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Dynamic interactions between membrane and cytoskeleton components are crucial for T cell antigen recognition and subsequent cellular activation. We report here that the membrane-microfilament linker ezrin plays an important role in these processes. First, ezrin relocalizes to the contact area between T cells and stimulatory antigen-presenting cells (APCs), accumulating in F-actin-rich membrane protrusions at the periphery of the immunological synapse. Second, T cell receptor (TCR)-mediated intracellular signals are sufficient to induce ezrin relocalization, indicating that this protein is an effector of TCR signaling. Third, overexpression of the membrane binding domain of ezrin perturbs T cell receptor clustering in the T cell-APC contact area and inhibits the activation of nuclear factor for activated T cells (NF-AT).

Drosophila thoracic mechanosensory bristles originate from cells that are singled out from 'proneural' groups of competent epithelial cells. Neural competence is restricted to individual sensory organ precursors (SOPs) by Delta/Notch-mediated 'lateral inhibition', whereas other cells in the proneural field adopt an epidermal fate. The precursors of the large macrochaetes differentiate separately from individual proneural clusters that comprise about 20-30 cells or as heterochronic pairs from groups of more than 100 cells, whereas the precursors of the small regularly spaced microchaetes emerge from even larger proneural fields. This indicates that lateral inhibition might act over several cell diameters; it was difficult to reconcile with the fact that the inhibitory ligand Delta is membrane-bound until the observation that SOPs frequently extend thin processes offered an attractive hypothesis. Here we show that the extension of these planar filopodia--a common attribute of wing imaginal disc cells--is promoted by Delta and that their experimental suppression reduces Notch signalling in distant cells and increases bristle density in large proneural groups, showing that these membrane specializations mediate long-range lateral inhibition.

Abstract. Overexpression in insect cells of the full coding sequence of the human membrane cytoskeletal linker ezrin (1-586) was compared with that of a NHEterminal domain (ezrin 1-233) and that of a COOHterminal domain (ezrin 310-586). Ezrin (1-586), as well as ezrin (1-233) enhanced cell adhesion of infected Si x ) cells without inducing gross morphological changes in the cell structure. Ezrin (310-586) enhanced cell adhesion and elicited membrane spreading followed by microspike and lamellipodia extensions by mobilization of Sf9 cell actin. Moreover some microspikes elongated into thin processes, up to 200 #m in length, resembling neurite outgrowths by a mechanism requiring microtubule assembly. Kinetics of videomicroscopic and drug-interference studies demonstrated that mobilization of actin was required for tubulin assembly to proceed. A similar phenotype was observed in CHO cells when a comparable ezrin domain was transiently overexpressed. The shortest domain promoting cell extension was localized between residues 373-586. Removal of residues 566-586, involved in in vitro actin binding (Turunen, O., T. Wahlstr6m, and A. Vaheri. 1994. J. Cell Biol. 126:1445-1453, suppressed the extension activity. Coexpression of ezrin (1-233) with ezrin (310-586) in the same insect cells blocked the constitutive activity of ezrin COOH-terminal domain. The inhibitory activity was mapped within ezrin 115 first NH2-terminal residues. We conclude that ezrin has properties to promote cell adhesion, and that ezrin NHe-terminal domain negatively regulates membrane spreading and elongation properties of ezrin COOH-terminal domain.

Ezrin plays a key role in coupling signal transduction to cortical cell organization. This actin-membrane linker undergoes a series of conformational changes that modulate its interactions with various partners and its localization in membrane or cytosolic pools. Its mobility and exchange rates within and between these two pools were assessed by two-photon fluorescence recovery after photobleaching in epithelial cell microvilli. Analysis of ezrin mutants with an altered actin-binding site revealed three ezrin membrane states of different mobilities and exchange properties, reflecting sequential association with membrane components and F-actin in the context of a fast overall turnover. Ezrin is a member of the ezrin͞radixin͞moesin (ERM) family of actin-membrane linkers that play a key role in coupling signal transduction pathways with the maintenance of the cortical cytoskeleton architecture and its dynamic response to external stimuli (1, 2). ERM linkers concentrate in cell-surface structures rich in actin such as microvilli and filopodia, and their impaired expression or inactivation severely alters cell surface morphology, motility, and adhesion (1).Ezrin is present in membrane and soluble pools, and its dynamic localization between these two compartments is an important determinant of its activity. In the cytoplasm, ezrin is mainly present in a so-called ''masked'' form, in which intramolecular interactions between the N and C termini block its binding sites (3). Conformational masking is also achieved by similar interactions in an intermolecular head-to-tail fashion (4). The formation of the fully active state of cytoskeletonmembrane linker of ezrin͞radixin͞moesin (ERM) proteins is thought to be achieved by a succession of conformational changes that involve binding to PIP 2 and membrane͞peripheral proteins (intercellular adhesion molecules, CD44͞43, and EBP50), as well as the phosphorylation of a Thr present in the actin-binding site (5-11). Deactivation of ERM linkers might be triggered by dephosphorylation, because in vivo dephosphorylation correlates with microvilli breakdown (12, 13).The two-way exchange between the cytosolic and membrane pools plays a crucial role, because it controls not only the relative amount of ezrin molecules in the active membrane form but also the relative amount of time spent by each molecule at the membrane and the total flux of ezrin turnover. However, the exact sequence of these events, as well as the lifetimes of the different conformation states, is unknown and poses a fundamental challenge to understanding the morphogenetic properties of ezrin. Key parameters controlling these different states could be either static (relative amounts of active vs. inactive forms at a given time point) or dynamic (residence times, mobilities, fluxes).In the present work, mobility studies resolving ezrin membrane and cytosol contributions have been performed in epithelial LLC-PK1 cells. These cells display, on their apical surface, microvilli that are well characterized ultrastructurally a...

Human recombinant ezrin, or truncated forms, were coated in microtiter plate and their capacity to bind actin determined. F-actin bound ezrin with a K d of 504 ؎ 230 nM and a molecular stoichiometry of 10.6 actin per ezrin. Ezrin bound both ␣-and ␤/␥-actin essentially as F-form. F-actin binding was totally prevented or drastically reduced when residues 534 -586 or 13-30 were deleted, respectively. An actin binding activity was detected in amino-terminal constructs (ezrin 1-310 and 1-333) provided the glutathione S-transferase moiety of the fusion protein was removed. Series of carboxyl-terminal truncations confirmed the presence of this actinbinding site which bound both F-and G-actin. The Fand G-actin-binding sites were differently sensitive to various chemical effectors and distinct specific ezrin antibodies. The internal actin-binding site was mapped between residues 281 and 333. The association of ezrin amino-terminal fragment to full-length ezrin blocked Factin binding to ezrin. It is proposed that, in full-length ezrin, the F-actin-binding site required the juxtaposition of the distal-most amino-and carboxyl-terminal residues of the ezrin molecule.Proteins, located at the interface between the plasma membrane and the cytoskeleton, are essential elements involved in cell plasticity, and are expected to possess association properties tuned by both intra-and extracellular regulators. Ezrin is a protein linker between the cortical skeleton and the plasma membrane (1, 2), and, in polarized epithelial cells, colocalizes with actin predominantly in apical microvilli (1,(3)(4)(5)(6)(7)(8)(9)(10). With talin, ezrin is part of the superfamily of protein 4.1-like proteins sharing a homologous NH 2 -terminal domain (11)(12)(13)(14). With radixin (15, 16) and moesin (17, 18), which share ϳ70% homology, ezrin define the ERM 1 family to which merlin (19,20), is also related. Ezrin NH 2 -terminal domain is reported to interact with the plasma membrane, while the COOH-terminal domain would link the actin cytoskeleton (21). In some cell types, ERMs associate with CD44, a transmembrane receptor for hyaluronan (22) through regulation by the rho GTPase pathway (23).In multicellular organisms, the relative level of ERM expression is tissue specific (4, 16, 24 -28). However, ERMs are coexpressed and play a redundant role in most cell lines since major phenotypic alterations were only observed when the expression of all three ERMs was down-regulated (29). The subcellular redistribution of ERMs upon cell has been best studied in gastric parietal cells (2,8). The elongation of ezrin-enriched secretory microvilli is linked to ezrin phosphorylation on serine and threonine residues (30), and ezrin acts as a protein kinase A anchoring protein in these cells (31). Ezrin is also tyrosinephosphorylated (32-35) on two major sites (36) and a differential sensitivity to various growth factors exists between ERMs (37).Ezrin can self-associate and, through interactions between two domains, the N-and C-ERMADs, form dimers or oligomers, a proper...

SummaryThe ezrin, radixin and moesin (ERM) proteins regulate cell membrane architecture in several cellular contexts. Current models propose that ERM activation requires a PtdIns(4,5)P 2 -induced conformational change, followed by phosphorylation of a conserved threonine. However, how these inputs contribute in vivo to orchestrate ERM activation is poorly understood. We addressed this issue by evaluating the contribution of PtdIns(4,5)P 2 and phosphorylation to the regulation of moesin during Drosophila development. Unexpectedly, we found that a form of moesin that cannot be phosphorylated displayed significant activity and could substitute for the endogenous product during wing morphogenesis. By contrast, we also show that PtdIns(4,5)P 2 binding is essential for moesin recruitment to the membrane and for its subsequent phosphorylation. Our data indicate that PtdIns(4,5)P 2 acts as a dosing mechanism that locally regulates ERM membrane recruitment and activation, whereas cycles of phosphorylation and dephosphorylation further control their activity once they have reached the cell cortex.