Sbf and Rab35 are required for Myosin II organization. (A) Still frames from two-color live imaging of embryo expressing endogenous CRISPR:GFP:Rab35 (green) and mCherry:Sqh (red). Arrowhead indicates Rab35 compartment that precedes Myosin II localization. (B) Time lag between Rab35 and Myosin II appearance. Experiment was replicated with Rab35 in either the mCherry/red (left) or GFP/green (right) channel. GFP:Sqh or mCherry:Sqh was used in the opposing channel. n = 96 (GFP:Rab35) and 36 (mCh:Rab35) compartments. (C) Percentage of colocalization between Rab35 compartments and Myosin II over time after formation of a Rab35 compartment. n = 163 compartments. (D) Still images of Myosin II and plasma membrane (PM) outline marker in control, Sbf shRNA, and Rab35 shRNA embryos (Resille:GFP, mCherry:Sqh). Asterisk marks big cell adjacent to “selfish” cells. (E) Mean Myosin II fluorescence tissue intensity during ventral furrow formation in control, Sbf shRNA, and Rab35 shRNA embryos. (F–H) The distribution of Myosin II intensities and cell area sizes in individual control (F), Rab35 shRNA (G), and Sbf shRNA cells (H). n = 67 (control), 173 (Rab35 shRNA), and 89 (Sbf shRNA) cells. (I–K) Line plot of Myosin II fluorescence intensity along A–P axis during ventral furrow formation in control (I), Rab35 shRNA (J), and Sbf shRNA (K). (L) Mean Myosin II fluorescence intensity in 20–40 µm² big cells during ventral furrow formation. n = 61 (control), 65 (Rab35 shRNA), and 22 (Sbf shRNA) cells. (M) Standard deviation of average Myosin II intensity in control, Rab35 shRNA, and Sbf shRNA cells. Scale bars in A and D represent 5 μm. In bar plots, error bars indicate standard errors. Statistical significance was calculated using a Mann–Whitney U test (E and L) and a Student’s t test (M). *, P < 0.05; **, P < 0.05; ***, P < 0.0005. In A and D, embryos are oriented with anterior up and posterior down.