consistent with previous findings that cells expressing similar APC mutants become polyploid over time (Fodde et al., 2001; Tighe et al., 2004). To examine the possibility that APC1–1,450 expression gives rise to polyploid cells as a result of failed cytokinesis, we moni- tored the behavior of chromosomes in time-lapse videos using an E-YFP-histone 2B (H2B) fusion. Cells with chromosomes aligned in metaphase were identified and filmed as they proceeded through anaphase (Fig. 2 and Video 1, available at http://www .jcb.org/cgi/content/full/jcb.200703186/DC1). After chromosome segregation in control cells, cytokinetic ingression was observed in brightfield images (Fig. 2 B, 18 min; arrow). Control cells initiated a furrow, completed cytokinesis, and returned to their interphase state 30 min after anaphase began. In many cells expressing APC1–1,450, we observed no evidence of furrow initiation after anaphase (Fig. 2, C and D; and Video 2), and chromosomes became tightly juxtaposed 10 min after segregation, which is consistent with a collapse of the anaphase spindle (Fig. 2 C, 28 min). Chromosomes began to decondense, and nuclei formed close to one another as cells returned to their interphase state. We believe that this behavior gives rise to the binucleate (i.e., polyploid) cells we observed in Fig. 1 A. Consistent with cyto- kinetic failure, staining of cells with antibodies against γ-tubulin revealed binucleate and multinucleate interphase cells with two or more centrosomes (unpublished data). We conclude that the expression of APC1–1,450 results in binucleated cells as a result of failures to carry out cytokinesis before exiting mitosis. We suspected that defects in spindle microtubules were responsible for the failed cytokinesis in cells expressing APC1–1,450 for at least two reasons. First, an allelic series of APC mutants with increasingly severe defects in mitotic spindle organization correlated with the number of bi- and multinucleated cells (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/ jcb.200703186/DC1; and not depicted; Green et al., 2005). The appearance of bi- and multinucleated cells is also increased in colorectal tumor cell lines with truncating mutations in APC (Fig. S1 A, SW480). Second, we observed that compared with control cells, large regions of the cortex of cells expressing APC1–1,450 lack growing microtubule plus ends marked with EB1-GFP, and these spindles undergo increased rotations with respect to the spindle axis (Fig. 3, A–C; arrows; and compare Video 4 with Video 3). Failed spindle anchoring blocks cytokinesis As furrow initiation is specified by the mitotic spindle, we were particularly intrigued by the observation that microtubules make less contact with the cell cortex, and spindles appear to undergo considerable rotation when EB1-GFP was monitored in cells expressing APC1–1,450 (Video 4). We reasoned that the rotation of anaphase spindles may impact the ability of cells to initiate a cytokinetic furrow. To test this possibility, we compared the be- havior of the mitotic apparatus with respect to the onset of ana- phase and furrow initiation in control cells with cells expressing APC1–1,450. Metaphase cells expressing GFP fusions to tubulin, Figure 2. Expression of APC1–1,450 results in cytokinetic failure. (A–D) EYFP–H2B was transiently expressed in the indicated cell lines. Metaphase cells were ﬁlmed, and select time points from videos (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200703186/DC1) were formatted to show ﬂuorescence (A and C) or the ﬂuorescence images overlaid with brightﬁeld images (B and D). Images were recorded every minute; time 0 was set as the last time point before anaphase onset. The arrow in B indicates furrow ingression, and a corresponding arrow in D shows the lack of change at the cortex at the same time point after anaphase begins. APC MUTANTS LEAD TO TETRAPLOID CELLS IN VIVO • CALDWELL ET AL. 1111