Peng I, Fischman D A
Department of Cell Biology and Anatomy, Cornell University Medical College, New York.
Cell Motil Cytoskeleton. 1991;20(2):158-68. doi: 10.1002/cm.970200208.
The incorporation of actin into myofibrils has been examined in a cell-free system [Bouché et al.: Journal of Cell Biology 107:587-596, 1988; Goldfine et al.: Cellular and Molecular Biology of Muscle Development, 1989]. Actin was translated in a reticulocyte lysate in the presence of 35S-methionine (35S-actin) or purified from muscle and labeled with fluorescein-5-isothiocyanate (FITC-actin). Myofibrils were incubated with either 35S-actin or FITC-actin and then analyzed by gel electrophoresis or fluorescence microscopy. When myofibrils were incubated with FITC-actin monomer in the reticulocyte lysate buffer, strong fluorescent labeling was observed in Z-band regions and less so in I-bands. No fluorescence was detected in non-overlap regions of A-bands. Confocal microscopic analysis of these myofibrils indicated that FITC-actin was distributed evenly across the diameter of the myofibrils. These observations suggest that actin incorporation in the reticulocyte lysate buffer occurred at sites in the sarcomere which contain actin. In contrast, FITC-actin showed a variety of non-physiological incorporation patterns when incubated with myofibrils in the presence of an isotonic buffer (I-buffer). However, when ATP was added to I-buffer, FITC-actin showed a pattern of incorporation into myofibrils similar to that seen in the reticulocyte lysate buffer. Immunoblots indicated that actin of native size was released from myofibrils during incubation in the reticulocyte lysate buffer. No actin release was detected when the myofibrils were incubated in I-buffer lacking ATP. We used this system to compare the incorporation of actin isoforms into myofibrils. Both alpha- and beta-actins exhibited incorporation into the myofibrils but there was a three-fold greater incorporation of the alpha isoform. We propose that the differential affinities of actin isoforms for myofibrils and other cytoskeletal structures could provide a mechanism for actin isoform targeting within the cytoplasm.
肌动蛋白向肌原纤维中的掺入已在无细胞体系中进行了研究[布歇等人：《细胞生物学杂志》107:587 - 596，1988年；戈德芬等人：《肌肉发育的细胞与分子生物学》，1989年]。肌动蛋白在网织红细胞裂解物中于35S - 甲硫氨酸存在的情况下进行翻译（35S - 肌动蛋白），或者从肌肉中纯化并标记异硫氰酸荧光素（FITC - 肌动蛋白）。将肌原纤维与35S - 肌动蛋白或FITC - 肌动蛋白一起孵育，然后通过凝胶电泳或荧光显微镜进行分析。当肌原纤维在网织红细胞裂解物缓冲液中与FITC - 肌动蛋白单体一起孵育时，在Z带区域观察到强烈的荧光标记，而在I带区域则较弱。在A带的非重叠区域未检测到荧光。对这些肌原纤维的共聚焦显微镜分析表明，FITC - 肌动蛋白均匀分布在肌原纤维的直径上。这些观察结果表明，在网织红细胞裂解物缓冲液中肌动蛋白的掺入发生在肌节中含有肌动蛋白的部位。相比之下，当在等渗缓冲液（I缓冲液）存在的情况下将FITC - 肌动蛋白与肌原纤维一起孵育时，它显示出各种非生理性的掺入模式。然而，当向I缓冲液中加入ATP时，FITC - 肌动蛋白显示出一种与在网织红细胞裂解物缓冲液中所见相似的掺入到肌原纤维中的模式。免疫印迹表明，在网织红细胞裂解物缓冲液中孵育期间，天然大小的肌动蛋白从肌原纤维中释放出来。当肌原纤维在缺乏ATP的I缓冲液中孵育时，未检测到肌动蛋白释放。我们利用这个系统比较了肌动蛋白异构体向肌原纤维中的掺入情况。α - 肌动蛋白和β - 肌动蛋白均显示掺入到肌原纤维中，但α异构体的掺入量大三倍。我们提出，肌动蛋白异构体对肌原纤维和其他细胞骨架结构的不同亲和力可能为肌动蛋白异构体在细胞质内的靶向定位提供一种机制。
