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  • Proteomic resolution of IGFN1 complexes reveals a functional interaction with the actin nucleating protein COBL.

Proteomic resolution of IGFN1 complexes reveals a functional interaction with the actin nucleating protein COBL.

Experimental cell research (2020-08-10)
Tobias Cracknell, Steinar Mannsverk, Angus Nichols, Adam Dowle, Gonzalo Blanco
ABSTRACT

The Igfn1 gene produces multiple proteins by alternative splicing predominantly expressed in skeletal muscle. Igfn1 deficient clones derived from C2C12 myoblasts show reduced fusion index and morphological differences compared to control myotubes. Here, we first show that G:F actin ratios are significantly higher in differentiating IGFN1-deficient C2C12 myoblasts, suggesting that fusion and differentiation defects are underpinned by deficient actin remodelling. We obtained pull-downs from skeletal muscle with IGFN1 fragments and applied a proteomics approach. The proteomic composition of IGFN1 complexes identified the cytoskeleton and an association with the proteasome as the main networks. The actin nucleating protein COBL was selected for further validation. COBL is expressed in C2C12 myoblasts from the first stages of myoblast fusion but not in proliferating cells. COBL is also expressed in adult muscle and, as IGFN1, localizes to the Z-disc. We show that IGFN1 interacts, stabilizes and colocalizes with COBL and prevents the ability of COBL to form actin ruffles in COS7 cells. COBL loss of function C2C12-derived clones are able to fuse, therefore indicating that COBL or the IGFN1/COBL interaction are not essential for myoblast fusion.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Mouse IgG−Agarose, (Suspension in 0.5 M NaCl containing preservative.)
Sigma-Aldrich
Protease Inhibitor Cocktail, for use in purification of Histidine-tagged proteins, DMSO solution
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
Anti-COBL antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution, Ab2
Sigma-Aldrich
Monoclonal Anti-GAPDH−Peroxidase antibody produced in mouse, clone GAPDH-71.1, purified immunoglobulin, buffered aqueous solution
Sigma-Aldrich
RIPA Buffer
Sigma-Aldrich
Anti-V5 antibody produced in rabbit, IgG fraction of antiserum