Fig. 3. Kinetics analysis of chromosome condensation in time-lapse sequences acquired using widefield optics. Images of embryos coexpressing GFP:histone H2B and GFP:g-tubulin were collected on a Nikon E800 upright microscope (Nikon Instruments, Melville, NY) using a 60 ´ 1.4 N.A. Plan Apo objective lens (no auxiliary magnification) and an Orca ER CCD camera (Hamamatsu Photonics, Bridgewater, NJ) without binning. (A) Representative images of GFP:histone in a single nucleus before scaling. Times are with respect to nuclear envelope breakdown (NEBD). (Scale bar: 5 mm.) (B) Kinetic plot generated by plotting the percentage of pixels below each of the indicated thresholds (the condensation parameter) as a function of time. For each condition, the average value of the condensation parameter for each threshold measured from nine sequences time-aligned with respect to NEBD is plotted (error bars = SE). Primary condensation (dark gray), secondary condensation (light gray), and the intervening pause (white) are indicated.

Fig. 4. Condensin targets to chromosomes during prophase in Aurora B-depleted embryos. (A) Control and AIR-2-depleted prophase embryos fixed and stained for DNA, the condensin subunit SMC-4, and AIR-2. AIR-2 is absent from chromosomes in the depleted embryos, but SMC-4 is detected on chromosomes. (B) Selected stills from time-lapse sequences of control and AIR-2-depleted embryos expressing a GFP fusion with the Caenorhabditis elegans homolog of the hCAP-G2 subunit of condensin (also see Movie 2). (Scale bars: 5 mm.)

Movie 1. Spinning disk confocal microscopy was used to image chromosome condensation in control embryos and embryos depleted of CeCENP-C, CeCENP-A, and SMC-4. Three examples of time-lapse sequences of the sperm pronuclei for each condition (as indicated) are shown.

Movie 2. Condensin targeting in embryos depleted of the Aurora B kinase, AIR-2. Living embryos expressing GFP:F55C5.4, the C. elegans homolog of the hCAP-G2 subunit of condensin, were imaged by spinning disk confocal microscopy. GFP condensin localizes throughout mitosis, from prophase until telophase, in both control (Upper) and AIR-2 depleted (Lower) embryos.