核受体相互作用蛋白在协调成肌细胞融合的侵袭小体形成中的创新作用。
The Innovative Role of Nuclear Receptor Interaction Protein in Orchestrating Invadosome Formation for Myoblast Fusion.
作者信息
Chen Hsin-Hsiung, Lin Chia-Yang, Han Ya-Ju, Huang Yun-Hsin, Liu Yi-Hsiang, Hsu Wan-En, Tsai Li-Kai, Lai Hsing-Jung, Tsao Yeou-Ping, Huang Hsiang-Po, Chen Show-Li
机构信息
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Department of Neurology, National Taiwan University Hospital, Taipei, Taiwan.
出版信息
J Cachexia Sarcopenia Muscle. 2024 Dec;15(6):2559-2573. doi: 10.1002/jcsm.13598. Epub 2024 Sep 25.
BACKGROUND
Nuclear receptor interaction protein (NRIP) is versatile and engages with various proteins to execute its diverse biological function. NRIP deficiency was reported to cause small myofibre size in adult muscle regeneration, indicating a crucial role of NRIP in myoblast fusion.
METHODS
The colocalization and interaction of NRIP with actin were investigated by immunofluorescence and immunoprecipitation assay, respectively. The participation of NRIP in myoblast fusion was demonstrated by cell fusion assay and time-lapse microscopy. The NRIP mutants were generated for mechanism study in NRIP-null C2C12 (termed KO19) cells and muscle-specific NRIP knockout (NRIP cKO) mice. A GEO profile database was used to analyse NRIP expression in Duchenne muscular dystrophy (DMD) patients.
RESULTS
In this study, we found that NRIP directly and reciprocally interacted with actin both in vitro and in cells. Immunofluorescence microscopy showed that the endogenous NRIP colocalized with components of invadosome, such as actin, Tks5, and cortactin, at the tips of cells during C2C12 differentiation. The KO19 cells were generated and exhibited a significant deficit in myoblast fusion compared with wild-type C2C12 cells (3.16% vs. 33.67%, p < 0.005). Overexpressed NRIP in KO19 cells could rescue myotube formation compared with control (3.37% vs. 1.00%, p < 0.01). We further confirmed that NRIP directly participated in cell fusion by using a cell-cell fusion assay. We investigated the mechanism of invadosome formation for myoblast fusion, which depends on NRIP-actin interaction, by analysing NRIP mutants in NRIP-null cells. Loss of actin-binding of NRIP reduced invadosome (enrichment ratio, 1.00 vs. 2.54, p < 0.01) and myotube formation (21.82% vs. 35.71%, p < 0.05) in KO19 cells and forced NRIP expression in KO19 cells and muscle-specific NRIP knockout (NRIP cKO) mice increased myofibre size compared with controls (over 1500 μm, 61.01% vs. 20.57%, p < 0.001). We also found that the NRIP mRNA level was decreased in DMD patients compared with healthy controls (18 072 vs. 28 289, p < 0.001, N = 10 for both groups).
CONCLUSIONS
NRIP is a novel actin-binding protein for invadosome formation to induce myoblast fusion.
背景
核受体相互作用蛋白（NRIP）功能多样，可与多种蛋白质相互作用以执行其多种生物学功能。据报道，NRIP缺乏会导致成年肌肉再生过程中肌纤维尺寸变小，这表明NRIP在成肌细胞融合中起关键作用。
方法
分别通过免疫荧光和免疫沉淀试验研究NRIP与肌动蛋白的共定位和相互作用。通过细胞融合试验和延时显微镜观察证明NRIP参与成肌细胞融合。构建NRIP突变体用于在NRIP基因敲除的C2C12细胞（称为KO19）和肌肉特异性NRIP基因敲除（NRIP cKO）小鼠中进行机制研究。使用GEO谱数据库分析杜氏肌营养不良（DMD）患者中NRIP的表达。
结果
在本研究中，我们发现NRIP在体外和细胞内均直接与肌动蛋白相互作用。免疫荧光显微镜显示，在C2C12分化过程中，内源性NRIP与侵袭小体的成分如肌动蛋白、Tks5和皮质肌动蛋白在细胞尖端共定位。与野生型C2C12细胞相比，构建的KO19细胞在成肌细胞融合方面表现出显著缺陷（3.16%对33.67%，p<0.005）。与对照组相比，KO19细胞中过表达的NRIP可挽救肌管形成（3.37%对1.00%，p<0.01）。我们通过细胞-细胞融合试验进一步证实NRIP直接参与细胞融合。通过分析NRIP基因敲除细胞中的NRIP突变体，我们研究了侵袭小体形成促进成肌细胞融合的机制，该机制依赖于NRIP-肌动蛋白相互作用。NRIP肌动蛋白结合的丧失减少了KO19细胞中侵袭小体的形成（富集率，1.00对2.54，p<0.01）和肌管形成（21.82%对35.71%，p<0.05），并且在KO19细胞和肌肉特异性NRIP基因敲除（NRIP cKO）小鼠中强制表达NRIP与对照组相比增加了肌纤维尺寸（超过1500μm，61.01%对20.57%，p<0.001）。我们还发现，与健康对照组相比，DMD患者中NRIP mRNA水平降低（18072对28289，p<0.001，两组N均为10）。
结论
NRIP是一种新型的肌动蛋白结合蛋白，可促进侵袭小体形成以诱导成肌细胞融合。
Zotero 插件