Molecular Mechanisms Of Myofibril Assembly And Function

肌原纤维组装和功能的分子机制

• 批准号: 6823051
• 负责人: Robert Horowits
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6823051
• 关键词: actin binding protein; biomechanics; cardiac myocytes; chick embryo; electron microscopy; embryo /fetus tissue /cell culture; genetic library; genetic screening; green fluorescent proteins; molecular assembly /self assembly; muscle disorders; muscle proteins; myocardium; myofibrils; myogenesis; protein protein interaction; protein purification; protein structure function; striated muscles; structural biology; yeast two hybrid system

N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. To discover more about N-RAP's role in myofibril assembly , we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell. From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: alpha-actinin, filamin-2, and Krp1 (also called sarcosin). In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved. N-RAP contains an N-terminal LIM domain (N-RAP-LIM), C-terminal actin-binding super repeats homologous to nebulin (N-RAP-SR), and nebulin-related simple repeats in between the two (N-RAP-IB); these three regions of N-RAP were expressed as His-tagged recombinant proteins. We detected significant alpha-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to colocalize with alpha-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly. Based on biochemical data, immunofluorescence analysis of cultured embryonic chick cardiomyocytes, and the targeting and phenotypic effects of individual GFP-tagged regions of N-RAP, we previously 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 N-RAP-LIM, N-RAP-IB, or N-RAP-SR regions of N-RAP inhibited alpha-actinin assembly into Z-lines, expression of either the N-RAP-LIM-IB fusion or the N-RAP-IB-SR fusion permitted normal alpha-actinin organization. In contrast, the N-RAP-LIM-SR fusion inhibited alpha-actinin organization into Z-lines, indicating that the IB region is critical for Z-line assembly. While permitting normal Z-line assembly, N-RAP-LIM-IB and N-RAP-IB-SR decreased sarcomeric actin staining intensity; however, the effects of N-RAP-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, N-RAP-LIM-IB was consistently retained in mature Z-lines, while mature Z-lines without significant N-RAP-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. The present work strongly supports the N-RAP scaffolding model of premyofibril assembly, for the first time providing a molecular framework for understanding the initial steps used by muscle cells to build the contractile machinery responsible for force and motion. Abnormalities in assembling myofibrils and in linking them to other cellular structures are believed to underlie many diseases of skeletal muscles and the heart. Linkage analysis identified chromosome 10q24-26, a region including the N-RAP gene, as a disease locus for dilated cardiomyopathy (DCM). This past year we described the sequence, genomic structure and expression of human N-RAP, as well as an initial screen to determine whether N-RAP mutations cause cardiomyopathy. Human expressed sequence tag databases were searched with the published 3528 bp mouse N-RAP open reading frame (ORF). Putative cDNA sequences were interrogated by direct sequencing from cardiac and skeletal muscle RNA. We identified two human N-RAP isoforms with ORFs of 5085 bp (isoform C) and 5190 bp (isoform S), encoding products of 193-197 kDa. Genomic database searches localized N-RAP to human chromosome 10q25.3 and matched isoforms C and S to 41 and 42 exons. Only isoform C was detected in human cardiac RNA; in skeletal muscle, approximately 10% is isoform C and approximately 90% is isoform S. We investigated apparent differences between human N-RAP cDNA and mouse sequences. Two mouse N-RAP isoforms with ORFs of 5079 and 5184 bp were identified with ~ 85% similarity to human isoforms; previously published mouse sequences include cloning artifacts truncating the ORF. Murine and human isoforms have similar gene structure, tissue specificity, and size. N-RAP is especially conserved within its LIM domain and super repeat regions. We expressed both N-RAP isoforms and the previously described truncated N-RAP in embryonic chick cardiomyocytes. All constructs targeted to myofibril precursors and the cell periphery, and inhibited myofibril assembly. Several human N-RAP polymorphisms were detected, but none were unique to cardiomyopathy patients. N-RAP is highly conserved and exclusively expressed in cardiac and skeletal muscle. Abnormalities in the N-RAP gene remain excellent candidate causes for cardiac and skeletal myopathies.

N-RAP 是一种肌肉特异性蛋白，集中在骨骼肌的肌腱连接处和心肌的闰盘处，与肌原纤维的组装有关。为了更多地了解 N-RAP 在肌原纤维组装中的作用，我们使用酵母双杂交系统在小鼠骨骼肌 cDNA 文库中筛选能够在真核细胞中结合 N-RAP 的蛋白质。从酵母双杂交实验中，我们能够鉴定出三种新的 N-RAP 结合伴侣：α-肌动蛋白、filamin-2 和 Krp1（也称为肌氨酸）。体外结合测定用于验证这些相互作用并鉴定所涉及的 N-RAP 结构域。 N-RAP 包含 N 端 LIM 结构域 (N-RAP-LIM)、与 Nebulin 同源的 C 端肌动蛋白结合超级重复序列 (N-RAP-SR) 以及两者之间与 Nebulin 相关的简单重复序列 (N-RAP-IB)； N-RAP 的这三个区域被表达为带有 His 标签的重组蛋白。我们检测到显着的α-辅肌动蛋白与N-RAP-IB和N-RAP-LIM结合，细丝蛋白与N-RAP-SR结合，以及Krp1与N-RAP-SR和N-RAP-IB结合。在培养的鸡心肌细胞的肌原纤维组装过程中，N-RAP 和细丝蛋白似乎与细胞周边附近发现的最早的肌原纤维前体中的 α-肌动蛋白共定位，以及这些结构横向融合时形成的新生肌原纤维中。相比之下，Krp1 直到组装过程的后期才被定位，此时它出现在似乎横向融合的肌原纤维的外围。结果表明，N-RAP 结合伴侣的顺序募集可能在肌原纤维组装过程中发挥重要作用。 基于生化数据、培养胚胎鸡心肌细胞的免疫荧光分析以及 N-RAP 单个 GFP 标记区域的靶向和表型效应，我们之前提出了一种启动肌原纤维组装的新模型，其中 N-RAP 将 α-肌动蛋白和肌动蛋白组织成前肌原纤维 I-Z-I 复合物。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 三个区域中的两个的构建体）并观察对 α-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 N-RAP-LIM、N-RAP-IB 或 N-RAP-SR 区域会抑制 α-辅肌动蛋白组装成 Z 系，但 N-RAP-LIM-IB 融合物或 N-RAP-IB-SR 融合物的表达允许正常的 α-辅肌动蛋白组织。相比之下，N-RAP-LIM-SR 融合抑制 α-辅肌动蛋白组织成 Z 线，表明 IB 区域对于 Z 线组装至关重要。在允许正常 Z 线组装的同时，N-RAP-LIM-IB 和 N-RAP-IB-SR 降低了肌节肌动蛋白染色强度；然而，根据形态学评估和肌动蛋白染色强度的定量测量估计，N-RAP-LIM-IB 对肌动蛋白组装的影响明显更严重。此外，N-RAP-LIM-IB 始终保留在成熟的 Z 系中，而经常观察到没有显着 N-RAP-IB-SR 掺入的成熟 Z 系。我们得出的结论是，N-RAP 超级重复对于培养的胚胎鸡心肌细胞中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 支架方面也发挥着重要作用。目前的工作强烈支持前肌原纤维组装的 N-RAP 支架模型，首次提供了一个分子框架，用于理解肌肉细胞构建负责力和运动的收缩机制的初始步骤。 据信，肌原纤维组装以及将其与其他细胞结构连接的异常是许多骨骼肌和心脏疾病的基础。连锁分析确定染色体 10q24-26（包含 N-RAP 基因的区域）是扩张型心肌病 (DCM) 的疾病位点。去年，我们描述了人类 N-RAP 的序列、基因组结构和表达，以及确定 N-RAP 突变是否导致心肌病的初步筛选。使用已发表的 3528 bp 小鼠 N-RAP 开放阅读框 (ORF) 搜索人类表达序列标签数据库。通过对心肌和骨骼肌 RNA 进行直接测序来询问推定的 cDNA 序列。我们鉴定了两种人类 N-RAP 亚型，ORF 分别为 5085 bp（亚型 C）和 5190 bp（亚型 S），编码产物为 193-197 kDa。基因组数据库搜索定位于人类染色体 10q25.3 的 N-RAP，并将亚型 C 和 S 与 41 和 42 外显子相匹配。在人类心脏 RNA 中仅检测到同种型 C；在骨骼肌中，大约 10% 是同种型 C，大约 90% 是同种型 S。我们研究了人类 N-RAP cDNA 和小鼠序列之间的明显差异。两个小鼠 N-RAP 同工型的 ORF 分别为 5079 和 5184 bp，与人类同工型的相似度约为 85%；先前发表的小鼠序列包括截断 ORF 的克隆伪影。鼠类和人类亚型具有相似的基因结构、组织特异性和大小。 N-RAP 在其 LIM 结构域和超级重复区域内尤其保守。我们在胚胎鸡心肌细胞中表达了 N-RAP 亚型和之前描述的截短 N-RAP。所有构建体都针对肌原纤维前体和细胞外周，并抑制肌原纤维组装。检测到了几种人类 N-RAP 多态性，但没有一个是心肌病患者所独有的。 N-RAP 高度保守，仅在心肌和骨骼肌中表达。 N-RAP 基因异常仍然是心肌病和骨骼肌病的极好候选原因。