Biochimica et Biophysica Acta 1833 (2013) 2334–2347

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Biochimica et Biophysica Acta

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Stimulation of in vivo nuclear transport dynamics of and its co-factors IQGAP1 and Rac1 in response to DNA replication stress

Michael A. Johnson 1, Manisha Sharma, Myth T.S. Mok 2, Beric R. Henderson ⁎

Westmead Institute for Cancer Research, The University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia article info abstract

Article history: Actin, a constituent of the , is now recognized to function in the nucleus in transcription, Received 21 November 2012 chromatin remodeling and DNA replication/repair. Actin shuttles in and out of the nucleus through the action of Received in revised form 31 May 2013 transport receptors importin-9 and exportin-6. Here we have addressed the impact of cell cycle progression and Accepted 4 June 2013 DNA replication stress on actin nuclear localization, through study of actin dynamics in living cells. First, we Available online 13 June 2013 showed that thymidine-induced G1/S phase cell cycle arrest increased the nuclear levels of actin and of two factors that stimulate actin polymerization: IQGAP1 and Rac1 GTPase. When cells were exposed to hydroxyurea to induce Keywords: Nuclear actin DNA replication stress, the nuclear localization of actin and its regulators was further enhanced. We employed live IQGAP1 cell photobleaching assays and discovered that in response to DNA replication stress, GFP-actin nuclear import and Rac1 export rates increased by up to 250%. The rate of import was twice as fast as export, accounting for actin nuclear Nuclear transport accumulation. The faster shuttling dynamics correlated with reduced cellular retention of actin, and our data im- FRAP plicate actin polymerization in the stress-dependent uptake of nuclear actin. Furthermore, DNA replication stress induced a nuclear shift in IQGAP1 and Rac1 with enhanced import dynamics. Proximity ligation assays revealed that IQGAP1 associates in the nucleus with actin and Rac1, and formation of these complexes increased after hy- droxyurea treatment. We propose that the replication stress checkpoint triggers co-ordinated nuclear entry and trafficking of actin, and of factors that regulate actin polymerization. © 2013 Elsevier B.V. All rights reserved.

1. Introduction actin and of specific actin co-factors are likely determinants of the propensity of actin to polymerize in the nucleus. Actin is an important cytoskeletal and is vital for maintenance Nuclear transport of actin is tightly regulated. Transport of mole- of cell architecture and processes such as cell adhesion and migration [1]. cules through the nuclear pore complex (NPC) occurs through two In the interphase nucleus, actin and various actin-associated co-factors mechanisms: passive diffusion or active, energy-dependent transport. also function in chromatin remodeling and DNA repair, and regulate Passive diffusion, due to physical limits of the NPC channel, is generally the activity of all three RNA polymerases [2–4]. The forms of actin within restricted to molecules no larger than 40 kDa. Actin, at 42 kDa, is on the nucleus remain enigmatic, yet there is a growing consensus that actin the cusp of this size limit and is purported to passively diffuse through can form unconventional nuclear polymers [5–8]. Details of how nuclear the NPC [10]. Although actin contains two conserved CRM1-targeted actin polymers are regulated remain poorly defined, nevertheless recent nuclear export sequences (NESs), CRM1 seems to indirectly affect reviews have described how such polymers may assemble in the nucleus actin nuclear export [11,12]. Exportin 6 (Exp6) appears to be the exclu- [5,8,9]. Actin concentration and the availability of co-factors are two key sive export receptor for actin, which translocates actin through a influences on actin polymerization. Thus, changes in nuclear levels of piggy-back mechanism via profilin [11,13]. Similarly for nuclear import, actin does not contain a conserved nuclear localization signal (NLS) – an amino acid sequence recognized by the nuclear import receptor Abbreviations: FRAP, fluorescence recovery after photobleaching; HU, hydroxyurea; complex importin-α/β – and was initially proposed to be transported IQGAP1, IQ-domain GTPase-activating protein 1; IR, ionizing radiation; N-WASp, neural- into the nucleus via its binding partner cofilin [14].Thisnotionofa – Wiskott Aldrich syndrome protein; Rac1, Ras-related C3 botulinum toxin substrate 1; cofilin-mediated piggy-back of actin was bolstered by the recent identi- ROI, region of interest; UV, ultraviolet fi – fi ⁎ Corresponding author at: Westmead Millennium Institute, Darcy Road (PO Box 412), cation of importin 9 (Ipo9) as an import receptor of actin co lin com- Westmead, NSW 2145, Australia. Tel.: +61 2 9845 9057; fax: +61 2 9845 9102. plexes under normal cell culture conditions [13]. E-mail address: [email protected] (B.R. Henderson). The concentration of nuclear actin amongst different species and 1 Current address: Kolling Institute of Medical Research, University of Sydney, Royal cell types remains fairly equivalent (reviewed [15]). However, little North Shore Hospital, St Leonards, NSW, Australia. is known of the conditions and factors that influence nuclear entry of 2 Current address: Institute of Digestive Disease, Li Ka Shing Institute of Health Sciences, Department of Medicine and Therapeutics, The Chinese University of Hong actin or that regulate its polymerization in the nucleus. This is becoming Kong, Hong Kong, China. a topic of broad interest [2,5,9]. Several cellular stress conditions, such

0167-4889/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2013.06.002 M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347 2335 as DMSO treatment, heat-shock and ATP depletion, were reported to phalloidin (0.5 μg/ml) was used to label F-actin and Hoechst dye was increase nuclear actin levels and to induce the formation of aberrant used to stain chromatin, each purchased from Sigma. rods, or crystals, of nuclear actin (reviewed [15]). These aberrant struc- Secondary antibodies used were anti-mouse Alexa-Fluor-405 (1:50), tures are relevant in certain pathological conditions such as myopathies anti-mouse or anti-rabbit Alexa-Fluor-488 (1:500) and anti-mouse or [16]. Inhibition of nuclear actin export through silencing Exp6 expression anti-rabbit AlexaFlour-594 (1:1500) (Molecular Probes). or by blocking CRM1 activity also induced nuclear actin rod formation pGFP-β-actin was a gift from Dr. Walter Witke (Rheinische Friedrich- [17–19]. Blocking nuclear import of actin also affects the transcriptional Wilhelms-Universität, Bonn, Germany). pΔCMV-EGFP constructs of activity of RNA Polymerase II [13]. Thus, a regulated and continual ex- G13R and R62D non-polymerizing actin mutants were gifts from Naoki change of actin between nucleus and cytoplasm appears to be essential Watanabe [35]. pEGFP-tagged IQGAP1-WT was a gift from Dr. Kozo for normal physiological functioning of the cell. Kaibuchi [36] and pIQGAP1-N-flag (1–863 aa) from Dr. Robert Grosse Previous reports by our team and others have noted the cell [37]. Rac1 plasmids were gifts from Dr. Mark Phelps [23,38].mCherry cycle-regulated nuclear translocation of specific actin-associated was a gift from Dr. Roger Tsien [39]. pmCherry-actin was made from cytoskeletal [20–24], and there are clues from the literature the wild-type GFP-tagged plasmid by replacing the GFP with mCherry. that actin itself might be regulated in a similar manner [25–28]. For This plasmid was confirmed by sequencing and restriction mapping. instance, in an in vitro nuclear assembly assay actin began to accrue Small amounts of plasmid (1 μg pGFP-actin, 1 μgpmCherry-actin in the nucleus after the formation of nuclear pores yet prior to DNA 400 ng pGFP-Rac1 and 2 μg pGFP-IQGAP1) DNA were transfected to synthesis [26], and BrdU-tagged S phase cells within an asynchronous minimize the amount of ectopically-expressed GFP-tagged protein. population were found to display the highest levels of nuclear actin [28]. 2.3. Immunoprecipitation and immunoblot analysis We observed a nuclear accumulation of the cytoskeletal regulator IQGAP1 in cells at G1/S phase of the cell cycle [21]. IQGAP1 is a principal Immunoprecipitation was performed as previously described [40]. regulator of actin filament rearrangements at the plasma membrane Briefly, total cell lysates from GFP-Rac1(L61-SAAX)-transfected 10T1/2 [29]. In conjunction with small GTPases Rac1 and Cdc42, IQGAP1 con- cells (Fig. 8) or nuclear fractions of pGFP-actin-transfected 10T1/2 cells trols actin filaments via diverse actions that include actin bundling, (Fig. S4) were mixed with 2 μg of H-109 IQGAP1 polyclonal antibody, barbed-end capping, and binding to actin branching and nucleating GFP polyclonal antibody (Invitrogen) or rabbit IgG (Sigma) and incubat- proteins, such as N-WASp and Diaphanous 1 [30,31].Conversely,N- ed overnight at 4 °C with continuous end-over-end mixing. Then, 30 μl WASp has also been detected in the nucleus [32] and found to induce of Protein A-Sepharose (CL4B, GE Healthcare Bio-Sciences) was added actin polymerization in nuclear extracts in vitro [33]. Considering to the immuno-mix at 4 °C for 2 h. 1% inputs and the immunocomplexes these broad attributes of actin regulation, we hypothesized that were subjected to SDS-PAGE, followed by immunoblotting with the IQGAP1 might act as a potential regulator of nuclear actin through its as- indicated antibodies. For Western-blot analysis, cell extracts were dena- sembly of actin-regulatory binding partners. In this study, the dynamics tured in sample buffer (100 mm Tris–HCl, pH 6.8, 20% glycerol, 0.01% of actin, along with IQGAP1 and its effector Rac1, was analyzed in bromophenol blue, 10% β-mercaptoethanol, 5% SDS). 40–60 μgoftotal response to DNA replication arrest by Fluorescence Recovery After protein extracts was loaded per lane and resolved by SDS-PAGE and Photobleaching (FRAP) assays to determine changes in nuclear trans- transferred onto nitrocellulose membrane (Millipore Corp.). The mem- port kinetics in live cells. We present the first detailed comparison of branes were blocked using 5% skim milk powder in PBS containing shifts in the nuclear import/export rates of these related proteins and 0.2% Tween 20 for 1 h at room temperature followed by primary anti- of their interaction in the nucleus and co-ordinated response to cellular body incubation for 2 h at room temperature using indicated antibodies. DNA replication stress. Incubation with secondary horseradish peroxidase-conjugated anti- bodies (1:5000; Sigma) for 1 h was followed by detection by enhanced 2. Materials and methods chemiluminescence (Amersham Biosciences).

2.1. Cell culture, reagents and transfection 2.4. Microscope image acquisition and fluorescence recovery after photobleaching (FRAP) analysis NIH 3T3 and 10T1/2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and Cells were washed, fixed and immunostained as previously de- antibiotics (penicillin and streptomycin) at 37 °C in 5% CO2 humidified scribed [41], then visualized by fluorescence microscopy. Cells express- atmosphere. Cells were grown on glass cover-slips in 6-well dishes ing excessive amounts of GFP-tagged protein were disregarded, as too (Nunc) or, for live imaging, 2 well chamber slides (Nunc) and treat- were cells displaying abnormal morphologies. For basic fluorescence ments were performed 24 h post-seeding. Transfection of plasmids image analysis for subcellular localization studies, cell samples were ob- was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as served under an Olympus BL51 fluorescence microscope for scoring previously described [34]. Cells were arrested in G0 by incubating purposes. For advanced fluorescence image analysis, cells were visual- cells in DMEM supplemented with 0.5% FCS for 48 h, or at early S ized through a 60× oil immersion lens using Olympus FV1000 confocal phase by treatment with hydroxyurea (HU) (2.0 μmol/ml, Calbiochem) laser scanning microscope with images processed using Fluoview and thymidine (2.5 μmol/ml, Sigma) for 20 and 24 h, respectively. Version 1.6a software, or cells were visualized through a 100 × 1.4 nu- merical aperture oil immersion lens with an inverted Olympus IX-70 mi- 2.2. Antibodies and plasmids croscope (DeltaVision Image Restoration Microscope; Applied Precision/ Olympus) and a photometrics CoolSnap QE camera. We acquired 10–20 The following antibodies and dilutions were used for immunofluo- serial optical sections of 0.2–0.5 μm. The images were then deconvolved rescence (IF) and Western blot (WB): β-actin monoclonal antibody (IF and generated volume projections of the entire z-series using DeltaVision 1:150 (PF fixation) & 1:1000 (MeOH fixation) and WB 1:2000; Sigma SoftWoRx software (version 3.4.4.) The images were compiled in Adobe AC-74), flag monoclonal antibody (IF 1:2000; Sigma M2), GFP mono- Photoshop CS5. clonal antibody (WB 1:2000; Roche), GFP polyclonal antibody (Invitrogen FRAP was performed as previously described [42].Briefly, FRAP #A11122), Topo II monoclonal antibody (Calbiochem #NA14; WB 1:800), analysis of nuclear import kinetics was performed on NIH 3T3 cells monoclonal antibody (Sigma #T0198; WB 1:2000), IQGAP1 expressing moderate levels of GFP-tagged plasmids at 42–48 h (GFP- monoclonal antibody (WB 1:2000; BD Biosciences #610611) and actin, GFP-IQGAP1) or 28–36 h (GFP-Rac1) post-transfection in a

IQGAP1 polyclonal antibody (IF 1:150; Santa Cruz H-109). FITC- humidified CO2 chamber at 37 ° C. The analysis was performed using 2336 M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347 an Olympus FV1000 confocal laser scanning microscope. For FRAP, each 2.6. Nuclear–cytoplasmic fractionation and F-actin sedimentation analysis cell was scanned with low laser power to minimize loss of fluorescence during the pre-bleach, and the region of interest (ROI) then photo- Nuclear–cytoplasmic fractionation of pGFP-actin-transfected C3H/ bleached at 100% laser power. 10T1/2 cells was performed as previously described [21]. Actin fraction- ation was performed as described [43], with slight adjustments. Briefly, nuclei from pGFP-actin-transfected C3H/10T1/2 cells were separated as 2.4.1. FRAP data acquisition previously described [21] but the nuclear extract was prepared in actin Each FRAP transport experiment commenced with 3 pre-bleach lysis buffer (50 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 20 mM image scans followed by bleaching ~90% of the nucleus for ~6 s (for HEPES, pH 7.9) and incubated on ice for 30 min. Nuclear extracts import), or the cytoplasm for ~8 s (for export). Images were collected were then centrifuged for 60 min at 100,000 ×g at 4 °C. The resulting as previously described [42]. For FRAP of diffuse nuclear GFP-actin, supernatant (S) contains monomeric G-actin while the pellet (P) con- GFP-actin in IQGAP1-N-induced nuclear polymers or cytoplasmic stress tains F-actin. The pellet was sonicated, and then equal amounts of fibers a ROI 1.5 μm in diameter was selected across the nucleus or stress each fraction were subjected to SDS-PAGE followed by immunoblotting. fibers and bleached for 250 ms and images during the recovery phase were taken at ~2 s intervals. Average intensities in all regions of interest 2.7. Statistical analysis including the background signal were calculated using Olympus Fluoview Version 1.6a software, before exporting data into Microsoft Statistical analysis was performed with Microsoft Excel using Students Excel (2007). unpaired t-test. Where statistics are indicated, * denotes p b 0.05; ** de- notes p b 0.01; *** denotes p b 0.001. All experiments were performed three times, unless indicated otherwise. 2.4.2. FRAP data analysis FRAP analysis of different nuclear or cytoplasmic regions was based on direct localized recovery of fluorescence intensity. To normalize and 3. Results compare the rates of nuclear transport between different cell samples, the fluorescence data before and after bleaching was expressed as a 3.1. Nuclear actin levels increase in response to DNA replication stress ratio of nuclear/cytoplasmic fluorescence for nuclear import or cyto- plasmic/nuclear fluorescence for nuclear export. The pre-bleach ratios Results from previous reports suggest that nuclear levels of actin were then set to 100% to normalize between samples. Finally, the first may be regulated by DNA replication events [25,26,28]. In this post-bleach image was set to time 0, with successive time points context, we recently described the G1/S-dependent nuclear entry converging towards 100% fluorescence recovery. Differences in initial of IQGAP1, an actin-binding regulatory protein, which we showed post-bleach mean values may be exaggerated due to the large differ- to be a component of the DNA replication machinery [21].In ence in early transport rates (Fig. 2) and a slight delay between fluores- order to determine whether actin nuclear localization is linked to S cence bleach and image capture. The fraction of protein that contributes phase of the cell cycle, we transiently expressed GFP-actin in NIH 3T3 to the recovery is called the ‘mobile’ fraction and the protein that does cells and employed fluorescence microscopy to analyze untreated/ not is called the ‘immobile’ fraction. The recovery curves shown are asynchronous cells or cell cycle-arrested cells blocked at (i) G0 by each based on an average of 8–15 cells and representative of at least serum starvation or (ii) late G1/early S phase by treatment with thymi- two independent experiments. Details are previously described [42]. dine or hydroxyurea (HU). The subcellular distribution of GFP-actin To compare initial nuclear transport rates, linear regression analysis of was assessed visually by immunofluorescence microscopy, scoring cell the values from the first 32 s was performed in Excel. The average staining patterns as predominantly cytoplasmic (C > N), uniformly recovery curves were analyzed in GraphPad Prism 5 (GraphPad Software nuclear–cytoplasmic (N = C), or predominantly nuclear (N > C). We Inc., CA, USA) and fitted to a two-phase model. found that a small proportion (~8%) of untreated cells displayed partial nuclear staining of GFP-actin, and this level of nuclear staining dimin- ished to only 2% when cells were arrested in G0 (Fig. 1A and B), 2.5. Proximity ligation assay supporting recent data by others [28]. In contrast, the proportion of cells with partial or exclusively nuclear GFP-actin jumped significantly A Duolink (Olink Bioscience, Uppsala, Sweden) in situ co-IP after G1/S arrest; the replication stress agent HU had an even greater ef- assay was performed as follows. NIH3T3 cells were plated in an 8 fect than thymidine (Fig. 1A and B, and Supp. Fig. S1). The G1/S phase well chamber slide to ~60% confluency. After 24 h cells were treat- increase in nuclear actin we detected is very similar to that recently ob- ed with hydroxyurea (20 μlof0.1Mstock/ml)for16h.Todetect served for IQGAP1 [21] and other cytoskeletal proteins [20,22,24]. Inter- associations between endogenous IQGAP1 and endogenous actin, estingly, no nuclear actin rods were detected after these treatments, cells were fixed with 4% paraformaldehyde for 20 min and washed which differ to that seen in response to other modes of cellular stress, three times with PBS. Cells were permeabilized with 0.2% Triton- such as DMSO or heat-shock treatment [15]. The impact of these drugs X-100 for 10 min and washed three times with PBS. Cells were on cell cycle arrest was con firmed as recently shown [21,40]. then blocked with Duolink blocking reagent and subsequent steps The labeling of endogenous actin with commercial AC-74 were followed according to manufacturer's instruction. For detec- mAb showed disparate patterns after different fixation methods: tion of association between endogenous IQGAP1 and ectopic GFP- cytoplasmic/filamentous after MeOH fixation and nuclear/speckled tagged actin or Rac1, cells were fixed with 3.7% formalin for after paraformaldehyde (PF) fixation (Figs. 1C and S2A). The AC-74 20 min and washed three times with PBS, and then processed staining patterns are comparable to that reported for the 2G2 actin as described above. Dilutions of antibodies used were: IQGAP1 mAb raised against the profilin–actin complex [7]. Indeed the speckled polyclonal antibody (H-109) 1:150; actin monoclonal antibody nuclear staining of AC-74 is strikingly similar to that described for 2G2 (AC-74) 1:100; and anti-GFP antibody (Roche) 1:1000. Cell images (Fig. 1C). The fluorescence intensity of endogenous nuclear actin AC- were acquired using a confocal microscope (Olympus FV1000) with 74 staining, normalized against DAPI, increased ~50% after G1/S arrest 60× oil objective. In order to score the nuclear dots, each image of (Fig. 1D), although changes in the nuclear fraction were harder to detect cells was stacked in the z-plane (5 stacks) at 0.5 micron step size. by Western blot possibly due to the known shuttling of actin (Fig. S3A). Only the image cross section showing middle of the nucleus was The nuclear GFP-actin displayed a more consistent cell cycle-dependent used for scoring Duolink positive dots. up-regulation after different fixations (Fig. S2A), and was therefore a M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347 2337

Fig. 1. Nuclear actin increases after G1/S arrest. (A) Representative confocal cell images showing the three nuclear–cytoplasmic distribution scoring criteria of GFP-actin in NIH 3T3 cells that are asynchronous/untreated, or arrested at either G0 (SS, serum starved) or G1/S phase (thy and HU). (B) Graph shows the % cells with nuclear GFP-actin in experiments from A. Black bars are % cells with N > C GFP-actin distribution and gray bars are % cells with N = C GFP-actin distribution. (C) Deconvolution fluorescence cell images of AC-74 antibody staining in paraformaldehyde (PF)-fixed cells. (D) Graph represents nuclear fluorescence AC-74 staining intensity. Cells left asynchronous or treated with thymidine or HU, were fixed with PF and stained with AC-74 mAb. The asynchronous sample was standardized to 1 and thymidine and HU samples compared appropriately. AU = arbitrary units. N, nuclear; C, cytoplasmic.

more tenable method to investigate nuclear actin by fluorescence N/C equilibrium of GFP-actin recovery after drug treatment, consistent microscopy. with a substantial decrease in cytoplasmic retention (Fig. 2E, left panel). The data therefore suggest the possibility that replication stress 3.2. DNA replication stress reduces anchorage of actin and stimulates might disengage actin from cytoplasmic retention and stimulate its dynamic actin shuttling between nucleus and cytoplasm rate of movement into the nucleus. In order to test for similar changes in the opposite direction, we To determine if the nuclear accumulation of actin after DNA replica- next analyzed nuclear export rates of GFP-actin, as shown in Fig. 2A tion arrest was due to a change in nuclear import/export rates, we and C. For export, the entire cytoplasmic GFP-actin fluorescence investigated changes in GFP-actin nuclear transport in live cells after S signal was bleached and its recovery from the unbleached nuclear phase arrest using confocal microscopy and fluorescence recovery GFP-actin was monitored. GFP-actin displayed a steady but slower after photobleaching (FRAP) assay (Fig. 2A and B). The GFP tag increases export rate compared to import in untreated cells (Fig. 2C–D). Some- the molecular size of actin (~70 kDa; Fig. 2F) beyond the 40 kDa what surprisingly, we observed a similar response to DNA replication diffusion size limit of the NPC, ensuring that only active transport is stress for nuclear export as was seen for import, with the GFP-actin measured [13].Wefirst applied FRAP to measure kinetics of nuclear export rate increasing ~200% after drug-induced DNA replication import, wherein the nuclear fluorescence of GFP-actin in transfected arrest (Fig. 2D, right panel). Moreover, significant reductions in nuclear cells was bleached and its recovery calculated as a ratio of nuclear: GFP-actin retention were measured after thymidine and HU treatments cytoplasmic (N/C) fluorescence (see Fig. 2A for cell images pre- and compared to untreated cells (Fig. 2E, right panel). Integrity of GFP-actin post-bleaching, and Fig. 2Bforquantification of the fluorescence recov- and its expression was confirmed by Western blot (Fig. 2F). These ery after bleaching). The FRAP recovery curve data revealed a moderate results reveal for the first time that DNA replication stress leads to rate of nuclear import and a high degree of cytoplasmic retention sharp increases in actin nuclear import/export rates, and reduction of (for details see Methods) consistent with the live cell import kinetics actin retention in both cytoplasmic and nuclear compartments, indicating of GFP-actin previously reported by Dopie et al. [13]. However, in a global mobilization of actin after DNA replication arrest. The rate of response to drug induced S phase arrest, a dramatic shift in GFP-actin GFP-actin nuclear import was faster than export after each drug treat- kinetics was discovered. The nuclear import rate of GFP-actin increased ment, which explains why we observed an increase in nuclear pools by ~200–300% after DNA replication arrest compared to untreated cells of actin despite the enhanced shuttling activity. The key point to note is (Fig. 2D, left panel). In parallel, there were major differences in the final that the increased nuclear localization of actin is not due to nuclear 2338 M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347

Fig. 2. Nuclear shuttling of actin becomes highly dynamic after DNA replication arrest. (A) Nuclear import kinetics of GFP-actin was analyzed by FRAP. Nuclear import (top panels) was determined by bleaching the entire nucleus and monitoring fluorescence recovery for ~8 min. Nuclear export (bottom panels) was determined by bleaching the entire cytoplasm for ~8 s and monitoring fluorescence recovery for 8 min. Cell images are representative from FRAP experiments. (B) Graph indicates mean nuclear import recovery curves for GFP-actin in asynchronous cells, or in thymidine and HU G1/S-arrested cells. (C) Graph indicates mean nuclear export recovery curves. (D) Nuclear import and export rates are expressed as the change in ratio of N/C and C/N fluorescence, respectively. Rates were determined from the initial recovery slope (0–32 s). (E) Graph of GFP-actin cytoplasmic (import) and nuclear (export) immobile fractions. * denotes p b 0.05; ** denotes p b 0.01; *** denotes p b 0.001. (F) Total cell extract from GFP-actin expressing cells was subjected to SDS-PAGE and analyzed by Western blot with GFP (left blot)orAC-74(right blot)mAbs.

retention, but in fact reflects an overall increase in the dynamic rate of compared the non-polymerizing actin mutants G13R and R62D, and nuclear shuttling. This may have implications for the rapid regulation found that G13R displayed a nuclear–cytoplasmic distribution more of actin nuclear positioning and function (see Discussion). comparable to wild-type actin before and after DNA replication stress (see Supp. Fig. S2; [43]). Thus, the G13R mutant was tested for differ- 3.3. The G13R polymerization-mutation alters the dynamic response of ences in transport rate in live cell photobleaching assays (Fig. 3A–C). actin to thymidine treatment, but not to HU-induced replication stress FRAP analysis revealed that the G13R GFP-actin mutant (GFP-G13R) displayed a similar nuclear import rate to WT-actin in untreated cells A recent actin FRAP study by Dopie et al. [13] indicated that intro- (Fig. 3D). Surprisingly, GFP-G13R displayed no significant change in im- duction of the R62D mutation, which prevents actin polymerization, port rate or cytoplasmic retention after thymidine treatment (Fig. 3E,G, modestly increased nuclear import and export rates. This could H), in contrast to wild-type GFP-actin which became very dynamic indicate that monomeric actin is less well retained at cytoskeletal in thymidine treated cells. However, in cells exposed to HU-induced or nuclear structures and thus shuttles more effectively. Our FRAP DNA replication stress, GFP-G13R responded more like wild-type actin recovery curve data agreed with the general rate of GFP-actin nuclear with a faster import rate and reduced cytoplasmic retention, albeit import described by Dopie et al. [13], and also revealed a high degree not to the same extent as WT-actin (Fig. 3F–H). These new findings of retention in the cell. To test for the influence of polymerization, we indicate that actin polymerization activity contributes to the nuclear M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347 2339

Fig. 3. Nuclear shuttling of monomeric mutant actin becomes highly dynamic after DNA replication arrest. (A) Fluorescence images of transfected cells expressing GFP-actin wild-type (WT; left) or a non-polymerizing mutant, G13R (right). Cells were co-stained with phalloidin-FITC or AC-74 mAb. (B) The % cells expressing predominantly nuclear (N > C) or nuclear–cytoplasmic (N = C) GFP-actin are shown, comparing WT with G13R actin in untreated, thymidine- or HU-arrested cells. (C) FRAP assay to determine nuclear import rates of GFP-G13R-actin in living NIH 3T3 cells. Nuclear fluorescence was bleached and recovery quantified over an 8 min period. A graph of the mean nuclear import recovery curves for GFP-G13R-actin in cells subjected to DNA replication stress, or not, is shown. Nuclear import was determined as in Fig. 2.(D–F) Results of nuclear import FRAP assays to compare import rates of WT and G13R-mutant actin. The recovery curve graphs of G13R vs. WT actin for each respective cell condition are shown. (G) Comparison of nuclear import rates of GFP-actin WT (left) and G13R (right) during the first 32 s recovery post-bleaching. (H) Comparison of cytoplasmic immobile fractions of WT (left) and G13R (right) actin. * denotes p b 0.05; ** denotes p b 0.01. translocation of actin in thymidine growth-arrested cells, but is not consistent with prior reports of exogenous Rac1 [23], which was required for nuclear entry of actin in response to HU-activation of shown to enter the nucleus at G1/S and continued to accumulate into the DNA replication stress checkpoint. late G2.

3.5. Comparison of nuclear import/export rates of IQGAP1 and Rac1 in 3.4. Nuclear entry of IQGAP1 and Rac1 correlates with nuclear translocation response to DNA replication stress in live cells of actin Given that IQGAP1 and Rac1 increase in the nucleus in response to We next compared the localization and transport kinetics of a G1/S phase arrest, we compared the nuclear import and export rates factors comprising the IQGAP1-driven actin polymerization machinery. of each factor by FRAP in live cells (Fig. 5). The initial import rate (first We extend our previous study of endogenous IQGAP1 nuclear localiza- 32 s) of GFP-IQGAP1 did not significantly change (Fig. 5B and D). tion [21] by analysis of the dynamics of ectopically-expressed GFP- In contrast, GFP-Rac1 nuclear import increased significantly after S IQGAP1. The cellular distributions of GFP-tagged IQGAP1 and Rac1 phase arrest (Fig. 5G and I). The extent of nuclear recovery for GFP- were determined by microscopy in cells arrested in G0 or G1/S phase tagged IQGAP1 and Rac1 improved in S phase-arrested cells, indicating (as in Figs. 1–3). IQGAP1 and Rac1 each displayed a striking increase an overall modest reduction in cytoplasmic retention for these factors in nuclear accumulation when cells were arrested at G1/S by thymidine (Fig. 5EandJ,black bars). or HU treatment (Fig. 4). Interestingly, only Rac1 nuclear pools in- In the reverse direction, we found that the nuclear export rate of creased in G0 cells (Fig. 4B). The data supports our previous observation IQGAP1 was not significantly altered (Fig. 5C), whereas Rac1 nuclear for cell cycle regulation of nuclear endogenous IQGAP1 [21],andis export was significantly reduced in response to replication stress 2340 M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347

Fig. 4. G1/S arrest induces nuclear accumulation of IQGAP1 and Rac1. Representative confocal cell images of the three nuclear–cytoplasmic distribution scoring criteria of (A) GFP-IQGAP1 and (B) GFP-Rac1 in NIH 3T3 cells that are asynchronous/untreated, or arrested in G0 (SS, serum starved) or at G1/S phase, as previously described. Representative confocal cell images of GFP-expressing cells under each condition are shown on the left. Bar graphs of cells counts for N/C distributions of each cofactor (black bars N > C cells; gray bars N = C cells) are shown on the right. U, untreated; SS, serum-starved cells in G0; T, thymidine-treated cells in G1/S; H, hydroxyurea-treated cells in G1/S.

(Fig. 5H, white bars). To summarize these findings, it appears that when To determine how quickly cytoplasmic actin can be imported into S phase is stalled, IQGAP1 nuclear staining increases due to a combina- the nucleus and incorporated into IQGAP1-N-induced actin polymers, tion of reduced cytoplasmic anchorage and a modest increase in nuclear we bleached the entire nucleus and monitored the fluorescence shuttling. On the other hand, the Rac1 nuclear increase was more recovery of small regions of interest (ROIs) on nuclear polymers directly attributable to altered transport dynamics, i.e. enhanced nuclear over ~550 s (Fig. 6D; Video 3). GFP-actin was imported and rapidly import and decreased export. incorporated into the induced actin polymeric structures. This implies that the IQGAP1-N-induced actin polymers are dynamic structures 3.6. Evidence for role of IQGAP1 in actin polymerization in the nucleus with high turnover rate of actin, consistent with previous claims that nuclear actin polymers are dynamic [47]. Furthermore, it demon- We next asked whether IQGAP1 can induce polymerization of strates an intimate link between cytoplasmic and nuclear pools of nuclear actin in vivo. We transiently expressed in cells the N-terminal actin, and suggests that once imported, actin may readily be utilized actin-binding fragment of IQGAP1 (IQGAP1-N), which almost exclu- by nuclear-localized actin polymerizing factors. sively localizes to the nucleus [21],and48hlaterfixed the cells for mi- croscopy. The over-expression of IQGAP1-N induced phalloidin-stained 3.7. Evidence for DNA replication stress induced interaction of IQGAP1 nuclear actin structures in a small percentage of transfected cells with actin in the nucleus (Fig. 6A and B). GFP-actin also formed similar nuclear structures when co-expressed (Fig. 6A). Deconvolution microscopy z-stacks confirmed Given that IQGAP1 and actin increase in the nucleus after DNA that these polymeric actin structures were nuclear (Videos 1 and 2). replication arrest, we applied the Duolink (Olink, Sweden) proximity Significantly, the percentage of IQGAP1-N-transfected cells displaying ligation assay to determine if these proteins interact. In this assay, the FITC-phalloidin-stained polymeric nuclear actin was increased by NIH 3T3 cells are first stained with primary antibodies against endog- 300–400% after DNA replication arrest yet remained unchanged in G0 enous IQGAP1 and actin, and then subjected to hybridization with a cells (Fig. 6B). Furthermore, GFP-Rac1 also decorated the IQGAP1- chimeric DNA-antibody secondary reagent, followed by successive N-induced nuclear actin polymers (Fig. 6C). While these experi- rounds of primer binding, polymerization and detection of potential ments should be interpreted with caution due to the transient over- complexes. A positive Duolink signal (red dot) represents a site expression of an actin-binding domain, nonetheless the same approach of protein–protein interaction. Using this assay we detected a very has been used by other studies with different actin co-factors [44–46] clear interaction between endogenous IQGAP1 and actin in the and is consistent with the idea that IQGAP1 can contribute to nuclear nucleus of cells, as determined by acquisition and scoring of selected actin polymerization in vivo. confocal cross-sections (Fig. 7A). Orthographic representations show M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347 2341

Fig. 5. G1/S arrest alters nuclear transport dynamics of GFP-tagged IQGAP1 and Rac1. NIH 3T3 cells were transfected with plasmids pGFP-IQGAP1 (see panels A–E) and pGFP-Rac1 (panels F–J). Cells were left untreated/asynchronous or arrested at S phase (as in Fig. 4). Representative FRAP confocal cell images of GFP-expressing cells under each condition are shown for both IQGAP1 and Rac1 on the left. Graphs of FRAP recovery curves for GFP-IQGAP1 (B,C) and GFP-Rac1 (G,H) show nuclear import (black bars) and export (white bars) kinetics, as analyzed in Fig. 2. Graphs of early import (black bars) and export (white bars) transport rates for IQGAP1 (D) and Rac1 (I) were determined as in Fig. 2. Graphs of cytoplasmic (import/black bars) and nuclear (export/white bars) immobile fractions for GFP-IQGAP1 (E) and GFP-Rac1 (J) are shown.

that the Duolink dots reside within the nucleus (Fig. 7B). The detection the nucleus of transfected cells by Duolink assay (Fig. 7D–F) and of specific nuclear Duolink signals (in the 5–10 and >10 categories) by immunoprecipitation (Fig. S4). actually increased from ~15% in untreated cells to ~50% in HU-treated cells (Fig. 7C). Importantly, 100% of cells in the background controls 3.8. Evidence for interdependent regulation of nuclear actin, IQGAP1 (cells exposed only to the IQGAP1 antibody or the actin antibody, but and Rac1 not to both) displayed only 0–5 Duolink dots per sample. The result is the first convincing evidence that cellular IQGAP1 and actin associate in Rac1 and IQGAP1 co-localize and associate at plasma membrane sites the nucleus. Moreover, a similar although less pronounced interaction [48]. Activated Rac1 regulates IQGAP1-mediated actin reorganization was observed between endogenous IQGAP1 and ectopic GFP-actin in in cell:cell adhesion [49],cellmigration[50,51] and cell polarization 2342 M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347

Fig. 6. Actin-binding N-terminus of IQGAP1 stimulates formation of polymeric nuclear actin. (A) Deconvolution fluorescence images of NIH 3T3 cell expressing IQGAP1-N (stained red) and co-stained with phalloidin-FITC or co-transfected with GFP-actin. (B) Graph shows % of cells expressing IQGAP1-N that also display phalloidin-FITC stained polymeric nuclear actin in asynchronous cells (blue) or cells arrested in G0 (gray) or G1/S by thymidine (green)orHU(red). * denotes p b 0.05. (C) Deconvolved fluorescence images of NIH 3T3 cells co-expressing IQGAP1-N and GFP-Rac1, showing co-localization between Rac1 and IQGAP1 at the IQGAP1-N directed polymers. (D) Representative cell images of a FRAP assay of imported cytoplasmic GFP-actin being incorporated into IQGAP1-N-induced nuclear polymers. Photo-bleaching of whole nucleus was performed as previously for GFP-actin import. Scale bars, 5 μm.

[36,52]. These cytoplasmic actin-regulators display similar cell cycle- to dominant-negative GFP-Rac1(N17) or GFP alone (Fig. 8E and F). This dependent nuclear shuttling properties ([21,23] and Figs. 5 and 6)and observation is consistent with nuclear complex formation. indeed may associate within the nucleus [53]. To investigate whether Rac1 and IQGAP1 form a nuclear complex in vivo, we employed a Rac1 3.9. Co-ordinated nuclear entry of actin and co-factors mutant which displays near complete nuclear localization pattern due to a cystine → serine mutation in the C-terminal CAAX motif (Fig. 8A; The nuclear translocation responses of actin, IQGAP1 and Rac1 [23]). First, we immunoprecipitated GFP-Rac1(L61-SAAX) and endoge- after 20 h HU treatment were remarkably similar, yet their transport nous IQGAP1 from lysates of pGFP-Rac1(L61-SAAX)-transfected cells rates differed. Cells were therefore treated with HU and fixed at pro- left untreated or with HU. We could consistently detect an increase of gressively increasing time points to test for possible differences in the IQGAP1 in GFP-Rac1(L61-SAAX) immunoprecipitates, and conversely timing of nuclear entry, which might yield insights into their regulated only slight increases of GFP-Rac1(L61-SAAX) in IQGAP1 immunopre- mobilization. The percentage of cells displaying nuclear-localized cipitates, after HU treatment compared to IgG control (Fig. 8B). Next, GFP-actin, endogenous IQGAP1 or GFP-Rac1, at indicated times of HU we used the Duolink assay (described above in Section 3.7) and in treatment, was scored by fluorescence microscopy (Fig. 8G). GFP-actin transfected NIH 3T3 cells we could identify a small but specific pool and IQGAP1 began to accumulate in nuclei within 1 h of HU, rising of cells displaying interaction between endogenous IQGAP1 and the significantly at 3–6 h post-HU and maintained similar levels up to 9 h. GFP-Rac1(L61-SAAX) in the nucleus (see Fig. 8C). The % cells with posi- GFP-Rac1 was slower to accumulate in the nucleus, and significant tive Duolink signal increased after HU treatment (~8% to 20%) (Fig. 8D). changes occurred only after 9 h. These data demonstrate that actin IQGAP1 binds exclusively to the GTP-bound active (L61) form and IQGAP1 become mobilized at similar times post-HU treatment, of Rac1 [48]. We therefore asked whether Rac1 GTPase activity or nuclear while Rac1 mobilization occurs later, implicating the possible sequen- accumulation affects the localization of IQGAP1 or actin in DNA tial assembly of an IQGAP1-centric nuclear actin complex (Fig. 8G). replication-arrested cells. To address this, we transiently expressed different Rac1 mutants and quantified their influence on nuclear 4. Discussion localization (N > C) of mCherry-actin or endogenous IQGAP1 in transfected cells. Indeed, constitutively-active GFP-Rac1(L61) and This is the first study to compare the nuclear transport dynamics nuclear-targeted GFP-Rac1(L61-SAAX) significantly enhanced nuclear of actin with specific actin regulatory co-factors in living cells. staining of both actin and IQGAP1 in thymidine-arrested cells, compared We discovered a dramatic accumulation of nuclear actin during DNA M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347 2343

Fig. 7. DNA replication stress induces formation of nuclear complexes between IQGAP1 and actin. NIH 3T3 cells were treated ± HU for 16 h and processed with the in-cell immunostaining Duolink assay to detect potential protein interactions. (A) Representative images of nuclei (stained blue with DAPI) from cells treated with primary antibodies to detect either endogenous IQGAP1 or endogenous actin (individual antibodies applied as controls), and both antibodies together. Following application of the Duolink oligo-antibody hybrids, primers, rolling circle am- plification and detection reagent (see Methods), the Duolink positive dots that represent protein interactions were detected within the nucleus by confocal microscope. (B) Orthographic representation of HU-treated cell probed for IQGAP1 and endogenous actin. In order to score accurately the nuclear Duolink dots, only nuclear trans-sections from confocal were measured (see bottom panel). Yellow line indicates where the cut-through section is positioned. (C) Graph showing the % cells with 0–5, 6–10 or >10 Duolink dots are shown, where control samples (only one primary antibody used) were 100% in the 0–5 background category. More than 120 cells were scored per sample. (D) NIH 3T3 cells were transfected with pGFP-actin-WT plasmid and treated ± HU for 16 h. Cells were processed as above by Duolink assay to detect association between endogenous IQGAP1 and ectopic GFP-actin. (E) Orthographic representation of HU-treated cell probed for IQGAP1 and GFP-actin. Yellow line indicates where the cut-through section is positioned. (F) Graph showing control samples showed 100% cells in the 0–5Duolink dot category. 75–100 cells were scored per sample. All data collated from at least two separate experiments.

replication arrest which coincided with the nuclear entry of IQGAP1 and response to DNA replication arrest, increasing by ~1.7–2.5-fold. The Rac1, but also confirmed the recent finding that nuclear actin expres- rate of GFP-actin nuclear import exceeded its rate of export, and this sion is diminished in quiescent cells [28]. A recently published RNAi finding, coupled with a detectable loss of cellular retention, explains screen found that knockdown of several actin-regulatory factors could the substantial increase of nuclear actin. induce nuclear accumulation of actin, which included not only previously identified actin transporters, such as Exp6 and profilin, but also cell cycle 4.1. A change in the balance of actin polymer stability and actin dynamics regulatory [17]. This is consistent with earlier reports that imply after DNA replication arrest that nuclear actin levels may be regulated by cell cycle-dependent cues [25–28]. Here, we define for the first time a link between DNA replication The immobile fractions of actin decreased significantly after DNA arrest in late G1/early S phase and the nuclear influx of actin, demonstrat- replication arrest (Fig. 2). This likely results from monomeric G-actin ing that both endogenous and ectopically expressed GFP-actin increase either becoming “transport competent”, due to post-translational mod- in the nucleus after S phase arrest (thymidine) and in response to DNA ifications within itself or its binding partners (e.g. actin transport facili- replication stress (HU treatment). The actin nuclear accumulation was tators and polymerization mediators cofilin or profilin), or a greater rate specific and not caused by spurious changes in nuclear pore structure or of turnover of mature cytoplasmic F-actin with subsequent increases in nuclear envelope integrity, as validated by testing of the β- shut- the availability of G-actin. The phosphorylation of cofilin and profilin can tling protein [54], which did not accumulate in the nucleus after S phase stimulate their interaction with actin [55,56], and evaluation of changes arrest (data not shown). Moreover, we show that DNA replication stress in the activity of cofilin or profilin after thymidine or HU-induced arrest induces the formation of IQGAP1–actin and IQGAP1–Rac1 complexes in could prove interesting in future. On the other hand, the SUMOylation the nucleus (Figs. 7 and 8). status of actin may change under DNA replication arrest, or throughout The nuclear transport kinetics of actin was recently investigated by the cell cycle. SUMOylation of nuclear actin may restrict the export of Dopie et al. [13] in the same cell model as this study. It is reassuring actin [57]. Whether SUMO modifications mediate actin polymerization that our FRAP analysis of GFP-actin nuclear import and export rates in within the nucleus and in consequence alter its retention is not yet untreated/asynchronous NIH 3T3 cells is comparable to that of the known. Dopie study (Fig. 2; [13]). Dopie et al. also demonstrated that tagging The fact that nuclear actin dynamics increased, and immobile actin with large fluorophores does not alter its nuclear shuttling pools of actin decreased, after DNA replication arrest infers that any dynamics. Our live cell FRAP assays have revealed for the first time pre-existing nuclear polymeric actin pools are unlikely to be stabi- that the nuclear shuttling kinetics of actin is markedly altered in lized, nor would there be a greater retention of actin at cytoplasmic 2344 M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347

Fig. 8. Evidence for interdependent regulation of nuclear actin with IQGAP1 and Rac1. (A) Fluorescence images of GFP-Rac1(L61-SAAX)-expressing cells ± HU co-stained for IQGAP1. (B) GFP-Rac1(L61-SAAX) or endogenous IQGAP1 was immunoprecipitated with specific antibodies from cell extracts ± HU. Inputs (1%) and immunoprecipitate samples were separated by 8% SDS-PAGE, transferred to membrane and probed for IQGAP1 (top panel) or GFP (bottom panel). Graphs represent relative band intensities of IQGAP1 in GFP-Rac1 IP lanes (left graph) and GFP-Rac1 in IQGAP1 IP lanes (right graph) normalized to IgG control. (C) To further demonstrate that endogenous IQGAP1 interacts with ectopic GFP-Rac1(L61-SAAX) in the nucleus, a Duolink assay was performed as described in the legend to Fig. 7. Cell images show the nucleus (stained blue with DAPI) and positive Duolink signals (red dots). Nuclear staining of Duolink dots was scored from confocal cross-sections (between 60 and 100 cells per sample) and reveal a HU-induced association between IQGAP1 and the GFP-Rac1. Cells treated with IQGAP1 or GFP antibodies alone displayed only 0–5 Duolink dots (background). (E and F) The effect of GFP alone or Rac1 mutants on subcellular distributions of (E) IQGAP1 and (F) ectopic mCherry-actin were assessed in thymidine-arrested NIH 3T3 cells. The % of cells with N > C fluorescence were scored. (G) Time-course experiment monitoring the nuclear localizations of GFP-actin, endogenous IQGAP1 and GFP-Rac1 after addition of HU. Graph compares the total % of cells displaying nuclear localization of each factor at indicated times post-HU.

locations (e.g. cell:cell/cell:matrix adhesions, stress fibers) or nuclear rapidly utilized between the two compartments, but also supports the sites (such as chromatin, transcription complexes or DNA repair idea that nuclear polymeric actin exhibits a rapid turnover rate. machinery). Indeed, the turnover of GFP-actin in cytoplasmic stress The ectopic expression of actin can alter certain cellular characteris- fibers and artificially-induced nuclear polymeric actin structures tics in vivo. Excessive amounts of ectopic GFP-actin can alter cell mor- was found to increase in thymidine-treated cells (see Supp. Fig. S5). phology [58], and when GFP-actin accounts for >30% of actin within This is supported by observations that the fluorescence recovery of dif- filaments certain characteristics such as filament sliding velocity become fuse nuclear GFP-actin was greater after photo-bleaching strips across irregular [59].InFig.S3B,wefoundthattheamountofGFP-actinineither the nucleus (similar to that performed by McDonald et al. [47])in G-actin or F-actin fractions was no different to endogenous actin. This S phase-arrested cells than in untreated cells. The non-polymerizing suggests that the amount of GFP-actin in our system does not dramati- G13R actin mutant was more dynamic in the nucleus than wild-type cally alter the behavior of endogenous actin. In vivo actin probes such actin and its dynamics/retention did not change after thymidine arrest as Lifeact are becoming more widely accepted methods of studying (see Supp. Fig. S5). These observations suggest that there is no change actin, however they do not come problem-free [60–62]. In future studies in retention of monomeric actin at nuclear sites. Indeed, we could not it is likely that specific probes for monomeric and polymeric actin will be detect any discernible differences of either endogenous or GFP-actin required to study both forms of actin [62], alongside GFP-actin. Currently in G-actin and F-actin fractions in nuclear extracts after HU treatment GFP-actin remains the best approach to measuring actin dynamics (Supp. Fig. S3B). We propose that DNA replication arrest leads to a directly in vivo. greater turnover of nuclear polymeric actin which in turn could contrib- ute to the change in nuclear GFP-actin mobility observed in our FRAP 4.2. A link between DNA replication stress checkpoint and nuclear actin experiments. We also showed that imported GFP-actin is quickly incor- porated into the IQGAP1-N-induced nuclear polymeric actin structures The levels of nuclear actin can increase in response to several (Fig. 6). This not only illustrates an intimate link between cytoplasmic forms of cellular stress, such as that induced by heat shock and ATP and nuclear actin pools, showing that actin is readily transferrable and depletion (reviewed [15]). In our study, we used HU, which depletes M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347 2345 cells of dNTPs and thereby blocks DNA synthesis; it causes not just a 4.4. Implications for a functional nuclear actin complex in arrested cells: stalling of replication forks, but triggers a mild DNA damage response. roles in S phase and stalled replication forks HU is therefore widely studied for its activation of the DNA replica- tion stress checkpoint. It is interesting that other actin-regulatory Direct evidence for a role of actin in DNA replication or the replication proteins, such as IQGAP1 and Rac1, also accumulate in the nucleus stress checkpoint in mammalian cells has yet to be directly shown. after DNA replication stress. Previously, DNA replication arrest and However, insights can be gained from viral and bacterial studies on UV-induced DNA damage were reported to provoke the N-WASp actin, and evidence from mammalian studies showing that cytoskeletal activating protein Nck to rapidly translocate into the nucleus, and proteins associate with DNA replication factors. Viruses have been the resulting depletion of cytoplasmic Nck led to the disintegration of shown to not only stimulate nuclear actin polymerization, but to employ actin stress fibers [63]. It is possible that this might partly contribute to polymeric actin for replication purposes [69,70]. Bacterial may also the stress-dependent decrease in cytoplasmic retention of actin seen in have roles in DNA replication. For instance, MreB, the bacterial actin this study. More recently, polymeric nuclear actin was implicated in sta- homolog, was shown to mediate chromosome segregation [71] and bilizing the DNA repair proteins Ku70/80 at IR- and UV-induced sites of regulate DNA Topoisomerase (Topo) IV activity; in the latter case, poly- damaged DNA. Nuclear actin polymers may therefore stimulate more ef- meric MreB stimulated while monomeric MreB inhibited Topo IV activity fective repair of damaged DNA [4], potentially through the sequestration [72]. of repair proteins or via scaffolding and stabilizing DNA at damaged sites. Certain cytoskeletal proteins have been found in the nu- It remains to be determined to what extent actin might actually accumu- cleus and to associate with DNA replication machinery and mediate late at repair protein complexes at damaged DNA. In our study, the use of progression through S phase. XVIb co-locates with proliferating HU had the most profound impact on actin transport dynamics (Fig. 2), cell nuclear antigen (PCNA) and its overexpression slows S phase and it will be interesting in future studies to determine the effect of progression [44]. We previously showed that nuclear localized IQGAP1 more potent and genotoxic DNA damage agents on the actin nuclear interacts with the DNA replication apparatus through binding to PCNA translocation response. and replication protein A subunit RPA32, and that silencing of IQGAP1 expression caused a delay in progression through S phase [21]. Roles for Rac1 in DNA replication and repair are only beginning to 4.3. Co-ordinated translocation of actin cytoskeletal regulators after DNA be established. Rac1 was recently found to associate with -B1 replication arrest: tight control of a nuclear IQGAP1–Rac1 complex and several histones as well as Topo II [53]. Signaling of Rac1 was required for a DNA damage response induced by Topo II poisons [73]. The assembly of an IQGAP1–Rac1 complex at the plasma membrane Furthermore, IR-induced DNA damage stimulated Rac1 activity, inhibi- is important for actin rearrangements during cell migration and polari- tion of which attenuated integrity of the G2/M cell cycle checkpoint zation [48], raising the possibility of a similar functionality in the nucle- [74]. We speculate that Rac1 regulates these events not only via signal- us. In this context, we note that ectopic expression of the actin-binding ing mechanisms, but directly through association with nuclear binding N-terminus of IQGAP1 induced formation of GFP-actin polymeric struc- partners such IQGAP1 (Fig. 8). Rac1 is likely to act as the distinct tures, which stained positive with phalloidin (Fig. 6). Moreover, IQGAP1 regulator of an actin–IQGAP1–Rac1 complex. Rac1 mediates the nuclear and actin were observed in the sedimentation fraction of nuclear localization of several proteins [75,76], and ectopic expression of nuclear- extracts (which contain actin polymers), and these increased modestly targeted active Rac1 increased the percentage of cells displaying high after replication arrest (Fig. S3B). The N-terminus of IQGAP1 bundles levels of nuclear IQGAP1 or mCherry-actin in cells arrested at S phase actin filaments [64,65] and binds N-WASp [66,67], a cytoskeletal com- (Fig. 8). This suggests that active nuclear Rac1 may act to retain IQGAP1 ponent shown to be ubiquitously nuclear and to stimulate actin poly- and actin within the nucleus. Thus, Rac1 possesses distinct nuclear activ- merization in nuclear extracts in vitro [33]. We therefore speculate ities, with roles expected to involve DNA replication and/or replication that nuclear-localized IQGAP1 acts upon nuclear polymeric actin either stress checkpoint mediation. through its actin-bundling/cross-linking activity, which is exaggerated when the predominantly nuclear-localized IQGAP1-N is over-expressed, 4.5. Conclusions and implications or via an N-WASp-mediated mechanism. These nuclear actin polymers also increased after DNA replication arrest, presumably facilitated by the The coordinated recruitment of different actin-polymerization co- coordinated import of actin, endogenous IQGAP1 and Rac1. In future factors to the nucleus after induction of DNA replication arrest may be studies it would be interesting to test whether IQGAP1 associates with required to generate actin polymers for specific processes including N-WASp, or other actin-polymerizing factors like Dia1 [68],toinfluence initiation and/or progression of DNA replication [21], chromatin remod- nuclear actin polymerization. eling [77], cell cycle stage-specific transcription of genes [3,78] or DNA The formation of an actin polymerizing complex within the nucleus repair. It is therefore plausible that the replication stress-induced nuclear would likely be under tight control. Actin and IQGAP1 were mobilized uptake of these factors may regulate the DNA replication machinery at similar times post-HU treatment, while Rac1 mobilization occurs (e.g. through protection of stalled replication forks) and provide some later, implicating the sequential assembly of an IQGAP1-centric nuclear type of feedback control of the replication process. The differences in actin complex (Fig. 8). This is strongly supported by our Duolink assay nuclear entry timing and dynamics of actin, IQGAP1 and Rac1 reveal data which identify for the first time an interaction between IQGAP1 that distinct mechanisms regulate the nuclear shuttling of cytoskeletal and actin in the nucleus, with evidence for an increase in complex factors with important consequences for the rate of assembly of actin formation after HU-induced stress (Fig. 7). The FRAP approach allowed polymerization complexes. This would allow for distinct regulation of us to compare the basis for elevated levels of nuclear actin, Rac1 and these cytoskeletal-turned-nucleoskeletal co-factors to induce actin poly- IQGAP1 after replication arrest. Increased IQGAP1 nuclear localization mer formation within the nucleoplasmic environment in response to a reflected a considerable reduction in its anchorage in the cytoplasm range of stimuli. with only modest changes in import rate (Figs. 4 and 5; [21]). However, Supplementary data to this article can be found online at http:// it is interesting that Rac1 and actin were directed to the nucleus through dx.doi.org/10.1016/j.bbamcr.2013.06.002. increased rates of import. Whereas for export, actin dynamics increased but Rac1 export rate significantly decreased upon replication arrest. Acknowledgements Our novel findings of Rac1 nuclear transport kinetics may explain the previous observation that nuclear Rac1 levels increase during Many thanks to those who supplied plasmid DNA and to the mem- early S phase and peak at G2/M [23]. bers of the Henderson lab for helpful comments. This work was funded 2346 M.A. Johnson et al. / Biochimica et Biophysica Acta 1833 (2013) 2334–2347 by grants to B.R.H. from the National Health and Medical Research [30] D.T. Brandt, R. Grosse, Get to grips: steering local actin dynamics with IQGAPs, EMBO Rep. 8 (2007) 1019–1023. Council (NH&MRC) of Australia, the Cancer Council of New South [31] A. Pelikan-Conchaudron, C. Le Clainche, D. Didry, M.-F. Carlier, IQGAP1 is a Wales and the Cancer Institute New South Wales (CINSW). B.R.H. is calmodulin-regulated barbed end capper of actin filaments: possible implications a Senior Research Fellow of the NH&MRC and supported by a career in its function in cell migration, J. Biol. 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