Multiple roles for RhoA during T cell transendothelial migration

Abstract

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.

Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells

“…This site has been previously reported to regulate ARHGEF2 activity (40). Moreover, ARHGEF2-dependent RhoA activity also has been shown to regulate the uropod of migrating T cells (71), in accordance with the report by Vang et al (88) that PDE8 inhibition caused a decrease in T-cell motility. In the current study, the combination of PDEs 1, 7, and 8 showed the largest increase in ARHGEF2 phosphorylation.…”

“…A number of the biological processes identified by GO analysis have been previously reported to be regulated by cAMP. These include mRNA splicing (64)(65)(66), spindle organization (67,68), fibroblast migration (69,70), lamellipodium assembly (71,72), apoptosis (73)(74)(75)(76), ATM signaling (77,78), gene silencing via microRNA (79)(80)(81)(82), T-cell selection (83), and cytoskeletal reorganization (84). This study suggests which molecular substrates might participate in the regulation of these processes.…”

The effects of modifying RhoA and Rac1 activities on heterotypic contact inhibition of locomotion

“…While a constitutively active form of Rac1 persists at cell contacts, a loss of both Rac1 and active Rac1 from newly formed MDCK cell contacts occurs within 5 min of E-cadherin recruitment [39]. RhoA activity would then increase and could result in membrane retraction via ROCK and myosin light chain (p-MLC) phosphorylation [40]. The mechanism determining which of the two RhoA pathways is activated at the leading edge is not known but may depend on the localization of effectors and/or the levels of RhoA activity [40].…”

“…RhoA activity would then increase and could result in membrane retraction via ROCK and myosin light chain (p-MLC) phosphorylation [40]. The mechanism determining which of the two RhoA pathways is activated at the leading edge is not known but may depend on the localization of effectors and/or the levels of RhoA activity [40].…”

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor, Modulation by Allosteric Factors, and the Uncompetitive Inhibitor Brefeldin A

“…Low molecular weight guanine-nucleotide-binding (G) proteins of the Ras superfamily, also known as small GTPases for their GTP hydrolysis activity, are single-subunit proteins activated by diverse extracellular stimuli and implicated in a number of crucial cellular processes including cell proliferation, differentiation, apoptosis, cytoskeletal organization, cell polarity, and vesicular trafficking (1)(2)(3)(4). G proteins act as molecular switches and cycle between a GDP-bound inactive and a GTPbound active conformation that regulate their ability to interact with downstream effectors and regulatory proteins.…”