Clock is not a component of Z-bands.
The process of Z-band assembly begins with the formation of small Z-bodies composed of a complex of proteins rich in alpha-actinin. As additional proteins are added to nascent myofibrils, Z-bodies are transformed into continuous bands that form coherent discs of interacting proteins at the boundaries of sarcomeres. The steps controlling the transition of Z-bodies to Z-bands are not known. The report that a circadian protein, Clock, was localized in the Z-bands of neonatal rat cardiomyocytes raised the question whether this transcription factor could be involved in Z-band assembly. We found that the anti-Clock antibody used in the reported study also stained the Z-bands and Z-bodies of mouse and avian cardiac and skeletal muscle cells. YFP constructs of Clock that were assembled, however, did not localize to the Z-bands of muscle cells. Controls of Clock's activity showed that cotransfection of muscle cells with pYFP-Clock and pCeFP-BMAL1 led to the expected nuclear localization of YFP-Clock with its binding partner CeFP-BMAL1. Neither CeFP-BMAL1 nor antibodies directed against BMAL1 localized to Z-bands. A bimolecular fluorescence complementation assay (VC-BMAL1 and VN-Clock) confirmed the absence of Clock and BMAL1 from Z-bands, and their nuclear colocalization. A second anti-Clock antibody stained nuclei, but not Z-bands, of cells cotransfected with Clock and BMAL1 plasmids. Western blots of reactions of muscle extracts and purified alpha-actinins with the two anti-Clock antibodies showed that the original antibody cross-reacted with alpha-actinin and the second did not. These results cannot confirm Clock as an active component of Z-bands. © 2012 Wiley Periodicals, Inc.
Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
Jushuo
J
Wang
Dipak K
DK
Dube
Jennifer
J
White
Yingli
Y
Fan
Jean M
JM
Sanger
Joseph W
JW
Sanger
D000185:Actinin / Q000378:metabolism*; D000818:Animals; D056926:CLOCK Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D051381:Rats; D014162:Transfection
D000185:Actinin; D056926:CLOCK Proteins