MOLECULAR MECHANISMS OF MYOFIBRIL ASSEMBLY AND FUNCTION

肌原纤维组装和功能的分子机制

• 批准号: 6289016
• 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/6289016
• 关键词: actin binding protein; electron microscopy; extracellular matrix; genetic mapping; human genetic material tag; human tissue; immunofluorescence technique; laboratory mouse; molecular cloning; muscle proteins; muscle tension; myofibrils; nucleic acid repetitive sequence; protein isoforms; protein sequence; protein structure function; stoichiometry; striated muscles

Previously, we had discovered N-RAP, a novel, muscle specific protein with homology to nebulin. The C-terminal half of N-RAP contains more than two complete actin-binding super repeats similar to those found in nebulin. The N-terminus contains the consensus sequence of a cysteine rich LIM domain, which binds talin in vitro. Our work had localized N- RAP at the myotendon junction in skeletal muscle and at the intercalated disk in cardiac muscle, leading us to hypothesize that the N-RAP domains may be involved in linking the ends of myofibrils to specialized protein complexes found beneath the sarcolemma. In the past year, we used a variety of methods to test this hypothesis. Double immunofluorescent staining of cultured embryonic chick cardiomyocytes for N-RAP and either alpha-actinin, titin, or vinculin showed that myofibrillogenesis and cell contact formation are both associated with targeting of N-RAP to the longitudinal ends of cardiac myocytes. In addition, preliminary results from experiments in which defined regions of N-RAP were expressed in chick cardiomyocytes as fusions with green fluorescent protein (GFP) suggest that the N-RAP LIM domain is responsible for targeting the molecule to myofibril ends and cell contacts. Meanwhile, GFP-tagged N-RAP super repeats are incorporated into sarcomeric actin filaments. We found that, at the ultrastructural level, N-RAP localization is complementary to that of nebulin in skeletal muscle. Even though both proteins contain actin-binding super repeats, nebulin is exclusively found in the sarcomeres, while N-RAP is confined to the terminal bundles of actin filaments at the myotendon junction. These results are consistent with our model of N-RAP as a modular link between the terminal actin filaments of myofibrils and the protein complexes located beneath the cell membrane, with the N-RAP LIM domain targeting the molecule to the ends of the muscle cell. Furthermore, we found that N-RAP binds muscle LIM protein (MLP) in vitro and that the targeting of N-RAP to the intercalated disks in mice lacking MLP is imprecise. The results suggest that N-RAP targeting to the cardiac intercalated disk is influenced by its interaction with MLP. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherentes region of the intercalated disk membrane, while N-RAP extends ~100 nm further towards the interior of the cell. Furthermore, N-RAP remains tightly bound to myofibrils and fascia adherentes during biochemical purification, consistent with it being a key constituent in the mechanical link between these two structures. - muscle, heart, myofibril, nebulin, N-RAP

此前，我们发现了 N-RAP，一种与 nebulin 同源的新型肌肉特异性蛋白。 N-RAP 的 C 端一半包含两个以上完整的肌动蛋白结合超级重复序列，与星云蛋白中发现的类似。 N 末端包含富含半胱氨酸的 LIM 结构域的共有序列，可在体外结合踝蛋白。我们的工作将 N-RAP 定位在骨骼肌的肌腱连接处和心肌的闰盘上，这使我们推测 N-RAP 结构域可能参与将肌原纤维末端与肌膜下方发现的特殊蛋白质复合物连接起来。在过去的一年里，我们使用了多种方法来检验这个假设。对培养的胚胎鸡心肌细胞进行 N-RAP 和 α-肌动蛋白、肌联蛋白或纽蛋白的双重免疫荧光染色表明，肌原纤维发生和细胞接触形成均与 N-RAP 靶向心肌细胞纵向末端有关。此外，N-RAP 的特定区域在鸡心肌细胞中表达为与绿色荧光蛋白 (GFP) 融合的实验的初步结果表明，N-RAP LIM 结构域负责将该分子靶向肌原纤维末端和细胞接触。同时，GFP 标记的 N-RAP 超级重复序列被整合到肌节肌动蛋白丝中。我们发现，在超微结构水平上，N-RAP 定位与骨骼肌中的星云蛋白定位是互补的。尽管两种蛋白均含有肌动蛋白结合超级重复序列，但 Nebulin 只存在于肌节中，而 N-RAP 仅限于肌腱连接处的肌动蛋白丝末端束。这些结果与我们的 N-RAP 模型一致，N-RAP 是肌原纤维末端肌动蛋白丝与位于细胞膜下方的蛋白质复合物之间的模块化连接，其中 N-RAP LIM 结构域将分子靶向肌细胞末端。此外，我们发现 N-RAP 在体外结合肌肉 LIM 蛋白（MLP），并且 N-RAP 对缺乏 MLP 的小鼠闰盘的靶向是不精确的。结果表明，N-RAP 靶向心脏闰盘受到其与 MLP 相互作用的影响。小鼠心肌的双重免疫金标记显示，纽蛋白位于紧邻闰盘膜的筋膜粘附区域的位置，而 N-RAP 则向细胞内部进一步延伸约 100 nm。此外，N-RAP 在生化纯化过程中仍然与肌原纤维和筋膜粘附紧密结合，这与它作为这两种结构之间机械连接的关键成分一致。 - 肌肉、心脏、肌原纤维、星云蛋白、N-RAP