Abstract
We describe a strategy for exploring the function of protein-protein interactions in striated muscle in vivo. We describe our experience using this strategy to study the interaction of UNC-112 (kindlin) with PAT-4 (integrin linked kinase). Random mutagenesis is used to generate a collection of mutants that are screened for lack of binding or gain of binding using a yeast 2-hybrid assay. The mutant proteins are then expressed in transgenic C. elegans to determine their ability to localize in the sarcomere. We emphasize two advantages of this strategy: (1) for studying the interaction of protein A with protein B, when protein A can interact with multiple proteins, and (2) it explores the function of an interaction rather than the absence of, or reduced level of, a protein as can be obtained with null mutants or knockdown by RNAi. We propose that this method can be generalized for studying the meaning of a protein-protein interaction in muscle for any system in which transgenic animals can be generated and their muscles can be imaged.
Highlights
UNC-112 (KINDLIN) AND PAT-4 (ILK) The model genetic system C. elegans is an excellent platform for learning new information about striated muscle, including new components of the sarcomere, requirements for assembly and maintenance of the sarcomere, and mechanisms of the regulation of the contraction/relaxation cycle (Waterston, 1988; Moerman and Fire, 1997; Moerman and Williams, 2006; Qadota and Benian, 2010)
In C. elegans the myofibrils are organized such that all thick filament organizing centers (M-lines) and thin filament attachment sites are anchored to the muscle cell membrane
EVALUATION OF THE MEANING OF PROTEIN-PROTEIN INTERACTION In vivo If our model was correct, we reasoned that an UNC-112 mutant that cannot bind to PAT-4, would not localize to muscle adhesion sites in vivo
Summary
UNC-112 (KINDLIN) AND PAT-4 (ILK) The model genetic system C. elegans is an excellent platform for learning new information about striated muscle, including new components of the sarcomere, requirements for assembly and maintenance of the sarcomere, and mechanisms of the regulation of the contraction/relaxation cycle (Waterston, 1988; Moerman and Fire, 1997; Moerman and Williams, 2006; Qadota and Benian, 2010). We used the cytoplasmic tail of PAT-3 (β-integrin) as bait to screen a yeast 2-hybrid library of C. elegans cDNAs, and recovered as the only interacting protein, UNC-112 (Qadota et al, 2012).
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