Twf1 but not Aim21 and Bsp1 require interaction with CP for proper localization to CME sites. (A) Organization of Aim21, Bsp1, and Twf1 domains. (B) Top: Live-cell fluorescence microscopy showing increased recruitment of Aim21-GFP to endocytic sites in cells with the CPI motif of Aim21 mutated (SDY1472), or cells lacking Cap1 (SDY1478). Middle: Live-cell fluorescence microscopy showing increased recruitment of Bsp1-GFP to endocytic sites in cells with the CPI motif of Bsp1 mutated (SDY1516), or cells lacking Cap1 (SDY1570). Bottom: Live-cell fluorescence microscopy showing no recruitment of Twf1-GFP to endocytic sites in cells with the CPI motif of Twf1 mutated (SDY1517) or cells lacking Cap1 (SDY1572). Scale bars, 1 µm. (C) Quantification of Aim21-GFP peak fluorescence intensity at endocytic patches in WT, aim21CPI*, and cap1∆ cells. From left to right, mean peak patch/cytosol ratio = 17.61, 30.18, 37.07, and n = 50 for all groups. Error bars, mean with 95% CI. **P ≤ 0.01, ****P ≤ 0.0001. (D) Quantification of Bsp1-GFP peak fluorescence intensity at endocytic patches in WT, bsp1CPI*, and cap1∆ cells. From left to right, mean peak patch/cytosol ratio = 17.50, 25.02, 28.06, and n = 50 for all groups. Error bars, mean with 95% CI. *P ≤ 0.05, ****P ≤ 0.0001. (E) Live-cell fluorescence microscopy of cells coexpressing (top) Aim21CPI*-GFP, (middle) Bsp1CPI*-GFP, and (bottom) Twf1CPI*-GFP with Cap1-mCherry, respectively (SDY1519, SDY1521, SDY1523). Aim21CPI*-GFP and Bsp1CPI*-GFP show strong colocalization with Cap1-mCherry, while Twf1CPI*-GFP shows no colocalization with Cap1-mCherry. Endocytic patches used for construction of kymographs to the right are indicated by arrowheads. Scale bars, 1 µm. (F) Live-cell fluorescence microscopy showing decreased recruitment of Twf1-GFP to endocytic sites in cells with the α-tentacle of Cap1 mutated (SDY1588), the CPI motifs of Aim21 and Bsp1 mutated (SDY1611), or with the mutations combined (SDY1627). Scale bar, 1 µm. (G) Quantification of Twf1-GFP peak fluorescence intensity at endocytic patches in WT, cap1*, aim21CPI*/bsp1CPI*, and aim21CPI*/bsp1CPI*/cap1* cells. From left to right, the mean peak patch/cytosol ratio = 25.06, 5.15, 12.49, 4.43, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, ****P ≤ 0.0001.
Figure 5.

Twf1 but not Aim21 and Bsp1 require interaction with CP for proper localization to CME sites. (A) Organization of Aim21, Bsp1, and Twf1 domains. (B) Top: Live-cell fluorescence microscopy showing increased recruitment of Aim21-GFP to endocytic sites in cells with the CPI motif of Aim21 mutated (SDY1472), or cells lacking Cap1 (SDY1478). Middle: Live-cell fluorescence microscopy showing increased recruitment of Bsp1-GFP to endocytic sites in cells with the CPI motif of Bsp1 mutated (SDY1516), or cells lacking Cap1 (SDY1570). Bottom: Live-cell fluorescence microscopy showing no recruitment of Twf1-GFP to endocytic sites in cells with the CPI motif of Twf1 mutated (SDY1517) or cells lacking Cap1 (SDY1572). Scale bars, 1 µm. (C) Quantification of Aim21-GFP peak fluorescence intensity at endocytic patches in WT, aim21CPI*, and cap1∆ cells. From left to right, mean peak patch/cytosol ratio = 17.61, 30.18, 37.07, and n = 50 for all groups. Error bars, mean with 95% CI. **P ≤ 0.01, ****P ≤ 0.0001. (D) Quantification of Bsp1-GFP peak fluorescence intensity at endocytic patches in WT, bsp1CPI*, and cap1∆ cells. From left to right, mean peak patch/cytosol ratio = 17.50, 25.02, 28.06, and n = 50 for all groups. Error bars, mean with 95% CI. *P ≤ 0.05, ****P ≤ 0.0001. (E) Live-cell fluorescence microscopy of cells coexpressing (top) Aim21CPI*-GFP, (middle) Bsp1CPI*-GFP, and (bottom) Twf1CPI*-GFP with Cap1-mCherry, respectively (SDY1519, SDY1521, SDY1523). Aim21CPI*-GFP and Bsp1CPI*-GFP show strong colocalization with Cap1-mCherry, while Twf1CPI*-GFP shows no colocalization with Cap1-mCherry. Endocytic patches used for construction of kymographs to the right are indicated by arrowheads. Scale bars, 1 µm. (F) Live-cell fluorescence microscopy showing decreased recruitment of Twf1-GFP to endocytic sites in cells with the α-tentacle of Cap1 mutated (SDY1588), the CPI motifs of Aim21 and Bsp1 mutated (SDY1611), or with the mutations combined (SDY1627). Scale bar, 1 µm. (G) Quantification of Twf1-GFP peak fluorescence intensity at endocytic patches in WT, cap1*, aim21CPI*/bsp1CPI*, and aim21CPI*/bsp1CPI*/cap1* cells. From left to right, the mean peak patch/cytosol ratio = 25.06, 5.15, 12.49, 4.43, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, ****P ≤ 0.0001.

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