Spinning disk confocal time-lapse imaging of the first mitosis in a C. elegans embryo expressing GFP-KNL-2 (strain OD31) in which KNL-1 has been depleted by RNAi. Discrete foci of GFP signal within the nucleus coalesce along curvilinear chromosomes during prophase. After nuclear envelope breakdown, chromosomes fail to congress to the metaphase plate, but GFP-KNL-2 localization is evident. As seen after CeCENP-A depletion and CeCENP-C depletion (CIL 28779 and CIL 28782, respectively), foci of GFP-KNL-2 formed along the spindle after anaphase onset. The video is ∼11 min long, and the frame is ∼30 µm top to bottom. Images were acquired at 10-s intervals (see Materials and methods), and corresponding data are shown in Fig. 3 in J Cell Biol. 2007 Mar 12;176(6):757-63. Epub 2007 Mar 5.
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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