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Cell-cell fusion is an indispensable process in the conception, development and physiology of multicellular organisms. Here we demonstrate a direct and noncanonical role for dynamin, best known as a fission GTPase in endocytosis, in cell-cell fusion. Our genetic and cell biological analyses show that dynamin colocalizes within the F-actin-enriched podosome-like structures at the fusogenic synapse, which is required for generating invasive membrane protrusions and myoblast fusion in vivo, in an endocytosis-independent manner. Biochemical, negative stain EM and cryo-electron tomography (cryo-ET) analyses revealed that dynamin forms helices that directly bundles actin filaments by capturing multiple actin filaments at their outer rim via interactions with dynamin's proline-rich domain.GTP hydrolysis by dynamin triggers disassembly of the dynamin helix, exposes the sides of the actin filaments, promotes dynamic Arp2/3-mediated branched actin polymerization, and generates a mechanically stiff actin network. Thus, dynamin functions as a unique actin-bundling protein that enhances mechanical force generation by the F-actin network in a GTPase-dependent manner. Our findings have universal implications for understanding dynamin-actin interactions in various cellular processes beyond cell-cell fusion. 3Cell-cell fusion is essential for the conception, development, regeneration and physiology of multicellular organisms 1-3 . A common theme underlying cell-cell fusion events ranging from insects to mammals is the presence of actin-propelled invasive protrusions at the site of fusion, known as the fusogenic synapse 4-8 . Such invasive protrusions were first identified as part of an F-actin-enriched podosome-like structure (PLS) in Drosophila myoblast fusion 7 . The PLS is generated by an "attacking" fusion partner, the fusion-competent myoblast (FCM), which drills multiple invasive protrusions into the "receiving" fusion partner, the muscle founder cell, to promote cell membrane juxtaposition and fusion pore formation 7,9,10 . Although each protrusion has a diameter similar to that of a filopodium, these protrusions are mechanically stiffer and are capable of triggering mechanosensitive responses in the receiving cell 11 . Mechanoresponsive accumulations of myosin II and spectrin in the receiving cell, in turn, increase the cortical tension of the receiving cell, thus generating resisting forces against the invasive protrusions and bringing the two cell membranes to close proximity to promote cell fusion 11,12 .The invasive protrusions from the attacking cells are propelled by Arp2/3-mediated branched actin polymerization, which generates short actin filaments more suitable for mechanical work than long and linear filaments 9 . However, how the branched actin filaments are organized to generate mechanically stiff membrane protrusions during fusion is unknown.Dynamin (Dyn) is a large GTPase best known for its role in endocytosis. Dynamin catalyzes membrane fission by forming rings/helices around necks of a budding endocytic ve...

Skeletal muscles can regenerate over the lifetime from resident muscle stem cells (MuSCs). Interactions between MuSCs and extracellular matrix (ECM) proteins are essential for muscle regeneration. The best-known receptors for ECM proteins are integrins, a family composed of twenty-some heterodimeric combinations of an αand a βsubunit. β1integrin (encoded by Itgb1) is required for quiescence, proliferation, migration, and fusion of Pax7 + MuSCs in the mouse model. β3integrin (encoded by Itgb3) has been reported to be critical for the myogenic differentiation of C2C12 myoblasts, and Itgb3 germline mutant mice were shown to regenerate few if any myofibers after injury. To investigate the autonomous role of Itgb3 in the myogenic lineage in vivo, we conditionally inactivated a floxed Itgb3 allele (Itgb3 F ) by constitutive Pax7-Cre and tamoxifen-inducible Pax7-CreERT2 drivers. Unexpectedly, we found no defects in muscle regeneration in both conditional knockout models. In vitro studies using Itgb3 mutant myoblasts or RNAi knockdown of Itgb3 in myoblasts also did not reveal a role for myogenic differentiation. As β1and β3integrins share ECM ligands and downstream signaling effectors, we further examined Itgb3's role in a Itgb1 haploid background.Still, we found no evidence for an autonomous role of Itgb3 in muscle regeneration in vivo. Thus, while Itgb3 is critical for the differentiation of C2C12 cells, the regenerative defects reported for the Itgb3 germline mutant are not due to its role in the MuSC. We conclude that if β3integrin does have a role in Pax7 + MuSCs, it is compensated by β1and/or another βintegrin(s).