Description

6-cell sand dollar embryo expressing three tandem GFPs fused to the ensconsin microtubule-binding domain (EMTB-3G; Faire et al., 1999). 10 µM nocodazole was added at time 00:00, at which time three of four macromeres have entered anaphase; the east cell enters anaphase shortly thereafter (03:00). In all cells, numerous astral microtubules, pointing in all directions, persist >5 min after nocodazole addition. Stable microtubules may approach the cortex in west and south cells (03:00), but none can be seen to do so in north and east cells; all commence furrowing at a normal time and complete cytokinesis. Note that in all cases, stable microtubules become brighter because EMTB-3G liberated by disassembly becomes available for binding. 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, which is tolerated well. Movie corresponds to Fig 3C and fourth segment of video 3 in J Cell Biol. 2009 Dec 14;187(6):831-45.