIM to measure FRET efficiency between GFP and Cy3 (Parsons et al, 2005). The detection of FRET between a GFP donor and a Cy3 by FLIM requires a spatial separation between the fluorophores of no more than 9 nm (Bastiaens and Jovin, 1996) and results in a shortening of the GFP (donor) fluorescence lifetime ( ).…”

Section: N-ermad(e244k) Specifically Affects the Activity Of The Smalmentioning

confidence: 99%

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Activated Ezrin Promotes Cell Migration through Recruitment of the GEF Dbl to Lipid Rafts and Preferential Downstream Activation of Cdc42

Prag

Parsons

Keppler

et al. 2007

MBoC

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Establishment of polarized cell morphology is a critical factor for migration and requires precise spatial and temporal activation of the Rho GTPases. Here, we describe a novel role of the actin-binding ezrin/radixin/moesin (ERM)-protein ezrin to be involved in recruiting Cdc42, but not Rac1, to lipid raft microdomains, as well as the subsequent activation of this Rho GTPase and the downstream effector p21-activated kinase (PAK)1, as shown by fluorescence lifetime imaging microscopy. The establishment of a leading plasma membrane and the polarized morphology necessary for random migration are also dependent on ERM function and Cdc42 in motile breast carcinoma cells. Mechanistically, we show that the recruitment of the ERM-interacting Rho/Cdc42-specific guanine nucleotide exchange factor Dbl to the plasma membrane and to lipid raft microdomains requires the phosphorylated, active conformer of ezrin, which serves to tether the plasma membrane or its subdomains to the cytoskeleton. Together these data suggest a mechanism whereby precise spatial guanine nucleotide exchange of Cdc42 by Dbl is dependent on functional ERM proteins and is important for directional cell migration.

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Spatially Distinct Binding of Cdc42 to PAK1 and N-WASP in Breast Carcinoma Cells

Cited by 86 publications

References 58 publications

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

Multiple roles for RhoA during T cell transendothelial migration

Activated Ezrin Promotes Cell Migration through Recruitment of the GEF Dbl to Lipid Rafts and Preferential Downstream Activation of Cdc42

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