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Mol Biol Cell
2019 Jan 01;301:82-95. doi: 10.1091/mbc.E18-02-0133.
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Comprehensive analysis of formin localization in Xenopus epithelial cells.
Higashi T
,
Stephenson RE
,
Miller AL
.
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Reorganization of the actin cytoskeleton is crucial for cellular processes, including cytokinesis and cell-cell junction remodeling. Formins are conserved processive actin-polymerizing machines that regulate actin dynamics by nucleating, elongating, and bundling linear actin filaments. Because the formin family is large, with at least 15 members in vertebrates, there have not been any comprehensive studies examining formin localization and function within a common cell type. Here, we characterized the localization of all 15 formins in epithelial cells of Xenopus laevis gastrula-stage embryos. Dia1 and Dia2 localized to tight junctions, while Fhod1 and Fhod3 localized to adherens junctions. Only Dia3 strongly localized at the cytokinetic contractile ring. The Diaphanous inhibitory domain-dimerization domain (DID-DD) region of Dia1 was sufficient for Dia1 localization, and overexpression of a Dia1 DID-DD fragment competitively removed Dia1 and Dia2 from cell-cell junctions. In Dia1 DID-DD-overexpressing cells, Dia1 and Dia2 were mislocalized to the contractile ring, and cells exhibited increased cytokinesis failure. This work provides a comprehensive analysis of the localization of all 15 vertebrate formins in epithelial cells and suggests that misregulated formin localization results in epithelial cytokinesis failure.
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.
ALFERT,
The development of polysomaty in rat liver.
1958, Pubmed
ALFERT,
The development of polysomaty in rat liver.
1958,
Pubmed
Acharya,
Mammalian Diaphanous 1 Mediates a Pathway for E-cadherin to Stabilize Epithelial Barriers through Junctional Contractility.
2017,
Pubmed
Aijaz,
Binding of GEF-H1 to the tight junction-associated adaptor cingulin results in inhibition of Rho signaling and G1/S phase transition.
2005,
Pubmed
Al Haj,
Distribution of formins in cardiac muscle: FHOD1 is a component of intercalated discs and costameres.
2015,
Pubmed
Alberts,
Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein beta subunits and the yeast response regulator protein Skn7.
1998,
Pubmed
Alberts,
Diaphanous-related Formin homology proteins.
2002,
Pubmed
Alberts,
Identification of a carboxyl-terminal diaphanous-related formin homology protein autoregulatory domain.
2001,
Pubmed
Anderson,
Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.
1988,
Pubmed
Arnold,
Rho GTPases and actomyosin: Partners in regulating epithelial cell-cell junction structure and function.
2017,
Pubmed
Bartolini,
Formins and microtubules.
2010,
Pubmed
Bohnert,
Formin-based control of the actin cytoskeleton during cytokinesis.
2013,
Pubmed
Braga,
The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts.
1997,
Pubmed
Breitsprecher,
Formins at a glance.
2013,
Pubmed
Carramusa,
Mammalian diaphanous-related formin Dia1 controls the organization of E-cadherin-mediated cell-cell junctions.
2007,
Pubmed
Castrillon,
Diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene.
1994,
Pubmed
Chalkia,
Origins and evolution of the formin multigene family that is involved in the formation of actin filaments.
2008,
Pubmed
Chang,
cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.
1997,
Pubmed
Cheng,
Aurora B regulates formin mDia3 in achieving metaphase chromosome alignment.
2011,
Pubmed
Fujiwara,
Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells.
2005,
Pubmed
Grikscheit,
Formins at the Junction.
2016,
Pubmed
Grikscheit,
Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1.
2015,
Pubmed
Guillemot,
Paracingulin regulates the activity of Rac1 and RhoA GTPases by recruiting Tiam1 and GEF-H1 to epithelial junctions.
2008,
Pubmed
Hara,
Cell Boundary Elongation by Non-autonomous Contractility in Cell Oscillation.
2016,
Pubmed
Hartsock,
Adherens and tight junctions: structure, function and connections to the actin cytoskeleton.
2008,
Pubmed
Henty-Ridilla,
Accelerated actin filament polymerization from microtubule plus ends.
2016,
Pubmed
Higashi,
Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis.
2016,
Pubmed
Higashi,
Flightless-I (Fli-I) regulates the actin assembly activity of diaphanous-related formins (DRFs) Daam1 and mDia1 in cooperation with active Rho GTPase.
2010,
Pubmed
Higgs,
Phylogenetic analysis of the formin homology 2 domain.
2005,
Pubmed
Hoffman,
Giant and binucleate trophoblast cells of mammals.
1993,
Pubmed
Homem,
Exploring the roles of diaphanous and enabled activity in shaping the balance between filopodia and lamellipodia.
2009,
Pubmed
Homem,
Diaphanous regulates myosin and adherens junctions to control cell contractility and protrusive behavior during morphogenesis.
2008,
Pubmed
Imamura,
Bni1p and Bnr1p: downstream targets of the Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae.
1997,
Pubmed
Ishizaki,
The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.
1996,
Pubmed
Kishi,
Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI).
1993,
Pubmed
Kohno,
Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae.
1996,
Pubmed
Kovar,
The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin.
2003,
Pubmed
Krug,
Tight junction, selective permeability, and related diseases.
2014,
Pubmed
Kühn,
Formins as effector proteins of Rho GTPases.
2014,
Pubmed
Lammers,
The regulation of mDia1 by autoinhibition and its release by Rho*GTP.
2005,
Pubmed
Larkin,
Clustal W and Clustal X version 2.0.
2007,
Pubmed
Lecuit,
E-cadherin junctions as active mechanical integrators in tissue dynamics.
2015,
Pubmed
Levayer,
Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis.
2011,
Pubmed
Li,
Dissecting requirements for auto-inhibition of actin nucleation by the formin, mDia1.
2005,
Pubmed
Li,
The mouse Formin mDia1 is a potent actin nucleation factor regulated by autoinhibition.
2003,
Pubmed
Mabuchi,
A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs.
1993,
Pubmed
,
Echinobase
Margall-Ducos,
Liver tetraploidization is controlled by a new process of incomplete cytokinesis.
2007,
Pubmed
Matsui,
Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.
1996,
Pubmed
Meng,
Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts.
2008,
Pubmed
Miller,
The contractile ring.
2011,
Pubmed
Nekrasova,
Desmosome assembly and dynamics.
2013,
Pubmed
Nezami,
Structure of the autoinhibitory switch in formin mDia1.
2006,
Pubmed
Nusrat,
Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia.
1995,
Pubmed
Otomo,
Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain.
2005,
Pubmed
Otomo,
Structural basis of Rho GTPase-mediated activation of the formin mDia1.
2005,
Pubmed
Perrière,
WWW-query: an on-line retrieval system for biological sequence banks.
1996,
Pubmed
Pruyne,
Role of formins in actin assembly: nucleation and barbed-end association.
2002,
Pubmed
Pulimeno,
PLEKHA7 is an adherens junction protein with a tissue distribution and subcellular localization distinct from ZO-1 and E-cadherin.
2010,
Pubmed
Rao,
Formin-mediated actin polymerization at cell-cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair.
2016,
Pubmed
Ratheesh,
Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens.
2012,
Pubmed
Reyes,
Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin.
2014,
Pubmed
Rivero,
A comparative sequence analysis reveals a common GBD/FH3-FH1-FH2-DAD architecture in formins from Dictyostelium, fungi and metazoa.
2005,
Pubmed
Rose,
Structural and mechanistic insights into the interaction between Rho and mammalian Dia.
2005,
Pubmed
Ryu,
Regulation of cell-cell adhesion by Abi/Diaphanous complexes.
2009,
Pubmed
Sahai,
ROCK and Dia have opposing effects on adherens junctions downstream of Rho.
2002,
Pubmed
Saitou,
The neighbor-joining method: a new method for reconstructing phylogenetic trees.
1987,
Pubmed
Schneider,
NIH Image to ImageJ: 25 years of image analysis.
2012,
Pubmed
Sedzinski,
Emergence of an Apical Epithelial Cell Surface In Vivo.
2016,
Pubmed
Severson,
A Formin Homology protein and a profilin are required for cytokinesis and Arp2/3-independent assembly of cortical microfilaments in C. elegans.
2002,
Pubmed
Spadaro,
Tension-Dependent Stretching Activates ZO-1 to Control the Junctional Localization of Its Interactors.
2017,
Pubmed
Staus,
Enhancement of mDia2 activity by Rho-kinase-dependent phosphorylation of the diaphanous autoregulatory domain.
2011,
Pubmed
Takeichi,
Dynamic contacts: rearranging adherens junctions to drive epithelial remodelling.
2014,
Pubmed
Takeya,
The mammalian formin FHOD1 is activated through phosphorylation by ROCK and mediates thrombin-induced stress fibre formation in endothelial cells.
2008,
Pubmed
Terry,
Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis.
2011,
Pubmed
Tolliday,
Rho1 directs formin-mediated actin ring assembly during budding yeast cytokinesis.
2002,
Pubmed
Van Itallie,
Architecture of tight junctions and principles of molecular composition.
2014,
Pubmed
Watanabe,
mDia2 induces the actin scaffold for the contractile ring and stabilizes its position during cytokinesis in NIH 3T3 cells.
2008,
Pubmed
Watanabe,
Cooperation between mDia1 and ROCK in Rho-induced actin reorganization.
1999,
Pubmed
Watanabe,
Rho and anillin-dependent control of mDia2 localization and function in cytokinesis.
2010,
Pubmed
Watanabe,
Loss of a Rho-regulated actin nucleator, mDia2, impairs cytokinesis during mouse fetal erythropoiesis.
2013,
Pubmed
Watanabe,
p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin.
1997,
Pubmed
Wu,
Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast.
2006,
Pubmed
Xu,
Crystal structures of a Formin Homology-2 domain reveal a tethered dimer architecture.
2004,
Pubmed
Yasuda,
Cdc42 and mDia3 regulate microtubule attachment to kinetochores.
2004,
Pubmed
Zhou,
Formin-1 protein associates with microtubules through a peptide domain encoded by exon-2.
2006,
Pubmed
Zihni,
Tight junctions: from simple barriers to multifunctional molecular gates.
2016,
Pubmed