(A) 3D reconstruction of immunofluorescence images. Arrow indicates mitotic cell. Arrowhead indicates the corresponding apical footprint. DNA: Hoechst 33342, actin: Alexa488-phalloidin, tight junctions: anti-ZO-1. (B) Quantification of the perimeter of the apical footprint of mitotic cells compared to interphase cells. The apical footprint was determined from anti-ZO1 immunofluorescence. Each data point represents the ratio between the apical perimeter of a mitotic cell and the average apical perimeter of 4 of its interphase neighbors. n = 10. (C) 3D reconstruction of immunofluorescence images. Arrow indicates mitotic cell. Arrowhead indicates the corresponding apical footprint. DNA was labeled with Hoechst 33342. Organoids transduced with MRLC2-mScarlet were used to report on myosin localization. This process can generate mosaic organoids, in which only a subset of cells expresses the transgene, allowing for assignment of the myosin localization to specific cells. (D) Frames from time-lapse imaging of a mitotic cell in a live organoid in which microtubule plus-ends are labeled with EB3-GFP. (E) Fluorescent image of a metaphase cell in a live organoid, in which DNA is fluorescently labeled with H2B-mScarlet. The membranes of a subset of cells within the organoid have been labeled with GFP by inducing low levels of recombination of the R26mTmG reporter (for example, see [Packard et al., 2013]) with an inducible, pan-intestinal epithelial Cre (Vil1CreER). Arrowheads indicate thin membranous processes that maintain the connection of the mitotic cell to the basal surface. (F) Fluorescent images of anaphase cells in live Vil1CreER; R26mTmG organoids, induced as in Figure 2—figure supplement 1E to stochastically label a subset of cell membranes in the organoid. Arrowheads indicate membranous processes. Far right panel represents a later time point of the cell shown in Figure 2—figure supplement 1E. Images scaled with ɣ adjustment. Although membranous processes are inherited by only one daughter during division in the developing kidney (Packard et al., 2013), in the intestine we found that both daughters inherited processes (23/25 anaphases have at least one process per daughter cell). Consistent with symmetric inheritance of these processes, we observed that daughters re-established full contact with the basal surface at highly similar rates after division: daughters reestablished full contact with the basal surface within 4 ± 4 min (SD) of one another. (G) Quantification of anaphase spindle orientation compared to the plane of the epithelium following vehicle and Latrunculin A treatment, on a 0–90° scale, n = 10. (H) Quantification of chromosome movements in organoids following mitotic arrest and induced mitotic exit. Mitotic arrest was induced by 45 min treatment with S-trityl-L-cysteine (STLC). Mitotic exit was subsequently induced by pharmacological disruption of the spindle assembly checkpoint (SAC, using the Mps1 inhibitor AZ3146) or cyclin-dependent kinase (CDK, using the CDK inhibitor RO-3306). Each arrow corresponds to the position of the DNA from one cell, before and after SAC/CDK inhibition, normalized to the total apical-basal distance of the epithelium. Arrowheads point towards the end position. The end point was defined as chromosome decondensation for the +SAC and +CDK inhibitors conditions. Since the control case does not undergo mitotic exit, the end point was defined as the end of the experiment, after 3 hr of imaging, which is substantially before the organoid begins to die from the treatment. n = 5, ***: p<0.001, ANOVA of distances moved (end position – start position). (I) Schematic of assay used to assess chromosome movements in organoids following mitotic arrest with STLC, pharmacological disruption of the cytoskeleton, and induction of mitotic exit with the SAC inhibitor AZ3146 (Figure 2H and I). (J) Frames from time-lapse imaging of cells in live organoids treated with the Plk1 inhibitor BI2536 to inhibit cytokinesis. DNA was labeled with H2B-mScarlet. Membrane (labeled with R26mTmG) shows absence of cytokinetic furrow ingression. Arrowheads indicate daughter nuclei. Time following BI2536 addition is indicated. We note a significant delay in the observation of pharmacological effects on the organoids compared to cultured cells, allowing for initial chromosome alignment and satisfaction of the SAC before the effects of the BI2536 were observed. Plk1 inhibition was used to inhibit cytokinesis since blebbistatin, a myosin II inhibitor, did not disrupt cytokinesis at the limits of its solubility in this system. Scale bars, 10 µm.