Figure S1. YOP-1 localizes to the peripheral ER as well as the nuclear envelope. (A) Spinning-disk confocal optics were used to image embryos (n = 7) coexpressing RFP:SP-12 and GFP:YOP-1. A representative central confocal section of RFP:SP-12 (left), GFP:YOP-1 (middle), and a color overlay of the two signals (right) in a mitotic embryo is shown. Arrowheads point to a region of the nuclear envelope, illustrating the relative exclusion of YOP-1 compared to the lumenal marker SP-12 from this ER domain. Bar, 10 μm. (B) Spinning-disk confocal optics were used to image embryos (n = 8) expressing GFP:YOP-1. Representative images of interphase (left) and metaphase (middle) embryos are shown. Bar, 10 μm. (C) Reorganization of the ER into thick tubules and mitotic clusters does not require microtubules. Metaphase control (left) and
rab-5(RNAi) (right) embryos were fixed and stained for microtubules. Projections of deconvolved 3D data sets are shown. Bar, 10 μm. (D) Single central (left) and cortical (right) confocal sections of a GFP:SP-12-expressing embryo that entered mitosis after depolymerization of the microtubule cytoskeleton with 10 uM nocodazole. Bar, 10 µm. (E) The defect in ER structure in RAB-5-depleted embryos is not caused by a defect in the organization of the actomyosin cytoskeleton. Spinning-disk confocal optics were used to image the cortex in control (n = 6),
rab-5(RNAi) (n = 6), and RFP:RAB-5Q78L-expressing (n = 6) embryos that were expressing a GFP fusion with the actin binding domain of Drosophila melanogaster moesin (GFP:Moe). Cortical (left) and medial (right) sections of metaphase embryos are shown. The organization of the filamentous actin in the cortical cap appears similar under all three conditions. Bar, 10 um. (F and G) Spinning disk confocal optics were used to image the cortex in control (n = 6),
rab-5(RNAi) (n = 6), and
chc-1(RNAi) (n = 6) embryos expressing a GFP fusion with the heavy chain of cytoplasmic myosin II (GFP:NMY-2). Representative cortical (left) and medial (right) sections acquired during the establishment of polarity (F) and at metaphase (G) are shown. Bars, 10 μm.