PRC1SPD-1 depletion prevents the formation of midbody microtubule bundles and Aurora BAIR-2 targeting to the intercellular bridge. (A) Central plane confocal images of control (top; n = 8 embryos) and PRC1spd-1(RNAi) (bottom; n = 9) embryos expressing GFP–β-tubulin and mCherry-histone. Kymographs of the GFP–β-tubulin signal in the midbody region are also shown. Times are seconds after furrow initiation. (B) Central plane confocal images of control (n = 5) and PRC1spd-1(RNAi) (n = 5) embryos expressing GFP–Aurora BAIR-2 along with the mCherry-tagged plasma membrane probe. Times are seconds after furrow initiation. (C) The central region of confocal images of control (n = 18) and PRC1spd-1(RNAi) embryos (n = 7) expressing a GFP-tagged plasma membrane probe and mCherry-Mklp1ZEN-4 are shown at different time points after furrow initiation. (D) Deconvolved wide-field image (single z plane) of a control embryo stained for MyosinNMY-2 and MKLP1ZEN-4 along with tubulin and DNA (not depicted). DNA condensation and midbody compaction indicate that this is an abscission phase embryo whose furrow retracted during the freeze-crack fixation. Insets show MKLP1ZEN-4 at the midbody (white arrowhead) and overlapping with MyosinNMY-2 on the midbody ring. (E, top) Deconvolved wide-field images of control and PRC1spd-1(RNAi) embryos fixed during constriction phase (left) or abscission phase (right) and stained for tubulin (green), DNA, and Mklp1ZEN-4. Images are 2-µm projections through the central region of the embryo. (bottom) Traces show line scans (20 × 70 pixels) drawn across the center of control and PRC1spd-1(RNAi) embryos (n = 6 control and 6 PRC1spd-1(RNAi) embryos for each phase imaged in a single experiment). Intensity values were normalized by dividing by the mean fluorescence intensity in the cytoplasm near the cell periphery. a.u., arbitrary unit. White boxes on the low magnification images in B, D, and E mark the location of the region shown at higher magnification in the images at the bottom. Bars, 5 µm.
