Pappas Christopher T, Bhattacharya Nandini, Cooper John A, Gregorio Carol C
Departments of Cell Biology and Anatomy and *Molecular and Cellular Biology, The University of Arizona, Tucson, AZ 85721-0106, USA.
Mol Biol Cell. 2008 May;19(5):1837-47. doi: 10.1091/mbc.e07-07-0690. Epub 2008 Feb 13.
The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant "molecular ruler" nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160-164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160-164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.
横纹肌中肌动蛋白丝的带刺末端锚定在Z盘内，并由CapZ封端；这种蛋白质在体外可阻止肌动蛋白的聚合和解聚。细肌丝的成熟长度可能由跨越细肌丝长度的巨大“分子尺”伴肌动蛋白确定。在此，我们报告在印迹覆盖、固相结合、色氨酸荧光和SPOTs膜分析中，CapZ与伴肌动蛋白的C末端（模块160 - 164）特异性相互作用。伴肌动蛋白模块160 - 164与CapZ的结合并不影响CapZ在体外封端肌动蛋白丝的能力，这与我们的观察结果一致，即CapZ的两个C末端肌动蛋白结合区域对于其与伴肌动蛋白的相互作用都不是必需的。使用小干扰RNA敲低鸡骨骼肌肌管中的伴肌动蛋白会导致组装的CapZ减少，而且引人注目的是，肌动蛋白丝带刺末端的均匀排列丧失。这些数据表明，伴肌动蛋白通过与CapZ的直接相互作用将细肌丝带刺末端的位置限制在Z盘。我们提出了一种新的Z盘结构分子模型，其中伴肌动蛋白与来自相邻肌节细肌丝的CapZ相互作用，从而在肌节之间提供结构联系。
