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Snapshot: Formins Christian Baarlink, Dominique Brandt, and Robert Grosse University of Marburg, Marburg 35032, Germany

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SnapShot: Formins Christian Baarlink, Dominique Brandt, and Robert Grosse University of Marburg, Marburg 35032, Germany Formin Regulators Localization Cellular Function Disease Association DIAPH1/DIA1 RhoA, RhoC Cell cortex, Polarized cell migration, microtubule stabilization, Autosomal-dominant nonsyndromic deafness (DFNA1), myeloproliferative (mDia1) phagocytic cup, phagocytosis, axon elongation defects, defects in T lymphocyte traffi cking and proliferation, tumor cell mitotic spindle invasion, defects in natural killer lymphocyte function DIAPH2 Cdc42 Kinetochore Stable microtubule attachment to kinetochore for Premature ovarian failure (mDia3) chromosome alignment DIAPH3 Rif, Cdc42, Filopodia, Filopodia formation, removing the nucleus from Increased chromosomal deletion of gene locus in metastatic tumors (mDia2) Rac, RhoB, endosomes erythroblast, endosome motility, microtubule DIP* stabilization FMNL1 (FRLα) Cdc42 Cell cortex, Phagocytosis, T cell polarity Overexpression is linked to leukemia and non-Hodgkin lymphoma microtubule- organizing center FMNL2/FRL3/ RhoC ND Cell motility Upregulated in metastatic colorectal cancer, chromosomal deletion is FHOD2 associated with mental retardation FMNL3/FRL2 Constituently Stress fi bers ND ND active DAAM1 Dishevelled Cell cortex Planar cell polarity ND DAAM2 ND ND ND Overexpressed in schizophrenia patients Human (Mouse) FHOD1 ROCK Stress fi bers Cell motility FHOD3 ND Nestin, sarcomere Organizing sarcomeres in striated muscle cells Single-nucleotide polymorphisms associated with type 1 diabetes FMN1 Spire* Adherens junctions, Forming intercellular junctions Oligodactylism and partial renal agenesis microtubules FMN2 Spire* Chromosomes Asymmetric spindle positioning in meiotic Female infertility, birth defects oocytes, cytokinesis Delphilin ND Postsynapse Synaptic transmission Defects in Induction of long-term depression and optokinetic adaptation INF1/FHDC1 ND Microtubules Microtubule stabilization ND INF2 Cdc42 ER, apical zone, slit ER structure, cell polarity, transcytosis, and Mutated in patients with focal segmental glomerulosclerosis diaphragm lumen formation Bni1 Rho3, Rho1, Bud tip, bud neck Cytokinesis, cell polarity, actin cables *Negative regulation Fus3, Bud6, ND, not determined Prk1 Budding Bnr1 Rho3, Rho4, Bud neck Cytokinesis, cell polarity, actin cables Bud14* Yeast Cdc12 ND Contractile ring Cytokinesis For3 Rho3, Cdc42, Cell tip, polarisome Cell polarity, actin cables Bud6 Fission Fus1 ND Projection tip Mating, cell wall degradation, membrane fusion 172 Cell 142, July 9, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.06.030 See online version for legend and references. SnapShot: Formins Christian Baarlink, Dominique Brandt, and Robert Grosse University of Marburg, Marburg 35032, Germany Many fundamental processes of eukaryotic cells, such as cell motility, cytokinesis, polarity, and morphogenesis, require reorganization of the plasma membrane. The Rho family of GTPases orchestrates these changes in cellular shape by triggering the rearrangement of the cytoskeleton. Specifically, the disassembly and assembly of microtubule and actin filaments leads to the production of cellular invaginations or protrusions required for the given physiological process. The largest family of Rho-GTPase effectors is represented by the formins, which are conserved across plants, animals, and fungi. Formins can nucleate the polymerization of actin filaments, accelerate the elongation of actin filaments, and stabilize microtubules. Therefore, formins play an essential role in remodeling the cytoskeleton to drive changes in cell shape. This SnapShot describes the formins in humans (with a subset of their mouse counterparts) and yeast, including diseases associated with mutations or defects in these proteins. In addition, the SnapShot illustrates major signaling pathways in the cell that lead to the activation of formins and thus the reorganization of the cytoskeleton. Formin Activation In humans, formins are represented by 15 different family members that can be divided into 7 subgroups. In contrast, budding and fission yeasts contain 2 and 3 formins, respectively. The signature domain of formins is the highly conserved formin homology 2 (FH2) domain, which is often required and sufficient to promote actin assembly. The FH1 domain, which precedes the catalytic FH2 domain, binds profilin-actin to accelerate elongation of actin filaments. In the prototypic formin, the N-terminal FH3 domain forms autoinhibitory interactions with the C-terminal DAD region (Dia autoregulatory domain). Activated Rho-GTPases help to relieve this inhibition by interacting with the GTPase- binding domain (GBD). Once activated, the exposed catalytic FH2 domain forms an antiparallel dimer to promote processive incorporation of actin monomers into the barbed or “+” end of the growing actin filament, which is therefore called a “leaky cap.” Formin Signaling Pathways Extracellular cues can trigger rearrangements of the cytoskeleton by stimulating receptors on the cell surface, such as G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and integrins. For example, the mitogen lysophosphatidic acid (LPA) activates specific GPCRs, which subsequently switch on various GEFs (guanine exchange factors), such as LARG (leukemia-associated Rho-GEF). GEFs in turn regulate the activity of RhoA-GTPases (yellow ovals), which bind and activate specific formins (white ovals). Activated LARG switches on RhoA, which can bind several formins, including mDia1 and DAAM1. FHOD1 and mDia1 regulate bleb-associated cell motility involving ROCK (Rho-associated kinase) and myosin. Myosin is critical for the retraction or deflation of the bleb, and mDia1 appears to regulate myosin through feedback mechanisms involving activation of LARG and RhoA/ROCK signaling. DAAM1 (dishevelled-associated activator of morphogenesis) plays a key role in the noncanonical Wnt signaling pathway for planar cell polarity; DAAM1 interacts with Dishevelled (Dvl) downstream of frizzled heptahelical receptors. The Rho-GTPases Rif and Cdc42 activate the mouse formin mDia2, which stimulates the production of filopodia (slender cytoplasmic projections at the leading edge of migrating cells). ABI1 (Abl interactor 1) promotes the formation of filopodia by recruiting mDia2 to the filopodia tips. In contrast, DIP (Dia-interacting protein) negatively regulates filopodia formation by inhibiting mDia2. Cdc42 can also activate FRLα to catalyze actin assembly during the formation of phagocytic cups when Fc-γ (fragment, crystallizable γ) stimulates phagocytosis. In addition, IQGAP1 (IQ motif containing GTPase-activating protein 1) recruits mDia1 to phagocytic cups when CR3 (complement receptor 3) triggers phagocytosis. Other GEFs switch on the Rho-GTPases RhoB and RhoD, which activate mDia2 and hDia2C (a DIAPH2 splice variant), respectively. These formins cooperate with Src tyrosine kinases to promote the motility of endosomes. In addition to their role in actin polymerization, some formins (e.g., mDia1, mDia2, and INF1) are implicated in assembling, orienting, and stabilizing microtubules. For example, mDia binds to EB1 and APC (adenomatous polyposis coli) to promote microtubule stabilization at the leading edge of the cell during polarized cell migration (i.e., at the lamella). The formins FMN1 and mDia1 also regulate cell polarity. FMN1 associates with α-catenin to promote actin polymerization, which is required for forming and stabilizing adherens junctions. ABI1 and mDia1 colocalize with β-catenin at cell-cell junctions. Another important consequence of actin assembly through formins is the transcriptional activation of the serum response factor (SRF) coactivator MAL (MRTF-A). Binding to actin monomers inhibits MAL. Thus, actin polymerization relieves this inhibition, allowing MAL to localize and stay in the cell nucleus. Once in the nucleus, MAL promotes the gene expression of many factors associated with the cytoskeleton, including actin, myosin heavy and light chain 9, tropomyosin 1, vinculin, β1-integrin, and Cys-rich protein 61. REFERENCES Brown, E.J., Schlöndorff, J.S., Becker, D.J., Tsukaguchi, H., Uscinski, A.L., Higgs, H.N., Henderson, J.M., Pollak, M.R., and Tonna, S.J. (2010). Mutations in the formin gene INF2 cause focal segmental glomerulosclerosis. Nat. Genet. 42, 72–76. Chesarone, M.A., DuPage, A.G., and Goode, B.L. (2010). Unleashing formins to remodel the actin and microtubule cytoskeletons. Nat. Rev. Mol. Cell Biol. 11, 62–74. Kitzing, T.M., Wang, Y., Pertz, O., Copeland, J.W., and Grosse, R. (2010). Formin-like 2 drives amoeboid invasive cell motility downstream of RhoC. Oncogene 29, 2441–2448. Madrid, R., Aranda, J.F., Rodríguez-Fraticelli, A.E., Ventimiglia, L., Andrés-Delgado, L., Shehata, M., Fanayan, S., Shahheydari, H., Gómez, S., Jiménez, A., et al. (2010). The formin INF2 regulates basolateral-to-apical transcytosis and lumen formation in association with Cdc42 and MAL2. Dev. Cell 18, 814–827. Miralles, F., Posern, G., Zaromytidou, A.I., and Treisman, R. (2003). Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113, 329–342. Palazzo, A.F., Cook, T.A., Alberts, A.S., and Gundersen, G.G. (2001). mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat. Cell Biol. 3, 723–729. Paul, A.S., and Pollard, T.D. (2009). Review of the mechanism of processive actin filament elongation by formins. Cell Motil. Cytoskeleton 66, 606–617. Pruyne, D., Evangelista, M., Yang, C., Bi, E., Zigmond, S., Bretscher, A., and Boone, C. (2002). Role of formins in actin assembly: nucleation and barbed-end association. Science 297, 612–615. Sagot, I., Rodal, A.A., Moseley, J., Goode, B.L., and Pellman, D. (2002). An actin nucleation mechanism mediated by Bni1 and profilin. Nat. Cell Biol. 4, 626–631. Watanabe, N., Madaule, P., Reid, T., Ishizaki, T., Watanabe, G., Kakizuka, A., Saito, Y., Nakao, K., Jockusch, B.M., and Narumiya, S. (1997). p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J. 16, 3044–3056. 172.e1 Cell 142, July 9, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.06.030.
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  • Supplemental Figure Legends

    Supplemental Figure Legends

    Supplemental Figure Legends: Supplemental Figure 1. Disruption of DIAPH1 gene expression and example of ingressions found in DIAPH1-deficient cultures. A) Western blot analysis of HaCaT cells transduced with non-targeting (NT) and DIAPH1-directed (Dia1) shRNA via lentivirus, cultured in low or high calcium media for 20hrs. Relative abundance indicated with respect to control samples. B) Western blot analysis of control cells (CTL) and those carrying a disrupted DIAPH1 locus (CRISPR) at two different passages. Relative abundance to controls is indicated. C) NGS analysis of genomic DNA flanking CRISPR-editing site and variant frequency. D) Example of DIAPH1-deficient tissue growth into the underlying collagen substrate. Hematoxylin and eosin stain of cross- sectioned, paraffin-embedded tissue fixed 10 days after raising to an air-liquid interface. Scale bar= 20 microns. Yellow arrow indicates basal keratinocyte layer. Red arrows indicate distinct ingressions. Supplemental Figure 2. Organotypic cultures treated with shRNA, ectopic mDia1 constructs and ancillary RNA-seq data. A) Hematoxylin and eosin (H&E) staining and keratin-10 (KRT10) staining of organotypic cultures transduced with control or Dia1- targeted shRNA. Counterstained with Hoechst for DNA. Dotted line indicates keratinocyte/collagen boundary. Scale bars = 20μm. B) Map of mDia1 construct introduced to CRISPR-edited Dia1 knockdown (Dia1KD) cells via lentivirus. Numerals are referenced in subsequent panels. FL= full length. C and D) Western blot analysis of ectopic constructs blotted with anti-DIAPH1 (C) or anti-Cherry (D). Residual Scarlet signal indicated by arrowhead. Irrelevant lanes of western blot lanes were dimmed for clarity. E) RNA-seq analysis of DIAPH1 transcripts in CRISPR-edited Dia1KD cells.