Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions is unclear. Here, by examining the mammalian fusogenic synapse, where invasive protrusions are generated to promote cell membrane juxtaposition and fusion, we have uncovered the mechanism underlying invasive protrusion formation. We show that two nucleation-promoting factors for the Arp2/3 complex, WAVE and N-WASP, exhibit different localization patterns in the protrusions. Whereas WAVE is closely associated with the plasma membrane at the leading edge of the protrusive structures, N-WASP is enriched with WIP along the actin bundles in the shafts of the protrusions. During protrusion initiation and growth, the Arp2/3 complex nucleates branched actin filaments to generate low-density actin clouds in which the large GTPase dynamin organizes the new branched actin filaments into bundles, followed by actin-bundle stabilization by WIP, the latter functioning as an actin-bundling protein. Disruption of any of these components results in defective protrusions and failed myoblast fusion in cultured cells and mouse embryos. Together, our study has revealed the intricate spatiotemporal coordination between two nucleation-promoting factors and two actin-bundling proteins in building invasive protrusions at the mammalian fusogenic synapse and has general implications in understanding invasive protrusion formation in cellular processes beyond cell–cell fusion.

侵袭性膜突起在各种细胞过程中起着核心作用。与丝状伪足不同，侵袭性突起在机械上是刚性的，并由支链肌动蛋白聚合推动。然而，支链肌动蛋白丝如何组织以产生手指状侵袭性突起尚不清楚。在这里，通过检查哺乳动物融合突触，其中产生侵袭性突起以促进细胞膜并列和融合，我们揭示了侵袭性突起形成的机制。我们表明 Arp2/3 复合物的两种成核促进因子 WAVE 和 N-WASP 在突起中表现出不同的定位模式。WAVE 与突起结构前缘的质膜密切相关，而 N-WASP 则沿着突起轴中的肌动蛋白束富含 WIP。在突起起始和生长过程中，Arp2/3 复合物使支链肌动蛋白丝成核以产生低密度肌动蛋白云，其中大 GTP 酶动力蛋白将新的支链肌动蛋白丝组织成束，然后通过 WIP 实现肌动蛋白束稳定，后者作为肌动蛋白束蛋白发挥作用。这些成分中的任何一个破坏都会导致培养细胞和小鼠胚胎中的突起缺陷和成肌细胞融合失败。总之，我们的研究揭示了两种成核促进因子和两种肌动蛋白束缚蛋白在哺乳动物融合突触处构建侵袭性突起过程中错综复杂的时空协调，并对理解细胞间融合后细胞过程中侵袭性突起的形成具有一般意义。