Spinning disk confocal time-lapse imaging of the first mitosis in a C. elegans embryo expressing GFP-KNL-2 in which CeCENP-A has been depleted by RNAi in strain OD31. Diffuse nuclear GFP signal is evident during prophase. However, unlike the control (CIL 28778), no discrete GFP foci were seen until anaphase onset, at which point foci of GFP-KNL-2 formed along the spindle and on the chromatin. The video is ∼18 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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