SEQUENTIAL APPEARANCE OF MUSCLE-SPECIFIC PROTEINS IN MYOBLASTS AS A FUNCTION OF TIME AFTER CELL-DIVISION - EVIDENCE FOR A CONSERVED MYOBLAST DIFFERENTIATION PROGRAM IN SKELETAL-MUSCLE
SEQUENTIAL APPEARANCE OF MUSCLE-SPECIFIC PROTEINS IN MYOBLASTS AS A FUNCTION OF TIME AFTER CELL-DIVISION - EVIDENCE FOR A CONSERVED MYOBLAST DIFFERENTIATION PROGRAM IN SKELETAL-MUSCLE
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DOI：
10.1002/cm.970290102
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发表时间：
1994-01-01
影响因子：
--
通讯作者：
HOLTZER, H
中科院分区：
文献类型：
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作者：
LIN, ZX;LU, MH;HOLTZER, H
基于一个保守的分化程序管理的方式类似于病毒衣壳组装程序的骨骼肌肌节的组装的假设，我们已经定义了时间和空间分布的单核成肌细胞终末细胞分裂后的时间的函数的10个肌肉特异性蛋白质。在鸡胚胸肌单层培养物中鉴定有丝分裂中的单个细胞，然后通过视频延时显微镜在选定的时间点（有丝分裂后0-24 h）进行鉴定，然后固定用于免疫荧光染色。为了方便起见，成肌细胞被称为x-h-old，以定义它们相对于有丝分裂“出生日期”的年龄。所有在富含丝裂原的培养基中培养6小时的成肌细胞都是结蛋白（+），但只有50%的成肌细胞对α-肌动蛋白、肌钙蛋白-I、α-肌动蛋白、MyHC、zeugmatin、肌联蛋白或星云蛋白呈阳性。到有丝分裂后15小时，约80%以上的蛋白质都呈阳性。这7种肌原纤维蛋白的上调似乎是随机的，因为许多成肌细胞是α-辅肌动蛋白（+）或zeugmatin（+），但MyHC（-）或肌联蛋白（-），而其他成肌细胞是肌钙蛋白-I+或MyHC（+），但α-辅肌动蛋白（-）或α-肌动蛋白（-）。在15小时的成肌细胞，这些收缩蛋白被组织成非横纹肌原纤维（NSMFs）。相反，横纹肌原纤维（SMFs），NSMFs表现出可变的化学计量的肌节蛋白，这些没有组织成任何一致的模式。在这个成熟阶段，发生了另外两个变化：（1）微管网络重组成平行束，驱动成肌细胞成为极化的针状细胞;（2）肌膜变得具有融合能力。有丝分裂后15至24小时（或更晚）发生了从NSMFs到SMFs的转变，并与肌桥蛋白，特别是MyBP-C（C蛋白）的晚期出现相关。一个或一串类似于2 μ长的肌节的出现总是以肌粒蛋白和MyBP-C定位于发育中的A带中的成熟位置为特征。后一组A带蛋白可能是组装程序中的限速蛋白。绝大多数的成肌细胞染色阳性结蛋白和肌原纤维蛋白之前，而不是之后，融合形成肌管。这种体外肌肉特异性蛋白质的连续出现完全重现了体内肌节和肢芽成肌细胞以及MyoD转化为成肌细胞的非肌肉细胞中的肌原纤维组装步骤。我们认为，这种细胞自主成肌细胞分化程序可能会被阻止在不同的控制点永生化肌细胞系。(C)1994 Wiley-Liss，Inc.
Based on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle-specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0-24 h h postmitosis) by video time-lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x-h-old to define their age relative to their mitotic ''birthdate.'' All 6 h myoblasts that emerged in a mitogen-rich medium were desmin(+) but only 50% were positive for a alpha-actin, troponin-I, alpha-actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up-regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were alpha-actinin(+) or zeugmatin(+) but MyHC(-) or titin(-) whereas others were troponin-I+ or MyHC(+) but alpha-actinin(-) or alpha-actin(-). In 15-h-old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle-shaped cells; and (2) the sarcolemma became fusion-competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP-C (C protein). The emergence of one, or a string of similar to 2 mu long sarcomeres, was invariably characterized by the localization of myomesin and MyBP-C to their mature positions in the developing A-bands. The latter group of A-band proteins may be rate-limiting in the assembly program. The great majority of myoblasts stained positively for desmin and myofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle-specific proteins in vitro fully recapitulates myofibrillar assembly steps in myoblasts of the myotome and limb bud in vivo, as well as in nonmuscle cells converted to myoblasts by MyoD. We suggest that this cell-autonomous myoblast differentiation program may be blocked at different control points in immortalized myogenic cell lines. (C) 1994 Wiley-Liss, Inc.