Leep1 overexpression alters cytoskeletal rearrangement. (A) Cells expressing low or high levels of GFP-Leep1 (cells 1–4, low expressers; cells 5 and 6, high expressers) were fixed and stained with Alexa Fluor 555–labeled phalloidin. Three-dimensional reconstructions were computed from confocal sections. (B) Cells expressing a control vector (pDM304), Leep1, or truncations and mutation of Leep1 were fixed and stained with Alexa Fluor 555–labeled phalloidin. Three-dimensional reconstructions were computed from confocal sections. Two Leep1^1-672 cells are separated by a white dashed line. (C) Scanning electron micrographs of representative cells expressing a control vector (pDM304) or the full-length or truncated Leep1. (D) Colocalization of RFP-Leep1 and GFP–myosin VII (MVII). (E) TRITC-dextran uptake in cells expressing high and low levels of GFP-Leep1. Images were acquired 30 min after incubation. (F) Quantification of dextran uptake in the high and low expressers. The scatter plots show data points with means and SEM (n = 15, 15, 18, 18, 20, 20, 18, and 18 for the samples listed from left to right). Significance was determined by one-way ANOVA with Dunn’s post-test (NS, P > 0.05; ***, P < 0.001). (G and H) Scatter plots showing negative correlation between fluorescence intensity (I) corresponding to GFP-Leep1 (G) or GFP-Leep1^1-672 (H) and TRITC-dextran (Pearson correlation coefficient = −0.86, top; −0.63, bottom). Scale bar = 5 µm. A.U., arbitrary units.