Proximity to sites of exocytosis and cell polarity signaling does not influence the rate of late steps in the endocytic pathway. (A) Maximum-intensity projections of z-stacks (top) of polarized and depolarized cells paired with radial kymographs of individual endocytic events from medial focal plane epifluorescence videos of the same cells (bottom) endogenously expressing Sla1-GFP (green) and Abp1-RFP (magenta). Kymograph generation and analysis were performed in mother cells. (B) Quantification of the ratio of the number of Abp1-RFP sites to Sla1-GFP sites from maximum-intensity projections of z-stacks of 50 polarized and 50 depolarized cells. Analysis was performed in mother cells. A two-tailed P value from a Mann–Whitney U test as in Fig. 7 C (U = 1051) is displayed. The median and interquartile ranges are denoted with error bars. (C) Sla1-GFP and Abp1-RFP lifetimes measured in mother cells of 40 polarized cells and 40 depolarized cells (five representative sites per cell). Two-tailed P values from Mann–Whitney U tests (U_Sla1 = 19,732, U_Abp1 = 17,429) with the null hypothesis that the lifetimes are identical are displayed. The median and interquartile ranges are denoted with error bars. (D) Maximum-intensity projections of cells endogenously expressing GFP-Sec4 before and 5 min after 17-fold dilution into isotonic imaging media (control, left) or water (right). (E) Maximum-intensity projections of a cell endogenously expressing Sla1-GFP (green) and Abp1-RFP (magenta) before and 5 min after 17-fold dilution into water. (F) Radial kymographs of individual endocytic events in small-budded mother cells from medial focal plane videos of cells endogenously expressing Sla1-GFP (green) and Abp1-RFP (magenta) before and 5 min after 17-fold dilution into water. (G) Quantification of the ratio of the number of Abp1-RFP sites to Sla1-GFP sites from maximum-intensity projections of z-stacks of 30 small-budded mother cells before and after osmotic shock. A two-tailed P value from a Mann–Whitney U test as in Fig. 7 C (U = 441.5) is displayed. The median and interquartile ranges are denoted with error bars. (H) Sla1-GFP and Abp1-RFP lifetimes measured in 30 small-budded mother cells before osmotic shock and 30 cells after osmotic shock (five representative sites per cell). Two-tailed P values from Mann–Whiney U tests as in (C; U_Sla1 = 1,678, U_Abp1 = 2,998) are displayed. The median and interquartile ranges are denoted with error bars. (I) Maximum-intensity projections of a wild-type cell endogenously expressing Sla1-mCherry (magenta) and Ede1-GFP (green) before and 10 min after treatment with BFA. (J) Quantification of the ratio of the number of Sla1-mCherry sites to Ede1-GFP sites from maximum-intensity projections of z-stacks of 21 large-budded mother cells before and after BFA treatment. A two-tailed P value from a Mann–Whitney U test as in Fig. 7 C (U = 132.5) is displayed. The median and interquartile ranges are denoted with error bars. (K) Circumferential kymographs around the mother cortex from medial focal plane videos of cells endogenously expressing Sla1-mCherry (magenta) and Ede1-GFP (green) before and 10 min after treatment with BFA. (L) Percentage of 375 Ede1 patches from 22 large-budded mother cells before and 477 Ede1 patches from 24 large-budded mother cells after BFA treatment that persist, are partially captured, or turn over during a 4-min video as in Fig. 7 D. A two-tailed P value from a chi-square test as in Fig. 7 D (chi-square = 10.77, 2 degrees of freedom) is displayed. ns, not significant.