(A) Top: Myo3-EGFP or Myo5-EGFP patches in WT cells, bottom: kymographs. Scalebar: 1 μm. (B) Inward movement (top) and number of molecules (bottom) of Myo3-EGFP and Myo5-EGFP over time at endocytic sites. Traces are aligned in time based on co-alignment with Abp1-mCherry, and plotted such that t = 0 is the peak in Rvs167 molecule numbers (scission). Shading represents 95% confidence interval. (C) Average Sla1-EGFP centroid inward movement in WT, myo3Δ and myo5Δ. Traces are plotted so that the initial position is y = 0, and are aligned in time to the onset of inward movement. Shading represents 95% confidence interval. One of two independent replicates for each genotype is shown. WT and myo5Δ trajectories are the same as in Figure 1. (D) The median number of Myo3-EGFP and/or Myo5-EGFP molecules over the lifetime of an endocytic site are plotted for several haploid and diploid genotypes (gray boxes). Error bars reflect SEM. Molecule numbers were compared using 2-sided z-tests. (E) Sla1-EGFP centroid inward movement is plotted over time for WT and MYO5 duplication cells. (F) Sla1-EGFP centroid movement in WT haploids, WT diploids and diploids carrying one or no MYO5 alleles. (G) Sla1 centroid inward movement speed is plotted as a function of the total number of myosins per endocytic site (the sum of the median number of Myo3 and Myo5 molecules). Purple datapoints originate from haploid cells, blue from diploids. The datapoints are labeled with their genotype. Horizontal errorbars are SEM, vertical errorbars SE. mov. = movement, dupl. = duplication, NS = not significant, *: p≤0.05, **: p≤0.01, ***: p≤0.001.