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

Themuscle 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 humanMLP or its ablation in transgenic mice pro-motes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying mo-lecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bun-dles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown howMLP cross-links actin filaments into both unipolar andmixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecu-lar fluorescence complementation (BiFC) assays revealedMLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link betweenMLP self-associa-tion 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 mainte-nance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking. The three vertebrate cysteine-rich proteins (CRP1, CRP2, andCRP3, the last also known as MLP, for muscle LIM protein) define a subset of LIM domain-containing proteins which have been associated with a wide range of cellular processes, includ