Association of emerin with nuclear and cytoplasmic actin is regulated in differentiating myoblasts

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Abstract

Emerin is a nuclear envelope protein whose biological function remains to be elucidated. Mutations of emerin gene cause the Emery-Dreifuss muscular dystrophy, a neuromuscular disorder also linked to mutations of lamin A/C. In this paper, we analyze the interaction between emerin and actin in differentiating mouse myoblasts. We demonstrate that emerin and lamin A/C are bound to actin at the late stages of myotube differentiation and in mature muscle. The interaction involves both nuclear α and β actins and cytoplasmic actin. A serine–threonine phosphatase activity markedly increases emerin–actin binding even in cycling myoblasts. This effect is also observed with purified nuclear fractions in pull-down assay. On the other hand, active protein phosphatase 1, a serine–threonine phosphatase known to associate with lamin A/C, inhibits emerin–actin interaction in myotube extracts. These data provide evidence of a modulation of emerin–actin interaction in muscle cells, possibly through differentiation-related stimuli.

Section snippets

Materials and methods

Cell culture and differentiation. C2C12 mouse myoblasts were grown in DMEM supplemented with 10% fetal calf serum. Myoblast cultures approaching confluence were shifted in differentiation medium (DMEM plus 1% fetal calf serum) and allowed to differentiate for the specified time points [16].

Transfection of cycling C2C12 myoblasts. A pEGFP-actin vector coding for cytoplasmic β actin was purchased from Clontech. Exponentially growing C2C12 myoblasts were transfected by using the FuGene

Results and discussion

We previously demonstrated that emerin is highly expressed in differentiated mouse and human myotubes and that both nuclear and cytoskeleton localization of emerin can be observed in these cells [15]. Emerin has been reported to interact with actin in C2C12 mouse myoblasts and to bind α actin in in vitro blot overlay assay [23]. The study here reported was aimed at the evaluation of emerin–actin interaction in differentiating muscle cells. To this purpose, emerin was immunoprecipitated from

Acknowledgements

The authors thank P. Sabatelli and I. Mura for the technical assistance. This work was supported by grants: E.C. project Myo-Cluster, Contract No. QLG1-1999-00870; P.F. 83/2001 “Ministero della Salute,” Italy; Fondazione Carisbo P.B. “Diagnosi, ricerca e trattamento nelle distrofie muscolari,” Italy.

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    These authors contributed equally to this work.

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