Higashi T et al. (2016), Maintenance of the Epithelial Barrier and Remod...

XB-ART-52303

Curr Biol 2016 Jul 25;2614:1829-42. doi: 10.1016/j.cub.2016.05.036.

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

Higashi T , Arnold TR , Stephenson RE , Dinshaw KM , Miller AL .

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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.

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Species referenced: Xenopus laevis
Genes referenced: cad h2bc21 lsr marveld2 mrc1 mtor racgap1 tjp1 vcl

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	Graphical Abstract
	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 0.1Ã MMR containing 10 Î¼M 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) (Bâ²), and 3D view (Bâ³). 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 Bâ²) to the basal side (bottom). Time, min:s. Asterisks in (B) and at 0:00 in (Bâ²) indicate chromosomes (red), which are not visible at other time points in (Bâ²). (C and D) Embryos expressing mRFP-ZO-1 (red) were injected with 5 nl of 0.1Ã MMR (C) or 100 mM EGTA (D) into the blastocoel, mounted in 10 Î¼M fluorescein (green), and observed. Upper panels show 3D view; lower panels show 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 represent 20 Î¼m. See also Movies S1 and S2.
	Figure 2. AJs and TJs Remain Continuous and Connected to the Contractile Ring during Cytokinesis (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 (Aâ²) (yellow rectangle in the en face view) are shown. 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-cad-3ÃGFP (pseudocolored red, AJs) and Lifeact-mRFP (pseudocolored green, F-actin). Projected multi-plane en face images (B) and side views at the cleavage plane (Bâ²; yellow rectangle in the en face view) are shown. Note that the cytokinetic ring remains connected to AJs throughout cytokinesis. For side views, top is apical and bottom is basal. Scale bars represent 10 Î¼m. 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-cad-3ÃmCherry 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 represent 10 Î¼m. (C) E-cad-3ÃmCherry FRAP data fitted with a double exponential curve and graph of average mobile fractions. The numbers of cells (n) quantified are interphase cells (n = 23), dividing cells/furrow (n = 17), and dividing cells/polar (n = 12). (DâF) FRAP data of Î²-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, and 14 (D); n = 23, 18, and 10 (E); and n = 34, 34, and 21(F). (G) Vinculin-3ÃGFP in a dividing cell. White asterisks indicate daughter cells, yellow arrowheads indicate accumulation of Vinculin-3ÃGFP at the cleavage furrow, and blue brackets indicate magnified areas of Vinculin-3ÃGFP and mCherry-membrane shown below the cell view. (H) Quantification of Vinculin-3ÃGFP and mCherry-membrane intensity at the cleavage furrow of dividing cells (n = 14). Error bars represent SEM. Statistics: unpaired Student's t test, âââp < 0.0001 (CâF); paired Student's t test, âââp < 0.001, âp < 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-3ÃGFP 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-3ÃGFP 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 represent 10 Î¼m. (D and E) Live imaging of embryos expressing mRFP-ZO-1 (red, TJs) and E-cad-3ÃGFP (green, AJs) without (D) or with (E) expression of Vinculin D1. Arrowheads show that ZO-1 (yellow arrowheads) and E-cadherin (white arrowheads) are maintained at the cleavage furrow in control cells (D) or reduced in Vinculin-D1-expressing cells (E). Scale bars represent 20 Î¼m. (Dâ² and Eâ²) 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 (Dâ²) and that both E-cadherin and ZO-1 are maintained (Dâ²) or reduced (Eâ²) at ingressing furrow region (yellow arrows). In (Eâ²), 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-cad-3ÃGFP at the furrow (green) and polar region (orange) in control (H) and Vinculin-D1-expressing cells (I). n = 12. Error bars represent SEM. Statistics: two-tailed paired Studentâs t test. âp < 0.05; ââp < 0.005; âââp < 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). The scale bar represents 20 Î¼m. (B) Live imaging of an embryo expressing GFP-Tricellulin (green) and mRFP-ZO-1 (red). The scale bar represents 20 Î¼m. (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 nâs indicate daughter cells and neighbor cells, respectively. Scale bars represent 20 Î¼m (left panels) and 5 Î¼m (right panels). (D) Live imaging of a dividing cell expressing MgcRacGAP-3ÃGFP (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 (Dâ²) and can be seen at the basal side of the midbody in side views (white arrows; Dâ³). Asterisks and nâs indicate daughter cells and neighbor cells, respectively. The scale bars represent 20 Î¼m (left panels) and 5 Î¼m (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). The scale bar represents 20 Î¼m. (B) Live imaging of an embryo expressing Angulin-1-3ÃGFP (green) and mRFP-ZO-1 (red). The scale bar represents 20 Î¼m. (C) Live imaging of a dividing cell expressing Angulin-1-3ÃGFP (green) and mRFP-ZO-1 (TJs, red). Note that two punctate Angulin-1-3ÃGFP spots appear at the cell vertices of the new interface between neighbor cells (white arrows). Asterisks and nâs indicate daughter cells and neighbor cells, respectively. The scale bars represent 20 Î¼m (left panels) and 5 Î¼m (right panels). (D) Live imaging of a dividing cell expressing Angulin-1-3ÃGFP (green), mCherry-Tricellulin (red), and mem-TagBFP (membrane, blue). Note that Angulin-1-3ÃGFP puncta formation precedes that of mCherry-Tricellulin (white arrows). Asterisks and nâs indicate daughter cells and neighbor cells, respectively. The scale bars represent 20 Î¼m (left panels) and 5 Î¼m (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-1-3ÃGFP (green) or mCherry-Tricellulin (red) reached half max (see Figure S5) are plotted. Half max intensity point of Angulin-1-3ÃGFP at first tTJ is defined as t = 0. Closed and open circles indicate average time points of Angulin-1 (green) and Tricellulin (red) recruitment at first and second tTJs, respectively. Horizontal gray lines indicate time span from Angulin-1 recruitment (green x) to Tricellulin recruitment (red x) for first and second tricellular junctions for individual cells. Note that Tricellulin recruitment is later than Angulin-1 for both tTJs. Error bars represent SEM. Statistics: two-tailed paired Studentâs t test; p = 0.013 (first tTJ) and 0.016 (second 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. The scale bar represents 20 Î¼m. (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.

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