Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

Abstract

The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00651-14.

ACKNOWLEDGMENTS

This work was supported by the National Research Fund (FNR; C10/BM/784171-HUMCRP), the Fondation Cancer (FC/2013/03), and the Ministry of Culture, Higher Education and Research (REC-LBMV-20100902) of Luxembourg.

We are grateful to Tom Kerppola (University of Michigan Medical School) and Chang-Deng Hu (Purdue University, USA) for sharing BiFC plasmids, pFlag-Cerulean and pHA-Venus (A206K and I152L). We thank David Kovar (University of Chicago) and Simone Niclou (CRP-Santé, Luxembourg) for the gift of recombinant fascin and C2C12 cells, respectively. We also thank Petr Nazarov (CRP-Santé, Luxembourg) for his support in statistical analyses and Angela Tavares Furtado for technical assistance.