SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the γ-tubulin–mediated addition of centriolar microtubules

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Figure S3. Establishing a timeline of events during the first mitotic division. Embryos coexpressing GFP:γ-tubulin and GFP:histone H2B were mounted and filmed without compression using spinning disk confocal and DIC optics as described (see Materials and methods). A GFP z series (11 planes at 1-µm intervals) as well as a single, central plane DIC image were acquired every 20 s. GFP images were acquired using 100-ms exposures at ∼40% laser power with a 60× 1.4 NA oil objective and no binning. Fluorescence images are maximum intensity projections of the z series. Representative stills of the fluorescence and DIC channels illustrate key events (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200709102/DC1). Schematics to the right illustrate the landmark events scored in the image sequences and their mean time of their occurrence in seconds relative to cytokinesis onset (t = 0; n > 5 embryos for each event scored). Note that the time given for centriole separation is when this event becomes detectable at the light level. Errors are the standard deviation. Arrowheads indicate segregating chromosome masses (anaphase meiosis I, meiosis II, and mitosis), separated centrosomes adjacent to the sperm pronucleus (centriole separation), nuclear envelope breakdown (NEBD; lower pronucleus), and cleavage furrow (cytokinesis onset). Note that onset of cytokinesis (bottom panel, arrowhead) is clearly visible in the DIC image. Bar, 10 μm.