Colley N J, Tokuyasu K T, Singer S J
Department of Biology, University of California at San Diego, La Jolla 92093.
J Cell Sci. 1990 Jan;95 ( Pt 1):11-22. doi: 10.1242/jcs.95.1.11.
Previous reports on skeletal muscle myogenesis have shown that postmitotic spindle-shaped myoblasts express muscle-specific proteins, some of which are organized into nascent myofibrils. However, we show that, in skeletal muscle cultures derived from 12-day chick embryos, by 6 h after plating the predominant mononucleated cell type that expresses muscle-specific proteins is a round cell. These round myoblasts appear to precede spindle-shaped myoblasts in development, since the latter are more abundant in later cultures and contain larger amounts of muscle proteins and more highly organized myofibrils. By double immunofluorescence microscopy using antibodies specific for the muscle proteins titin, myosin heavy chain (MHC) and zeugmatin we find that 18 h after plating approximately 20% of the round myoblasts that are titin-positive are negative for myofibrillar MHC and zeugmatin. On the other hand, all spindle-shaped myocytes that are positive for titin are also positive for myofibrillar MHC and zeugmatin. These results suggest that titin expression precedes that of myofibrillar MHC and zeugmatin in the non-synchronized round myoblasts, and is consistent with earlier suggestions that titin may function as an initial organizer of myofibrillar proteins during myogenesis. Immunofluorescence data indicate that the earliest localization of the myofibrillar proteins titin, MHC, zeugmatin and alpha-actinin in the round myoblasts is surrounding the nucleus with no immunofluorescent labeling of the cytoplasm or near the plasma membrane. Furthermore, pairwise double immunofluorescence experiments show that these four myofibrillar proteins are all co-localized, at the light-microscopic level of resolution, in irregular patterns that may appear in either a punctate or a basket-like distribution. These labeling patterns around the nucleus are resistant to extraction with Triton X-100, suggesting that the proteins are associated in a stable array. These Triton X-100-resistant assemblies in round myoblasts appear to be composed solely of structural myofibrillar proteins, since the non-structural myofibrillar protein creatine kinase (CK) does not colocalize with the other myofibrillar proteins. These results indicate that in early myoblasts myofibrillar proteins form stable pre-myofibrillar assemblies surrounding the nucleus, and raise the possibility that these initial assemblies may play an organizing role during subsequent early stages of myofibrillogenesis.
先前关于骨骼肌肌生成的报道表明，有丝分裂后的梭形成肌细胞表达肌肉特异性蛋白，其中一些蛋白组装成新生的肌原纤维。然而，我们发现，在源自12日龄鸡胚的骨骼肌培养物中，接种后6小时，表达肌肉特异性蛋白的主要单核细胞类型是圆形细胞。这些圆形成肌细胞似乎在发育过程中先于梭形成肌细胞出现，因为后者在后期培养物中更为丰富，含有更多的肌肉蛋白和组织更高度有序的肌原纤维。通过使用针对肌肉蛋白肌联蛋白、肌球蛋白重链（MHC）和接合素的特异性抗体进行双重免疫荧光显微镜观察，我们发现接种后18小时，约20%的肌联蛋白阳性圆形成肌细胞对肌原纤维MHC和接合素呈阴性。另一方面，所有肌联蛋白阳性的梭形成肌细胞对肌原纤维MHC和接合素也呈阳性。这些结果表明，在未同步化的圆形成肌细胞中，肌联蛋白的表达先于肌原纤维MHC和接合素，这与早期的推测一致，即肌联蛋白在肌生成过程中可能作为肌原纤维蛋白的初始组织者发挥作用。免疫荧光数据表明，肌原纤维蛋白肌联蛋白、MHC、接合素和α-辅肌动蛋白在圆形成肌细胞中的最早定位是围绕细胞核，细胞质或质膜附近无免疫荧光标记。此外，成对的双重免疫荧光实验表明，在光学显微镜分辨率水平上，这四种肌原纤维蛋白均共定位，呈不规则模式，可能呈点状或篮状分布。细胞核周围的这些标记模式对Triton X-100提取具有抗性，表明这些蛋白以稳定的阵列形式结合。圆形成肌细胞中这些对Triton X-100有抗性的组装体似乎仅由结构性肌原纤维蛋白组成，因为非结构性肌原纤维蛋白肌酸激酶（CK）不与其他肌原纤维蛋白共定位。这些结果表明，在早期成肌细胞中，肌原纤维蛋白在细胞核周围形成稳定的前肌原纤维组装体，并增加了这些初始组装体在随后肌原纤维形成早期阶段可能发挥组织作用的可能性。
