Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis

Abstract

Epithelial integrity and barrier function must be maintained during the complex cell shape changes that occur during cytokinesis in vertebrate epithelial tissue. Here, we investigate how adherens junctions and bicellular and tricellular tight junctions are maintained and remodeled during cell division in the Xenopus laevis embryo. We find that epithelial barrier function is not disrupted during cytokinesis and is mediated by sustained tight junctions. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that adherens junction proteins are stabilized at the cleavage furrow by increased tension. We find that Vinculin is recruited to the adherens junction at the cleavage furrow, and that inhibiting recruitment of Vinculin by expressing a dominant-negative mutant increases the rate of furrow ingression. Furthermore, we show that cells neighboring the cleavage plane are pulled between the daughter cells, making a new interface between neighbors, and two new tricellular tight junctions flank the midbody following cytokinesis. Our data provide new insight into how epithelial integrity and barrier function are maintained throughout cytokinesis in vertebrate epithelial tissue.

Keywords: FRAP; Vinculin; Xenopus laevis; adherens junctions; contractile rings; cytokinesis; epithelial barrier function; tight junctions; tricellular tight junctions.

Figure 1. Barrier function is maintained during Xenopus epithelial cytokinesis

A. Experimental setup for fluorescent tracer penetration assay. Gastrula-stage embryos expressing mRFP-ZO-1 (TJs) and mCherry-H2B (chromosomes) were mounted in 0.1X MMR containing 10 μM fluorescein (tracer dye) and observed. B. Fluorescent tracer penetration assay of a representative dividing cell. Three views of the same region of interest are shown: en face view (B), side view of the region indicated with yellow rectangles in B (B’) and 3D view (B’’). Note that the TJ labeled by mRFP-ZO-1 (red) is initially pulled basally, but fluorescein (green) at apical side (top) does not breach through the TJ (yellow arrowheads in B’) to the basal side (bottom). Time, min:sec. Asterisks in B and at 0:00 in B’ indicate chromosomes (red), which are not visible at other time points in B’. C and D. Embryos expressing mRFP-ZO-1 (red) were injected with 5 nl of 0.1x MMR (C) or 100 mM EGTA (D) into the blastocoel, mounted in 10 μM fluorescein (green) and observed. Upper panels, 3D view; lower panels, side view. Note that fluorescein tracer breaches the TJ in D (EGTA-treated), but not in C (control). Arrows and arrowheads indicate bicellular and tricellular junctions, respectively. Scale bars, 20 μm. See also Movies S1 and S2.

Figure 2. The contractile ring ingresses anisotropically from basal to apical and remains continuous and connected to cell-cell junctions

A. Live imaging of TJs and the cytokinetic contractile ring in embryos expressing mRFP-ZO-1 (red, TJs) and Lifeact-GFP (green, F-actin). Projected multi-plane en face images (A) and side views at the cleavage plane (A’) (yellow rectangle in the en face view). Note that the cytokinetic ring remains connected to TJs throughout cytokinesis. B. Live imaging of AJs and the cytokinetic contractile ring in embryos expressing E-cadherin-3xGFP (pseudocolored red, AJs) and Lifeact-mRFP (pseudocolored green, F-actin). Projected multi-plane en face images (B) and side views at the cleavage plane (B’) (yellow rectangle in the en face view). Note that the cytokinetic ring remains connected to AJs throughout cytokinesis. For side views, top is apical, bottom is basal. Scale bars, 10 μm. See also Movies S3 and S4.

Figure 3. AJ proteins, but not TJ proteins, are stabilized at the cleavage furrow of dividing cells and vinculin is recruited to the cleavage furrow

A. Diagram depicting locations of FRAP measurements in interphase (purple circle) and dividing cells (green circle: cleavage furrow, orange circle: polar region). B. Representative examples of E-cadherin-3xmCherry FRAP. Cell images (left) show a frame taken from a time-lapse movie. The colored dashed circles indicate the bleached areas, the red lines indicate the locations used to generate the kymographs (right), and the white asterisks indicate the two daughter cells. A FIRE lookup table was applied to the kymographs; time (horizontal axis) and bleach time points are indicated. Scale bars, 10 μm. C. E-cadherin-3xmCherry FRAP data fitted with a double exponential curve and graph of average mobile fractions. The number of cells (n) quantified is: interphase cells (n=23), dividing cells/furrow (n=17), dividing cells/polar (n=12). D-F. FRAP data of β-catenin-GFP (D), mCherry-claudin-6 (E), and mRFP-ZO-1 (F) fitted with a single exponential curve and graph of average mobile fractions. n=21, 19, 14 (D), n=23, 18, 10 (E), and n=34, 34, 21(F). G. Vinculin-3xGFP in a dividing cell. White asterisks indicate daughter cells, yellow arrowheads indicate accumulation of Vinculin-3xGFP at the cleavage furrow, blue brackets indicate magnified areas of Vinculin-3xGFP and mCherry-membrane shown below the cell view. H. Quantification of Vinculin-3xGFP and mCherry-membrane intensity at the cleavage furrow of dividing cells (n=14). Error bars, S.E.M. Statistics, unpaired t-test, ***p<0.0001 (C-F), paired t-test, ***p<0.001, *p<0.05 (H). See also Figures S1 and S2.

Figure 4. Dominant negative vinculin abolishes cell-cell junction reinforcement at the cleavage furrow and accelerates ingression

A. Domain structure of Xenopus laevis vinculin. Pro, proline-rich region. B and C. Live imaging of Vinculin-3xGFP in interphase (B) and dividing (C) cells of embryos expressing vinculin D1 in a mosaic manner. D1 indicates vinculin D1-expressing cells, which are identified with a lineage tracer (mCherry-H2B, not shown). Note that vinculin-3xGFP intensity at bicellular and tricellular AJs is reduced in B, and the localization of vinculin at cleavage furrow (yellow arrowheads) is abolished when the neighbor cell expresses vinculin D1 (white arrowhead). Asterisks in C indicate daughter cells. Scale bars, 10 μm. D and E. Live imaging of embryos expressing mRFP-ZO-1 (red, TJs) and E-cadherin-3xGFP (green, AJs) without (D) or with (E) expression of vinculin D1. Yellow and white arrowheads show that ZO-1 and E-cadherin are maintained at the cleavage furrow in control cells (D) or reduced in vinculin D1-expressing cells (E), respectively. Scale bars, 20 μm. D’ and E’. Kymographs of the furrow region shown by yellow rectangles in D and E. Note that E-cadherin (green) completes invagination (white arrow) before ZO-1 (red) in D’ and that both E-cadherin and ZO-1 are maintained (D’) or reduced (E’) at ingressing furrow region (yellow arrows). In E’, two vertices move apart after division (asterisk) because the dividing cell underwent a Type I division (see Figure 7). F and G. Time that it takes cells to reach 25, 50, 75, and 100% ingression are shown for control (F) and vinculin D1-expressing (G) cells. Whiskers indicate the minimum and maximum, boxes indicate the 25 and 75 percentiles, and vertical line indicates the median. n=12. H and I. Normalized fluorescence intensity of mRFP-ZO-1 and E-cadherin-3xGFP at the furrow (green) and polar region (orange) in control (H) and vinculin D1-expressing cells (I). n=12. Error bars, S.E.M. Statistics, two-tailed paired Student’s t-test. *, p<0.05; **, p<0.005; ***, p<0.0005. See also Figure S3 and Movie S5.

Figure 5. Two nascent tTJs are formed after cytokinesis

A. Immunofluorescence staining of embryos using anti-tricellulin (green) and anti-ZO-1 (red). Scale bar, 20 μm. B. Live imaging of an embryo expressing GFP-tricellulin (green) and mRFP-ZO-1 (red). Scale bar, 20 μm. C. Live imaging of a dividing cell expressing GFP-tricellulin (green) and mRFP-ZO-1 (TJs, red). Note that two punctate GFP-tricellulin spots appear at the cell vertices of the new interface between neighbor cells (white arrows). Asterisks and “n”s indicate daughter cells and neighbor cells, respectively. Scale bar, 20 μm (left panels), 5 μm (right panels). D. Live imaging of a dividing cell expressing MgcRacGAP-3xGFP (midbody, green) and mCherry-tricellulin (tTJs, red) (D). Note that the two tricellulin puncta are located on either side of the midbody in en face views (D’) and can be seen at the basal side of the midbody in side views (white arrows) (D’’). Asterisks and “n”s indicate daughter cells and neighbor cells, respectively. Scale bar, 20 μm (left panels), 5 μm (right panels). See also Movie S6.

Figure 6. Angulin-1/LSR recruitment to the new tTJs precedes tricellulin recruitment

A. Immunofluorescence staining of an embryo using anti-angulin-1 (green) and anti-ZO-1 (red). Scale bar, 20 μm. B. Live imaging of an embryo expressing angulin-1-3xGFP (green) and mRFP-ZO-1 (red). Scale bar, 20 μm. C. Live imaging of a dividing cell expressing angulin-1-3xGFP (green) and mRFP-ZO-1 (TJs, red). Note that two punctate angulin-1-3xGFP spots appear at the cell vertices of the new interface between neighbor cells (white arrows). Asterisks and “n”s indicate daughter cells and neighbor cells, respectively. Scale bar, 20 μm (left panels), 5 μm (right panels). D. Live imaging of a dividing cell expressing angulin-3xGFP (green), mCherry-tricellulin (red) and mem-TagBFP (membrane, blue). Note that angulin-3xGFP puncta formation precedes that of mCherry-tricellulin (white arrows). Asterisks and “n”s indicate daughter cells and neighbor cells, respectively. Scale bar, 20 μm (left panels), 5 μm (right panels). E. Kymograph of the formation of the two tTJs shown in D (yellow rectangle in D). Note that angulin-1 (green) appears earlier than the tricellulin (red). F. Timing of recruitment of angulin-1 and tricellulin to the two nascent tTJs. Time points when the fluorescence intensity of angulin-1-3xGFP (green) or mCherry-tricellulin (red) reached half max (see Figure S5) are plotted. Half max intensity point of angulin-1-3xGFP at first tTJ is defined as t=0. Closed and open circles indicate average time points ± s.d. of angulin-1 (green) and tricellulin (red) recruitment at 1st and 2nd tTJs, respectively. Horizontal gray lines indicate time span from angulin-1 recruitment (green x) to tricellulin recruitment (red x) for 1st and 2nd tricellular junctions for individual cells. Note that tricellulin recruitment is later than angulin-1 for both tTJs. Error bars, S.E.M. Statistics, two-tailed paired Student’s t-test, p=0.013 (1^st tTJ) and 0.016 (2^nd tTJ). See also Figures S4 and S5, and Movie S7.

Figure 7. Three types of packing patterns for daughter and neighboring cells after vertebrate epithelial cytokinesis

A. Long-term live imaging of cell packing patterns after cytokinesis in embryos expressing mRFP-ZO-1 (pseudocolored white, TJs) and GFP-H2B (pseudocolored blue, chromosomes). Projected multi-plane en face images are shown. Scale bar, 20 μm. B. Scheme of the three types of cell packing patterns observed after cell division. Each color (magenta for Type I, yellow for Type II and cyan for Type III) corresponds to the color shown in A. C. Model of epithelial cell cytokinesis in Xenopus (vertebrate) and Drosophila (invertebrate). See also Figure S6 and Movie S8.