BackgroundNuclear 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.MethodsThe 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.ResultsIn 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 μm2, 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).ConclusionsNRIP is a novel actin‐binding protein for invadosome formation to induce myoblast fusion.

中文翻译：

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核受体相互作用蛋白在协调成肌细胞融合侵袭体形成中的创新作用


背景核受体相互作用蛋白（NRIP）用途广泛，可与多种蛋白质结合以执行其多种生物学功能。据报道，NRIP 缺陷会导致成体肌肉再生过程中肌纤维变小，这表明 NRIP 在成肌细胞融合中发挥着至关重要的作用。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。通过细胞融合测定和延时显微镜证实了 NRIP 参与成肌细胞融合。生成 NRIP 突变体用于 NRIP 无效 C2C12（称为 KO19）细胞和肌肉特异性 NRIP 敲除（NRIP cKO）小鼠的机制研究。利用GEO图谱数据库分析杜氏肌营养不良症（DMD）患者中NRIP的表达。结果在本研究中，我们发现NRIP在体外和细胞内与肌动蛋白直接相互作用。免疫荧光显微镜显示，在 C2C12 分化过程中，内源性 NRIP 与肌动蛋白、Tks5 和 cortactin 等入侵体成分共定位于细胞尖端。与野生型 C2C12 细胞相比，KO19 细胞的成肌细胞融合表现出显着缺陷（3.16% vs. 33.67%，p < 0.005）。与对照相比，KO19 细胞中过表达的 NRIP 可以挽救肌管形成（3.37% vs. 1.00%，p < 0.01）。我们通过细胞-细胞融合实验进一步证实NRIP直接参与细胞融合。我们通过分析 NRIP 缺失细胞中的 NRIP 突变体，研究了成肌细胞融合的侵袭体形成机制，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侵袭体（富集比，1.00 vs. 2.54，p < 0.01）和肌管形成（21.82% vs. 35.71%，p < 0.05）在 KO19 细胞中，以及在 KO19 细胞和肌肉特异性 NRIP 敲除 (NRIP cKO) 小鼠中强制表达 NRIP，与对照组相比，肌纤维尺寸增加（超过 1500 μm2，61.01% vs. 20.57%，p < 0.001）。我们还发现，与健康对照相比，DMD 患者的 NRIP mRNA 水平降低（18 072 vs. 28 289，p < 0.001，N = 10，两组）。 结论 NRIP 是一种新型肌动蛋白结合蛋白，用于侵袭体形成诱导成肌细胞融合。