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.

XIRP2, an Actin-Binding Protein Essential for Inner Ear Hair-Cell Stereocilia

“…Both the long isoform1 and the short isoforms are predicted to include a LIM domain (http://prosite.expasy.org/). In cardiomyocytes, Muscle LIM Protein self-associates with one of its LIM domains and binds actin filaments with another (Hoffmann et al, 2014). Thus XIRP2 short isoforms may interact with each other and bind actin filaments.…”

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics

“…Briefly, increasing amounts (0.2 to 8 µM) of TWD1 1-180 or TWD1 1-339 protein were incubated with prepolymerized F-actin (4 µM) under different pH conditions in the presence or absence of CaCl 2 (6303 mM) and centrifuged at 150,000g (Hoffmann et al, 2014a). The resulting pellet and supernatant fractions were analyzed by SDS-PAGE and Coomassie blue staining (or immunoblot analyses using anti-TWD1; Wang et al, 2013).…”

“…The resulting pellet and supernatant fractions were analyzed by SDS-PAGE and Coomassie blue staining (or immunoblot analyses using anti-TWD1; Wang et al, 2013). The influence of TWD1 on AF polymerization kinetics was investigated in classical fluorimetric assays using pyrene-labeled actin filaments (3 mM) with 12 mM TWD1 1-339 6 12 mM NPA (Hoffmann et al, 2014a). The increase in pyrene fluorescence accompanying actin polymerization was recorded over 1000 s using a PTI QM-4 QuantaMaster fluorimeter.…”

“…For filament lifetime, the definition of Henty et al (2011) was used. Actin bundling and debundling frequencies were analyzed as described by Hoffmann et al (2014a). At least 100 filaments from at least 20 different cells in 10 or more hypocotyls were selected.…”

Animal Models of Congenital Cardiomyopathies Associated With Mutations in Z-Line Proteins

“…In addition, MLP contains a nuclear localization signal (NLS) (Boateng et al, ) and shuttles to the nucleus, where it interacts with MyoD, myogenin, and myogenic regulatory factor 4 (MRF4) (Kong et al, ). MLP has been shown to be involved in various processes, including (i) maintenance of cytoskeletal structure through its interaction with various cytoskeletal proteins and by acting as a scaffolding protein (Arber et al, ; Knöll et al, , ); (ii) actin cytoskeleton dynamics through binding to filamentous actin (F‐actin) (Hoffmann et al, ) and cofilin‐2 (Papalouka et al, ); (iii) mechanosensation and mechanotransduction (Knöll et al, ; Boateng et al, ; Boateng et al, ), although this is controversial (Gehmlich et al, ; Gunkel et al, ; Gehmlich et al, ); (iv) calcium handling and myofilament contractility (Esposito et al, ; Su et al, ; Gupta et al, ; Kemecsei et al, ); and (v) myofibrillogenesis, promoting myogenic differentiation (Arber et al, ; Kong et al, ; Vafiadaki et al, ). The critical role of MLP for normal cardiac function is highlighted by the development of cardiac hypertrophy followed by DCM and progressive heart failure in MLP knockout (KO) mice (Arber et al, ; Esposito et al, ) as well as the association of a number of CSRP3 mutations with human DCM (Knöll et al, ; Mohapatra et al, ; Hershberger et al, ) and HCM (Geier et al, ; Bos et al, ; Geier et al, ).…”