826 J. zyxwvutsrqponml V. F. Coumans and C. G. dos Remedios Electrophoresis 1998, 19, 826-833 Joelle V. F. Coumans Cristobal zyxwvutsrqpo G. dos Remedios Actin-binding proteins in mouse C2 myoblasts and myotubes: A combination of affinity chromatography Department of Anatomy and Histology, m e University of Sydney, Sydney, Australia This paper analyzes proteins expressed in a mouse muscle precursor cell line (C2 myoblasts) and compares them with those observed in differentiated myo- tubes from the same cell line. We observed hundreds of proteins in myoblasts using IPG two-dimensional gel electrophoresis but this number is greatly reduced using Mini-Leak (divinylsulfone-activated agarose) affinity chromatog- raphy. Two kinds of affinity columns were prepared. One contained a chemi- cally modified monomeric actin bound to the affinity matrix. The second matrix contained a high-affinity actin-binding protein (DNase I) which was bound to the actin Mini-Leak column to block specific sites on actin. Actin- binding proteins in homogenates of myoblasts or myotubes were passed through the affinity columns and eluted under high salt conditions. The Mini- Leak affinity medium itself appeared to have little ability to bind proteins. Our two-dimensional (2-D) gels identified a small number of proteins and we are currently focusing our attention on a particular protein spot which could corre- spond to cofilin. Comparison of myoblast and myotube proteins using affinity chromatography shows no qualitative, clearly identifiable differences but the analysis is still in progress. These findings are discussed in relation to reports in which the myoblast-myotube transformation was associated with the up-regulation or zyxwvu de zyxwvut now synthesis of more than ten proteins. 1 Introduction In striated muscle the sarcomere is the functional unit of the myofibrils. Each sarcomere represents a highly ordered macromolecular structure and its formation re- quires coordinated synthesis of a range of constituent proteins. Thin filaments (mainly actin), the thick fila- ments (mainly myosin), and their associated proteins are then assembled into the sarcomere. In muscle precursor cells (myoblasts) the thin filaments are not arranged in regular arrays but form the cytoskeleton. Regulation of actin polymerization and filament length of both sarco- meric and cytoskeletal thin filaments is an important process but little is known about the molecular mechan- isms involved. A large number of actin-binding proteins (ABPs) have been identified [l]. Some maintain a pool of monomeric actin in the cytoplasm by acting as a “buffer”. Some are involved in the regulation of filament assembly, while others stabilize and link actin filaments to each other or to other cellular components [2-51. In 1986, Shimizu and Obinata [6] showed that zyxwvut 4O-5O0/o of the total cytoplasmic actin in embryonic skeletal muscle tissue is in a monomeric form. Some of this G-actin may be present in myoblasts in association with ABPs. Correspondence: Dr. Joelle Coumans, Institute for Biomedical Research, Muscle Research Unit, Department of Anatomy & Histology, F13, The University of Sydney, Sydney 2006, Australia (Tel: +61-2- 9351-6543; Fax: +61-2-9351-2813; E-mail: joelle@anatomy.usyd. edu.au) Abbreviations: ABP, actin-binding protein; DNase I, deoxyribonuclease I; DTE, dithioerythritol; F-actin, filamentous actin; G-actin, mono- meric actin; PEG, polyethylene glycol Keywords: Actin / Actin-binding proteins / C2 myoblast / C2 myo- tubes / Divinylsulfone-activated agarose (Mini-Leak) / Two-dimen- sional polyacrylamide gel electrophoresis / Affinity chromatography Recently, we reported the development of an affinity chromatography method using monomeric actin bound to a column of divinylsulfone-activated agarose (Mini- Leak) [7]. An important characteristic of this matrix (compared to the more conventional cyanogen-bromide- activated agarose) is its high capacity to retain proteins, even at extreme pH [8]. Here we use this technique to isolate G-ABPs from mouse C2 myoblasts and myo- tubes. Since we have suggested that the actin-DNase I complex shares conformational properties of both G- and F-actin [7, 91, the binding of DNaseI to the actin Mini-Leak matrix prior to the application of myoblast or myotube extracts may isolate ABPs that bind to monomers with an F-actin-like conformation. This last strategy may also enable us to distinguish between ABPs that bind to the DNaseI-binding site in contrast to other sites on the actin monomer. DNaseI [lo] and tropomod- ulin 111-131 have the unusual property of being able to bind to the pointed end of F-actin (referring to the orientation of myosin subfragment 1 “arrowheads” when they bind to an actin filament), whereas most other ABPs bind to the opposite or barbed end of the monomer. ABPs which bind strongly to actin monomers can be eluted from the actin-Mini-Leak columns with and without DNase I and analyzed using two-dimensional electrophoresis (2-DE). By studying myoblasts and myo- tubes, we expected to identify ABPs which may play a role in their differentiation. This was timely because Cor- bett et zyxwvu al. [14] recently used co-electrophoresis to show that there are more than 200 proteins from mouse car- diac tissues which comigrate with human cardiac pro- teins. This then raises the prospect of characterizing ABPs involved in human myofibrillogenesis, some of which may be novel. zyxw 0 WILEY-VCH Verlag GmbH, 69451 Weinheim, 1998 0173-0835/98/0505-0826 $17.50+.50/0