Zhukarev V, Sanger J M, Sanger J W, Goldman Y E, Shuman H
Department of Physiology, Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Cell Motil Cytoskeleton. 1997;37(4):363-77. doi: 10.1002/(SICI)1097-0169(1997)37:4<363::AID-CM7>3.0.CO;2-5.
Phalloidin staining of muscle does not reflect the known disposition of sarcomeric thin filaments. Quantitative image analysis and steady-state fluorescence polarization microscopy are used to measure the local intensity and orientation of tetramethyl rhodamine-labeled phalloidin (TR-phalloidin) in skinned myofibrils. TR-phalloidin staining of isolated skeletal myofibrils labeled while in rigor reveals fluorescence that is brighter at the pointed ends of the thin filaments and Z lines than it is in the middle of the filaments. In cardiac myofibrils, phalloidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor myofibrils, except in 0.2-micron dark areas on either side of the Z line. Extraction of myosin or tropomyosin-troponin molecules does not change the nonuniform staining. To test whether long-term storage in glycerol changes the binding of phalloidin to thin filaments in myofibrils, minimally permeabilized (briefly skinned) myofibrils, or myofibrils stored in glycerol for at least 7 days (glycerol extraction) were compared. TR-phalloidin was well ordered throughout the sarcomere in briefly skinned skeletal and cardiac myofibrils, but TR-phalloidin bound to the Z line and pointed ends of thin filaments was randomly oriented in glycerol-extracted myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extraction. In relaxed skeletal myofibrils with sarcomere lengths greater than 3.0 microns, staining was nearly uniform all along the actin filaments. Exogeneous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825-833) in and along the myofibril bind rhodamine phalloidin uniformly. Our results support the hypothesis that nebulin can block the binding of phalloidin to actin in skeletal myofibrils and nebulette can block phalloidin binding to cardiac thin filaments.
肌肉的鬼笔环肽染色并不反映肌节细肌丝的已知分布情况。采用定量图像分析和稳态荧光偏振显微镜来测量经透皮处理的肌原纤维中四甲基罗丹明标记的鬼笔环肽（TR-鬼笔环肽）的局部强度和方向。对处于僵直状态时标记的分离骨骼肌原纤维进行TR-鬼笔环肽染色，结果显示细肌丝尖端和Z线处的荧光比肌丝中部更亮。在心肌原纤维中，无论处于舒张状态还是僵直状态，鬼笔环肽染色在细肌丝全长上都是均匀的，但在Z线两侧0.2微米的暗区除外。提取肌球蛋白或原肌球蛋白-肌钙蛋白分子并不会改变这种不均匀染色。为了测试在甘油中长时间储存是否会改变鬼笔环肽与肌原纤维中细肌丝的结合，对轻度通透（短暂透皮）的肌原纤维或在甘油中储存至少7天的肌原纤维（甘油提取）进行了比较。在短暂透皮的骨骼肌和心肌原纤维中，TR-鬼笔环肽在整个肌节中排列良好，但在甘油提取的肌原纤维中，与Z线和细肌丝尖端结合的TR-鬼笔环肽是随机取向的，这表明甘油提取后细肌丝的末端变得无序。在肌节长度大于3.0微米的舒张骨骼肌原纤维中，肌动蛋白丝上的染色几乎是均匀的。从Z线聚合的外源裸露肌动蛋白丝（Sanger等人，1984年：《细胞生物学杂志》98:825 - 833）在肌原纤维内和沿肌原纤维排列，均匀结合罗丹明鬼笔环肽。我们的结果支持这样的假设，即伴肌动蛋白可以阻止鬼笔环肽与骨骼肌原纤维中的肌动蛋白结合，而细伴肌动蛋白可以阻止鬼笔环肽与心肌细肌丝结合。
