Steady-state nuclear actin levels are determined by export competent actin pool

Steady-State Nuclear Actin Levels are Determined byExport Competent Actin Pool

Kari-Pekka Skarp, Guillaume Huet, and Maria K. Vartiainen*Program in Cell and Molecular Biology, Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00014, Helsinki, Finland

Received 3 May 2013; Revised 22 May 2013; Accepted 23 May 2013Monitoring Editor: Laura Machesky

A number of studies in the last decade have irrevocablypromoted actin into a fully fledged member of thenuclear compartment, where it, among other crucialtasks, facilitates transcription and chromatin remodel-ing. Changes in nuclear actin levels have been linked todifferent cellular processes: decreased nuclear actin toquiescence and increased nuclear actin to differentia-tion. Importin 9 and exportin 6 transport factors areresponsible for the continuous nucleocytoplasmic shut-tling of actin, but the mechanisms, which result inmodulated actin levels, have not been characterized. Wefind that in cells growing under normal growth condi-tions, the levels of nuclear actin vary considerably fromcell to cell. To understand the basis for this, we haveextensively quantified several cellular parameters whileat the same time recording the import and export ratesof green fluorescent protein (GFP)-tagged actin. Sur-prisingly, our dataset shows that the ratio of nuclear tocytoplasmic fluorescence intensity, but not nuclearshape, size, cytoplasm size, or their ratio, correlatesnegatively with both import and export rate of actin.This suggests that high-nuclear actin content is main-tained by both diminished import and export. Thehigh nuclear actin containing cells still show highmobility of actin, but it is not export competent, sug-gesting increased binding of actin to nuclear complexes.Creation of such export incompetent actin pool wouldensure enough actin is retained in the nucleus andmake it available for the various nuclear functionsdescribed for actin. VC 2013 Wiley Periodicals, Inc.

Key Words: actin; nucleus; nuclear transport; actin poly-merization; nucleo-cytoplasmic shuttling

Introduction

Actin is an integral component of the cytoskeletal appa-ratus involved in cellular motility. In addition to the

main structural role in the cytoskeleton, it is currentlyknown that actin exists also in the nucleus. In this compart-ment, the exact form and functions of actin have been elu-sive although the presence of many cytoplasmic actin-binding proteins (ABPs) [Suetsugu and Takenawa, 2003;Yoo et al., 2007; Miki et al., 2009] suggest a role not com-pletely unlike from the one played in the cytoplasm. How-ever, large classical F-actin structures such as stress fibers arenot found in the nucleoplasm [de Lanerolle and Serebryan-nyy,2011]. In fact, many of the roles assumed by actin inthe nucleus are specific to that compartment. For example,it is known that actin decorates many of the nuclear com-plexes involved in gene expression from RNA polymerases[Hofmann et al., 2004; Hu et al., 2004; Philimonenkoet al., 2004] to the actual messenger ribonucleoprotein par-ticles (mRNPs) traveling out of the nucleus [Kukalev et al.,2005; Obrdlik et al., 2008] and has been linked to themovement of chromosomal loci [Chuang et al., 2006].Actin together with actin-related proteins (Arps) are com-mon binding partners of many chromatin remodeling com-plexes facilitating transcription [Oma and Harata,2011]and many nuclear actin tasks also require myosins, which inthe cytoplasm cooperate with actin in cargo transport[Pestic-Dragovich et al., 2000; Ye et al., 2008; de Lanerolleand Serebryannyy, 2011]. In summary, actin maintains gen-eral transcription while at the same time retaining the capa-bility to inflict very specific regulation of individual genes,such as serum response factor target genes [Vartiainen et al.,2007]. The involvement of actin in gene expression hasbeen recently reviewed in these articles [de Lanerolle andSerebryannyy, 2011; Huet et al., 2012; Percipalle, 2013].

Despite the important role of actin in various nuclearprocesses, the polymerization status of nuclear actin is stilllargely unclear. Recent biochemical data are clarifying thisissue at least in chromatin remodeling complexes. In theINO80, complex actin is monomeric and its barbed end isnot accessible for polymerization. Instead, the pointed end

Abbreviations: ABP, actin-binding proteins; FRAP, fluorescencerecovery after photobleaching.*Address correspondence to: Maria K. Vartiainen; Program in Cell

and Molecular Biology, Institute of Biotechnology, University ofHelsinki, Viikinkaari 9, 00014 Helsinki, Finland.E-mail: [email protected]

Published online 11 July 2013 in Wiley Online Library(wileyonlinelibrary.com).

RESEARCH ARTICLECytoskeleton, October 2013 70:623–634 (doi: 10.1002/cm.21116)VC 2013 Wiley Periodicals, Inc.

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of actin appears to regulate the interaction between INO80and chromatin [Kapoor et al., 2013]. Arps may have a cru-cial role in maintaining actin in the monomeric form in thechromatin remodelers. At least yeast Arp4 and Arp8 influ-ence actin polymerization in vitro, and may help to keepactin monomeric within the INO80 complex [Fenn et al.,2011].

Nevertheless, circumstantial evidence has for a long timesuggested that nuclear actin would also be capable of form-ing polymers. For example, many of the nuclear processesinvolved in gene expression that harness actin one way oranother are disturbed on application of drugs affecting actinpolymerization [McDonald et al., 2006; Ye et al., 2008]. Inaddition, many proteins that either bind to actin filamentsor regulate their polymerization have been linked to geneexpression. These include for example N-WASp [Miyamotoet al., 2011], coronin-2a [Huang et al., 2011], cofilin[Obrdlik and Percipalle, 2011], and several motor proteinmyosins [de Lanerolle and Serebryannyy, 2011].

Also light microscopy methods with fluorescently labeledactin have been used to gain insight into the type of actin tak-ing action in the nuclei of living cells. Earlier, nuclear actinwas shown to have similar two phased curve in fluorescencerecovery after photobleaching (FRAP) experiments in thenucleus as well as in the cytoplasm, representing monomericand polymeric pools, respectively [McDonald et al., 2006].Recently, in a significantly longer nuclear FRAP experimentwith GFP-actin and GFP-actin-R62D, which cannot poly-merize, three recovery phases were found with the wild typeand two with the unpolymerizable mutant. This suggeststhat nuclear actin exists at least in three forms of differentmobility and while the first two can be explained classicallyas monomers and polymers, the third phase might corre-spond to actin bound by nuclear complexes. In support ofthis, a longer phase was also present for the mutant lackingthe traditional second phase [Dopie et al., 2012]. Recentdata have extended these studies by using probes that specifi-cally detect polymeric actin. The Mullins laboratorydesigned fluorescent probes based on various actin-bindingdomains found in the eukaryotic genome. A probe using theRPEL domains from MRTF-A recognizing monomeric actinlocalized to nuclear speckles. Another probe based on trun-cated utrophin calponin homology domains with reducedbundling capabilities (Utr-230) recognized actin filaments insmall distinct puncta. Interestingly, the latter did not colocal-ize with any tested markers for nuclear bodies and were dis-placed by chromatin. The actin structures labeled with thisprobe may therefore form part of the viscoelastic structure ofthe nucleoplasm [Belin et al., 2013]. Another study usingLifeact peptide demonstrated transient actin polymerizationin the nucleus that was dependent on formins mDia1 andmDia2. These structures also stained with phalloidin, andplayed an important role in the activation of SRF cofactorMRTF-A [Baarlink et al., 2013]. This data suggest the pres-ence of formin compatible actin monomers in the nucleus

perhaps in the form of polymerization competent profilin–actin complex. More studies are needed to understand thedifferences in the polymeric actin structures visualized bythese two approaches. Nevertheless, they provide the firstglimpse of polymerized endogenous nuclear actin.

Despite the size of actin, which is close to the passive dif-fusion limit of the nuclear pore, active machinery seems togovern both nuclear import and export of actin. Nuclearexport of actin is mediated by the transport factor exportin6 [Stuven et al., 2003] and import by importin 9 [Dopieet al., 2012]. Depletion of importin 9 decreases nuclearactin levels and also impairs transcription in an actin-dependent manner. Therefore, active nuclear import isrequired to maintain nuclear actin levels and to sustaintranscription [Dopie et al., 2012]. The discovery that“enough” actin in the nucleus is required for transcriptionis interesting in the context that low levels of nuclear actinhave been linked to cell quiescence in epithelial cells. There,information transmitted from the ECM by laminin-111 isrealized in the nuclear compartment as actin depletionresulting in cell quiescence [Spencer et al., 2011]. Theopposite phenomenon has been observed in myeloid celldifferentiation model HL-60, where after induction, aninflux of actin into the nucleus takes place [Xu et al.,2010]. More precisely, actin is recruited to several gene lociwhile also being required along with Brg1 to activate amyeloid-specific gene [Xu et al., 2011]. In steady-state cells,actin has been shown to shuttle continuously between thenucleus and the cytoplasm and the actin monomers seem tobe limiting factor in both import and export [Dopie et al.,2012]. Which process(es) is=are affected in the case ofincreased=decreased nuclear actin levels are not known.

Here, we have examined the steady-state distribution ofactin between the nuclear and cytoplasmic compartmentsin cells growing under normal culture conditions and dis-playing varied nuclear actin levels. By measuring both thenuclear import and export rates, as well as cellular parame-ters, we find that the levels of nuclear actin inversely corre-late with the nucleocytoplasmic shuttling speed. Thissuggests that the amounts of nuclear actin are maintainedby binding to nuclear complexes and thereby restricting theexport competent pool of actin.

Results

Systematic Analysis of Cellular Parameters thatAffect Nuclear Import and Export of Actin

As aforementioned, actin monomers are constantly trans-ported between the nucleus and the cytoplasm, and theamount of nuclear actin varies in certain cell fates as well asaffects transcription. In this context, it is interesting to notethat the amount of nuclear actin markedly varies already incells under normal cell culture conditions (Fig. 1A).Because nuclear actin dynamics are linked to such vital

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cellular processes, we wanted to understand what kind ofcellular parameters might affect and perhaps regulate thisongoing process. This knowledge is essential to fully under-stand how nuclear actin levels may regulate important cellfate decisions.

In our laboratory, we have developed tools to quantita-tively measure nuclear import (FRAP) and export

(fluorescence loss in photobleaching (FLIP)) rates in livingcells using fluorescent tags with confocal microscopy. Thesemethods have already been successfully applied to GFP-actin as well as other constructs and fluorescent tags andvalidated GFP-actin as a tool to study nuclear actin trans-port [Dopie et al., 2012]. Thus, we transiently transfectedcells with GFP-actin and the unpolymerizable mutant

Fig. 1. (A) NIH 3T3 mouse fibroblasts and MCF-7 human epithelial breast cancer cells transfected with GFP-actin. Fluores-cence signal intensities in both compartments have been quantified and the ratio on nucleus=cytoplasmic (N=C) fluorescence calcu-lated. Scale bar, 10 lm. (B–E) Total cellular GFP-actin fluorescence signal intensity compared to N=C fluorescence in NIH 3T3 (Band C) and MCF-7 (D and E) cells. The regression line was represented and the Pearson product-moment correlation coefficient (q)was calculated for each set of data compared. An asterisk indicates a statistically significant correlation. Averages in each graph repre-sent mean signal intensities 6 std, n 5 98 (B), n 5 51 (C), n 5 48 (D), n 5 57 (E). Fluorescence intensity does not drive actinlocalization in either of the cell models.

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GFP-actin-R62D and applied this methodology to thewide spectrum of cells of different expression levels foundon a NIH 3T3 or MCF-7 cell culture plate (Fig. 1A). NIH3T3 is a mouse fibroblast cell line exhibiting an elegantactin cytoskeleton [Jainchill et al., 1969] while MCF-7 is ahuman epithelial cell line used to study breast cancer [Souleet al., 1973]. To cover a range of parameters possibly affect-ing actin transport, we also recorded cell physiologicalparameters for each individual cell such as shape of nucleus(round or elongated) and size of nucleus=cytoplasm inaddition to fluorescence intensity in both compartments.With sufficient n (�40–100), these parameters can then becompared to each other and to rates of transport to uncoverunderlying correlations.

Initially, we wondered whether the expression level itselfmight affect the localization of the GFP-actin construct, forexample, by pushing the construct more toward the nucleus,for example, by saturating the export process. The range ofnuclear=cytoplasm (N=C) ratio in NIH 3T3 varied between0.69 and 2.74 for the wild type (Fig. 1B) and 0.55 and 2.59for the R62D mutant (Fig. 1C) and in MCF-7 cells between0.27 and 1.71 (Fig. 1D) and 0.34 and 1.99 (Fig. 1E), respec-tively. When the N=C fluorescence intensity ratio is com-pared to the total cell fluorescence intensity, no obvioustrend is visible either with the wild-type GFP-actin (Fig. 1B)or with the R62D mutant (Fig. 1C) in NIH 3T3 cells (seeMaterials and Methods for the details of data and statisticalanalysis). Similarly, with MCF-7 cells, our range of expres-sion levels does not facilitate ectopic accumulation of theprotein (Figs. 1D and 1E) and further validates GFP-actinfor the study of fluctuating nuclear actin levels in steady-statecells. In NIH 3T3 cells, the fluorescence range of wild-typeactin is wider than with the R62D mutant while the oppositeis true in MCF-7 cells. It is possible this reflects the incon-venience the cell has to suffer due to overexpression and thecell-type specific preferences for various types of actin.

Nuclear Transport Rates of Actin do notCorrelate with Nucleus Shape, Nucleus Size,Cytoplasm Size, or N=C Size Ratio

We then started to systematically compare the parametersrecorded to actual import and export rates of GFP-actinand GFP-actin-R62D. For schematics of the assay andexample cells, see Fig. 2A. In NIH 3T3 cells, wild-typeimport rate varied between 0.022 and 0.060 (Fig. 2B) andexport between 0.002 and 0.012 (Fig. 2D). For the R62Dmutant, the values are 0.023–0.089 (Fig. 2C) and 0.004–0.022 (Fig. 2E), respectively. In MCF-7 cells, the importand export range for the wild type was 0.024–0.082 (Fig.4E) and 0.006–0.031 (Fig. 4F) and for R62D 0.026–0.095(Fig. 4G) and 0.004–0.029 (Fig. 4H). As reported earlier,GFP-actin-R62D was transported faster than the wild-typeGFP-actin [Dopie et al.2012].

Again, the total cell fluorescence does not significantlycorrelate with either, import (Figs. 2B and 2C) or export

rate (Figs. 2D and 2E), confirming that the nucleocytoplas-mic transport machinery remains largely intact by theexpression levels used. This was true also for MCF-7 cells(data not shown).

Actin-dependent nuclear elongation has been linked togene expression at least in T-cells [Gupta et al., 2012],while Brg1, a chromatin remodeling complex binding actinhas been shown to be responsible for nuclear deformationindependent of cytoplasmic influences [Imbalzano et al.,2013]. Also, cells expressing GFP-actin-R62D mutantappear to be slightly elongated due to the X versus Y ratioof their nuclei radii on average is 1.45 6 0.11 versus 1.356 0.09 (P 5 0.00705) of the wild type. This prompted usto investigate the role of nuclear shape in import and exportof actin. Our data show no correlation between the shapeof the nucleus and these processes (Figs. 3A and 3B). Inaddition to general cell size and shape, actin has also beenlinked to the machinery responsible for nuclear size throughnesprins, which anchor cytoplasmic actin structures to lam-ins at the nuclear envelope [Chambliss et al., 2013]. Thus,we next investigated whether the area of the nucleus or thecytoplasm or their N=C area ratio might correlate withnuclear actin import or export. This was not the case andactin is steadily transferred across the nuclear enveloperegardless of these parameters (Figs. 3C–3H).

Actin Shuttling Rates Correlate Inversely withFluorescent N=C Ratio

Finally, we compared the N=C ratio of fluorescence inten-sities to nuclear import and export rates. Surprisingly, GFP-actin is imported into the nucleus faster in cells with lowfluorescence N=C ratio, that is, in cells with less actin inthe nucleus compared to cytoplasm (Fig. 4A). Fast transportrate for lower fluorescence N=C ratio was confirmed to bethe case also in the export of GFP-actin (Fig. 4B), suggest-ing a scenario where relatively low nuclear actin concentra-tion is due to expedited import and export. Inversely,relatively high nuclear actin concentration is maintained byboth diminished import and export. Transport rates corre-lated inversely with N=C ratio also with the export of GFP-actin-R62D (Fig. 4D), which is unable to polymerize.However, despite the similar trend, the import rate of theR62D mutant versus N=C ratio was not statistically signifi-cant (Fig. 4C). Importantly, the same scenario takes placein MCF-7 cells both with the wild type and R62D (Figs.4E–4H) with also here the R62D import rate versus N=Cratio not being statistically significant.

Intranuclear Mobility of Actin does notCorrelate with Nuclear Actin Levels

If cells were to adjust their actin N=C ratio by modulatingthe size of transport competent G-actin pool, our datawould suggest that the increased amount of actin in thenuclei of high N=C ratio cells is less mobile than in cells


Fig. 2. (A) Illustration of import (upper panels) and export (lower panels) assays. Representative images of cells are shown at dif-ferent time-points indicated in seconds. Pre: before bleaching; Post: immediately after bleaching. For export assays, the cytoplasm wascontinuously bleached. Fluorescence increase (import assay) or decrease (export assay) was measured at the level of the whole nucleus.Illustrative cartoons are shown below each picture. Scale bar, 10 lm. (B–E) Total cellular GFP-actin fluorescence signal intensitycompared to import and export rates of GFP-actin (B and D) and GFP-actin-R62D (C and E). The regression line was representedand the Pearson product-moment correlation coefficient (q) was calculated for each set of data compared. An asterisk indicates a stat-istically significant correlation. Averages in each graph represent mean signal intensities and transport rates 6 std, n 5 98 (B), n 551 (C), n 5 38 (D), n 5 35 (E). Nucleocytoplasmic transport remains unaffected by the range of actin expression used.


Fig. 3. Shape of the nucleus (A,B), area of the nucleus (C,D), cytoplasmic area (E,F), and nuclear to cytoplasmic (N=C) ratio ofareas (G,H) compared to the import (A,C,E,G) and export (B,D,F,H) of GFP-actin. The regression line and the Pearson product-moment correlation coefficient (q) were determined for each compared dataset. An asterisk indicates a statistically significant correlation.Averages in each graph represent mean shape (index of X-Y radii of nuclei) or area and transport rates 6 std, n 5 98 (A,C,E,G) and n5 38 (B,D,F,H). Neither parameter seems to affect import of GFP-actin.


Fig. 4. Import and export of GFP-actin (A,B,E,F) and GFP-actin-R62D (C,D,G,H) compared to nucleus=cytoplasm (N=C)fluorescence ratio in NIH 3T3 (A–D) and MCF-7 (E-H) cells. The regression line was represented and the Pearson product-moment correlation coefficient (q) was calculated for each set of data compared. An asterisk indicates a statistically significant correla-tion. Averages in each graph represent mean signal intensities and transport rates 6 std, n 5 98 (A), n 5 38 (B), n 5 51 (C), n 535 (D), n 5 48 (E), n 5 54 (F), n 5 57 (G), and n 5 38 (H). An obvious trend suggests import and export is fast in cells of lowN=C ratio and low in cells of high N=C ratio.


Fig. 5. (A) Illustration of intranuclear and cytoplasmic mobility assays. Representative images of cells are shown at differenttime-points indicated in seconds (s). Pre: before bleaching; Post: immediately after bleaching. Fluorescence recovery was measured atthe level of the bleached area, and in the case of intranuclear FRAP normalized to the whole nuclear intensity to exclude the effectsof import=export. Illustrative cartoons are shown below each picture. Scale bar, 10 lm. (B–E) Nucleus=cytoplasm (N=C) fluorescenceratio compared to the intranuclear and intracytoplasmic mobility of actin. The mobility of actin was assessed through the measure-ment of the half-time recovery (t1=2) of actin signal (B and D) and the immobile actin fraction (C and E) in the nucleus (B and C)or the cytoplasm (D and E) of NIH 3T3 cells. Both parameters were compared to N=C ratio of actin. The regression line was repre-sented and the Pearson product-moment correlation coefficient (q) was calculated for each set of data compared. Averages in eachgraph represent mean signal intensities and rates of mobility 6 std, n 5 29 (B,C), n 5 20 (D,E). The mobility of actin in bothnucleus and cytoplasm remains similar regardless of the relative amount of actin in the nucleus compared to cytoplasm.


with lower N=C ratio. To directly test this, we designed anintranuclear FRAP assay aimed at measuring intranuclearmobility. In this assay, we bleach an area in the center of thenucleus symmetrical to the shape of the nucleus and exclud-ing nucleoli. In contrast to the import FRAP assay, The X-Y diameters of the area are only one quarter of the nucleusto ensure the immediate recovery is intranuclear. To ensurethis further, the bleaching time is minimized and recoveryis normalized to whole nuclear fluorescence, which shouldexclude the effect of import and export from the assay (Fig.5A). We did not find any significant correlation betweenthe intranuclear mobility and actin N=C ratio (Figs. 5Band 5C). Moreover, the cytoplasmic mobility of actin didnot correlate with nuclear actin levels (Figs. 5D and 5E).Thus, our data support a model where cell maintains highnuclear actin concentration by diminishing both importand export by means other than modifying global cellularG-actin pool. At the same time, there exists a pool of actinwithin the nucleus capable of high mobility despite beingexcluded from export.

Discussion

Sufficient amount of nuclear actin is required for transcrip-tion [Dopie et al., 2012], and dramatic changes in nuclearactin amounts have been linked to both quiescence[Spencer et al., 2011] and differentiation [Xu et al., 2010].However, the mechanisms resulting in varied nuclear actinlevels have not been characterized. Basically, it is possiblefor the cell to regulate the amount of nuclear actin in actin-dependent and actin-independent manner. The latter con-cerns regulating the transport machinery itself for example,the expression levels and=or activity of exportin 6 andimportin 9. This scenario seems to take place at least in theXenopus oocyte, where the large actin pool needed to sup-port the huge nuclei of these cells is maintained by post-transcriptional down-regulation of exportin 6 [Bohnsacket al., 2006]. The actin-dependent regulation involves theregulation of nuclear and cytoplasmic G-actin pools, thesizes of which form a limiting factor for nucleocytoplasmictransport, because actin is carried across the nuclear enve-lope as a monomer [Dopie et al., 2012]. This step involvesthe actin treadmilling regulating small ABPs, cofilin, andprofilin, which also play a role in import [Dopie et al.,2012] and export [Stuven et al., 2003] of actin, respectively.In addition to polymerization, the availability of actinmonomers will also depend on other binding events ofactin. In the nucleus, this means association of actin withmany gene expression machineries. Indeed, FRAP experi-ments have suggested that over 50% of actin is relativelystably bound to nuclear complexes [Dopie et al., 2012].The actin-dependent and independent possibilities do notexclude each other and could be used to induce effects ofdifferent magnitude. Intuitively, actin-dependent regulationof nuclear actin import and export might allow a faster

reaction to cellular needs, because it is nongenetic and theself-regulatory nature makes the circuit smaller while simul-taneously avoiding disruption of other possible cargos ofexportin 6 and importin 9. Actin monomer pool hasalready been shown to regulate itself via Phactr family ofproteins, which can activate cofilin when actin monomerlevels are low, which in turn converts F-actin back to G-actin [Huet et al., 2013]. Because homeostasis favors feed-back loops, something similar might take place also in thenucleus.

Our data show that nuclear actin levels vary considerablyeven in cells growing under normal culture conditions (Fig.1). Importantly, the experimental set-up, for example, theuse of GFP-actin did not affect the scenario. At the presenttime, we do not know the biological difference between thecells demonstrating varied nuclear actin levels. It is temptingto speculate that the actin level might reflect the transcrip-tional activity of the cell. On the other hand, as we did notsynchronize the cells, the steady-state cell population willcontain cells from most cell cycle phases. None of the cellu-lar parameters (nuclear shape and size, cytoplasm size ornuclear to cytoplasm size ratio, Fig. 3) that we recorded cor-related with either nuclear actin levels or with actin transportrate. This information will be very useful in future studies toelucidate the different cellular states that affect nuclear actinlevels, by allowing the elimination of many parameters.

Intuitively, one could assume that increased nuclear actinwould be a result of either increased import and=ordecreased export, and vice versa for the decreased nuclearactin. In our cells, we found an intriguing steady-state situa-tion, where high nuclear actin containing cells displayedboth decreased import and decreased export. On the otherhand, low nuclear actin containing cells showed faster shut-tling in both directions (Fig. 4). This phenomenon wasapparent in both cell lines we studied and applied to bothnuclear import and export of wild-type actin and nuclearexport of the unpolymerizable actin mutant. However, nosignificant correlation was evident for the unpolymerizableactin mutant in the context of nuclear import in either cellline (Figs. 4C and 4G). This indicates that in the cyto-plasm, the availability of actin monomers above a certainthreshold does not affect import. This suggest that eitherthe import machinery becomes saturated or that only a sub-population of actin is shuttled in and out of the nucleus,and that this pool is not directly sensitive to the total actinmonomer content. Supporting this notion, the generalmobility of cytoplasmic actin as measured by FRAP did notcorrelate with nuclear actin levels (Figs. 5D and 5E).

Cells with increased nuclear actin displayed decreasednuclear export, suggesting that in these cells, actin was notexport competent. This could indicate that actin was eitherbound to nuclear complexes or polymerized and thereforenot available as a transport-competent monomer. To oursurprise, the intranuclear FRAP revealed that cells withhigh nuclear actin levels showed similar high mobility than


low actin containing cells (Figs. 5B and 5C). The exportincompetence of actin in high nuclear actin cells is notlikely due to increased polymerized actin, because at leastthe actin foci stained with the truncated utrophin CH-domain probe diffuse relatively slowly within the nucleus[Belin et al., 2013]. Many transcription associated proteinsshow very dynamic behaviors within the nucleus, with theirFRAP recovery profiles showing t1=2 values of some seconds[Carrero et al., 2003]. Our current FRAP techniques andmethods do not allow us to reliably distinguish this frommonomer actin diffusion. Moreover, the long-term FRAPexperiment which we had used previously to reveal the sta-ble pool of actin [Dopie et al., 2012], and where thenucleus is bleached completely void of fluorescence, andthe recovery depends solely on imported material, was notamenable for the large sample numbers required here. Atthe moment, we favor the hypothesis that in high nuclearactin cells, more actin is bound to nuclear complexes. How-ever, we cannot also rule out the possibility that actin issomehow else rendered export-incompatible, for instancethrough a post-translational modification. Increased bind-ing of actin to nuclear complexes, for example, to thoseengaged in transcription, would then decrease the availableactin monomers for export. If only a subpopulation of actinshuttles, this would then explain why also nuclear import isaffected in these cells.

GFP-actin is a commonly used tool to study actindynamics especially in the cytoplasm. Our data here andpreviously indicates that it is a good probe to measurenuclear actin levels [Dopie et al., 2012], and it has alsobeen used by us and others [McDonald et al., 2006] tostudy intranuclear dynamics of actin. The recent reportsutilizing other probes to visualize especially polymeric actinmust force us to reconsider the way we visualize nuclearactin. The finding that formins play a crucial role in nuclearactin polymerization creating “canonical” phalloidin stain-able filaments at least under some circumstances [Baarlinket al., 2013], suggest that GFP-actin might not be an idealprobe to study nuclear actin polymerization, as it is not avery good substrate for formin-mediated actin polymeriza-tion [Chen et al., 2012]. Nevertheless, the filamentous actinprobe developed by Mullins laboratory displayed similar-ities in its kinetic behavior to those measured previously forGFP-actin [McDonald et al., 2006; Belin et al., 2013].Also, the fluorescence recovery curves for their probeshowed t1=2 of 195 s, while our value for the second phase,which we deemed polymeric in our long FRAP assay was230 s [Dopie et al., 2012]. All three probes mentioned hereare likely to recognize at least partially different subpopula-tions of actin in the nucleus. In the future, it will be impor-tant to decipher how these subpopulations are related toeach other, and how their dynamics change, for example incells with varied nuclear actin levels.

Our data suggest a model, where at steady state, thenuclear actin levels are governed by export competent actin

monomers. The similarity of the results in two unrelatedcell lines, mouse fibroblastic cells and human epithelialcells, suggests the conservation of this regulation of nuclearactin levels in all cell types, at least in mammals. Becausewe have here only measured the import and export kineticsat steady-state, we do not know which event initiates thechange in nuclear actin. As cytoplasmic actin monomerslevels do not affect actin import rates, then merely theincreased binding of actin in the nucleus is sufficient toaffect both import and export processes thereby amplifyingthe effect. On the other hand, increased import ordecreased export might result in increased nuclear bindingof actin, which can then affect for example different geneexpression programs. This raises the question, whetherincreased=decreased actin is a consequence or a reason fordifferential nuclear activity. Detailed measurements of actinnucleocytoplasmic shuttling studies in a system, whichshows robust changes in nuclear actin content, and takinginto account the basic principles outlined here, will help toresolve this issue.

Materials and Methods

DNA Constructs

GFP-actin and GFP-actin-R62D expression vectors arebased on the pEGFP-C1 plasmid from Clontech. Humanbeta-actin cDNA with stop codon was inserted intoHindIII-KpnI sites.

Cell Culture and Transfection

NIH 3T3 and MCF-7 cells were cultured in DMEM(Sigma) supplemented with 10% FCS (GIBCO) and main-tained in 37�C with 5% CO2. NIH 3T3 cells (120,000) orMCF-7 cells (500,000) were plated on 35-mm Falcondishes. The day after plating, NIH cells were transfectedwith jetPRIMEVR (Polyplus) and MCF-7 with Lipofect-amine 2000VR (Invitrogen) according to manufacturer’sinstructions. The total amount of transfected DNA was 1lg while the actual amount of expression plasmids was nomore than 100 ng (NIH 3T3) or 200 ng (MCF-7) and theremaining was topped with an empty plasmid. The cellswere imaged the day after transfection.

Live-Cell Imaging

Leica TCS SP5 scanning confocal microscope was usedunder 37�C hood with 5% CO2. It is equipped with 230mW optically pumped semiconductor laser, which was setto 90% hardware laser power. All data were collected with a63 3 0.9 NA HCX APO L objective in 12-bit format andwith pinhole 1. Gain was kept at constant 1000 V throughall samples. Images were taken using 1.5% software laserpower while all bleaching was done with 100% percent SWpower using the “zoom in” function. The microscope wasoperated with LAS AF software version 2.6.0.7266, and all


data were collected in 12-bit format. Import and exportassays were performed as described in Dopie et al [2012].The intranuclear FRAP assay was done by bleaching oncean area corresponding to nuclear shape but only 1=4 of thediameters of the nucleus. Imaging settings for this assaywere 128 3 128 resolution, bidirectional 700-Hz scanning,line average 2 resulting in a frame rate of 0.208 s=frame.The background was first subtracted and the recovery in thebleached area normalized to the whole nuclear intensity toexclude the effect of import and export on intranuclearmobility. The data were fitted in OriginPro 8.6 to the oneexponential equation y 5 A1*(1 2 exp(-x=t1)) 1 y0, andthe t1=2 and immobile pool were calculated from the fittedparameters. The cytoplasmic FRAP was performed withsimilar settings except images were recorded at 256 3 256resolution and the bleached area was a 5-lm wide rectanglegoing across the cell. After background subtraction, thedata were normalized to a control cell to account forbleaching during image acquisition. The data were fitted asfor the intranuclear FRAP.

Statistical Analysis

Statistical analysis was performed with Microsoft Excel2010 v. 14.0.6112.5000. The data conformed to normaldistribution. Statistical significance was tested using t-test.The confidence interval was set at P 5 0.05 for the wholeset of experiments which corresponded to a P-value of0.00192 for each individual experiment when taking intoaccount the family-wise error rate by the Bonferroni proce-dure. The cut-off value for the Pearson product-momentcorrelation coefficient corresponding to this correctedP-value was then determined for each experiment using theappropriate degrees of freedom for the corresponding data-set (n-2).

Acknowledgements

The imaging was done at the Light Microscopy Unit, Insti-tute of Biotechnology. The authors thank Marko Crivaro forhelp with statistical analysis. The work in the laboratory ofMKV is funded by Academy of Finland, Sigrid Juselius foun-dation and ERC Starting grant 310930. K.-P.S. is funded bya fellowship from the Viikki Graduate School in Biosciences.G.H. is funded by a grant from the Finnish CulturalFoundation.

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Steady-state nuclear actin levels are determined by export competent actin pool