An F-actin–enriched protrusion resembling an invasive podosome promotes fusion pore formation between muscle founder cells and fusion-competent myoblasts.

The development and regeneration of skeletal muscles require the fusion of mononulceated muscle cells to form multinucleated, contractile muscle fibers. Studies using a simple genetic model, Drosophila melanogaster, have discovered many evolutionarily conserved fusion-promoting factors in vivo. Recent work in zebrafish and mouse also identified several vertebrate-specific factors required for myoblast fusion. Here, we integrate progress in multiple in vivo systems and highlight conceptual advance in understanding how muscle cell membranes are brought together for fusion. We focus on the molecular machinery at the fusogenic synapse and present a three-step model to describe the molecular and cellular events leading to fusion pore formation.

Cell-cell fusion is critical for the conception, development and physiology of multicellular organisms. Although cellular fusogenic proteins and the actin cytoskeleton are implicated in cell-cell fusion, whether and how they coordinate to promote plasma membrane fusion remain unclear. Here, we reconstituted a high-efficiency, inducible cell-fusion culture system in the normally non-fusing Drosophila S2R+ cells. Both fusogenic proteins and actin cytoskeletal rearrangements were necessary for cell fusion, and, in combination, were sufficient to impart fusion competence. Localized actin polymerization triggered by specific cell-cell or cell-matrix adhesion molecules propelled invasive cell membrane protrusions, which, in turn, promoted fusogenic protein engagement and plasma membrane fusion. This de novo cell-fusion culture system reveals a general role for actin-propelled invasive membrane protrusions in driving fusogenic protein engagement during cell-cell fusion.

Summary Dynamic rearrangements of the actin cytoskeleton play a key role in numerous cellular processes. In Drosophila, fusion between a muscle founder cell and a fusion competent myoblast (FCM) is mediated by an invasive, F-actin-enriched podosome-like structure (PLS). Here, we show that the dynamics of the PLS is controlled by Blown fuse (Blow), a cytoplasmic protein required for myoblast fusion but whose molecular function has been elusive. We demonstrate that Blow is an FCM-specific protein that co-localizes with WASP, WIP/Solitary and the actin focus within the PLS. Biochemically, Blow modulates the stability of the WASP-WIP complex by competing with WASP for WIP binding, leading to a rapid exchange of WASP, WIP and G-actin within the PLS, which, in turn, actively invades the adjacent founder cell to promote fusion pore formation. These studies identify a regulatory protein that modulates the actin cytoskeletal dynamics by controlling the stability of the WASP-WIP complex.

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Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage. Here, we report that α/β-spectrin (β is also called karst) dynamically accumulates and dissolves at the fusogenic synapse between fusing Drosophila muscle cells, where an attacking fusion partner invades its receiving partner with actin-propelled protrusions to promote cell fusion. Using genetics, cell biology, biophysics and mathematical modelling, we demonstrate that spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse. The transiently accumulated spectrin network functions as a cellular fence to restrict the diffusion of cell-adhesion molecules and a cellular sieve to constrict the invasive protrusions, thereby increasing the mechanical tension of the fusogenic synapse to promote cell membrane fusion. Our study reveals a function of spectrin as a mechanoresponsive protein and has general implications for understanding spectrin function in dynamic cellular processes.

Group I p21-activated kinases organize actin filaments in myoblasts into dense foci, which promote podosome invasion and subsequent myoblast fusion.

Cell–cell fusion is a fundamental cellular process that is essential for development as well as fertilization. Myoblast fusion to form multinucleated skeletal muscle myotubes is a well studied, yet incompletely understood example of cell–cell fusion that is essential for formation of contractile skeletal muscle tissue. Studies in this report identify several novel cytoskeletal events essential to an early phase of myoblast fusion among cultured murine myoblasts. During myoblast pairing and alignment, cortical actin filaments organize into a dense actin wall structure that parallels and extends the length of the plasma membrane of the bipolar, aligned cells. As fusion progresses, gaps appear within the actin wall at sites of vesicle accumulation, the vesicles pair across the aligned myoblasts, cell–cell contacts and fusion pores form. Inhibition of nonmuscle myosin IIA (NM-MHC-IIA) motor activity prevents formation of this cortical actin wall, as well as the appearance of vesicles at a membrane proximal location, and myoblast fusion. These results suggest that early formation of a subplasmalemmal actin wall during myoblast alignment is a critical event for myoblast fusion that supports bipolar membrane alignment and temporally regulates trafficking of vesicles to the nascent fusion sites during skeletal muscle myoblast differentiation.