Figure 4.
Leep1 regulates macropinocytic cup and pseudopod dynamics. (A) TRITC-dextran uptake in the WT and two independent knockout (ko) clones. Images were acquired after 30 min of incubation. (B) Quantification of dextran uptake (n = 60, 74, 60, 61, 64, and 58 for the samples listed from left to right). (C) Macropinocytosis competence determined by flow cytometry analysis of WT (red), ko6 (green), and ko18 (blue) cells incubated with TRITC-dextran for 30 min or WT cells incubated without TRITC-dextran (black). Each sample represents ∼100,000 cells. (D) Cell growth measured by generation time. (E) Time-lapse imaging of PHcrac-GFP in WT and leep1− cells. Arrows point to macropinocytic cups that closed. (F) Quantification of ruffle size (n = 120 for WT and 97 for leep1−). (G) Quantification of the frequency of macropinosome formation (n = 53 for WT and 58 for leep1−). (H) Quantification of phagocytosis of TRITC-labeled yeast. (I) Top: Trajectories of cells migrating under agarose against self-generated folate gradients (n = 60 for WT and 55 for leep1−). Bottom: Summary of the respective chemotaxis parameters. (J) Quantification of pseudopod split events in WT and leep1− expressing GFP-ArpC4 during under-agarose chemotaxis (n = 39 for WT and 34 for leep1−). Images on the left show representative tracks, with each pixel corresponding to the maximum pixel intensity for that location over the course of the video. Data in B, F, G, and J were from at least two independent experiments, and n represents the number of cells or events (for F) quantified. Graphs in C and I are from one representative experiment out of three independent experiments. Data in D, H, and the bottom panels of I were from three independent experiments and represent mean ± SD. The scatter plots show data points with means and SEM. Significance was determined by one-way ANOVA in B and t test with Welch’s correction in F, G, and J (NS, P > 0.05; ***, P < 0.001). Scale bar = 10 µm. A.U., arbitrary units; FMI, forward migration index.

Leep1 regulates macropinocytic cup and pseudopod dynamics. (A) TRITC-dextran uptake in the WT and two independent knockout (ko) clones. Images were acquired after 30 min of incubation. (B) Quantification of dextran uptake (n = 60, 74, 60, 61, 64, and 58 for the samples listed from left to right). (C) Macropinocytosis competence determined by flow cytometry analysis of WT (red), ko6 (green), and ko18 (blue) cells incubated with TRITC-dextran for 30 min or WT cells incubated without TRITC-dextran (black). Each sample represents ∼100,000 cells. (D) Cell growth measured by generation time. (E) Time-lapse imaging of PHcrac-GFP in WT and leep1− cells. Arrows point to macropinocytic cups that closed. (F) Quantification of ruffle size (n = 120 for WT and 97 for leep1−). (G) Quantification of the frequency of macropinosome formation (n = 53 for WT and 58 for leep1−). (H) Quantification of phagocytosis of TRITC-labeled yeast. (I) Top: Trajectories of cells migrating under agarose against self-generated folate gradients (n = 60 for WT and 55 for leep1−). Bottom: Summary of the respective chemotaxis parameters. (J) Quantification of pseudopod split events in WT and leep1− expressing GFP-ArpC4 during under-agarose chemotaxis (n = 39 for WT and 34 for leep1−). Images on the left show representative tracks, with each pixel corresponding to the maximum pixel intensity for that location over the course of the video. Data in B, F, G, and J were from at least two independent experiments, and n represents the number of cells or events (for F) quantified. Graphs in C and I are from one representative experiment out of three independent experiments. Data in D, H, and the bottom panels of I were from three independent experiments and represent mean ± SD. The scatter plots show data points with means and SEM. Significance was determined by one-way ANOVA in B and t test with Welch’s correction in F, G, and J (NS, P > 0.05; ***, P < 0.001). Scale bar = 10 µm. A.U., arbitrary units; FMI, forward migration index.

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