Spinning disk confocal time-lapse imaging of the first mitosis in a C. elegans embryo that has been depleted of KNL-2 by RNAi. GFP-histone and GFP-γ-tubulin were imaged (strain TH32). Pronuclear migration is slightly disrupted by a chromatin bridge resulting from failed meiotic segregation of the maternal chromatin. As seen in CeCENP-A depletion (CIL 28776), chromosome condensation is disrupted, chromosomes fail to form a proper metaphase plate, and the spindle poles move away from the chromatin masses prematurely. Anaphase segregation fails, and chromatin masses are left in the middle of the cell. Also note the meiotic defect that results in a defective oocyte pronucleus (left side of image at the beginning of the videos; this is the embryo anterior). The video is ∼16 min long, and the frame is ∼30 µm top to bottom. Images were acquired at 10-s intervals.
Images were acquired via a spinning disk confocal microscope (CSU10; McBain Instruments) mounted on an inverted microscope (TE2000e; Nikon). Strain TH32 coexpressing GFP-histone H2b and GFP–γ-tubulin was imaged using a 60× 1.4 NA plan Apo objective with 1.5× auxiliary magnification and a cooled CCD camera (Orca ER; Hamamatsu) binning 2 × 2. Strain OD31 expressing GFP–KNL-2 was imaged in the same manner without the 1.5× auxiliary magnification.
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