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

Figure 5. The transition from the Tlg2p- to the Sec7p-residing compartment requires GGA adaptors.

(A) 2D imaging of GFP-Tlg2p and Sec7-mCherry in gga1Δ gga2Δ cells. Representative intensity profiles of GFP-Tlg2p or Sec7-mCherry along a line in the merged images are indicated in the right lower panel. (B) Quantification of GFP-Tlg2p overlapping with Sec7-mCherry in wild-type and gga1Δ gga2Δ cells. Error bars indicate the SD from n ≥ 3 experiments (n > 30 puncta for each experiment). **p<0.01, unpaired t-test with Welch’s correction. (C, E) 4D super-resolution confocal live imaging microscopy (SCLIM) imaging of GFP-Tlg2p and Sec7-mCherry in wild-type cells (C) and gga1Δ gga2Δ cells (E).The time series of regions in the boxed areas in (C) are shown in the lower panels. Arrows and arrowheads denote the appearance and disappearance of each marker. (D, G) Time-course changes in relative fluorescence intensity of GFP-Tlg2p and Sec7-mCherry in the boxed areas in (C) or (E). (F) Time series of the region in the boxed area in (E). (H, I) Multi-angle magnified 3D views from (F). (J) 4D SCLIM imaging of GFP-Tlg2p, Gga2-mCherry, and Sec7-iRFP. The time series of regions in the boxed areas in (J) are shown in the lower panels. (K, L) Multi-angle magnified 3D views of time points from (J).Scale bars, 2.5μm.

Figure 5—source data 1. Data for graphs presented in Figure 5B.

Figure 5.

Figure 5—figure supplement 1. Localization of Tlg2p and Sec7p at different cell cycle stages.

Figure 5—figure supplement 1.

(A) Maximum intensity projections of Z stacks of S- or M-phase cell expressing Sec7-mCherry. The Z series was acquired through the entire cell at 0.2 μm intervals. Cells were synchronized at S- or M-phase by treating with 30 mM hydroxyurea or 50 μM nocodazole for 3 hr at 25°C. (B) Quantification of the number of Sec7-mCherry-positive puncta. Data show the mean ± SD (n = 10 cells). *p<0.05, unpaired t-test with Welch’s correction. (C) 2D imaging of GFP-Tlg2p and Sec7-mCherry in S- or M-phase cell. (D) Quantification of GFP-Tlg2p overlapping with Sec7-mCherry. Data show the mean ± SEM from n ≥ 3 experiments (n > 30 puncta for each experiment). (E) Lifetime of Sec7-mCherry in wild-type and gga1Δ gga2Δ cells. Data show the mean ± SD (n > 10 cisterna for each strain). **p<0.01, unpaired t-test with Welch’s correction.
Figure 5—figure supplement 1—source data 1. Data for graphs presented in Figure 5—figure supplement 1B.
Figure 5—figure supplement 1—source data 2. Data for graphs presented in Figure 5—figure supplement 1D.
Figure 5—figure supplement 1—source data 3. Data for graphs presented in Figure 5—figure supplement 1E.
Figure 5—figure supplement 2. Dynamics of Tlg1p and Tlg2p in wild-type and gga1Δ gga2Δ cells.

Figure 5—figure supplement 2.

(A, D) 2D imaging of GFP-Tlg1p and mCherry-Tlg2p in wild-type (A) or gga1Δ gga2Δ (D) cell. (B, E) A time series of the region boxed in (A, D) are shown. Arrows and arrowheads denote the appearance and disappearance of each marker. (C, F) Time course changes in relative fluorescence intensity of GFP-Tlg1p and mCherry-Tlg2p in wild-type (C) or gga1Δ gga2Δ (F) cell. (G) Plates showing the growth phenotype of gga1Δ cells expressing Gga2-mCherry. A dilution series of indicated cells were plated on YPD plate and incubated at 25°C and 37°C. (H) 2D imaging of GFP-Tlg2p and Gga2-mCherry. 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 right panel. Quantification of Gga2-mCherry overlapping with GFP-Tlg2p in wild-type cells is shown in the right panel. Scale bars, 2.5 μm.
Figure 5—figure supplement 2—source data 1. Data for graphs presented in Figure 5—figure supplement 2H.