Fus1-dependent actin accumulation at the prospective fusion site. (A) Homothallic h90 p^map3:tdTomato GFP-CHD strain. Arrowheads show the fusion site where actin gradually accumulates. Fusion between partner cells occurs at 100 min as shown by appearance of the tdTomato signal in the h⁻ cell. (B) LatA treatment reduces fusion efficiency of wild-type homothallic h90 strain. Mating cells were starved in MSL−N for 4 h, to allow pheromone response and shmooing, before addition of increasing concentrations of LatA (0, 50, and 200 µg/µl). Cells were immediately spotted on MSL−N 2% agarose pads (not containing LatA and thus diluting the LatA concentration) and incubated overnight at 25°C before imaging for fusion efficiency quantification. n > 200. (C) Homothallic h90 fus1Δ GFP-CHD strain. Cells grow toward each other but are unable to fuse. Though actin patches are present, no actin focus is detected. (D) Quantification of GFP-CHD intensity at the zone of cell contact and of p^map3-driven tdTomato intensity in the h⁻ partner cell in homothallic h90 wild-type mating pairs expressing both markers (as in A). Individual curves were aligned to fusion time and averaged. GFP-CHD intensity at the zone of cell contact in fus1Δ is also indicated, though no alignment could be performed as a result of fusion failure. Error bars are standard deviations. WT, wild type. Bars, 1 µm.