Arf6 (ADP ribosylation factor 6), activated by Arf-GEF (guanine nucleoside exchange factor), is involved in the membrane trafficking and actin-remodeling which are critical for maintenance of cell organization and activity and for fusion of myoblasts to form myotubes/myofibers. EFA6A (exchange factor for Arf6 type A) and BRAG2 (brefeldin A-resistant Arf-GEF 2) represent members of discrete subfamilies of Arf-GEF, while PIP5Kγ (phosphatidylinositol4-phosphate5-kinase γ) produces PI 4,5-bisphosphate (PIP2) and it is target for Arf6. In the present study, immunoreactive bands for Arf6, EFA6A, BRAG2 and PIP5Kγ were detected in immunoblots of skeletal muscle homogenates of mice at E18D (embryonic day 18), while the bands for Arf6, EFA6A and PIP5Kγ were reduced in density and no significant bands for BRAG2 were discerned at P1D (postnatal 1 day). No immunoblot bands for any of the molecules were eventually detected in skeletal fibers of adult mice. Immunoreactivities for endogenous Arf6, EFA6A and PIP5Kγ were visualized using immuno-light microscopy localized as periodic striations running perpendicular to the longitudinal axes of skeletal muscle fibers of mice at E18D and P1D. All the striations were co-immunoreactive for β-actin in double immunofluorescence microscopy, and the immunoreactivities were confined to thin myofilaments at sarcomeric I-domains in immuno-electron microscopy. On the other hand, immunoreactivities for Arf6, BRAG2 and PIP5Kγ were conspicuous on plasmalemma of myoblasts at E14D, while immunoreactivity for EFA6A was already distinct in striations perpendicular to myofibrils in myotubes at E14D. The present findings suggest three possibilities: involvement of EFA6A-activated Arf6 together with PIP5Kγ in maturation of myofibrils, movement of Arf6 and PIP5Kγ from the plasmalemma of myoblasts to myofibrils of myotubes, and that of BRAG2 to the cytoplasm of myotubes; and further a function of EFA6A independent of the activation of Arf6 in immature myofibrils. In addition, the involvement of Arf6, BRAG2 and PIP5Kγ in the fusion of myoblasts into myotubes was supported by the present finding.

中文翻译：

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Arf6及其激活物EFA6A和BRAG2及其效应子PIP5激酶γ在小鼠骨骼肌发育中的肌管原纤维和成肌细胞质膜上的离散定位模式。

由Arf-GEF（鸟嘌呤核苷交换因子）激活的Arf6（ADP核糖基化因子6）参与膜运输和肌动蛋白重塑，这对于维持细胞组织和活性以及成肌细胞融合形成肌管/肌纤维至关重要。 。EFA6A（A型Arf6的交换因子）和BRAG2（抗布雷菲德菌素A的Arf-GEF 2）代表Arf-GEF离散亚家族的成员，而PIP5Kγ（磷脂酰肌醇4-磷酸5-激酶γ）产生PI 4,5-双磷酸酯（PIP2 ），它是Arf6的目标。在本研究中，在E18D（胚胎第18天）的小鼠骨骼肌匀浆的免疫印迹中检测到Arf6，EFA6A，BRAG2和PIP5Kγ的免疫反应带，而Arf6，EFA6A和PIP5Kγ的带密度降低且没有明显的带。在出生后1天（P1D）可以识别出BRAG2的水平。最终在成年小鼠的骨骼肌中未检测到任何分子的免疫印迹带。使用免疫光显微镜观察内源性Arf6，EFA6A和PIP5Kγ的免疫反应性，该免疫光显微镜定位为周期性条纹，垂直于E18D和P1D小鼠骨骼肌纤维的纵轴。在双重免疫荧光显微镜检查中，所有条纹均对β-肌动蛋白具有共同免疫反应活性，并且在免疫电子显微镜检查中，免疫反应性局限于肌节I结构域的薄肌丝。另一方面，在E14D的成肌细胞质膜上对Arf6，BRAG2和PIP5Kγ的免疫反应性很明显，而在E14D的肌管中垂直于肌原纤维的条纹中EFA6A的免疫反应性已经很明显。目前的发现提出了三种可能性：EFA6A激活的Arf6与PIP5Kγ参与肌原纤维的成熟，Arf6和PIP5Kγ从成肌细胞质膜到肌管肌原纤维的运动，以及BRAG2到肌管细胞质的运动；EFA6A的功能还与未成熟肌原纤维中Arf6的激活无关。另外，本发现支持了Arf6，BRAG2和PIP5Kγ参与成肌细胞融合到肌管中。