FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2951532637> ?p ?o ?g. }

• W2951532637 endingPage "3231" @default.
• W2951532637 startingPage "3212" @default.
• W2951532637 abstract "Article2 November 2017free access Source DataTransparent process Initiation of DNA replication requires actin dynamics and formin activity Nikolaos Parisis orcid.org/0000-0002-5706-0122 IGMM, CNRS, Univ. Montpellier, Montpellier, France Laboratory of Functional Proteomics, INRA, Montpellier, France Search for more papers by this author Liliana Krasinska IGMM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author Bethany Harker IGMM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author Serge Urbach Functional Proteomics Platform (FPP), Institute of Functional Genomics (IGF), CNRS UMR 5203, INSERM U661, Montpellier, France Search for more papers by this author Michel Rossignol Laboratory of Functional Proteomics, INRA, Montpellier, France Search for more papers by this author Alain Camasses IGMM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author James Dewar Vanderbilt University, Nashville, TN, USA Search for more papers by this author Nathalie Morin CRBM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author Daniel Fisher Corresponding Author [email protected] orcid.org/0000-0002-0822-3482 IGMM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author Nikolaos Parisis orcid.org/0000-0002-5706-0122 IGMM, CNRS, Univ. Montpellier, Montpellier, France Laboratory of Functional Proteomics, INRA, Montpellier, France Search for more papers by this author Liliana Krasinska IGMM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author Bethany Harker IGMM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author Serge Urbach Functional Proteomics Platform (FPP), Institute of Functional Genomics (IGF), CNRS UMR 5203, INSERM U661, Montpellier, France Search for more papers by this author Michel Rossignol Laboratory of Functional Proteomics, INRA, Montpellier, France Search for more papers by this author Alain Camasses IGMM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author James Dewar Vanderbilt University, Nashville, TN, USA Search for more papers by this author Nathalie Morin CRBM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author Daniel Fisher Corresponding Author [email protected] orcid.org/0000-0002-0822-3482 IGMM, CNRS, Univ. Montpellier, Montpellier, France Search for more papers by this author Author Information Nikolaos Parisis1,2,†,‡, Liliana Krasinska1,‡, Bethany Harker1,†, Serge Urbach3, Michel Rossignol2, Alain Camasses1, James Dewar4, Nathalie Morin5 and Daniel Fisher *,1 1IGMM, CNRS, Univ. Montpellier, Montpellier, France 2Laboratory of Functional Proteomics, INRA, Montpellier, France 3Functional Proteomics Platform (FPP), Institute of Functional Genomics (IGF), CNRS UMR 5203, INSERM U661, Montpellier, France 4Vanderbilt University, Nashville, TN, USA 5CRBM, CNRS, Univ. Montpellier, Montpellier, France †Present address: Institut Jacques Monod, CNRS UMR7592, University Paris Diderot, Paris, France †Present address: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +33 434 359 964; E-mail: [email protected] EMBO J (2017)36:3212-3231https://doi.org/10.15252/embj.201796585 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP–importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. Synopsis Regulation of actin dynamics affects both the initiation and elongation steps of DNA replication in human cells and Xenopus egg extracts, through various mechanisms including nuclear transport and chromatin loading of replication factors. Nuclear actin dynamics are required for DNA replication in embryonic and somatic cell systems. Disrupting actin dynamics or inhibiting formin proteins blocks nuclear transport. Nuclear actin interactions with RanGTP–importin complexes control cargo release. Nuclear formin activity promotes chromatin localisation of replication factors. Introduction Mounting evidence suggests that nuclear actin is an important regulator of transcription in mammalian cells (Huet et al, 2012). Monomeric actin binds RNA polymerase complexes (Rando et al, 2002) and promotes transcription by all three RNA polymerases (Hofmann et al, 2004; Hu et al, 2004; Philimonenko et al, 2004). In epithelial cells, loss of nuclear actin triggers quiescence by disrupting binding of RNA polymerases to their transcription sites (Spencer et al, 2011). Nuclear actin is also involved in co-repressor eviction from promoters (Huang et al, 2011), and it can bind to gene regulatory regions (Miyamoto et al, 2011, 2013). The most studied transcriptional roles of nuclear actin are in the serum response, which is regulated by dynamics of actin nuclear transport and polymerisation. The involvement of nuclear actin dynamics in serum response factor (SRF)-dependent transcription is complex and remains incompletely understood. Nuclear actin levels are generally low and are regulated by active transport of monomers between the nucleus and cytoplasm (Stüven et al, 2003; Dopie et al, 2012), and by polymerisation (Vartiainen et al, 2007). Monomeric actin promotes export of the SRF cofactor MAL/MRTF, extinguishing SRF (Vartiainen et al, 2007). Yet both nuclear actin polymerisation, triggered by nuclear formin mDia2 (Baarlink et al, 2013), and depolymerisation, brought about via Met44 oxidation by MICAL-2 (Lundquist et al, 2014), can induce SRF-dependent transcription by depleting nuclear monomeric actin. The form of nuclear actin remains poorly characterised due to difficulties in staining nuclear actin with phalloidin (Grosse & Vartiainen, 2013) and the large amounts of actin in the cytoplasm. Polymeric nuclear actin is observed in several pathologies (de Lanerolle, 2012) and can be induced by various manipulations, including heat shock and DMSO treatment (Sanger et al, 1980; Iida et al, 1986); increasing nuclear actin concentrations (Stüven et al, 2003; Kalendová et al, 2014); activating nuclear mDia2 (Baarlink et al, 2013); overexpressing NLS-tagged IQGAP1 (Johnson et al, 2013) or supervillin (Serebryannyy et al, 2016); or knockdown of MICAL-2 (Lundquist et al, 2014). In specific settings, like the giant quiescent nuclei of amphibian oocytes, a filamentous actin network has scaffolding functions and links nuclear pore complexes to the nuclear interior (Clark & Rosenbaum, 1979; Gounon & Karsenti, 1981; Kiseleva et al, 2004; Feric & Brangwynne, 2013). In somatic cells, sub-populations of nuclear actin have distinct mobilities, suggesting existence of polymeric forms (McDonald et al, 2006; Dopie et al, 2012), and several regulators of actin polymerisation have been found in nuclei (Wu et al, 2006; Yoo et al, 2007; Khoudoli et al, 2008; Obrdlik & Percipalle, 2011; Miyamoto et al, 2013; Dopie et al, 2015). However, dynamic nuclear actin polymerisation has only been described upon serum stimulation of mouse fibroblasts (Baarlink et al, 2013). While roles of nuclear actin dynamics in transcription are well established, less is known about its involvement in other nuclear processes. Monomeric actin is also a functional component of the INO80 chromatin remodelling complex (Kapoor et al, 2013), which promotes both transcription and DNA replication (Conaway & Conaway, 2009; Kurat et al, 2017). Interestingly, replication stress strongly stimulates nuclear import of actin and actin regulators (Johnson et al, 2013). Whether nuclear actin dynamics is involved in DNA replication is not yet known. In the first step of DNA replication, licensing, pre-replication complexes (pre-RC) are assembled via the origin recognition complex (ORC). ORC, Cdc6 and Cdt1 recruit minichromosome maintenance (MCM) proteins 2–7, the main component of the replicative helicase. Pre-RC assembly is inhibited by CDK activity and does not require a nuclear envelope. In the second step, pre-RC are converted to pre-initiation complexes (pre-IC), which contain Cdc45, the sliding clamp PCNA and the replicative DNA polymerases. This step requires an intact nuclear envelope and activity of two key kinases, Cdc7 and CDK (for review, see Labib, 2010). Many of these mechanisms were identified by studying DNA replication in nuclei formed in Xenopus egg extracts (XEE; Arias & Walter, 2004), a system that has also been instrumental in identification of nuclear assembly pathways (Hetzer et al, 2005). Unlike Xenopus oocytes, which are quiescent but transcriptionally active, Xenopus eggs have undergone meiotic maturation, during which they acquire replication competence and transcription becomes repressed. Egg activation by fertilisation or calcium mobilisation triggers onset of rapid embryonic cell cycles that consist entirely of successions of S-phase and mitosis without intervening G1 or G2 phases, and in the total absence of transcription. XEE are undiluted extracts from calcium-activated eggs, and recapitulate early embryonic cell cycles in vitro upon the addition of demembranated sperm nuclei. Nuclei assemble autonomously before replicating, and resemble somatic cell nuclei in most respects, although they are transcriptionally silent and do not have a G1 phase. Highly concentrated nucleoplasmic extracts (NPE) of nuclei formed in XEE can promote DNA replication in the absence of a nuclear envelope (Walter et al, 1998), suggesting that nuclear transport is required to establish a threshold concentration of replication-promoting factors, possibly including CDK. Here, using XEE and human somatic cells, we show that actin dynamics is required for nuclear transport and initiation of DNA replication. Nuclear actin binds RanGTP–importin complexes, affecting importin–cargo release. Furthermore, nuclear formin activity promotes chromatin loading and activation of DNA replication factors, as well as replication elongation. Results Nuclear actin dynamics during the cell cycle To analyse the behaviour of nuclear actin during the cell cycle in human cells, we generated a tool for live nuclear actin imaging. We modified a camelid antibody-based probe, actin chromobody (Chromotek®), by appending a nuclear localisation signal (NLS—see Materials and Methods). An identical tool was independently developed recently (Plessner et al, 2015). We concurrently followed the DNA replication programme using a second chromobody to visualise endogenous PCNA, whose patterns change during S-phase progression (Burgess et al, 2012). We found that in U2OS cells, a dynamic network of actin filaments formed in most early G1 nuclei (Movie EV1; Fig 1A). Using confocal imaging and long exposures, we found that these G1 actin filaments could be stained with phalloidin (Fig 1A), which could also detect a G1 nuclear actin network in cells without any ectopic chromobody expression (Fig 1B). Chromobody-visualised filaments disassembled after an average of 200 min, in mid-late G1 (Fig 1C). Figure 1. Perturbing nuclear actin dynamics inhibits DNA replication in human somatic cells Early G1 U2OS cells expressing actin-NLS chromobody co-stained with phalloidin and DAPI (DNA). Scale bar, 5 μm. Serial confocal planes of an early G1 U2OS cell fixed with glutaraldehyde and stained with phalloidin and DAPI. Scale bar, 5 μm. Duration of early G1 nuclear actin network (mean ± SD, n = 135 cells from three independent experiments). Interphase U2OS cells expressing actin-NLS chromobody, treated with DMSO (Ctl) or SMIFH2 (50 μM) for 2 h, stained with mAb414 and DAPI. Scale bar, 5 μm. FACS analysis of U2OS cells synchronised in M-phase, released into G1 and treated with DMSO (Ctl) or SMIFH2 (50 μM). Quantification of EdU incorporation from two independent experiments after a 1-h pulse in U2OS cells expressing formin constructs (**P-value < 0.001; Student's t-test). Duration of PCNA foci in U2OS cells expressing PCNA chromobody, control or SMIFH2-treated (each dot represents the mean of 5–7 foci per cell, mean ± SD of 29 and 11 cells, respectively, per condition from two independent experiments; **P-value < 0.001; Student's t-test). Quantification of EdU incorporation from two independent experiments after a 1-h pulse in U2OS cells expressing actin-NLS mutants (***P-value < 0.0001; Student's t-test). Quantification (mean ± SEM) of EdU intensity from (H) normalised to the non-transfected cells. Download figure Download PowerPoint Next, in living cells, we investigated whether formins are involved in the regulation of nuclear actin dynamics. Specific formin inhibition with SMIFH2 induced stabilisation of long nuclear actin filaments in most cells (Fig 1D, Movies EV2 and EV3). Similar effects of SMIFH2 on actin filaments have been seen in a defined system with purified components in vitro (Rizvi et al, 2009), suggesting that formin inhibition prevents nucleation of new filaments but allows elongation of existing filaments. Actin dynamics is required for initiation and progression of DNA replication Since nuclear actin filaments were disassembled prior to S-phase, we surmised that their stabilisation by formin inhibition might affect DNA replication. To test this, we synchronised U2OS or HeLa cells in M-phase and released into mid-G1 before adding SMIFH2, and analysed entry into S-phase by flow cytometry (FACS). S-phase entry was dose dependently inhibited by SMIFH2 (Figs 1E and EV1A and B). We then altered endogenous formin activity by expressing a GFP-tagged mDia2 diaphanous autoregulatory domain (DAD), either specifically in the nucleus (nuc-DAD) or in the cytoplasm (cyt-DAD), or a nuclear-localised dominant negative mDia2 (nuc-dnDia; Baarlink et al, 2013). These constructs differentially affect formin activity and function: DAD overactivates endogenous mDia2, whereas dnDia2 inhibits it. Nuc-DAD increased the fraction of cells in S-phase significantly (Fig 1F), whereas cyt-DAD did not, implying that over-activating nuclear formins lengthens S-phase. Nuc-dnDia also slightly increased the S-phase fraction but this was not statistically significant (P = 0.08). These results suggest that interfering with nuclear formin activity might impede progression of DNA replication. To confirm this, we analysed the effects of SMIFH2 on dynamics of PCNA foci. In cells that were in S-phase, PCNA foci persisted upon SMIFH2 addition or nuc-DAD expression (Fig 1G; Movies EV4 and EV5). Arrested PCNA foci might indicate replication fork stalling, which can generate DNA damage. Indeed, immunofluorescence analysis showed that nuc-DAD expression or SMIFH2 treatment strongly increased the fraction of cells with γ-H2AX foci. Importantly, 100% of cells synchronised in S-phase and treated with SMIFH2 were γ-H2AX-positive (Fig EV1C and D), indicative of S-phase arrest. Click here to expand this figure. Figure EV1. Blocking formins inhibits DNA replication and general transcription and causes replication stress in human cells FACS analysis of M-phase-synchronised U2OS cells, released into G1 in the presence of increasing concentrations of SMIFH2. Cells were collected when control cells were in S-phase (+14 h). FACS analysis of HeLa cells, synchronised in M-phase and released into G1 as in Fig 1E, and treated with DMSO (Ctl) or SMIFH2, collected at the time points indicated. Asynchronous (AS) or double-thymidine block (DTB) S-phase-synchronised U2OS cells were treated for 2 h with DMSO, bleomycin (Bleo) or SMIFH2, and stained for γH2A.X. Corresponding FACS profiles are shown. Scale bar, 5 μm. Quantification of γH2A.X-positive cell number from experiment presented in (C), and in cells transiently transfected with mDia2 nuc-DAD or cyt-DAD constructs. Cells with > 10 foci were considered positive. Left: Immunofluorescent images of control or SMIFH2-treated (1 h pre-treatment, 1 h co-incubation) U2OS cells pulsed for 1 h with EU. Scale bar, 5 μm. Right: Quantification of EU signal intensity (n > 400). Crosses, mean values; whiskers, 10th and 90th percentiles. Left: Immunofluorescent images of U2OS cells transfected with GFP-mDia2 nuc-DAD or cyt-DAD constructs, pulsed for 1 h with EU. Right: Quantification of EU incorporation (n > 100, n = 61 and n = 47, respectively). Scale bar, 5 μm. Red arrows: examples of non-transfected cells; green arrows: examples of GFP-expressing (transfected) cells. Positions of arrows are the same in both panels. Graphs: crosses, mean values; whiskers, 10th and 90th percentiles. Source data are available online for this figure. Download figure Download PowerPoint To test whether altering nuclear actin dynamics without interfering with formins also affected DNA replication, we expressed nuclear-localised actin: wild-type (WT), or mutants S14C and G15S, which favour polymerisation, or the polymerisation-defective R62D (Appendix Fig S1). Nuclear WT and R62D actin expression led to a non-significant increase or decrease, respectively, in the S-phase fraction. In contrast, nuclear S14C and G15S mutants strongly increased the number of cells in S-phase (Fig 1H). Interestingly, cells expressing high levels of S14C and G15S also had significantly lower EdU signal intensity (Fig 1I). Thus, altering nuclear actin dynamics, derepressing nuclear formins, or inhibiting formins all impede S-phase progression. Since entry into S-phase requires E2F-dependent transcription of replication factors, we next analysed whether interfering with formin activity would affect global transcription, as assessed by 5-ethynyl-uridine (EU) incorporation into newly synthesised RNA. Indeed, SMIFH2 totally abolished general transcription (Fig EV1E), whereas neither nuc-DAD nor cyt-DAD constructs had significant effects (Fig EV1F). This suggests that the inhibition of global transcription by SMIFH2 might be responsible for the block in S-phase entry, but the observed defects in S-phase progression upon expression of nuclear formin mutants depend on a different mechanism. Inhibiting formins disrupts importin-dependent nuclear transport While SMIFH2 inhibits transcription, we also noticed that the nuclei were often smaller and misshapen after a 4-h treatment or longer (Fig 2A and B). This suggested that actin dynamics might be required for nuclear growth or transport, both of which are essential for DNA replication. To investigate this further, we used a well-established nuclear translocation assay. Figure 2. Formin inhibition abolishes nuclear import Confocal planes of nuclei of cells treated with DMSO (Ctl) or SMIFH2 (50 μM) for 4 h, stained with mAb414 and DAPI (DNA). Scale bar, 5 μm. Characterisation of nuclear morphology from (A) (mean ± SD of 20 cells from two independent experiments; ***P-value < 0.0001; Student's t-test). Scatter plot, lines are mean ± SD; box plots, whiskers: min and max values, crosses are means. Immunofluorescence images of RA-FLS fibroblasts, treated for 1 h with DMSO (Ctl), importazole (50 μM), CytD (40 μM) or SMIFH2 (50 μM), subsequently stimulated or not with IL-1β or TNF-α, stained for NF-κB. Scale bar, 20 μm. Quantification of the data presented in (C). Mean nuclear/cytoplasmic NF-κB intensity ratio (± SD) of two independent experiments using two different fibroblast sources; n ≥ 400 for each condition; CytD sample was lost in exp 2. Download figure Download PowerPoint In primary human fibroblasts, the p65 subunit of the ubiquitously expressed nuclear factor-κB (NF-κB) translocates to the nucleus upon stimulation with interleukin-1 beta (IL-1β) or tumour necrosis factor alpha (TNF-α), which cause degradation of the cytoplasmic inhibitor IκB, releasing the NLS of NF-κB. Cytokine treatment therefore bypasses effects of cytoplasmic actin disruption on cell shape and NF-κB regulation, which also impact on IκB (Németh et al, 2004; Sero et al, 2015). This allowed us to study effects of formin inhibition on NF-κB nuclear translocation itself. We also tested cytochalasin D (CytD), which binds the barbed (plus)-end of F-actin, arresting both polymerisation and depolymerisation (Schliwa, 1982). As a positive control for inhibition of nuclear transport, we used importazole, which distorts Ran–importin-β interactions, thereby inhibiting nuclear accumulation of importin and cargo (Soderholm et al, 2011). Cells were treated with drugs for 1 h, followed by 30-min cytokine stimulation. As expected, importazole strongly reduced NF-κB nuclear translocation (Fig 2C and D). CytD had no effect, whereas SMIFH2 did not significantly change cell or nuclear shape, but almost completely abolished NF-κB nuclear translocation (Fig 2C and D). Collectively, these data suggest that general nuclear transport is acutely sensitive to reduction in formin activity. Actin dynamics in Xenopus egg extracts To further characterise the defects in nuclear transport and DNA replication upon disruption of nuclear actin dynamics, we switched to Xenopus egg extracts (XEE). The advantage of this system is that the nuclear processes can be studied in a context that is independent of both transcription and cytoskeleton–environment interactions. First, to characterise nuclear actin regulators in this system, we analysed the combined nucleoskeleton and chromatin proteome of nuclei assembled in XEE by label-free high-resolution mass spectrometry. To identify proteins that associate with this fraction independently of DNA replication, we compared replicating nuclei with non-replicating nuclei assembled in the presence of purvalanol A (PA) to inhibit CDKs (Fig EV2A). We chose PA since it has high affinity for both CDK1 and CDK2 (Gray et al, 1998) and completely abolishes DNA replication in XEE (Echalier et al, 2012). Click here to expand this figure. Figure EV2. The proteome of replicating nuclei in XEE Replication time course of sperm chromatin in control and purvalanol A (PA)-treated egg extracts, with nuclei isolated for MS analysis at 50 min. Graphical representation of the identified proteome with relative quantitation data (mean values from three replicates). Full dataset, Dataset EV1. Volcano plot combining the fold change between control and CDK-inhibited conditions with their log10 P-values (Student's t-test). The most significantly differentially abundant proteins are highlighted. GO analysis using DAVID, showing the most highly enriched GO biological processes in each condition (full GO analysis, Dataset EV2). NE, not enriched. Western blots of chromatin fractions from control and PA-treated nuclei used for MS analysis. Download figure Download PowerPoint We identified 2610 non-redundant proteins (Fig EV2B and C; Appendix Fig S1B, Dataset EV1). Enriched biological processes included DNA metabolism, chromatin organisation and regulation of actin polymerisation (Fig EV2D; Dataset EV2). Specifically, we identified 55 actin regulators (Appendix Table S1), including actin filament nucleating factors such as formins and the Arp2/3 complex. Three formin homologues, diaph1 (mDia1), diaph3 (mDia2) and formin 2, were additionally found by homology searching using a database of the highly related Xenopus tropicalis (Dataset EV1, Appendix Table S2). These actin regulators did not require CDK activity for localisation to the insoluble fraction of nuclei, unlike chromatin recruitment of proteins involved in DNA replication, DNA repair and the S-phase checkpoint (Fig EV2B–E). Immunofluorescence analysis confirmed that many actin polymerisation regulators localised to replicating nuclei (Fig 3A). We also observed that actin factors were loaded onto chromatin at the pre-RC formation stage of DNA replication (Fig 3B), while nuclear actin was mostly insoluble (Fig 3C). The absence of tubulin (Fig 3C and Dataset EV1) confirmed the purity of our sample preparations. Figure 3. Dynamic nature of nuclear actin in Xenopus egg extract Immunofluorescence images of the actin regulators indicated, analysed 60 min after sperm head addition. Scale bar, 10 μm. Western blot analysis of the indicated replication and actin factors loaded onto chromatin at the indicated time points, in control conditions. Western blot analysis of cytoplasm (CP), whole nuclear (NC), nucleoplasmic (NP) and insoluble (P) fraction at 60-min time point during DNA replication, probed with antibodies against proteins indicated. Confocal images a control nucleus, formed in the presence of actin–Alexa Fluor 488 and stained for incorporated biotin-dUTP. Scale bar, 10 μm. Extract was supplemented with sperm nuclei and actin–Alexa Fluor 488; at 40 min, indicated drugs or Arp2/3 and VCA domain of WASP were added, and nuclei were analysed for fluorescent actin at 55 min. Long exposure time (2,000 ms) was needed to visualise nuclear actin in all conditions with the exception of CytD, jasplakinolide (exposure time 200 ms) and the formin inhibitor 2.4 (500 ms). Scale bar, 10 μm. Nuclei were allowed to form for 60 min before drugs (CytD, CD; SMIFH2, FH; latrunculin A, LA; 2.4 compound) or MICAL2 recombinant protein was added, then purified at 75 min. Soluble and insoluble nuclear fractions were blotted for the proteins indicated. Equal number of nuclei was used in each condition. Extract was supplemented with sperm nuclei; at 45 min, CytD (CD) was added and nuclei were analysed at 60 min and stained with phalloidin. Scale bar, 10 μm. Source data are available online for this figure. Source Data for Figure 3 [embj201796585-sup-0010-SDataFig3.pdf] Download figure Download PowerPoint To visualise nuclear actin directly as well as the effects of treatments modifying actin dynamics, we added trace concentrations (that are negligible compared with endogenous nuclear actin concentrations; see Materials and Methods) of fluorescently labelled actin protein to the extracts, prior to sperm chromatin addition. This revealed both diffuse and patterned intra-nuclear staining (Fig 3D and E), but, in contrast to a previous study (Krauss et al, 2003), we did not observe phalloidin-stained nuclear filaments, consistent with their absence in cells in S-phase. Next, we investigated the effects of recombinant actin regulatory proteins, as well as different drugs that modify actin dynamics on nuclear actin in XEE (Figs 3E and F, and EV3A). XEE contain around 50 mg/ml protein, of which 5–10% is actin. Thus, there is at least 100 μM actin in extracts. Since effective drug concentrations depend on adsorption, distribution and metabolism, we expected that several hundred micromolar concentrations of actin drugs would be required to elicit phenotypic effects in this system. In contrast, in cells, due to active import, drugs can routinely attain 1,000-fold higher concentrations than in the medium (Martinez Molina et al, 2013). Click here to expand this figure. Figure EV3. Actin dynamics is required for DNA replication A. Nuclei were allowed to form for 30 min before drugs (CytD, CD; SMIFH2, FH; latrunculin A, LA; jasplakinolide, Jpk; CytD and latrunculin A, CD/LA; SMIFH2 and latrunculin A, FH/LA) or Arp2/3 recombinant protein (in combination with VCA domain of WASP) was added, then purified at 45 min. Soluble and insoluble nuclear fractions were blotted for actin. Equal number of nuclei was used in each condition. B–F. DNA replication assessed in control extract, or extracts supplemented with CytD (CD), with or without latrunculin A (LA) (B); CytD (CD) with or without cofilin (C); CytD (CD), gelsolin, CytD and gelsolin (CD + Gel), jasplakinolide (Jspk), or CytD and jasplakinolide (CD + Jspk) (D); recombinant MICAL2 protein (E), or formin inhibitor 2.4 (F). Each panel is representative of multiple experiments: (B) 5 experiments; (C–F) 2 experiments. G. DNA replication analysed in control extract, or extracts supplemented with CytD, SMIFH2 or PA that was added at the time points indicated. Representative of two independent experiments. Source data are available online for this figure. Download figure Download PowerPoint SMIFH2 reduced levels of nuclear actin and control proteins in a similar manner to latrunculin A, which potently binds actin monomers and impedes filament assembly, while an unrelated formin inhibitor, compound 2.4 (Gauvin et al, 2009; Baarlink et al, 2013), had the opposite effect (Fig 3F). Therefore, different modes of formin inhibition may have contrasting effects on actin dynamics (of note: like phalloidin, 2.4 cannot penetrate into living cells and thus could not be used in the experiments described above" @default.
• W2951532637 created "2019-06-27" @default.
• W2951532637 creator A5009481547 @default.
• W2951532637 creator A5009888760 @default.
• W2951532637 creator A5017393337 @default.
• W2951532637 creator A5025876236 @default.
• W2951532637 creator A5061004429 @default.
• W2951532637 creator A5062612431 @default.
• W2951532637 creator A5078639309 @default.
• W2951532637 creator A5082152177 @default.
• W2951532637 creator A5090742550 @default.
• W2951532637 date "2017-11-02" @default.
• W2951532637 modified "2023-10-15" @default.
• W2951532637 title "Initiation of <scp>DNA</scp> replication requires actin dynamics and formin activity" @default.
• W2951532637 cites W116768201 @default.
• W2951532637 cites W1958099500 @default.
• W2951532637 cites W1964351412 @default.
• W2951532637 cites W1965718993 @default.
• W2951532637 cites W1967487834 @default.
• W2951532637 cites W1968050352 @default.
• W2951532637 cites W1968272833 @default.
• W2951532637 cites W1975484124 @default.
• W2951532637 cites W1976349517 @default.
• W2951532637 cites W1978028702 @default.
• W2951532637 cites W1984618049 @default.
• W2951532637 cites W1991366673 @default.
• W2951532637 cites W1993251792 @default.
• W2951532637 cites W1994774468 @default.
• W2951532637 cites W1995635845 @default.
• W2951532637 cites W1996233493 @default.
• W2951532637 cites W1999598776 @default.
• W2951532637 cites W2005758722 @default.
• W2951532637 cites W2009949081 @default.
• W2951532637 cites W2010496399 @default.
• W2951532637 cites W2011238489 @default.
• W2951532637 cites W2012083125 @default.
• W2951532637 cites W2018316000 @default.
• W2951532637 cites W2024060173 @default.
• W2951532637 cites W2024878957 @default.
• W2951532637 cites W2026993081 @default.
• W2951532637 cites W2027628919 @default.
• W2951532637 cites W2030923676 @default.
• W2951532637 cites W2033575518 @default.
• W2951532637 cites W2042627335 @default.
• W2951532637 cites W2045018569 @default.
• W2951532637 cites W2047245819 @default.
• W2951532637 cites W2049324563 @default.
• W2951532637 cites W2052178337 @default.
• W2951532637 cites W2057429454 @default.
• W2951532637 cites W2057925576 @default.
• W2951532637 cites W2063290334 @default.
• W2951532637 cites W2070127489 @default.
• W2951532637 cites W2074075770 @default.
• W2951532637 cites W2074536102 @default.
• W2951532637 cites W2075470041 @default.
• W2951532637 cites W2086347878 @default.
• W2951532637 cites W2088756859 @default.
• W2951532637 cites W2097930085 @default.
• W2951532637 cites W2097981550 @default.
• W2951532637 cites W2113126534 @default.
• W2951532637 cites W2115127473 @default.
• W2951532637 cites W2115308806 @default.
• W2951532637 cites W2119497951 @default.
• W2951532637 cites W2119691744 @default.
• W2951532637 cites W2124509928 @default.
• W2951532637 cites W2124599642 @default.
• W2951532637 cites W2124931400 @default.
• W2951532637 cites W2127198296 @default.
• W2951532637 cites W2128647012 @default.
• W2951532637 cites W2128889123 @default.
• W2951532637 cites W2139730706 @default.
• W2951532637 cites W2144245358 @default.
• W2951532637 cites W2147728748 @default.
• W2951532637 cites W2150759197 @default.
• W2951532637 cites W2151360504 @default.
• W2951532637 cites W2156139408 @default.
• W2951532637 cites W2158217645 @default.
• W2951532637 cites W2159452770 @default.
• W2951532637 cites W2160286292 @default.
• W2951532637 cites W2165321825 @default.
• W2951532637 cites W2166241889 @default.
• W2951532637 cites W2167260434 @default.
• W2951532637 cites W2167279371 @default.
• W2951532637 cites W2170079554 @default.
• W2951532637 cites W2171534430 @default.
• W2951532637 cites W2480088911 @default.
• W2951532637 cites W2538598113 @default.
• W2951532637 cites W2566377538 @default.
• W2951532637 cites W4249138713 @default.
• W2951532637 cites W4250686860 @default.
• W2951532637 cites W73930124 @default.
• W2951532637 doi "https://doi.org/10.15252/embj.201796585" @default.
• W2951532637 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5666611" @default.
• W2951532637 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28982779" @default.
• W2951532637 hasPublicationYear "2017" @default.
• W2951532637 type Work @default.
• W2951532637 sameAs 2951532637 @default.
• W2951532637 citedByCount "77" @default.

• next