MICAL1 G3W, WDR44 ΔC, and GRAF1/2 BAR-PH are dominant negative mutants. (A) Confocal stacks of transfected HeLa cells. GFP–MICAL1 ΔMO and GFP–MICAL1 G3W were associated with many puncta and tubules. (B) Confocal images of transfected HeLa cells. GFP–MICAL1 G3W colocalized with RFP-tagged GRAF2, Rab8a, and Rab10 on puncta and tubules. (C) Manders colocalization coefficients for the indicated proteins on GFP–MICAL1 G3W structures. n = 9–24 cells. (D) HA-GLUT4-GFP export in transfected 3T3-L1 adipocytes under resting conditions or after the addition of insulin (10 µg/ml, 30 min). n = 4–12. (E) Confocal images of transfected HeLa cells stained with α-WDR44 and α-Rab8. Unlike WDR44, endogenous Rab8 colocalized with GFP–MICAL1 G3W. (F) Percentage of transfected HeLa cells left untreated or after incubation with Cytochalasin D (0.5 µg/ml, 30 min), with endogenous (endo.) WDR44 tubules. n = 3–8. (G) Confocal stack of HeLa cells transfected with GFP–WDR44 ΔC showing its association with large peripheral patches but also, in a few cells, with a thin reticular network. (H) Confocal images of transfected HeLa cells. GFP–WDR44 ΔC recruited RFP-tagged Rab11a and Rab10 to amorphous peripheral patches and colocalized with Rab8a and Rab10 on intracellular puncta and tubules. (I) Manders colocalization coefficients for the indicated proteins on GFP–WDR44 ΔC structures. n = 20–31 cells. (J) Confocal images of transfected HeLa cells stained with α-MICAL1 and α-WDR44. Unlike MICAL1, WDR44 was recruited to sections of GRAF2 BAR-PH–positive membranes. (K) Confocal images of transfected HeLa cells. GRAF2 BAR-PH–GFP colocalized with RFP-tagged Rab8a and Rab10. (L) Manders colocalization coefficients for the indicated proteins on GRAF1/2 BAR-PH–GFP structures. n = 21–26 cells. (C, D, F, I, and L) Data are means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. (A, B, E, G, H, J, and K) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.