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

Abstract

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.

Quantification of integrin receptor agonism by fluorescence lifetime imaging

“…Data were analysed as previously described (Parsons et al, 2005;Prag et al, 2007). Briefly, bulk measurements of FRET efficiency (i.e.…”

Rac regulates the interaction of fascin with protein kinase C in cell migration

“…Data were analysed as previously described (Parsons et al, 2005;Prag et al, 2007). Briefly, bulk measurements of Forster resonance energy transfer (FRET) efficiency (i.e.…”

Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion

“…Dbl expression in mammary epithelial cells has been previously linked to transformation and stimulation of motility. [36][37][38] In this work we provide evidence that Dbl oncogene causes morphological changes, increases migratory behavior, and promotes transformation of MCF-10 A cells. Cells expressing Dbl also exhibit increase in Cdc42, Rac, and Rho activation in the absence of growth factor stimulation, compatible with the constitutive activation of GTPase exchange activity of Dbl oncogene.…”

“…One £ 10 5 cells were resuspended in serum-free medium and added to the upper chamber. Inserts, previously coated with fibronectin on the lower side, were incubated for 1, 2 and 4 h at 37 C with 700 ml of complete medium in the lower chamber. Cells on the inside of the inserts were removed with a cotton swab, and cells on the underside of the insert were fixed and stained with May-Grunwald/Giemsa.…”