Spinning disk confocal time-lapse imaging of the first mitosis in a C. elegans embryo expressing GFP-KNL-2 in strain OD31. As seen for GFP-CeCENP-A (CIL 28780), discrete foci of the GFP signal within the nucleus coalesce to form holocentric kinetochore plates during prophase. GFP image on left and DIC image on right. After nuclear envelope breakdown, chromosomes appear as paired lines and congress to the metaphase plate. At metaphase, sister kinetochores form two lines facing opposite spindle poles. During anaphase, foci of GFP-KNL-2 were seen to form in the spindle area. The video is ∼16 min long, and the frame is ∼30 µm top to bottom. DIC is shown on the right, and GFP is shown on the left. 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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