Littlefield Ryan, Fowler Velia M
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Biophys J. 2002 May;82(5):2548-64. doi: 10.1016/S0006-3495(02)75598-7.
The lengths of the actin (thin) filaments in sarcomeres directly influence the physiological properties of striated muscle. Although electron microscopy techniques provide the highest precision and accuracy for measuring thin filament lengths, significant obstacles limit their widespread use. Here, we describe distributed deconvolution, a fluorescence-based method that determines the location of specific thin filament components such as tropomodulin (Tmod) or probes such as phallacidin (a phalloidin derivative). Using Tmod and phallacidin fluorescence, we were able to determine the thin filament lengths of isolated chicken pectoralis major myofibrils with an accuracy and precision comparable to electron microscopy. Additionally, phallacidin fluorescence intensity at the Z line provided information about the width of Z lines. Furthermore, we detected significant variations in thin filaments lengths among individual myofibrils from chicken posterior latissimus dorsai and embryonic chick cardiac myocytes, suggesting that a ruler molecule (e.g., nebulin) does not strictly determine thin filament lengths in these muscles. This versatile method is applicable to myofibrils in living cells that exhibit significant variation in sarcomere lengths, and only requires a fluorescence microscope and a CCD camera.
肌节中肌动蛋白（细）丝的长度直接影响横纹肌的生理特性。尽管电子显微镜技术在测量细肌丝长度方面具有最高的精度和准确性，但重大障碍限制了它们的广泛应用。在此，我们描述了分布式反卷积，这是一种基于荧光的方法，可确定特定细肌丝成分（如原肌球蛋白（Tmod））或诸如鬼笔环肽（一种鬼笔毒素衍生物）等探针的位置。利用Tmod和鬼笔环肽荧光，我们能够确定分离的鸡胸大肌肌原纤维的细肌丝长度，其准确度和精密度与电子显微镜相当。此外，Z线处的鬼笔环肽荧光强度提供了有关Z线宽度的信息。此外，我们检测到来自鸡背阔肌后部和胚胎鸡心肌细胞的单个肌原纤维之间细肌丝长度存在显著差异，这表明在这些肌肉中，一种标尺分子（如伴肌动蛋白）并不严格决定细肌丝的长度。这种通用方法适用于肌节长度有显著变化的活细胞中的肌原纤维，并且只需要一台荧光显微镜和一台电荷耦合器件相机。
