Quantitative analysis of time series acquired with the LLSM over the full volume of 300–1000 yeast cells expressing a mixture of Snf7 and Snf7-eGFP with either Vps4-mCherry or Vps4E233Q-mCherry mutant, Vps24-eGFP with Vps4-mCherry or Vps4E233Q-mCherry, and Vps4-eGFP or Vps4E233Q-eGFP. Snf7-eGFP and Vps4-eGFP was also analyzed in pep12Δ mutants. The data presented are from all diffraction limited mobile objects (class I) detected in the periphery of cells (a, c, d) or in both peripheral and perivacuolar regions (b). (a) Cross-correlation of the fluorescence intensity (blue) and of the fluorescence intensity first derivative (orange) from Snf7-eGFP and Vps4-mCherry or from Vps24-eGFP and Vps4-mCherry. Data are from traces with lifetimes longer than 11 s and are expressed as average ± SD. (b) Plots showing the lifetime distribution (histogram) and corresponding cumulative frequency distribution (dotted curves) of Snf7-eGFP, Vps24-eGFP and Vps4-eGFP in WT cells and in the indicated mutants. The two-sample permutation test for differences between the medians was not significant. The number of tracked traces analyzed for each experiment is indicated. The inset showing a typical trace illustrates the definition of lifetime. (c) Plots showing the maximum accumulation (histogram) and corresponding cumulative frequency (dotted curve) distributions of fluorescent molecules of Snf7-eGFP, Vps24-eGFP and Vps4-eGFP in WT cells in the indicated mutants. Mutating Vps4 had minimal effects on the modes of maximum Snf7-eGFP recruitment (35 ± 12 and 30 ± 10, amplitude ± SD of the first fitted Gaussian, for wild-type and Vps4E233Q mutant, respectively) or of Vps24-eGFP (21 ± 5 and 17 ± 6; p<0.001, Kolmogorov-Smirnov and the two-sample permutation tests). Vps4E233Q or loss of Pep12 had a marked effect on the accumulation of Vps4-eGFP itself (from 24 ± 6 to 11 ± 3 and 12 ± 3 in wild-type Vps4, Vps4E233Q, and pep12Δ mutants, respectively; p<0.001). The inset of a typical trace illustrates the definition of maximum accumulation. (d) Averaged number of eGFP molecule traces per lifetime cohort, shown as mean ± 95th percentile confidence bound (shaded areas) for all traces above the local background threshold analyzed in (c). The data is for Snf7-eGPF, Vps24-eGFP and Vps4-eGFP expressed in the indicated wild type and mutant yeast cell strains. The Vps4-eGFP data from the pep12Δ mutant corresponds to traces likely to be associated with a single endocytic carrier; they correspond to events whose maximum accumulation of Vps4-eGFP molecules were within the 99th percentile of the first Gaussian distribution (Figure 4—figure supplement 10f). The complete data set is shown in Figure 4—figure supplement 10g.