The anisotropic diffusion of GFP::SP12 is not caused by the macroscopic reorganization of the ER morphology. (A) Brightfield and fluorescence images show the phenotypes of GFP::SP12; lpin-1(RNAi) embryos. Lipid droplets are reduced in the embryo, and the ER appears thready in milder conditions and becomes heavily patchy in severe cases. (B) Ctrl(RNAi) and rab-5(RNAi) wild-type embryos stained with α-RAB-5 antibody indicate that RAB-5 exists in punctate structures that are enriched in the anterior domain and around the centrosomes. Absence of these structures in the rab-5(RNAi) embryos shows that the RNAi is efficient in down-regulating the protein. Images are projection and deconvolved datasets. (C) Confocal images of the FLIP experiments showing a representative lpin-1(RNAi) embryo expressing GFP::SP12 photobleached in the anterior or the posterior domains (orange circles). Also see Video 8. (D) Graphs of the kinetics of fluorescence loss for the combined data of GFP::SP12; lpin-1(RNAi) embryos with the alignment to the time point of anisotropy onset. Three time points before the anisotropy onset are shown. Asterisks show the anisotropy onset. n_anterior = 5; n_posterior = 16. (E) Confocal images of the FLIP experiments showing a representative rab-5(RNAi) embryo expressing GFP::SP12 photobleached in the anterior or the posterior domains. Also see Video 9. (C and E) Graphs show the kinetics of fluorescence loss over time for the corresponding embryos. Gray bars indicate the prebleached first images; black bars indicate repeated bleaching in the remainder of the indicated cell cycle. The black arrowheads indicate NEBD; the white arrowheads indicate cytokinesis; gray shaded areas show the period of isotropic diffusion; asterisks show the anisotropy onset. (F) Graphs of the kinetics of fluorescence loss for the combined data of GFP::SP12; rab-5(RNAi) embryos with the alignment to the time point of anisotropy onset. Graphs are as described in D. n_anterior = 17; n_posterior = 21. Mean ± SD. Bars, 10 µm.