Discrete localization patterns of Arf6, and its activators EFA6A and BRAG2, and its effector PIP5kinaseγ on myofibrils of myotubes and plasma membranes of myoblasts in developing skeletal muscles of mice

• Published in: Acta histochemica Vol. 122; no. 3; p. 151513
• Main Authors: Chomphoo, Surang, Sakagami, Hiroyuki, Kondo, Hisatake, Hipkaeo, Wiphawi
• Format: Journal Article
• Language: English
• Published: Germany Elsevier GmbH 01.04.2020
• Subjects: ADP-Ribosylation Factors - metabolism; Animals; Arf6; BRAG2; Cell Membrane - metabolism; Developing skeletal muscle; EFA6A; Guanine Nucleotide Exchange Factors - metabolism; Mice; Mice, Inbred ICR; Muscle Fibers, Skeletal - metabolism; Muscle, Skeletal - growth & development; Muscle, Skeletal - metabolism; Myoblasts - metabolism; Myofibrils - metabolism; Phosphotransferases (Alcohol Group Acceptor) - metabolism; PIP5kinaseγ; Thin myofilament; Developing skeletal muscle; BRAG2; Thin myofilament; EFA6A; Arf6; PIP5kinaseγ
• ISSN: 0065-1281; 1618-0372
• DOI: 10.1016/j.acthis.2020.151513

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.