Wiskott-Aldrich syndrome proteins in the nucleus: aWASH with possibilities

Abstract

Actin and proteins that regulate its dynamics or interactions have well-established roles in the cytoplasm where they function as key components of the cytoskeleton to control diverse processes, including cellular infrastructure, cellular motility, cell signaling, and vesicle transport. Recent work has also uncovered roles for actin and its regulatory proteins in the nucleus, primarily in mechanisms governing gene expression. The Wiskott Aldrich Syndrome (WAS) family of proteins, comprising the WASP/N-WASP, SCAR/WAVE, WHAMM/JMY/WHAMY, and WASH subfamilies, function in the cytoplasm where they activate the Arp2/3 complex to form branched actin filaments. WAS proteins are present in the nucleus and have been implicated as transcriptional regulators. We found that Drosophila Wash, in addition to transcriptional effects, is involved in global nuclear architecture. Here we summarize the regulation and function of nuclear WAS proteins, and highlight how our work with Wash expands the possibilities for the functions of these proteins in the nucleus.

Keywords: JMY; WASH; WASP/N-WASP; WAVE/SCAR; WHAMM; Wiskott-Aldrich Syndrome; actin; nuclear architecture; nuclear organization; transcription.

Figure 1.

Schematics of WAS family protein function, domain topology, and activation. (A) The interaction between Arp2/3, actin, and the highly conserved VCA domain of WAS proteins is necessary and sufficient to induce actin polymerization off an existing filament of actin. The WH2 motif binds a G-actin monomer and the CA recruits and changes the conformation of Arp2/3 and interacts with an existing F-actin filament. (B) Topology diagrams of WAS family protein domains for human (H. sapiens), mouse (M. musculus), African clawed frog (X. laevis*), and fruit fly (D. melanogaster). All subfamilies contain a C-terminal VCA domain with class specific N-termini. Drosophila Whamy contains significant differences from both the mammalian WHAMM and JMY subfamilies. Domain Abbreviations: WH1 (WASP homology 1); RBD (Rho binding domain); PP (poly-proline); V/WH2 (Verprolin/WASP homology 2); C (Central/Connector); A (acidic); SHD (Scar homology domain); WHD1 (WASH homology domain 1); WHD2 (WASH homology domain 2); WMD (WHAMM membrane-interacting domain). Scale bar, 100 residues. *X. tropicalis for WHAMM/JMY. (C) Different modes of regulation for WAS family proteins. WASP/N-WASP proteins are auto-inhibited. This inhibition is released upon their direct binding to the Cdc42 small GTPase. WAVE/SCAR proteins interact indirectly with the Rac small GTPase. WAVE/SCAR proteins are trans-inhibited through their constitutive regulation by the WAVE Regulatory Complex (WRC). This inhibition is released upon Rac recruitment by the WRC subunit Sra1. The mechanism of WASH regulation is not yet clear, but appears to be context dependent. WASH proteins are not auto- or trans- inhibited. Nonetheless, WASH has been shown to bind directly to the Rho1 small GTPase, and has also been shown to associate with a WRC-like multiprotein complex (SHRC; Strumpellin, SWIP, CCDC53, FAM21).

Figure 2.

Nuclear roles for WASP/N-WASP, WAVE/SCAR, and JMY. (A) WASP nuclear localization is regulated by canonical NLS and NES import/export. De-convolved fluorescence micrographs of T_H1-skewed WASP^NULL cells rescued with the indicated WASP constructs. Expression from the full-length and ΔVCA constructs is found in both the nucleus and cytoplasm, whereas the ΔNLS is only found in the cytoplasm and the ΔNES2 is only found in the nucleus. (B) Nuclear-localized WASP in T_H1-skewed cells is responsible for expression of the T_H1 regulators IFNG and TBX21. Bar plot graph of gene expression quantitated by RT-qPCR in T_H1 WASP^NULL (UT) and rescued by full-length (FL), ΔNLS (delNLS), ΔNES2 (delNES2), and ΔVCA (delVCA) constructs compared to T_H0 expression levels showing that the T_H1 factors IFNG and TBX21 are not-regulated properly in ΔNLS T_H1 skewed cells. CSF2 is a non-T_H1-specific control. (C) Phosphorylation of N-WASP by the Src kinase, Fyn, leads to enhanced cytoplasmic and decreased nuclear accumulation of N-WASP. Fluorescent micrographs of COS-7 cells expressing ectopic constitutively active Fyn (FynCA) or dominant negative (FynDN) immunostained for anti-Fyn (red), anti-N-WASP (gray/green), and Phalloidin (blue) show that FynCA leads to more cytoplasmic N-WASP, whereas FynDN leads to more nuclear N-WASP. Quantification of N-WASP nuclear versus cytoplasmic N-WASP in COS-7 cells expressing FynCA and FynDN. (D) N-WASP can polymerize actin from nuclear lysates. Pyrene actin assay showing that a GST fusion to the N-WASP VCA domain can polymerize actin, however, it is unable to do so in the presence of the actin polymerization inhibitors cytochalasin D and Latrunculin A. (E) Wave1 is present in mouse C2C12 nuclei after transplantation into the germinal vesicle of Xenopus oocytes. Mouse somatic C2C12 nuclei were transplanted into Xenopus oocytes overexpressing HA-NLS-WAVE1. Immunofluorescence staining for anti-HA (WAVE1) showed WAVE1 co-localizing with active RNA polymerase II in these transplanted nuclei 24 hrs post-nuclear transfer. (F) Nuclear Wave1 is required for transcriptional reprogramming in Xenopus oocytes. Transcriptional activation of the embryonic gene, Oct4, is inhibited in the presence of antibodies against WAVE1 in transplanted somatic nuclei, as measured by QPCR. Expression of the housekeeping gene, Gapdh, was unaffected by the presence of α-WAVE1 antibodies. (G) Nuclear Wave1 is required for hox gene expression. Heat map showing the down-regulation of hox gene expression in WAVE1-morpholino (MO) injected embryos relative to control. (H) JMY and p300 function together to regulate p53-dependent transcription. Bax promoter-luciferase reporter assays were used to measure p53-dependent transcriptional activity. Co-expression of p53 with increasing levels of JMY resulted in a titratable increase in p53 activity that was further enhanced by the expression of p300. (I) The JMY NLS is required for damage-induced nuclear accumulation. Cells expressing GFP-JMY, but not a GFP-JMY truncation mutant removing its actin binding and NLS (GFP-JMYΔWWWCA), show accumulation of GFP signal in the nucleus in response to UV irradiation induced DNA damage. (J) Actin competes with Impα/β for binding to JMY. Actin and importins both bind to a C-terminal fragment of JMY containing tandem WH2 (WWW) motifs and a nested NLS sequence (WWWCA). Quantification of GST pulldowns assaying Impα/β binding to the GST-WWWCA in the presence of increasing concentrations of actin monomers. Permissions. (A–B) Reprinted from Sadhukan et al. (2014).³⁶ The Journal of Immunology 193:150-60. (C) Reprinted with permission from Suetsugu & Takenawa, J. Biol. Chem. 278(43):42515-23.³⁷ © The American Society for Biochemistry and Molecular Biology. Reproduced by permission of The American Society for Biochemistry and Molecular Biology. Permission to reuse must be obtained from the rightsholder. (D) © Macmillan Publishers Ltd: Nature Cell Biology. Reproduced by permission of Macmillan Publishers Ltd: Nature Cell Biology. Permission to reuse must be obtained from the rightsholder. Wu et al., Nat. Cell Biol. 8(7):756–63.⁴⁰ (E–G) © AAAS. Reproduced by permission of AAAS. Permission to reuse must be obtained from the rightsholder. From Miyamoto et al. (2013) Science 341(6149):1002-5.⁴⁵ (H) © Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from the rightsholder. Reprinted from Molecular Cell, Volume 4(3), Shikama et al., A Novel Cofactor for p300 that Regulates the p53 Response, pp. 365-376.⁴⁶ (I-J) © American Society for Cell Biology. Reproduced by permission of American Society for Cell Biology. Permission to reuse must be obtained from the rightsholder. From Actin binding to WH2 domains regulates nuclear import of the multifunctional actin regulator JMY, Zuchero et al., Mol. Biol. Cell 23:853, 2012.⁴⁷

Figure 3.

Nuclear localized WASH family proteins function as regulators of nuclear morphology and as transcription factors. (A) Wash accumulates in the nucleus of Drosophila cells in a temporally and spatially specific manner. Confocal projection of stage 7 embryos immunostained with anti-Wash show nuclear localization in specific mitotic domains (arrows) while remaining cytoplasmic in others (arrowheads) (left). Confocal projections of the nuclei of the salivary glands of 3^rd instar larvae immunostained with anti-Wash shows nuclear enrichment (right). (B) WASH exhibits different sub-cellular localizations in different sub-populations of haematopoietic stem cells. Micrographs (left) and western blot analysis of lysates (right) of WASH expression in long-term haematopoietic stem cells (LT-HSC; nuclear), short-term haematopoietic stem cells (ST-HSC; nuclear and cytoplasmic) and multipotent progenitor cells (MPP; cytoplasmic). Micrographs are co-stained with PI for nuclear visualization and DIC views are shown (left). (C) Purification of the Drosophila TRF2 complex showing the presence of Wash and its SHRC. Co-immunoprecipitation by TRF2 or DREF monoclonal antibodies purifies a complex containing Wash (p63), SWIP (p116) and Strumpellin (p118), in addition to ISWI, DREF, TRF2, and tubulin. (D) WASH knockout in LT-HSCs leads to reduced c-Myc expression, as well as reduced expression of its transcriptional targets. Bar plot graphs of gene expression levels measured by qPCR showing significantly decreased expression of c-Myc, and its target genes Tlr4, Tcerg1, Bub1b, and Ilf3, in cell-sorted WASH KO LT-HSCs. (E–G) wash mutant salivary glands have altered nuclear morphology compared to wildtype. Confocal projections of wildtype vs. wash mutant nuclei immunostained with anti-Lamin antibody shows crinkled, non-spherical wash nuclei (E). Micrographs of wildtype versus wash mutant salivary gland polytene chromosomes show misalignment and improper banding, as well as extremely fragile chromosomes (F). Three-Dimensional reconstruction of wildtype vs. wash mutant salivary gland nuclei hybridized with chromosome-specific paints (X chromosome: yellow, 2^nd chromosome: green, 3rd chromosome: red) showing less compact chromosome territories in wash mutant nuclei (G, left). Micrographs of wildtype verses wash mutant salivary gland nuclei immunostained with anti-HP1 (green; heterochromatin; left), anti-Coilin (green; Cajal bodies; middle), anti-Mtor (red; nuclear envelope protein; middle), anti-Fibrillarin (green; nucleolus; right), anti-MOF (red; X-chromosome; right) showing disrupted nuclear sub-compartments in wash mutant nuclei (G, right). (H) Wash increases chromatin accessibility in heterochromatin regions. Distribution of M.SssI-based chromatin accessibility in control RNAi and wash RNAi treated Drosophila S2 cells, showing increased accessibility in wash knockdown with no affect on transcription start site (TSS) chromatin regions. (I) Wash interacts with B-type Lamin at the nuclear periphery. Duolink proximity ligation assay in Drosophila salivary glands expressing GFP-Lamin using anti-Wash and anti-GFP antibodies shows amplification of Duolink signal at the nuclear periphery. Amplification signal is only observed when the 2 antibodies examined are within 30 nm. Permissions. (A, right) and (E–I) Reprinted from Current Biology, 25(6), Verboon et al., Wash Interacts with Lamin and Affects Global Nuclear Organization, pp. 804-810.⁵⁴ © Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from the rightsholder. (B, D) Reprinted with permission from: © Rockefeller University Press. Reproduced by permission of Rockefeller University Press. Permission to reuse must be obtained from the rightsholder. Reprinted from Xia et al. Journal of Experimental Medicine. 211:2119-2134. doi:10.1084/jem.20140169.⁵³ (C) © Macmillan Publishers Ltd: Nature. Reproduced by permission of Macmillan Publishers Ltd: Nature. Permission to reuse must be obtained from the rightsholder. Hochheimer et al., Nature 420:439–45.⁵⁸