The LIM Domains of WLIM1 Define a New Class of Actin Bundling Modules

Clément Thomas,Flora Moreau,Monika Dieterle,Céline Hoffmann,Sabrina Gatti,Christina Hofmann,Marleen Van Troys,Christophe Ampe,André Steinmetz

doi:10.1074/jbc.m703691200

Abstract

Actin filament bundling, i.e. the formation of actin cables, is an important process that relies on proteins able to directly bind and cross-link subunits of adjacent actin filaments. Animal cysteine-rich proteins and their plant counterparts are two LIM domain-containing proteins that were recently suggested to define a new family of actin cytoskeleton regulators involved in actin filament bundling. We here identified the LIM domains as responsible for F-actin binding and bundling activities of the tobacco WLIM1. The deletion of one of the two LIM domains reduced significantly, but did not entirely abolish, the ability of WLIM1 to bind actin filaments. Individual LIM domains were found to interact directly with actin filaments, although with a reduced affinity compared with the native protein. Variants lacking the C-terminal or the inter-LIM domain were only weakly affected in their F-actin stabilizing and bundling activities and trigger the formation of thick cables containing tightly packed actin filaments as does the native protein. In contrast, the deletion of one of the two LIM domains negatively impacted both activities and resulted in the formation of thinner and wavier cables. In conclusion, we demonstrate that the LIM domains of WLIM1 are new autonomous actin binding and bundling modules that cooperate to confer WLIM1 high actin binding and bundling activities.

Highlights

Binding proteins (ABPs).2 ABPs bind directly to G-actin and/or actin filaments to promote actin filament nucleation, polymerization/depolymerization, severing, stabilization, capping, as well as the assembly of actin networks and bundles [1, 2]
To further dissect the mechanism by which WLIM1 binds to, stabilizes, and bundles actin filaments, recombinant proteins were expressed in Escherichia coli with a His6 tag at the C terminus, affinity purified on a nickel-nitrilotriacetate-agarose matrix (Fig. 1B), and used in a series of in vitro experiments
The vast majority of the cells exhibited a diffuse fluorescent pattern or an only weak actin cytoskeleton labeling as illustrated in Fig. 2B for a WLIM1⌬LIM1-GFP expressing cell. Together these observations indicate that the interLIM spacer and the C-terminal domain are not essential for actin cytoskeleton targeting, whereas both LIM domains are important for efficient actin binding activity

Summary

Introduction

Binding proteins (ABPs).2 ABPs bind directly to G-actin and/or actin filaments to promote actin filament nucleation, polymerization/depolymerization, severing, stabilization, capping, as well as the assembly of actin networks and bundles [1, 2]. To better characterize the mechanism of action of WLIM1 and define the critical domains conferring the protein F-actin binding, stabilizing, and bundling activities, we generated four deletion variants (WLIM1⌬LIM1, WLIM1⌬IL, WLIM1⌬LIM2, and WLIM1⌬Ct) as well as two single LIM domains (LIM1 and LIM2).

Results

Conclusion