Clock Is Not a Component of Z-Bands Jushuo Wang, 1 Dipak K. Dube, 2 Jennifer White, 1 Yingli Fan, 1 Jean M. Sanger, 1 and Joseph W. Sanger 1 * 1 Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York 13210 2 Department of Medicine, College of Medicine, SUNY Upstate Medical University, Syracuse, New York 13210 Received 21 February 2012; Accepted 23 July 2012 Monitoring Editor: Pekka Lappalainen The process of Z-band assembly begins with the forma- tion of small Z-bodies composed of a complex of pro- teins rich in alpha-actinin. As additional proteins are added to nascent myofibrils, Z-bodies are transformed into continuous bands that form coherent discs of inter- acting 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 car- diomyocytes raised the question whether this transcrip- tion 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 con- structs 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 ab- sence 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. V C 2012 Wiley Periodicals, Inc Key Words: alpha-actinin, Z-bodies, Z-bands, clock, BMAL1 Introduction T he Z-bands of myofibrils are docking sites for many proteins with diverse roles, the best known of which anchor the myofilaments through links with titin and neb- ulin/nebulette filaments and membrane-associated proteins [Sanger and Sanger, 2008; Bennett, 2012]. Many proteins discovered more recently to be localized in Z-bands were identified through evidence of their binding to other sarco- meric proteins or through their association with skeletal and cardiac myopathies [Frank et al., 2006; Etard et al., 2008; Kn€ oll et al., 2011]. Their roles in muscle function are the focus of active investigation to determine their significance in normal and myopathic muscle function. A few Z-band proteins, e.g., Ankrd (ankyrin repeat protein); Muscle LIM (Lin11, Isl-1, Mec-3) protein; and myopalladin, have a dual localization in muscle moving between sarcomere and the nucleus [Otey et al., 2005; Lange et al., 2006; Kn€ oll et al., 2011] where they are thought to constitute a signaling path linking contractile activity and myofibril assembly with reg- ulation of transcription [Kn€ oll et al., 2011]. The circadian protein, Clock [Zhang and Kay, 2010; Lefta et al., 2011] was identified with an anti-Clock poly- clonal antibody as another protein that migrated from Z- bands to nuclei [Qi and Boateng, 2006; Boateng and Goldspink, 2008]. Both Western blot and immunofluores- cence analyses showed the movement of Clock into nuclei of neonatal rat cardiomyocytes under the influence of phenylephrine or increased contraction [Qi and Boateng, 2006; Boateng and Goldspink, 2008]. Our aim in this study was to determine at what stage of myofibrillogenesis the Clock protein was detected (premyofibril, nascent myofibril, or mature myofibril; [Sanger et al., 2010a]), and what its binding partners were in the Z-bands with a view to understanding how myofibrillogenesis is con- trolled. The anti-Clock antibody used by Qi and Boateng [2006] also stained the Z-bodies of premyofibrils, suggest- ing that Clock like alpha-actinin, was an early appearing protein components of myofibrillogenesis [Sanger et al., 2005, 2009, 2010a,b; Wang et al., 2005, 2011]. However, *Address correspondence to: Joseph W. Sanger, Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY 13210. E-mail: sangerjo@upstate.edu Published online in Wiley Online Library (wileyonlinelibrary.com). RESEARCH ARTICLE Cytoskeleton, Month 2012 000:000–000 (doi: 10.1002/cm.21058) V C 2012 Wiley Periodicals, Inc. 1 n