Cypher, a novel striated muscle specific cytoskeletal protein, has two splice isoforms, Cypher 1 and Cypher 2. Mice homozygous for both Cypher isoforms die perinatally with severe defects in striated muscle. Apparent heart failure is suggested by blood congestion in liver, and cardiac chambers, accompanied by ventricular dilation. Transmission Electronic Microscopy (TEM) studies indicated that striated muscle in mutant mice has severely disorganized Z-lines. This proposal will test the Cypher is a critical linker protein which serves to mediate structural integrity and signaling pathways at the Z-line via its interactions with alpha-actinin 2, alpha-cardiac actin, PKC, and other potential Z-line proteins. Cypher 2 may be partially redundant with Cypher l, and/or may act as an antagonist of Cypher l function.
The Specific Aims are: 1) To identify protein partners of Cypher utilizing the phage display system. Full length Cypherl and Cypher2 will be utilized as baits to screen a phage display adult heart cDNA library. 2) To determine the biological function of two Cypher splice isoforms, Cypher l and Cypher 2. We will investigate the function of Cypher 1 and Cypher 2 by creating mouse lines in which each isoform is selectively ablated. 3) To test physiological consequences of the inability of Cypher to bind to alpha-actinin-2 by generating mouse lines in which the essential amino acid for Cypher binding to alpha-actinin-2, Leu78, is mutated to Lys. 4) To further investigate mechanisms of interaction of Cypher l with PKC, we will perform extensive in vitro binding studies in combination with mutagenesis to determine critical residues within the Cypher l LIM domains which are required for PKC binding. For each mouse model in Aims 2 and 3, detailed biochemical and cellular analyses of resulting cardiac muscle phenotypes, and comprehensive physiological assessment of cardiac function will be performed.
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