Gregorio C C, Fowler V M
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA.
J Cell Biol. 1995 May;129(3):683-95. doi: 10.1083/jcb.129.3.683.
Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994, J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, alpha-actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (approximately 39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity. We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled.
原肌球蛋白调节蛋白是一种位于原肌球蛋白包被的肌动蛋白丝尖端部的封端蛋白，据推测它在调节横纹肌细肌丝的精确长度方面发挥作用（福勒，V.M.，M.A.苏斯曼，P.G.米勒，B.E.弗卢彻，和M.P.丹尼尔斯。1993年。《细胞生物学杂志》120:411 - 420；韦伯，A.，C.C.彭尼斯，G.G.巴布科克，和V.M.福勒。1994年，《细胞生物学杂志》127:1627 - 1635）。为了深入了解细肌丝组装机制以及原肌球蛋白调节蛋白在其中的作用，我们对原代鸡胚心肌细胞培养物中横纹肌原纤维形成过程中原肌球蛋白调节蛋白在细肌丝尖端部的出现时间、生物合成及组装机制进行了表征。我们的结果表明，原肌球蛋白调节蛋白并非与其他细肌丝蛋白协同组装。双重免疫荧光染色和超微结构免疫定位表明，在细肌丝组织成周期性的I带之后，原肌球蛋白调节蛋白才在细肌丝尖端部其特有的肌节位置掺入。实际上，原肌球蛋白调节蛋白的组装比所有其他已充分表征的研究过的肌原纤维蛋白都要晚，这些蛋白包括：肌动蛋白、原肌球蛋白、α - 辅肌动蛋白、肌联蛋白、肌球蛋白和C蛋白。然而，在稳态时，在整个肌原纤维组装过程中，相当一部分（约39%）的原肌球蛋白调节蛋白存在于可溶性池中。因此，一些横纹肌原纤维中不存在原肌球蛋白调节蛋白并非由于该蛋白数量有限。此外，从[35S]甲硫氨酸脉冲追踪实验获得的动力学数据表明，原肌球蛋白调节蛋白组装到肌原纤维中的速度比原肌球蛋白慢。这一观察结果，连同使用一种用于细肌丝组装的新型通透细胞模型所获得的结果，表明原肌球蛋白调节蛋白的组装依赖于原肌球蛋白与肌动蛋白丝的先前结合。我们得出结论，原肌球蛋白调节蛋白是心肌细胞中横纹肌原纤维组装的晚期标志物；其组装似乎与它们的成熟度相关。我们提出，原肌球蛋白调节蛋白在细肌丝组装后参与维持和稳定其最终长度。
