UBXN-2 interacts with CDC-48 and regulates spindle orientation in the one-cell C. elegans embryo. (A) Yeast two-hybrid assay showing UBXN-2 interaction with C. elegans CDC-48.1 and CDC-48.2. Cells expressing the indicated proteins were spotted on permissive or restrictive media. Growth on restrictive medium indicates interaction. (B) CDC-48 coimmunoprecipitates with UBXN-2. (C) Localization of UBXN-2 by antibody staining in control, ubxn-2(RNAi), and cdc-48.2(RNAi) two-cell embryos. UBXN-2 is cytoplasmic and nuclear in control embryos. (D) Still image from a wide-field time-lapse movie of a GFP::UBXN-2^R/mCherry::α-tubulin–expressing embryo (Video 1). Black arrowhead points to one of the centrosomes. (E) Top: DIC time-lapse recordings of control and ubxn-2(RNAi) embryos; bottom: schematic drawings. Black arrowheads point to centrosomes (red circles in the scheme). The black circles represent the pronuclei (referred to as PN in the figures), and the green dots represent the polar bodies. ubxn-2(RNAi) embryos had a weak or absent pseudocleavage. (F) Mitotic spindle orientation in one-cell embryos. Alignment of the centrosomes with the axis of polarity at NEBD and telophase, in control and ubxn-2(RNAi) embryos. Alignment along the antero-posterior axis corresponds to 0°. Each line represents an embryo. (G) Time interval (Δt = mean values in seconds ± SEM) from the exit of meiosis (appearance of the female pronucleus) to NEBD in the one-cell embryo, determined from DIC time-lapse movies. In all figures, numbers in italic (control-RNAi) indicate the number of embryos analyzed; anterior is on the left, posterior on the right. Bar, 5 µm.