EB1, or histone 2B were filmed for 1–2 h. In many cases, cells initiated anaphase and formed a cytokinetic furrow during the video. In control cells, we observed the consistent behavior of mitotic spindles documented in videos but more completely de- picted in the spindle histories. Spindle histories were created from traces of the cell cortex (circle) and spindle axis (a line) that were derived from each time frame of a video. The tracings were overlaid to provide an entire spindle history (Fig. 3, B–E; right). For example, in control cells during prometaphase and metaphase, spindles moved along their axis but infrequently ex- hibited large rotational deviations (Fig. 3 B and Video 5, avail- able at http://www.jcb.org/cgi/content/full/jcb.200703186/DC1). These movements decreased as cells entered anaphase and were quickly followed by furrow ingression and cytokinesis. We classified this stable behavior of control spindles as anchored (i.e., <15° of movement between time frames). Spindles in cells expressing APC1–1,450 exhibited two dis- tinct types of behaviors. In >80% of cells filmed, we observed unstable spindle positioning, with spindles more frequently slid- ing and undergoing large rotations relative to the spindle axis (Fig. 3 C, APC1–1,450a; and Video 6, available at http://www.jcb .org/cgi/content/full/jcb.200703186/DC1). The large movements of the spindle were observed equally in metaphase and anaphase cells. In many cases, anaphase was defective with the failed seg- regation of chromosomes (Video 2) or the reduced elongation of spindles. In <20% of cells, spindles behaved similarly to con- trols, exhibiting stable spindle anchoring, furrow ingression, and cytokinesis (Fig. 3 D, APC1–1,450b; and Video 7). Of the cells that initiated a cytokinetic furrow, there were no consistent changes in the timing of furrow completion between control cells and cells expressing APC1–1,450 (Fig. S2 A), arguing that APC mutants do not affect the timing of anaphase. To further examine the rela- tionship between spindle anchoring, anaphase onset, and the ini- tiation of furrow ingression, we collected >100 videos of mitosis for each cell type. We characterized the 68 videos in which we could measure each of the aforementioned parameters (see Materials and methods section Image and statistical analysis). More than 60% of control cells anchored their spindle, entered anaphase, and initiated furrow ingression. In stark contrast, cells expressing APC1–1,450 generally failed to anchor their spindles, and, although spindle elongation indicated entry into anaphase, <15% of cells initiated a cytokinetic furrow (Fig. 3 G). A direct tabulation of the relationship between spindle anchoring and fur- row initiation more precisely confirmed the trends in Fig. 3 G: regardless of the genotype, we observed a perfect correlation be- tween cells with partially anchored or unanchored spindles and failures to initiate a cytokinetic furrow (Fig. S2 B). Although we consider the failure to anchor the mitotic spindle the most likely explanation for the failure to induce the cytokinetic furrow, it remains possible that defects in chromo- some alignment previously reported in cells expressing APC1–1,450 could contribute to inhibition of the cytokinesis (Green and Kaplan, 2003). Indeed, findings in HeLa cells indicate that there is a connection between the rate of lagging chromo- somes in anaphase and cytokinetic failure (Shi and King, 2005). To address whether lagging chromosomes or spindle-anchoring defects contribute to cytokinetic failures in APC mutants, we turned to the previously characterized APC58–1,450 allele. This allele prevents the oligomerization of mutant and wild-type APC proteins, and spindles exhibit EB1-postive microtubule plus ends near the cell cortex (Fig. 3 A, arrows in APC58–1,450). Consistent with stable spindle anchoring, videos of cells ex- pressing APC58–1,450 and GFP-tubulin exhibit relatively normal spindle anchoring (Fig. 3 E, spindle history), and >50% of cells have normal cytokinetic ingression (compared with <15% of cells expressing APC1–1,450 and >60% for control cells; Fig. 3 G). Despite stable spindle anchoring, cells expressing APC58–1,450 have a high rate of misaligned chromosomes in metaphase (>70%) and anaphase cells (>30%), possibly as a result of a decrease in the pause frequency of growing microtubules (Green et al., 2005; and unpublished data). The presence of stably anchored spindles in cells with lagging anaphase chromosomes allowed us to assess whether misaligned chromosomes are sufficient to contribute to a defect in cytokinesis. Despite the obvious presence of chromo- somes near the midzone, we did not observe a high rate of bi- or multinucleate cells (Fig. S1 A). Moreover, examples of individual cells expressing H2B-YFP show that chromosomes left behind in the midzone are constricted by the ingressing furrow and clearly do not impede furrow ingression (Fig. 3 F, arrow in the 15-min time point). Thus, APC58–1,450 can be considered a sepa- ration of function allele (i.e., normal spindle anchoring and cyto- kinesis but defective chromosome alignment and segregation), and we conclude from this analysis that unanchored spindles but not lagging anaphase chromosomes correlate with a failure to induce cytokinesis in these cells. Min mice have aberrant mitoses Although genomic instability has been previously reported in early adenomas in both mice and humans (Shih et al., 2001), our in vitro data suggest that mitotic defects could occur in intestinal epithelium before the appearance of dysplasia or adenomas (i.e., in histologically normal tissue). Specifically, we predicted that changes in microtubule structure and dynamics could result in misoriented mitotic spindles within the gut that could contribute to cytokinetic failure. To test these predictions, we performed a series of analyses of mitoses in the intestinal epithelium of APCMin/+ mice (hereafter referred to as Min mice) and wild-type littermates at early time points after birth (7–13 wk before the appearance of polyps; Marshman et al., 2002). Importantly, the Min allele (APC1–850) gives rise to similar mitotic defects as ob- served for cells expressing APC1–1,450. Specifically, we observed that cells expressing APC1–850 have an increased incidence of multinucleated cells (fivefold increase), unanchored mitotic spindles (<25% anchored compared with >80% in controls), and a high incidence of anaphase cells that fail to induce a cyto- kinetic furrow (<20% anchored compared with >60% in con- trols; Figs. S1 A and S3). We first characterized small intestines of wild-type mice using antibodies against the apical marker ZO-1, the basolateral marker β-catenin to identify cell boundaries, and DAPI to visualize chromosomes. We observed mitotic cells positioned in crypts as expected, and we observed a shift of mitotic chromosomes away from the basement membrane (Fig. S4, A–C; [inset]; available at http://www.jcb.org/cgi/content/ full/jcb.200703186/DC1), a position that is consistent with the APC MUTANTS LEAD TO TETRAPLOID CELLS IN VIVO • CALDWELL ET AL. 1113