One of eight cells in a slightly compressed sand dollar embryo expressing three tandem GFPs fused to the ensconsin microtubule-binding domain (EMTB-3G; Faire et al., 1999). The embryo was treated with 20 µM trichostatin A (TSA), an inhibitor of HDAC6 (a tubulin deacetylase) that selectively disrupts dynamic microtubules in mammalian cells at time 00:00, before nuclear envelope breakdown. After an exceptionally long metaphase (>20 min) with no long astral microtubules, and during which one half of the spindle bifurcated, this cell entered anaphase approximately 26 min after TSA addition. Some astral microtubules regrew in anaphase, but none approach within 10 µm of the cortex. Sand dollar embryos were imaged with a laser scanning confocal microscope (Radiance 2000; Bio-Rad Laboratories) mounted on a compound microscope (E800; Nikon) using a 40× 1.3 NA Plan-Fluor oil lens. Room temperature was maintained at 14–17°C. Video corresponds to the fourth segment of Video 4 and Figure S4B of J Cell Biol. 2009 Dec 14;187(6):831-45.
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