Figure 3. Spindle anchoring is critical for cytokinesis. (A) Representative images from videos of the indicated cell lines cotransformed with EB1-GFP (complete videos can be found as Videos 3 and 4, available at http://www.jcb.org/cgi/content/full/jcb.200703186/DC1). Arrows indicate cortical EB1 comets found at the plus ends of microtubules. (B–E) Time points from videos of the indicated cell lines expressing tubulin-GFP are shown (t was set to 0 at the start of ﬁlming; complete videos can be found as Videos 5, 6, and 7 corresponding to B, C, and D, respectively). The two examples of cells expressing APC1–1,450 include one example of an unsuccessful cytokinesis (APC1–1,450a) and one of a successful cytokinesis (APC1–1,450b). Spindle histories are presented to the right of each time series. In brief, the cortex of the cell in each video frame was outlined with a circle, and the spindle position was marked with a line. A red line was used to indicate spindles that rotated out of the plane of focus or back into the plane of focus and, thus, could not be represented on the two-dimensional diagram. (F) A stable 293 cell line expressing APC58–1,450 was transfected with enhanced GFP–H2B and ﬁlmed from metaphase through the completion of cytokinesis. The arrow at the 15-min time point indicates furrow ingression (chromosomes are compressed) in the presence of lag- ging chromosomes. (G) 68 videos of each cell line expressing either tubulin-GFP, EB1-GFP, or EYFP-H2B were analyzed for spindle anchoring (<15° rota- tion between 2-min time points), initiation of anaphase (spindle elongation or chromosome separation), and initiation of a cleavage furrow as judged by ingression at the equatorial cortex. Bars, 10 μm. 1112 JCB • VOLUME 178 • NUMBER 7 • 2007