A potential Rab35 GEF, Sbf, displays apical localization during Drosophila ventral furrow formation. (A) Diagram of the imaging approach used to follow apical constriction of ventral furrow cells (left). Tangential z-slice of Resille:GFP; Spider:GFP from 2 μm below the apical surface to show cell outlines (right; image is duplicate of Fig. 4 A control for schematic purposes). (B) Structural domain schematic of Drosophila Sbf protein. (C) Still frames of embryos expressing EGFP:Sbf, EGFP:Sbf^ΔCC-PH, EGFP:Sbf^ΔDENN, and EGFP:DENN-GRAM during ventral furrow formation. (D) Time-lapse images of an embryo expressing EGFP:Sbf. Images depicting the lifetime of a Sbf compartment: initiation of Sbf compartment (0 s), elongation phase (67 s), maximum point (68 s), shrinkage (69 s), and termination of a compartment (96 s). Dashed circle marks the indicated Sbf compartment. (E) Distribution of Sbf compartmental lifetimes. n = 175 compartments. (F) Sbf compartments are colocalized with dextran immediately after injection. (G) Quantification of the number of dextran-labeled Sbf compartments during ventral furrow formation. n = 222 compartments. (H) Quantification of the number of Sbf compartments on the interface or apical surface per cell. n = 132 cells. Scale bars in A and C represent 5 μm, and scale bars in D and F represent 2.5 μm. Error bars indicate standard errors. Statistical significance was calculated using a Student’s t test. *, P < 0.05; ***, P < 0.0005. In A, C, D, and F, embryos are oriented with anterior up and posterior down.