Nuclear receptor interaction protein (NRIP) as a novel actin-binding protein involved in invadosome formation for myoblast fusion

To investigate the role of nuclear receptor interaction protein (NRIP) in myoblast fusion, both the primary myoblasts from muscle-specific NRIP-knockout mice and NRIP-null C2C12 cells (KO19 cells) exhibited a significant deficit in the fusion index during myogenesis; on the other hand, overexpressed NRIP in KO19 cells could rescue myotube formation. Furthermore, NRIP was found to interact with actin directly and reciprocally that is an invadosome component for myoblast fusion. Endogenous NRIP colocalized with components of invadosome such as F-actin, Tks5, and cortactin at the tips of cells during C2C12 differentiation, and exogenous NRIP was enriched with actin at the tip of attacking cells during myogenic fusion, implying that NRIP is a novel invadosome component. Using time-lapse microscopy and cell–cell fusion assays further confirmed NRIP directly participates in cell fusion through actin. Moreover, to map the domain of NRIP–actin binding, NRIP interacted with actin either through WD40 domains directly for binding or indirectly through the IQ domain for α-actinin 2 binding with actin. NRIP with actin binding was strongly correlated with invadosome formation and myotube fusion. Collectively, NRIP acts as a novel actin-binding protein through its WD40 or the IQ to form invadosomes that trigger myoblast fusion.