Myo2 stimulates polarisome formation during mating. (A) Left: Experimental setup. Right: MATa or MATa-myo2_RD cells expressing GFP-fusion to Pea2 (orange), Myo2 (not depicted), or Sec4 (not depicted) were mated with WT MATα cells, and the fluorescence intensity was recorded every 2 min. The time of cell fusion was set to t = 0. Right: Fluorescence intensity profiles during mating of MATa or MATa-myo2_RD cells expressing Myo2-GFP (n_WT = 13; n_myo2RD = 15), GFP-Pea2 (n_WT = 13; n_myo2RD = 14), or GFP-Sec4 (n_WT = 14; n_myo2RD = 13). The highest-intensity value of each single curve for each GFP fusion was normalized to 100 to then calculate the mean of the ensemble. Error bars are SD. Indicated is the significance of the difference between the values of GFP-Pea2 in WT and moy2_RD cells (yellow symbols), between Myo2-GFP and Myo2_RD-GFP (blue symbols), and between GFP-Sec4 in WT and myo2_RD cells (red symbols) at minutes −10, −22, and −54. (B) Fluorescence microscopy of Spa2-GFP–expressing MATa cells and Spa2-mCherry–expressing MATα cells during mating and cell fusion. The first appearance of Spa2 at the prospective mating projection differed between the genotypes of the mated cells. Upper panel: WT-WT. Middle panel: myo2_RD-myo2_RD. Lower panel: myo2_RD-WT. Each time series starts with the first visible Spa2 signal. DIC images of the contact sites are shown below. Scale bar: 5 µm. (C) Quantification of the data from B. Comparison of the dwell times of the Spa2-GFP/Spa2-mCherry fluorescence at the mating projections from first appearance to cell fusion: a-WT(Spa2-GFP) × α-WT(Spa2-mCherry): 42.3/42.3 min (n = 29); a-myo2_RD (Spa2-GFP) × α-myo2_RD (Spa2-mCherry): 56.5/55.1 min (n = 31); a-myo2_RD (Spa2-GFP) × α-WT (Spa2-mCherry): 40.3/53.9 min (n = 35). (D) Quantification of the data from B. Difference between the first appearance of Spa2-GFP and of Spa2-mCherry measured for each mating experiment of the different genotype combinations. Error bars are SD. (E) Left: Signal width of Spa2 fluorescence at the fusion site. Right: Total width of the fusion pores calculated from DIC pictures. Values were derived from matings of WT × WT cells and myo2_RD × myo2_RD cells, 2 min before fusion. Error bars are SD. (F) Efficiency of mating: MATa and MATα strains of identical genotypes were mated for 4 h at 30°C and plated on solid medium selecting for diploid zygotes. Colony numbers from WT matings were set to 100% efficiency. Error bars are SD. ns, not significant; ***, P < 0.001; **, P < 0.01; *, P < 0.05 (one-way ANOVA followed by Tukey’s multiple comparison test).