Figure 2. Polarized actin-dependent cell shape changes underlie division-coupled interspersion behaviors.

(A) Frames from time-lapse imaging of cytokinesis in an organoid expressing myosin regulatory light chain (MRLC)-mScarlet. (B) 3D reconstruction from live imaging of a cell dissociated from EB3-GFP organoids undergoing cytokinesis. EB3-GFP labeled organoids were used to facilitate identification of dissociated cells undergoing mitosis. Representative of 12/15 divisions. (C) Frames from SPIM of chromosome segregation in a live organoid. DNA: H2B-mScarlet. Arrowheads indicate mitotic chromosome masses. (D) Frames from confocal imaging of mitotic cells in live organoids treated with cytoskeletal inhibitors for 30 min before initiation of imaging. Membranes: R26^mTmG; DNA: SiR-DNA. Arrowheads: mitotic chromosomes. (E) Quantification of the distance of mitotic chromosomes from the apical surface of the organoid epithelium following treatment with cytoskeletal inhibitors, normalized to the total apical-basal height of the epithelium, n ≥ 10. ns: not significant; ***p<0.001, Student’s t-test. (F) Anaphase of mitoses shown in (D). Dashed lines underline anaphase chromosome masses. (G) Frames from time-lapse imaging of Vil1^CreERT2; R26^mTmG organoids in which recombination has been induced at low levels to label a subset of cell membranes in the organoid. The protrusive front of one daughter cell is indicated by an arrowhead. Note that the division occurred along the imaging plane, such that the other daughter cell is ‘behind’ the imaged daughter cell. Asterisk: nearby interphase cell that did not participate in the division. (H) Frames from confocal imaging of live organoids testing the cytoskeletal requirements for the basal movement of nascent nuclei (top, arrowheads indicate chromosomes) and elongation of the basal cell edge (bottom, arrowhead indicates basal edge of reinserting cell). A schematic of this experiment is shown in Figure 2—figure supplement 1I. DNA: SiR-DNA; Membrane: R26^mTmG; STLC: Eg5 inhibitor to induce mitotic arrest; SAC: spindle assembly checkpoint. (I) Quantification of DNA position before SAC inhibition (starting position), and at chromosome decondensation (end position), normalized to the total apical-basal distance of the epithelium. Arrowheads point towards the end position after mitotic exit. n ≥ 5, ns: not significant, ***: p<0.001, Student’s t-test of comparing end position and start position. Scale bars, 10 µm.

Figure 2—figure supplement 1. Polarized actin-dependent changes in cell shape during division in intestinal organoids.

(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 R26^mTmG reporter (for example, see [Packard et al., 2013]) with an inducible, pan-intestinal epithelial Cre (Vil1^CreER). 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 Vil1^CreER; R26^mTmG 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 R26^mTmG) 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.

Figure 2—video 1. Cytokinesis in the intestinal organoids.

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DOI: 10.7554/eLife.36739.011

MRLC-mGFP organoids imaged by spinning disc confocal with 60 X objective at 20 s time points.

Figure 2—video 2. Cytokinesis in the intestinal organoids.

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DOI: 10.7554/eLife.36739.012

MRLC-mScarlet organoids imaged by spinning disc confocal with 60X objective at 20 s time points.

Figure 2—video 3. Cytokinesis in the intestinal organoids.

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DOI: 10.7554/eLife.36739.013

Rare membrane-GFP cells in organoids generated by stochastic recombination of the R26^mTmG reporter as described in Figure 2—figure supplement 1E were imaged by spinning disc with 60X objective at 20 s time points. The division occurred along the imaging plane, such that the other daughter cell is ‘behind’ the imaged daughter cell. Note that cytokinesis is also accompanied by blebbing from the basal surface.

Figure 2—video 4. Furrow ingression in dissociated intestinal cells.

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DOI: 10.7554/eLife.36739.014

3D reconstruction of a single time point from live imaging of mitotic exit in cells dissociated from an EB3-GFP organoid. The reconstruction is rotated away from the viewer. Imaging was performed by spinning disc confocal with a 60X objective.

Figure 2—video 5. Spindle assembly and cell rounding during mitosis.

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DOI: 10.7554/eLife.36739.015

EB3-GFP organoids imaged from early prophase by spinning disc confocal with 60X objective at 20 s time points.

Figure 2—video 6. Cell rounding during mitosis in intestinal organoids.

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DOI: 10.7554/eLife.36739.016

Membrane-tomato labeled organoids (R26^mTmG in the absence of recombination) imaged by spinning disc confocal with 20X objective at 7 min time points.

Figure 2—video 7. Chromosome movements at mitotic onset in Latrunculin A-treated organoids.

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DOI: 10.7554/eLife.36739.017

DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.

Figure 2—video 8. Chromosome movements at mitotic onset in nocodazole-treated organoids.

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DOI: 10.7554/eLife.36739.018

DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points. The cell does not undergo chromosome segregation as it is unable to assemble a mitotic spindle.

Figure 2—video 9. Chromosome movements at mitotic onset in control organoids.

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DOI: 10.7554/eLife.36739.019

DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.

Figure 2—video 10. Cell reinsertion behavior.

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DOI: 10.7554/eLife.36739.020

A daughter cell within an organoid in which a subset of cells is labeled with membrane-GFP (R26^mTmG; Vil1^CreER induced at low levels) blebbing and extending protrusions to the basal surface following cytokinesis. Note that the division occurred along the imaging plane, such that the other daughter cell is ‘behind’ the imaged daughter cell. Imaged by spinning disc confocal with a 60X objective at 20 s time points.

Figure 2—video 11. Chromosome movements following induced mitotic exit in STLC-treated organoids.

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DOI: 10.7554/eLife.36739.021

DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.

Figure 2—video 12. Chromosome movements following induced mitotic exit in STLC and Latrunculin A-treated organoids.

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DOI: 10.7554/eLife.36739.022

DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.

Figure 2—video 13. Chromosome movements following induced mitotic exit in STLC and nocodazole treated organoids.

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DOI: 10.7554/eLife.36739.023

DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.