Ono Yasuko, Schwach Catherine, Antin Parker B, Gregorio Carol C
Department of Cell Biology and Anatomy, University of Arizona, 1501 N. Campbell Avenue-LSN 455, Tucson, AZ 85724, USA.
Dev Biol. 2005 Jun 15;282(2):336-48. doi: 10.1016/j.ydbio.2005.03.015.
Tropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, approximately 40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils.
原肌球蛋白（Tmods）是肌动蛋白丝尖端的封端蛋白家族。为了阐明Tmod1在新生肌原纤维形成过程中的功能意义，我们构建了Tmod1基因敲除的胚胎干细胞（ES细胞），并研究了它们向心肌细胞的分化。令人惊讶的是，野生型（WT）ES细胞在体外向心肌细胞的分化忠实地模拟了体内心肌细胞的分化，这使我们能够评估Tmod1基因敲除（KO）的肌原纤维的表型，而无需考虑Tmod1基因敲除小鼠的胚胎致死性。免疫荧光和电子显微镜研究表明，Tmod1基因敲除的心肌细胞呈圆形，形态不成熟，含有发育不全的肌原纤维，这些肌原纤维比WT细胞中的更短、更窄，且细肌丝更少。出乎意料的是，在大多数基因敲除细胞中，H区F-肌动蛋白染色模式出现明显间隙，表明存在长度一致的肌丝。这表明，除了封端蛋白外，还有其他机制负责维持心肌细胞中细肌丝的长度。同样出乎意料的是，约40%的基因敲除心肌细胞表现出收缩活性。我们的数据表明，分化的ES细胞是研究收缩蛋白功能特性的强大系统，并且Tmod1的功能对于肌原纤维形成的后期阶段以及肌原纤维的成熟至关重要。
