The C-terminal domain of EFA6A interacts directly with F-actin and assembles F-actin bundles

Eric Macia,Hong Wang,Christophe Le Clainche,Frederic Luton,Michel Franco,Mariagrazia Partisani,Sandra Lacas-Gervais

doi:10.1038/s41598-019-55630-9

Abstract

The Arf6-specific exchange factor EFA6 is involved in the endocytic/recycling pathway for different cargos. In addition EFA6 acts as a powerful actin cytoskeleton organizer, a function required for its role in the establishment of the epithelial cell polarity and in neuronal morphogenesis. We previously showed that the C-terminus of EFA6 (EFA6-Ct) is the main domain which contributes to actin reorganization. Here, by in vitro and in vivo experiments, we sought to decipher, at the molecular level, how EFA6 controls the dynamic and structuring of actin filaments. We showed that EFA6-Ct interferes with actin polymerization by interacting with and capping actin filament barbed ends. Further, in the presence of actin mono-filaments, the addition of EFA6-Ct triggered the formation of actin bundles. In cells, when the EFA6-Ct was directed to the plasma membrane, as is the case for the full-length protein, its expression induced the formation of membrane protrusions enriched in actin cables. Collectively our data explain, at least in part, how EFA6 plays an essential role in actin organization by interacting with and bundling F-actin.

Highlights

The ADP-ribosylation factor (Arf) family is comprised of 6 isoforms in mammalian cells, and is further subdivided into three classes based on primary sequence homology: Class I (Arf1/3), II (Arf4/5) and III (Arf[6])
We had previously shown that the C-terminal domain of EFA6A (EFA6-Ct) was required in vivo to reorganize the actin cytoskeleton and to promote the lengthening of actin-rich plasma membrane extensions[20,22,23]
The formation of actin bundles was visible by fluorescent microscopy after the addition of FITC-labeled phalloïdin (Fig. 1A compare h to g)

Summary

Introduction

The ADP-ribosylation factor (Arf) family is comprised of 6 isoforms in mammalian cells, and is further subdivided into three classes based on primary sequence homology: Class I (Arf1/3), II (Arf4/5) and III (Arf[6]). The human EFA6 family contains four tissue-specific EFA6 isoforms (EFA6A to D), which are encoded by four different genes They share a common domain organization structure consisting of a highly divergent N-terminal domain with unknown functions, a central catalytic Sec 7 domain, a PH domain responsible for the plasma membrane localization by interacting with PIP2 and a C-terminus (Ct) containing a coiled-coil domain surrounded by two proline rich regions and involved in actin remodeling. Despite their homology and overlapping tissue distribution, it remains to determine whether the different EFA6 proteins play specific roles. At the ultrastructural level, EFA6A-PH-Ct appears to connect actin filaments to the plasma membrane in these filopodia-like structures that it generates

Objectives

Results

Conclusion