Carroll Stefanie, Lu Shajia, Herrera Amy H, Horowits Robert
Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health/DHHS, Bethesda, MD 20892, USA.
J Cell Sci. 2004 Jan 1;117(Pt 1):105-14. doi: 10.1242/jcs.00847.
N-RAP is a muscle-specific protein with an N-terminal LIM domain (LIM), C-terminal actin-binding super repeats homologous to nebulin (SR) and nebulin-related simple repeats (IB) in between the two. Based on biochemical data, immunofluorescence analysis of cultured embryonic chick cardiomyocytes and the targeting and phenotypic effects of these individual GFP-tagged regions of N-RAP, we proposed a novel model for the initiation of myofibril assembly in which N-RAP organizes alpha-actinin and actin into the premyofibril I-Z-I complexes. We tested the proposed model by expressing deletion mutants of N-RAP (i.e. constructs containing two of the three regions of N-RAP) in chick cardiomyocytes and observing the effects on alpha-actinin and actin organization into mature sarcomeres. Although individually expressing either the LIM, IB, or SR regions of N-RAP inhibited alpha-actinin assembly into Z-lines, expression of either the LIM-IB fusion or the IB-SR fusion permitted normal alpha-actinin organization. In contrast, the LIM-SR fusion (LIM-SR) inhibited alpha-actinin organization into Z-lines, indicating that the IB region is critical for Z-line assembly. While permitting normal Z-line assembly, LIM-IB and IB-SR decreased sarcomeric actin staining intensity; however, the effects of LIM-IB on actin assembly were significantly more severe, as estimated both by morphological assessment and by quantitative measurement of actin staining intensity. In addition, LIM-IB was consistently retained in mature Z-lines, while mature Z-lines without significant IB-SR incorporation were often observed. We conclude that the N-RAP super repeats are essential for organizing actin filaments during myofibril assembly in cultured embryonic chick cardiomyocytes, and that they also play an important role in removal of the N-RAP scaffold from the completed myofibrillar structure. This work strongly supports the N-RAP scaffolding model of premyofibril assembly.
N-RAP是一种肌肉特异性蛋白，其N端有一个LIM结构域（LIM），C端有与伴肌动蛋白同源的肌动蛋白结合超重复序列（SR），两者之间还有伴肌动蛋白相关的简单重复序列（IB）。基于生化数据、对培养的胚胎鸡心肌细胞的免疫荧光分析以及N-RAP这些单独的绿色荧光蛋白标记区域的靶向和表型效应，我们提出了一种肌原纤维组装起始的新模型，其中N-RAP将α-辅肌动蛋白和肌动蛋白组织成前肌原纤维I-Z-I复合体。我们通过在鸡心肌细胞中表达N-RAP的缺失突变体（即包含N-RAP三个区域中的两个区域的构建体）并观察其对α-辅肌动蛋白和肌动蛋白组织成成熟肌节的影响，来测试所提出的模型。尽管单独表达N-RAP的LIM、IB或SR区域会抑制α-辅肌动蛋白组装到Z线中，但LIM-IB融合体或IB-SR融合体的表达允许α-辅肌动蛋白正常组织。相比之下，LIM-SR融合体（LIM-SR）抑制α-辅肌动蛋白组织到Z线中，表明IB区域对Z线组装至关重要。虽然允许正常的Z线组装，但LIM-IB和IB-SR降低了肌节肌动蛋白染色强度；然而，通过形态学评估和肌动蛋白染色强度的定量测量估计，LIM-IB对肌动蛋白组装的影响明显更严重。此外，LIM-IB始终保留在成熟的Z线中，而经常观察到没有明显IB-SR掺入的成熟Z线。我们得出结论，N-RAP超重复序列对于在培养的胚胎鸡心肌细胞的肌原纤维组装过程中组织肌动蛋白丝至关重要，并且它们在从完整的肌原纤维结构中去除N-RAP支架方面也起着重要作用。这项工作有力地支持了前肌原纤维组装的N-RAP支架模型。
