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membrane-ezrin-actin Report Summary

Project ID: 656442
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - membrane-ezrin-actin (Membrane-ezrin-actin interactions mediated by ezrin binding proteins in reconstituted systems)

Reporting period: 2016-01-01 to 2017-12-31

Summary of the context and overall objectives of the project

Ezrin, a member of the ezrin-radixin-moesin (ERM) protein family, plays a crucial role in linking the cellular membranes to the actin cytoskeleton. Ezrin participates in cell shape control that is involved in diverse cellular functions including cell adhesion and migration. It has been shown that ezrin malfunctioning has severe consequences in cancer progression and in embryonic development such as abnormal villus morphogenesis. Thus, it is important to understand how cells precisely regulate the membrane localization and function of ezrin. In cells ezrin is enriched in actin-rich plasma membrane structures such as filopodia and microvilli. In these membrane protrusions ezrin is located at the cytosolic side wherein the membrane has a negative mean curvature. This suggests that ezrin has a strong affinity for negatively curved membranes; in other words, ezrin may be a negative membrane curvature-sensing protein. However, ezrin is also associated with some intracellular vesicles including endosomes, wherein the membranes have a positive mean membrane curvature. Moreover, ezrin is also found at flat regions of the plasma membrane, such as at the cortex-membrane interface and at the surface of membrane blebs. How the same protein can be a positive and a negative membrane curvature sensor is a conundrum that had to be solved. In this project, we aimed to decipher bio-physical mechanisms underlying the enrichment of ezrin on curved membranes. By using cell biology and in vitro approaches combining lipid vesicles and purified ezrin, we showed that ezrin association with curved membranes requires a specific conformation of the protein or interaction with a curvature-sensitive partner.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

- To assess how the phosphorylation influences ezrin conformation and its binding to PIP2 membranes, we purified recombinant wild type ezrin (ezrinWT) and a phosphomimetic mutant where the threonine at position 567 was replaced by an aspartate (ezrinTD), mimicking the open configuration of ezrin.

- We showed that ezrinTD has a higher binding affinity to membranes containing PIP2 (a lipid to which ezrin interacts specifically in cells) compared to that of ezrinWT. The dissociation constant K_d is equal to 1.2 M and 4.2 M for ezrinTD and ezrinWT, respectively.

- By using large unilamellar vesicles (LUVs) combined with cryo-electron microscopy (cryo-EM), to our surprise, we observed that both ezrinWT and ezrinTD assemble in an anti-parallel manner to tether adjacent membranes. Furthermore, the distances measured between the centers of the globular domains of the two opposing ezrin molecules sandwiched between the lipid layers were different: 24.1 ± 1.3 nm for ezrinTD and 28.7 ± 1.2 nm for ezrinWT (error bar represents standard deviation).

- Inspired by the membrane tethering of ezrinWT and ezrinTD observed by cryo-EM, we measured their membrane tethering strengths by dual-micropipette experiments in which two opposing giant unilamellar vesicles (GUVs) were held by micropipettes and tethered by ezrinWT or ezrinTD. We found that the tethering energy of ezrinWT was on average higher than that of ezrinTD.

- We confirmed that the open configuration of ezrinTD can bridge actin filaments to membranes.

- We assessed ezrin binding to positively curved membranes by injecting ezrin adjacent to membrane nano-tubes (10nm-100nm in radius) pulled outward from PIP2-containing GUVs by optical tweezers. We observed that only the phosphomimetic mutant of ezrin, ezrinTD, senses positive membrane curvature, likely due to its different conformation compared to ezrinWT.

- To assess ezrin binding to negatively curved membranes, we performed that same GUV-nano-tubes experiments while encapsulating ezrin inside the GUVs. We showed that neither ezrinWT nor ezrinTD senses negative membrane curvature.

- By cell biology experiments, we identified IRSp53 is a binding partner of ezrin in cells. IRSp53 is involved in the initiation of filopodia and is enriched inside model membrane nanotubes, having negative membrane curvature. We demonstrated that both ezrinWT and ezrinTD interact directly with the I-BAR domain of IRSp53. And this interaction further facilitate the enrichment of ezrinWT or ezrinTD in I-BAR domain decorated membrane tubules.

- Overall, our work reveals new mechanisms, specific conformation or binding to a curvature sensor partner, for targeting curvature insensitive proteins to curved membranes. The results of this work is currently under reviewed in Proceedings of the National Academy of Sciences for publication.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We showed that ezrin by itself does not have preference to bind to negatively curved membranes. Given ezrin’s high abundance in cells, its curvature insensitivity prevents massive membrane deformation. Instead, the interaction with I-BAR domain proteins is required to facilitate the enrichment of ezrinWT or ezrinTD on membrane protrusions. We anticipate that the direct ezrin-I-BAR interaction together with the PIP2 clusters induced by the I-BAR domains synergistically enrich ezrin in the I-BAR domain-induced tubules. Given that IRSp53 contributes to the initiation of filopodia, we propose that IRSp53 recruits ezrin to strengthen the binding of actin filaments to the plasma membrane that in turn facilitates filopodia growth.

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