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. 2023 Jul 21;12:e84850. doi: 10.7554/eLife.84850

Figure 1. Localization of endocytosed α-factor at the Tlg2p-residing compartment.

(A) 2D imaging of A594-α-factor and GFP-Tlg2p. Arrowheads indicate examples of overlapping localization. Representative fluorescence intensity profiles along a line (direction from ‘a’ to ‘b’) are indicated in the lower panels. (B) Quantification of GFP-Tlg2p, GFP-Tlg1p, and Sec7-GFP overlapping with A594-α-factor. Data show the mean ± SEM from n ≥ 3 experiments (n > 30 puncta for each experiment). Different letters indicate significant differences at p<0.05 between the indicated times (i.e., no significant difference for a vs. a, significant difference for a vs. b with p<0.05), one-way ANOVA with Tukey’s post hoc test. Error bars indicate the standard SD from n ≥ 3 experiments (n ≥ 30 puncta for each experiment). (C) 3D super-resolution confocal live imaging microscopy (SCLIM) imaging of GFP-Tlg2p and Sec7-mCherry. White dashed lines indicate cell edges. (D) Multi-angle magnified 3D views of the boxed area and the representative fluorescence intensity profiles. Line scan as in (A) shown at right. (E) 3D SCLIM imaging of GFP-Tlg2p, Sec7-iRFP, and pHrode-α-factor; boxed areas shown magnified in (F–H). The images were acquired simultaneously at 5 min after pHrode-α-factor internalization. (F) Multi-angle magnified 3D views of the yellow-boxed area in (E). Line scan as in (A) shown at right. (G) Time series of region in the white-boxed area in (E). Arrows and arrowheads denote the appearance and disappearance of each marker. (H) Higher-magnification views of the red-boxed area in (G). Scale bars, 2.5 μm.

Figure 1—source data 1. Data for graphs presented in Figure 1B.

Figure 1.

Figure 1—figure supplement 1. Localization of α-factor, Sec7p, Tlg1p, and Tlg2p in wild-type cells.

Figure 1—figure supplement 1.

(A) 2D imaging of GFP-Tlg2p and FM4-64 in wild-type cells. (B, C) 2D imaging of A594-α-factor and Sec7-GFP (B) or GFP-Tlg1p (C) in wild-type cells. The images were acquired at 5, 10, or 20 min after A594-α-factor internalization. Yellow arrowheads indicate examples of overlapping localization. Red and green arrowheads indicate example of A594-α-factor or GFP signal, respectively. Higher-magnification views of the boxed area are shown in the right panels. Representative fluorescence intensity profiles along a line (direction from ‘a’ to ‘b’) in the merged images are indicated in the lower panels. (D) 2D imaging of GFP-Tlg1p and mCherry-Tlg2p in wild-type cells shown also in DIC at right. Yellow arrowheads indicate examples of overlapping localization. Red arrowheads indicate example of puncta predominantly localizing mCherry-Tlg2p. Higher-magnification views of the boxed area are shown in the right panels. Representative fluorescence intensity profiles along a line (direction from ‘a’ to ‘b’) in the merged images are indicated in the lower panels. (E) 2D imaging of A647-α-factor, GFP-Tlg1p, and mCherry-Tlg2p. The images were acquired at 5 min after A647-α-factor internalization. Yellow and white arrowheads indicate examples of puncta in which all fluorescent signals overlap. (F) Plates showing the growth phenotype of wild-type cells expressing pRS316 (vector only) or pRS-316-Sec7-iRFP. A dilution series of indicated cells were plated on SD plate lacking uracil and incubated at 25oC and 37oC. Scale bars, 2.5 μm.