Higashi T , Stephenson RE , Miller AL .

R01 GM112794 NIGMS NIH HHS

             cell-cell junction organization
             contractile ring

	Figure 1. Localization of 3xGFP-tagged Dia1, Dia2, and Dia3 in the X. laevis gastrula epithelium. A. Embryos expressing 3xGFP-tagged Dia1, Dia2, or Dia3 (green) and mRFP-ZO-1 (TJ marker; magenta) were live imaged using confocal microscopy, and z-stack images of formin alone (top panels) and merged with mRFP-ZO-1 (bottom panels) are shown. Note that Dia3 is strongly localized at the contractile ring of the dividing cell. B. The localization of Dia3 at the contractile ring is dependent on Rho binding. Embryos expressing 3xGFP-Dia1 WT or V187D (Rho-binding mutant; green) and mCherry-farnesyl (membrane probe; magenta) were imaged. Since the expression of Dia3 causes membrane deformation phenotypes (see Figs. 1A and S6), Dia3 was expressed at a lower level in these images. Note that the Dia3 V187D mutant cannot localize at the contractile ring. Scale bars, 10 µm.
	Figure 2. Localization of 3xGFP-tagged formin proteins in X. laevis gastrula-stage epithelium. Embryos expressing 3xGFP-tagged formin (green) and mCherry-farnesyl (membrane marker; magenta) were live imaged using confocal microscopy. Stacked images of formin alone (left panels) and merged with membrane (right panels) are shown. Note that Dia1, Dia2, Fhod1, and Fhod3 are strongly localized at cell-cell junctions. Scale bar, 10 µm.
	Figure 3. Comparison of the localization of junctional formins with TJ and AJ proteins. A-D. 3xGFP-tagged Dia1 (A), Dia2 (B), Fhod1 (C), and Fhod3 (D) (green) were expressed together with TagBFP-ZO-1 (TJ marker; blue) and PLEKHA7-mCherry (AJ marker; red) in gastrula-stage X. laevis embryos and imaged by confocal microscopy. Top views (left) and averaged side views (right) of cell-cell junctions (50 px x 16 junctions) are shown. Scale bars, 10 µm (left); 2 µm (right). E. Graphs indicate intensity profiles of formins (green solid line), ZO-1 (blue dotted line), and PLEKHA7 (red dotted line). Note that the intensity profiles of Dia1 and Dia2 are very similar to that of ZO-1 and that Fhod1 and Fhod3 have a peak at AJs as well as signal basal to the AJ. Error bars indicate s.d..
	Figure 4. Ala267 is necessary for junctional localization of Dia1. A. Domain structure and mutants of Dia1. G, GTPase-binding domain; DID, diaphanous inhibitory domain; DD, dimerization domain; FH, formin homology; DAD, diaphanous autoregulatory domain. B. Embryos expressing Dia1 WT or mutants (green) and mCherry-farnesyl (membrane probe; magenta) were observed. Note that the A267D/I842A mutant cannot localize at cell-cell junctions, but the I842A mutant can. Enlargements of cell-cell junctions (white boxes) are shown below. C. Embryo expressing Dia1 A267D (green) and mCherry-farnesyl (magenta). Note that the cell expressing Dia1 A267D at high level (asterisk) is enlarged probably due to cytokinesis failure. Scale bars, 10 µm (B); 40 µm (C).
	Figure 5. The DID-DD region is sufficient for junctional localization of Dia1. A. Fragments of Dia1. The localization of CT was not directly assessed because CT expression resulted in very large cell size (likely due to a cytokinesis defect) and compromised cell-cell junctions. B. Embryos expressing mCherry-tagged Dia1 fragments (pseudocolored green) and GFP-farnesyl (membrane probe; pseudocolored magenta) were observed. Note that DID-DD is localized at cell-cell junctions, but DID-DD A267D is not. Both NT and CT cause abnormally large cell size. Scale bars, 10 µm.
	Figure 6. Overexpressed Dia1 DID-DD displaces full-length Dia1 and Dia2 from cell-cell junctions but does not displace Fhod1 and Fhod3. A. Dia1 DID-DD was mosaically expressed in the 3xGFP-tagged Dia1-, Dia2-, Fhod1-, or Fhod3-expressing embryos. Note that Dia1 and Dia2 were removed from cell-cell junctions (arrowheads) between the DID-DD-expressing cells (see enlarged white boxes labeled 1) but not from that of control cells (see enlarged yellow boxes labeled 2), whereas Fhod1 and Fhod3 were not affected by Dia1 DID-DD expression. B and C. Dia1 DID-DD was mosaically expressed in Lifeact-GFP (B)- or Vinculin-3xGFP (C)- expressing embryos. Note that localization and intensity of F-actin and Vinculin are not altered in Dia1 DID-DD-expressing cells (asterisks). Scale bars, 20 µm.
	Figure 7. Dia1 and Dia2 are localized at the contractile ring in dividing Dia1 DID-DD overexpressing cells. A. Embryos expressing 3xGFP-Dia1 (upper panels) or 3xGFP-Dia2 39 (lower panels) in all cells and mCherry-Dia1 DID-DD mosaically were live imaged using confocal microscopy. Note that Dia1 and Dia2 are strongly localized at the contractile ring in the Dia1 DID-DD-overexpressing cells (yellow asterisks) but are not localized at the contractile ring in the non-expressing cells (white asterisks). B. Embryos expressing 3xGFP-Dia1 WT (upper panels) or 3xGFP-Dia1 V175D (lower panels) in all cells and mCherry-Dia1 DID-DD mosaically were live imaged. Note that Dia1 V175D (Rho-binding mutant) cannot localize at the contractile ring in Dia1 DID-DD overexpressing cells. Asterisks, daughter cells. Scale bars, 10 µm.
	Figure 8. Dia1 DID-DD-overexpressing cells exhibit cytokinesis defects. A. Control (left) or mCherry-Dia1 DID-DD (right) embryos expressing GFP-farnesyl (membrane probe) were fixed and stained with anti-GFP (pseudocolored red) and DAPI (cyan). Note that there are binucleate cells (arrows) in Dia1 DID-DD-overexpressing embryo. B. Live imaging of dividing cells in control (upper panels) and Dia1 DID-DD-overexpressing (lower panels) embryos using LifeactGFP (F-actin probe; green) and BFP-ZO-1 (TJ marker; blue). Note that in the Dia1 DID-DD- 41 overexpressing cells, the contractile ring is formed and regresses over time. C-D. Apical cell surface area (C) and percentage of binucleate cells (D) in the fixed embryos. (n=171 cells from 4 embryos (control) and 170 cells from 4 embryos (Dia1 DID-DD)). E. Success rate of cytokinesis in live-imaging. (n=12 cells from 3 embryos (control) and 32 cells from 3 embryos (Dia1 DID-DD)). p-values are 0.57 (C; t-test), 0.00035 (D; Fisher’s exact test), 0.0067 (E; Fisher’s exact test). Scale bars, 10 µm.
	FIGURE 1:. Localization of 3×GFP-tagged Dia1, Dia2, and Dia3 in the X. laevis gastrula epithelium. (A) Embryos expressing 3×GFP-tagged Dia1, Dia2, or Dia3 (green) and mRFP-ZO-1 (TJ marker; magenta) were live imaged using confocal microscopy; z-stack images of formin alone (top panels) and merged with mRFP-ZO-1 (bottom panels) are shown. Note that Dia3 is strongly localized at the contractile ring of the dividing cell. (B) The localization of Dia3 at the contractile ring is dependent on Rho binding. Embryos expressing 3×GFP-Dia1 WT or V187D (Rho-binding mutant; green) and mCherry-farnesyl (membrane probe; magenta) were imaged. Because the expression of Dia3 causes membrane deformation phenotypes (see A and Supplemental Figure S6), Dia3 was expressed at a lower level in these images. Note that the Dia3 V187D mutant cannot localize at the contractile ring. Scale bars: 10 µm.
	FIGURE 2:. Localization of 3×GFP-tagged formin proteins in X. laevis gastrula-stage epithelium. Embryos expressing 3×GFP-tagged formin (green) and mCherry-farnesyl (membrane marker; magenta) were live imaged using confocal microscopy. Stacked images of formin alone (left panels) and merged with membrane (right panels) are shown. Note that Dia1, Dia2, Fhod1, and Fhod3 are strongly localized at cell–cell junctions. Scale bar: 10 µm.
	FIGURE 3:. Comparison of the localization of junctional formins with TJ and AJ proteins. (A–D) 3×GFP-tagged Dia1 (A), Dia2 (B), Fhod1 (C), and Fhod3 (D) (green) were expressed together with TagBFP-ZO-1 (TJ marker; blue) and PLEKHA7-mCherry (AJ marker; red) in gastrula-stage X. laevis embryos and imaged by confocal microscopy. Top views (left) and averaged side views (right) of cell–cell junctions (50 pixels × 16 junctions) are shown. Scale bars: 10 µm (left); 2 µm (right). (E) Graphs indicate intensity profiles of formins (green solid line), ZO-1 (blue dotted line), and PLEKHA7 (red dotted line). Note that the intensity profiles of Dia1 and Dia2 are very similar to that of ZO-1 and that Fhod1 and Fhod3 have a peak at AJs as well as signal basal to the AJ. Error bars (vertical lines) indicate SD.
	FIGURE 4:. Ala-267 is necessary for junctional localization of Dia1. (A) Domain structure and mutants of Dia1. G, GTPase-binding domain; DID, Diaphanous inhibitory domain; DD, dimerization domain; FH, formin homology; DAD, Diaphanous autoregulatory domain. (B) Embryos expressing Dia1 WT or mutants (green) and mCherry-farnesyl (membrane probe; magenta) were observed. Note that the A267D/I842A mutant cannot localize at cell–cell junctions, but the I842A mutant can. Enlargements of cell–cell junctions (white boxes) are shown below. (C) Embryo expressing Dia1 A267D (green) and mCherry-farnesyl (magenta). Note that the cell expressing Dia1 A267D at high level (asterisk) is enlarged, probably due to cytokinesis failure. Scale bars: 10 µm (B); 40 µm (C).
	FIGURE 5:. The DID-DD region is sufficient for junctional localization of Dia1. (A) Fragments of Dia1. The localization of CT was not directly assessed, because CT expression resulted in very large cell size (likely due to a cytokinesis defect) and compromised cell–cell junctions. (B) Embryos expressing mCherry-tagged Dia1 fragments (pseudocolored green) and GFP-farnesyl (membrane probe; pseudocolored magenta) were observed. Note that DID-DD is localized at cell–cell junctions, but DID-DD A267D is not. Both NT and CT cause abnormally large cell size. Scale bars: 10 µm.
	FIGURE 6:. Overexpressed Dia1 DID-DD displaces full-length Dia1 and Dia2 from cell–cell junctions but does not displace Fhod1 and Fhod3. (A) Dia1 DID-DD was mosaically expressed in the 3×GFP-tagged Dia1-, Dia2-, Fhod1-, or Fhod3-expressing embryos. Note that Dia1 and Dia2 were removed from cell–cell junctions (arrowheads) between the DID-DD–expressing cells (see enlarged blue boxes) but not from that of control cells (see enlarged yellow boxes), whereas Fhod1 and Fhod3 were not affected by Dia1 DID-DD expression. (B, C) Dia1 DID-DD was mosaically expressed in Lifeact-GFP–expressing (B) or Vinculin-3×GFP–expressing (C) embryos. Note that localization and intensity of F-actin and Vinculin are not altered in Dia1 DID-DD–expressing cells (asterisks). Scale bars: 20 µm.
	FIGURE 7:. Dia1 and Dia2 are localized at the contractile ring in dividing Dia1 DID-DD–overexpressing cells. (A) Embryos expressing 3×GFP-Dia1 (top panels) or 3×GFP-Dia2 (bottom panels) in all cells and mCherry-Dia1 DID-DD mosaically were live imaged using confocal microscopy. Note that Dia1 and Dia2 are strongly localized at the contractile ring in the Dia1 DID-DD–overexpressing cells (yellow asterisks) but are not localized at the contractile ring in the nonexpressing cells (white asterisks). (B) Embryos expressing 3×GFP-Dia1 WT (top panels) or 3×GFP-Dia1 V175D (bottom panels) in all cells and mCherry-Dia1 DID-DD mosaically were live imaged. Note that Dia1 V175D (Rho-binding mutant) cannot localize at the contractile ring in Dia1 DID-DD–overexpressing cells. Asterisks, daughter cells. Scale bars: 10 µm.
	FIGURE 8:. Dia1 DID-DD–overexpressing cells exhibit cytokinesis defects. (A) Control (left) or mCherry-Dia1 DID-DD (right) embryos expressing GFP-farnesyl (membrane probe) were fixed and stained with anti-GFP (pseudocolored red) and DAPI (cyan). Note that there are binucleate cells (arrows) in Dia1 DID-DD–overexpressing embryos. (B) Live imaging of dividing cells in control (top panels) and Dia1 DID-DD–overexpressing (bottom panels) embryos using Lifeact-GFP (F-actin probe; green) and BFP-ZO-1 (TJ marker; blue). Note that in the Dia1 DID-DD–overexpressing cells, the contractile ring is formed and regresses over time. (C, D) Apical cell-surface area (C) and percentage of binucleate cells (D) in the fixed embryos (n = 171 cells from four embryos [control] and 170 cells from four embryos [Dia1 DID-DD]). (E) Success rate of cytokinesis in live imaging (n = 12 cells from three embryos [control] and 32 cells from three embryos [Dia1 DID-DD]). p values are 0.57 (C; t test), 0.00035 (D; Fisher's exact test), 0.0067 (E; Fisher's exact test). Scale bars: 10 µm.

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