Figure S2.
Osh2 uses PI4P counter-transport to sustain sterol extraction from ERSESs.(A) Graph representing the HA-GFP-D4H gold distance to the PM (GDPM) versus the length of the associated invagination (IL) for 40 endocytic invaginations. The GRP is calculated by dividing the GDPM by the IL. Red lines indicate the position of the PM (GRP = 0) and the position of the invagination tip (GRP = 1) as the invagination grows. An immuno-gold with GRP < 0.5 is defined to be at the base of the invagination, with 0.5 ≤ GRP < 0.8 at the invagination neck and GRP ≥ 0.8 at the invagination tip. The diagram on the right describes the parameters defined for each gold particle, cER, or invagination for a depicted invagination of IL = 150 nm. (B) Merged epifluorescence micrographs of WT and osh4Δ cells expressing GFP-D4H and Sec61-mCherry. Graphs of average ± SEM number of cortical GFP-D4H patches, total number of GFP-D4H foci, and their RFI normalized to the WT in the indicated strains, with the Student´s t test P values, are shown. Significant differences are shown in red. n > 45 cells or patches in three experiments. (C) Kymographs of GFP-D4H cortical patches in WT or osh2Δ cells either mock treated or treated with the indicated drugs. Pictures were taken every 5 s for 12 min, and drugs were added 2 min after initiation of the experiment (red line). (D) Time course of cortical GFP-D4H patch RFI, normalized to time 0, in WT or osh2Δ cells treated with FPM. The drug was added 2 min after initiation of the experiment (red dashed line). The Student’s t test P value in the last time recorded is indicated (n ≥ 10). (E) Graph of average ± SEM with Student´s t test P values of the GFP-D4H cortical patch intensity for the indicated strains, 1 h upon incubation with TBF or mock treated. RFI is normalized to the average of the mock-treated sample. (F) Graph of average ± SEM with Student’s t test P values of cortical GFP-D4H patch intensity of the indicated strains, before (0 min) or after 1-h treatment (60 min) with OSW-1. RFI is normalized to the average of the 0-min sample. Significant differences are in red. (G) Merged epifluorescence micrographs of the indicated strains expressing GFP-D4H and Sec61-mCherry before (0 min) or after 1-h treatment with OSW-1. The graph indicates average ± SEM of the percentage of cells with polarized GFP-D4H staining in the indicated strains. (H) Merged epifluorescence micrographs of WT or osh2Δ strains bearing either the empty plasmid (osh2Δ) or plasmids encoding the indicated OSH2 alleles expressing GFP-D4H and Sec61-mCherry. Individual channels and the merged micrographs of magnified areas showing GFP-D4H cortical patches associated with cER rims are shown. (I) Epifluorescence micrographs of yeast expressing PI4P (GFP-Osh2-PH) and PI(4,5)P2 probes (GFP-2xPH-PLCδ; Yu et al., 2004) in the indicated strains, and graphs of the average ± SEM with Student’s t test P values of the relative PM/cytosolic (Cyt.) RFI for the corresponding probes and indicated strain backgrounds.

Osh2 uses PI4P counter-transport to sustain sterol extraction from ERSESs. (A) Graph representing the HA-GFP-D4H gold distance to the PM (GDPM) versus the length of the associated invagination (IL) for 40 endocytic invaginations. The GRP is calculated by dividing the GDPM by the IL. Red lines indicate the position of the PM (GRP = 0) and the position of the invagination tip (GRP = 1) as the invagination grows. An immuno-gold with GRP < 0.5 is defined to be at the base of the invagination, with 0.5 ≤ GRP < 0.8 at the invagination neck and GRP ≥ 0.8 at the invagination tip. The diagram on the right describes the parameters defined for each gold particle, cER, or invagination for a depicted invagination of IL = 150 nm. (B) Merged epifluorescence micrographs of WT and osh4Δ cells expressing GFP-D4H and Sec61-mCherry. Graphs of average ± SEM number of cortical GFP-D4H patches, total number of GFP-D4H foci, and their RFI normalized to the WT in the indicated strains, with the Student´s t test P values, are shown. Significant differences are shown in red. n > 45 cells or patches in three experiments. (C) Kymographs of GFP-D4H cortical patches in WT or osh2Δ cells either mock treated or treated with the indicated drugs. Pictures were taken every 5 s for 12 min, and drugs were added 2 min after initiation of the experiment (red line). (D) Time course of cortical GFP-D4H patch RFI, normalized to time 0, in WT or osh2Δ cells treated with FPM. The drug was added 2 min after initiation of the experiment (red dashed line). The Student’s t test P value in the last time recorded is indicated (n ≥ 10). (E) Graph of average ± SEM with Student´s t test P values of the GFP-D4H cortical patch intensity for the indicated strains, 1 h upon incubation with TBF or mock treated. RFI is normalized to the average of the mock-treated sample. (F) Graph of average ± SEM with Student’s t test P values of cortical GFP-D4H patch intensity of the indicated strains, before (0 min) or after 1-h treatment (60 min) with OSW-1. RFI is normalized to the average of the 0-min sample. Significant differences are in red. (G) Merged epifluorescence micrographs of the indicated strains expressing GFP-D4H and Sec61-mCherry before (0 min) or after 1-h treatment with OSW-1. The graph indicates average ± SEM of the percentage of cells with polarized GFP-D4H staining in the indicated strains. (H) Merged epifluorescence micrographs of WT or osh2Δ strains bearing either the empty plasmid (osh2Δ) or plasmids encoding the indicated OSH2 alleles expressing GFP-D4H and Sec61-mCherry. Individual channels and the merged micrographs of magnified areas showing GFP-D4H cortical patches associated with cER rims are shown. (I) Epifluorescence micrographs of yeast expressing PI4P (GFP-Osh2-PH) and PI(4,5)P2 probes (GFP-2xPH-PLCδ; Yu et al., 2004) in the indicated strains, and graphs of the average ± SEM with Student’s t test P values of the relative PM/cytosolic (Cyt.) RFI for the corresponding probes and indicated strain backgrounds.

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