An invasive podosome-like structure promotes fusion pore formation during myoblast fusion.

Recent studies in Drosophila have implicated actin cytoskeletal remodeling in myoblast fusion, but the cellular mechanisms underlying this process remain poorly understood. Here we show that actin polymerization occurs in an asymmetric and cell type-specific manner between a muscle founder cell and a fusion-competent myoblast (FCM). In the FCM, a dense F-actin-enriched focus forms at the site of fusion, whereas a thin sheath of F-actin is induced along the apposing founder cell membrane. The FCM-specific actin focus invades the apposing founder cell with multiple finger-like protrusions, leading to the formation of a single-channel macro fusion pore between the two muscle cells. Two actin nucleation-promoting factors of the Arp2/3 complex, WASP and Scar, are required for the formation of the F-actin foci, whereas WASP but not Scar promotes efficient foci invasion. Our studies uncover a novel invasive podosome-like structure (PLS) in a developing tissue and reveal a previously unrecognized function of PLSs in facilitating cell membrane juxtaposition and fusion.

A critical function for the actin cytoskeleton in targeted exocytosis of prefusion vesicles during myoblast fusion.

Myoblast fusion is an essential step during muscle differentiation. Previous studies in Drosophila have revealed a signaling pathway that relays the fusion signal from the plasma membrane to the actin cytoskeleton. However, the function for the actin cytoskeleton in myoblast fusion remains unclear. Here we describe the characterization of solitary (sltr), a component of the myoblast fusion signaling cascade. sltr encodes the Drosophila ortholog of the mammalian WASP-interacting protein. Sltr is recruited to sites of fusion by the fusion-competent cell-specific receptor Sns and acts as a positive regulator for actin polymerization at these sites. Electron microscopy analysis suggests that formation of F-actin-enriched foci at sites of fusion is involved in the proper targeting and coating of prefusion vesicles. These studies reveal a surprising cell-type specificity of Sltr-mediated actin polymerization in myoblast fusion, and demonstrate that targeted exocytosis of prefusion vesicles is a critical step prior to plasma membrane fusion.

Insights into the sirtuin mechanism from ternary complexes containing NAD+ and acetylated peptide.

Sirtuin proteins comprise a unique class of NAD+-dependent protein deacetylases. Although several structures of sirtuins have been determined, the mechanism by which NAD+ cleavage occurs has remained unclear. We report the structures of ternary complexes containing NAD+ and acetylated peptide bound to the bacterial sirtuin Sir2Tm and to a catalytic mutant (Sir2Tm(H116Y)). NAD+ in these structures binds in a conformation different from that seen in previous structures, exposing the alpha face of the nicotinamide ribose to the carbonyl oxygen of the acetyl lysine substrate. The NAD+ conformation is identical in both structures, suggesting that proper coenzyme orientation is not dependent on contacts with the catalytic histidine. We also present the structure of Sir2Tm(H116A) bound to deacteylated peptide and 3'-O-acetyl ADP ribose. Taken together, these structures suggest a mechanism for nicotinamide cleavage in which an invariant phenylalanine plays a central role in promoting formation of the O-alkylamidate reaction intermediate and preventing nicotinamide exchange.