Stout Andrea L, Wang Jushuo, Sanger Jean M, Sanger Joseph W
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
Cell Motil Cytoskeleton. 2008 May;65(5):353-67. doi: 10.1002/cm.20265.
There are a large number of proteins associated with Z-bands in myofibrils, but the precise arrangements of most of these proteins in Z-bands are largely unknown. Even less is known about how these arrangements change during myofibrillogenesis. We have begun to address this issue using Sensitized Emission Fluorescence Resonance Energy Transfer (SE-FRET) microscopy. Cultured skeletal muscle cells from quail embryos were transfected to express fusions of alpha-actinin, FATZ, myotilin, or telethonin with cyan and yellow fluorescent proteins in various pair wise combinations. FATZ and myotilin were selected because previous biochemical studies have suggested that they bind to alpha-actinin, the major protein of the Z-band. Telethonin was selected for its reported ability to bind FATZ. Statistical analysis of data from FRET imaging studies yield results that are in agreement with published biochemical data suggesting that FATZ and myotilin bind to alpha-actinin near its C-terminus as well as to each other and that a region near the amino-terminus of FATZ is responsible for its interaction with telethonin. In addition, our analysis has revealed changes in the arrangement of alpha-actinin and FATZ that take place during the transition as the z-bodies of premyofibrils fuse to form the Z-bands of mature myofibrils. There was no evidence for a change in the arrangement of myotilin as z-bodies transformed into Z-bands. Myotilin is one Z-band protein that does not exhibit decreased dynamics as z-bodies fuse to form Z-bands. These FRET results from living cells support a stepwise model for the assembly of myofibrils.
肌原纤维中存在大量与Z带相关的蛋白质，但这些蛋白质在Z带中的精确排列在很大程度上尚不清楚。关于这些排列在肌原纤维形成过程中如何变化的了解更少。我们已开始使用敏化发射荧光共振能量转移（SE-FRET）显微镜来解决这个问题。用各种两两组合的方式转染鹌鹑胚胎培养的骨骼肌细胞，使其表达与青色和黄色荧光蛋白融合的α-辅肌动蛋白、FATZ、肌联蛋白或隐钙素。选择FATZ和肌联蛋白是因为先前的生化研究表明它们与Z带的主要蛋白质α-辅肌动蛋白结合。选择隐钙素是因为据报道它有结合FATZ的能力。FRET成像研究数据的统计分析结果与已发表的生化数据一致，表明FATZ和肌联蛋白在α-辅肌动蛋白的C末端附近相互结合，并且FATZ氨基末端附近的一个区域负责其与隐钙素的相互作用。此外，我们的分析揭示了在肌原纤维前体的z体融合形成成熟肌原纤维的Z带的转变过程中，α-辅肌动蛋白和FATZ排列的变化。没有证据表明随着z体转变为Z带，肌联蛋白的排列发生了变化。肌联蛋白是一种Z带蛋白，在z体融合形成Z带时其动力学不会降低。这些来自活细胞的FRET结果支持了肌原纤维组装的逐步模型。
