Marianne Martin

ERM proteins in cell adhesion and membrane dynamics

Ezrin, radixin and moesin, collectively known as the ERM proteins, are a group of closely related membrane-cytoskeleton linkers that regulate cell adhesion and cortical morphogenesis. ERM proteins can self-associate through intra- and inter-molecular interactions, and these interactions mask several binding sites on the proteins. ERM activation involves unfolding of the molecule, and allows the protein to bind to plasma membrane components either directly, or indirectly through linker proteins. The discovery that the tumour-suppressor NF2, also known as merlin/schwannomin, is related to ERM proteins has added a new impetus to investigations of their roles. This review discusses current understanding of the structure and function of members of the ERM family of proteins.

Ezrin is a cyclic AMP-dependent protein kinase anchoring protein.

cAMP‐dependent protein kinase (A‐kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A‐kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A‐kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin‐associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin‐related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14‐amino‐acid amphipathic α‐helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409–438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RIIimmunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A‐kinase to a region near the secretory canaliculus.

Calpain 3 deficiency is associated with myonuclear apoptosis and profoundperturbation of the IκBα/NF-κB pathway in limb-girdle musculardystrophy type 2A

Nature Med. 5, 503– 511 (1999). The top left corner of Fig. 1b on page 505 was cropped so that you could not view the calpain 3-stained nuclei in endomysia space. The corrected figure is shown below. We regret this error.

The Membrane-Microfilament Linker Ezrin Is Involved in the Formation of the Immunological Synapse and in T Cell Activation

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).

Delta-promoted filopodia mediate long-range lateral inhibition in Drosophila

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.

A Dual Involvement of the Amino-terminal Domain of Ezrin in F- and G-actin Binding

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 α- and β/γ-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 actin-binding site which bound both F- and G-actin. The F- and 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 F-actin 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.

Molecular analysis of microscopic ezrin dynamics by two-photon FRAP

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.

Membrane targeting of protein tyrosine phosphatase PTPL1 through its FERM domain via binding to phosphatidylinositol 4,5-biphosphate

PTPL1 is the largest known cytoplasmic protein tyrosine phosphatase (PTP) containing a FERM (four point-1, ezrin, radixin and moesin) domain. Enzyme localization and PTP-substrate specificity are thought to play crucial roles in the regulation of PTP activity, which determines their functions. Here we report that PTPL1 is predominantly localized at the apical face of plasma membrane enriched in dorsal microvilli when expressed in HeLa cells. By comparing localization of the full-length enzyme with its FERM domain or FERM-deleted PTPL1 construct, we first concluded that PTPL1-FERM domain is necessary and sufficient to address the wild-type enzyme at the membrane. Two potential phosphatidylinositol 4,5-biphosphate [PtdIns(4,5)P2]-binding motifs were identified within the PTPL1-FERM sequence. We further showed that mutation of both sites altered PTPL1 localization similarly to FERM domain deletion, and impaired its subcellular distribution as confirmed biochemically by cell-fractionation experiments. Using protein-lipid overlays, we demonstrated an interaction of the FERM domain of PTPL1 with PtdIns(4,5)P2, which was lost after mutation of potential PtdIns(4,5)P2-binding motifs. Moreover, neomycin, which masks PtdIns(4,5)P2 polar heads, was shown to decrease by 50% the association of PTPL1 with the cytoskeletal fraction. These results identify the crucial role of the FERM domain in PTPL1 intracellular targeting and demonstrate that localization of PTPL1 is regulated by phosphoinositide metabolism.

Differential roles of PtdIns(4,5)P2 and phosphorylation in moesin activation during Drosophila development

The 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)P2-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)P2 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)P2 binding is essential for moesin recruitment to the membrane and for its subsequent phosphorylation. Our data indicate that PtdIns(4,5)P2 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.

Exosomal sorting of the cytoplasmic domain of bovine leukemia virus TM Env protein.

Exosomes are small membrane vesicles that are released into the extracellular compartment as a consequence of fusion of multivesicular endosomes with the plasma membrane. To unravel the molecular basis of protein sorting into exosomes, we have made a chimeric protein containing the cytosolic domain of the transmembrane subunit of the viral Env protein of BLV and the ectodomain of CD8 (CDTM‐BLV–CD8). When expressed in K562 cells known to constitutively secrete exosomes, the chimera was found to be very efficiently targeted to the released vesicles. Very interestingly, the cytosolic domain of the Env protein contains peptide motifs potentially recognized by components of the ESCRT machinery that could be related to chimera sorting into the vesicles. Then, quantifying the chimera secretion, we investigated the site of exosome biogenesis in K562 cells using a pharmacological approach. We present different arguments indicating that CDTM‐BLV–CD8‐containing exosomes are likely formed from a recycling endosomal/TGN compartment.