ORIGINAL ARTICLE

Nicole Johnson1 . Matthew Krebs1 . Rosanne Boudreau .

Gisele Giorgi . Mark LeGros . Carolyn Larabell

Actin-filled nuclear invaginations indicate degree of cellde-differentiation

Received June 19, 2003; accepted June 29, 2003

Abstract For years the existence of nuclear actin has beenheavily debated, but recent data have clearly demon-strated that actin, as well as actin-binding proteins(ABPs), are located in the nucleus. We examined liveEGFP-actin-expressing cells using confocal microscopyand saw the presence of structures strongly resemblingactin filaments in the nuclei of MDA-MB-231 humanmammary epithelial tumor cells. Many nuclei had morethan one of these filamentous structures, some of whichappeared to cross the entire nucleus. Extensive analysis,including fluorescence recovery after photobleaching(FRAP), showed that all EGFP-actin in the nucleus ismonomeric (G-actin) rather than filamentous (F-actin)and that the apparent filaments seen in the nucleus areinvaginations of cytoplasmic monomeric actin. Immuno-localization of nuclear pore complex proteins shows thatsimilar invaginations are seen in cells that are notoverexpressing EGFP-actin. To determine whether thereis a correlation between increased levels of invagination inthe cell nuclei and the state of de-differentiation of the cell,we examined a variety of cell types, including live Xenopusembryonic cells. Cells that were highly de-differentiated,or cancerous, had an increased incidence of invagination,

while cells that were differentiated had few nuclearinvaginations. The nuclei of embryonic cells that werenot yet differentiated underwent multiple shape changesthroughout interphase, and demonstrated numeroustransient invaginations of varying sizes and shapes.Although the function of these actin-filled invaginationsremains speculative, their presence correlates with cellsthat have increased levels of nuclear activity.

Key words nucleus � nuclear invagination �de-differentiation � actin � FRAP � Xenopus

Introduction

The presence of actin in the nucleus has long been adisputed topic of cellular biology. The first paperssupporting the theory of nuclear actin were based uponWestern blot analyses (Ohnishi et al., 1963, 1964). Thesefindings were initially dismissed or discounted due tocytoplasmic contamination frequently associated withstudies of isolated cellular components (Rando et al.,2000; Pederson and Aebi, 2002). Other investigatorspersisted and found evidence of nuclear actin in a varietyof animal models, including Xenopus laevis and bovinenuclear extracts (Clark and Merriam, 1977; Clark andRosenbaum, 1979; Nakayasu and Kiyoshi, 1983; Onoet al., 1993; Wada et al., 1998). The observation of actinin manually isolated embryonic nuclei of Xenopus laeviswas the first to remove the criticism of cytoplasmiccontamination (Clark and Merriam, 1977; Clark andRosenbaum, 1979), thus reducing skepticism and sup-porting the existence of nuclear actin.

Continued studies throughout the years have helpedto further validate the presence of nuclear actin and

Nicole Johnson � Matthew KrebsAdvanced Light Source DivisionLawrence Berkeley National LaboratoryBerkeley, CA 94720, USA

Rosanne Boudreau . Gisele Giorgi . Mark LeGros .

Carolyn Larabell ( .*)Life Sciences DivisionLawrence Berkeley National LaboratoryBerkeley, CA 94720, USATel: (510) 486-5890, Fax: (510) 486-5664e-mail: larabel@itsa.ucsf.edu or calarabell@lbl.gov

Nicole Johnson � Matthew Krebs � Carolyn LarabellDepartment of AnatomyUniversity of California San Francisco, CA 94143, USA 1These authors contributed equally to this work

U.S. Copyright Clearance Center Code Statement: 0301–4681/2003/7107–414 $ 15.00/0

Differentiation (2003) 71: 414–424 r International Society of Differentiation 2003

begin to identify its functions. Goldstein et al. (1977a)postulated that because actin can passively diffuse fromthe cytoplasm into the nucleus, there was no specificfunction for nuclear actin. This was supported bygenetic studies that showed the presence of a nuclearexport signal on the actin gene (Wada et al., 1998),which, it has been argued, is there to help shuttle actinout of the nucleus as it leaks inside (Wada et al., 1998;Rando et al., 2000). However, there is now significantevidence to the contrary; it has been shown, forexample, that actin is involved with chromosomecondensation (Goldstein et al., 1977b; Rungger et al.,1979; Widlak et al., 2002). Injection of anti-actinantibodies into the cell nucleus (Rungger et al., 1979)has a profound effect, blocking both chromosomecondensation and progression of the cell throughmitosis. Cytoplasmic injection of these same antibodiesshowed no such effect. Actin has also been found to beinvolved with RNA nuclear-cytoplasmic transport as atype of chaperone (Nakayasu and Ueda, 1985; Kimuraet al., 2000), to have a regulatory effect on DNase I(Lazarides and Lindberg, 1974; Blikstad et al., 1978), andto form complexes with DNA in vitro (Miller et al., 1991).

Actin-binding proteins (ABPs) have also been found inthe nucleus, supporting the theory that actin is requiredfor normal nuclear functions (Ono et al., 1993; Van Ettenet al., 1994; Kato et al., 2001). One nuclear ABP thatassociates with actin is BAF (Brg-associated factor; Brg,Brahman-related gene), which interacts with chromatin.The BAF complex consists of an actin subunit and anactin-related protein (ARP). When the actin subunit isabsent from the complex, the activity of BAF is reducedand overall chromatin reorganization is altered (Zhaoet al., 1998; Rando et al., 2000, 2002; Olave et al., 2002).

Although there is mounting evidence supporting thepresence of nuclear actin, the mechanism by which itenters the nucleus remains unclear. A nuclear importsignal has not been found in the actin gene, but actinmonomers are small enough that they might passivelydiffuse through the nuclear pore complex (NPC) thattraverses the double-membrane nuclear envelope. Pro-teins such as cofilin have also been suggested as carrierproteins that could deliver actin monomers into thenucleus via nuclear pore interaction (Ono et al., 1993).Up-regulation of cofilin-associated actin movement intothe nucleus has been seen during periods of high cellularstress or increased nuclear activity (Yahara et al., 1996).Several investigators have showed that actin is alsoassociated with the NPC and, along with myosin, formsan ATP-driven contractile pore that can regulate thenuclear-cytoplasmic traffic of cells (Berrios and Fisher,1986; Schindler and Jiang, 1986; Berrios et al., 1991;Rakowska et al., 1998; Tonini et al., 1999). This leads tothe possibility that actin leaks into the nucleus duringtransport. Recently, invaginations of the nuclearenvelope were described in several cell types, and itwas suggested they provide an increased opportunity

for cytoplasmic-nuclear transfer of specific molecules(Fricker et al., 1997). If true, cells with invaginationsmight be expected to have more nuclear actin.

Whether nuclear actin is filamentous or monomericalso remains to be determined. Wada et al. (1998)treated cells with leptomycin B to inhibit nuclear exportand showed an accumulation of filamentous actin in thenucleus. Other investigators induced expression ofnuclear actin rods via DMSO treatment (Fukui, 1978;Fukui and Katsumaru, 1979; Sanger et al., 1980), heatshock (Welch and Suhan, 1985; Iida et al., 1986), andexposure to cytochalasin D (Yahara et al., 1982). Theinability to visualize nuclear actin with phalloidin,however, suggested it was not filamentous. Many yearspassed before Gonsior et al. (1999) found an antibodythat recognizes a specific conformational state of actin; itbinds to nuclear but not cytoplasmic actin, suggesting aconformational difference between nuclear and cytoplas-mic actin. These studies have led to ideas that the nucleuscontains short actin filaments, perhaps oligomers ratherthan polymers (Pederson and Aebi, 2002).

We used both confocal and multiphoton microscopy,along with 4-D imaging (3-D over time) of EGFP-actin,to examine the distribution and state of nuclear actin inlive cells. We also used fluorescence recovery afterphotobleaching (FRAP) to demonstrate that nuclearactin is monomeric and that the structures resemblingfilaments in the cell nuclei are actually invaginations ofactin-filled cytoplasm into the nucleus. Finally, weexamined a number of different cell types to determinewhether the presence and frequency of these actin-fillednuclear invaginations correlates with the state ofcellular de-differentiation and/or motility.

Methods

Cells and tissue culture

Cells were cultured in the appropriate media: NIH-3T3 cells inDMEM (ATCC, Manassas, VA) supplemented with 10% bovinecalf serum (BCS; GIBCO, Carlsbad, CA), MCF-10A cells inMEBM media (Clonetics, Cambrex, East Rutherford, NJ) supple-mented with 1mg/ml cholera toxin (Sigma-Aldrich, St. Louis, MO),5 mg/ml insulin, 1 ml/ml gentamycin, 1 ml/ml bovine pituitaryextract, 0.5 mg/ml hydrocortisone, and 0.01 mg/ml human epidermalgrowth factor (Clonetics), MDA-MB-231 and SW-480 cells inLeibovitz’s L-15 media supplemented with 10% fetal bovine serum(FBS; GIBCO), MCF-7 cells in MEME (ATCC) media supple-mented with 10% FBS and 0.01mg/ml bovine insulin (Sigma-Aldrich), and WI-38 cells in the same media as MCF-7 cells withoutsupplemented insulin. All cells were incubated at 371C in 5% CO2

except for MDA-MB-231 and SW-480 cells, which were incubatedin atmospheric CO2. Culture procedures included cell passaging inthe following media: MCF-10A cells in 0.05% trypsin (GIBCO)and cell dissociation solution (Sigma-Aldrich); SW-480, MCF-7,NIH-3T3, and MDA-MB-231 cells in 0.25% trypsin; and WI-38cells in 0.05% trypsin. All cells were rinsed in their dissociativesolution (� 1), incubated for 5min in fresh solution at 371C (15minfor MCF-10A cells), and trypsinization was stopped by addition ofmedia with 20% FBS. Cells were then transferred to sterile

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centrifuge tubes, spun at 4000 rpm for 5min, resuspended inappropriate media, and plated at subconfluent levels in standardcell culture flasks (Nunc, Corning, Worldwide). For imagingexperiments, all cells were grown on coverslips coated with vitrogen(Cohesion Technologies, Palo Alto, CA).

EGFP-actin

pEGFP-actin (Clontech, Palo Alto, CA) was transformed into XL1Blue MRF0 competent E. coli (Stratagene, La Jolla, CA) andamplified, and DNA was purified from bacteria cells using theQiagen (Valencia, CA) maxi kit. pEGFP-actin was stored in 10mMTris-HCl (pH 8) solution. Cells were removed from plates andtransferred to a sterile 15ml tube. Approximately 106 cells werecentrifuged for 5min at 4000 rpm, the supernatant was aspirated,and cells were resuspended in 360 ml of ICEB buffer (Harkin andHay, 1996). Twenty micrograms of EGFP-actin DNA were addedto the cell-ICEB solution in a Bio-Rad (Hercules, CA) 0.4 cmelectrode gap electroporation cuvette. After electroporation using aBio-Rad Gene Pulser II with a capacitance of 0.5 mF, voltage of0.25 kV, the cells were transferred to 10ml of fresh 371C ICEB,spun for 5min at 4000 rpm, and resuspended in supplementedmedia and plated on vitrogen-coated eight-well chamber slides.

Immunocytochemistry

Between 24 and 48hr after plating, cells were rinsed twice with 371CPBS and then fixed in 4% paraformaldehyde, 0.1% glutaraldehyde(Ted Pella, Redding, CA), and 0.1% Triton in sterile cytoskeletonbuffer (CSK; 100mM KCl, 3mM MgCl2, 5mM PIPES, 150mMsucrose [pH 6.8]) at 371C for 30min. Just prior to immunolabeling,cells were rinsed (5min� 3) in 10% goat serum (GS; GIBCO) and0.1% Triton in Superblock (SB; Pierce, Rockford, IL), and incubatedin a solution of 1:100 anti-human and 1:100 anti-mouse F(ab)2fragments (Jackson ImmunoResearch Laboratories, West Grove, PA),10% GS, and 0.1% Triton, in SB for 30min to block nonspecificlabeling. After rinsing, cells were incubated with anti-nuclear poreantibodies MAb414 (1:200; Covance, BabCo, Berkeley, CA) in SB,10% GS, and 0.1% Triton for 1hr, washed (5min� 3) with SB and0.1% Triton, incubated with goat anti-mouse AlexaFluor-488secondary antibodies (1:200; Molecular Probes, Eugene, OR) in SBand 10% GS for 1hr, and then washed (5min� 3) in SB. Cells weremounted in Prolong-Antifade (Molecular Probes) for imaging. Controlcells were labeled with Alexa-Fluor 488 secondary antibody alone.

Imaging

Cells in Figures 1 and 4 were imaged using a 1024-Bio-Radconfocal system (Bio-Rad Laboratories) using a krypton-argon

laser and appropriate GFP, rhodamine, and FITC filters andsettings. A Zeiss LSM-510 NLO imaging system (Carl Zeiss, Inc.,Thornwood, NY) equipped with a Ti-sapphire laser was used toimage cells in Figures 2, 3, and 6.

Fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching (FRAP) studies werecarried out to identify both the compartmentalization and form ofEGFP-actin expression in MDA-MB-231 cells. The measurementswere performed using a Zeiss 510 scanning laser confocalmicroscope using the 488 nm line of a 12mW argon ion laser andregion of interest capabilities of the Zeiss 510 software andhardware. Small regions of interest were selectively bleached andthe subsequent recovery of labeled actin was recorded. Thefluorescence recovery was measured using a laser power 0.01 timesthat used for photobleaching to minimize signal loss during thefluorescence recovery measurements. Similar bleach volumes wereused for filamentous actin, cytoplasmic G-actin, nuclear G-actin,and nuclear invagination-associated actin to allow for a qualitativecomparison of signal recovery by diffusion from unbleached regionsof the cell. For filamentous actin, the FRAP signal was given by thesum of pixels in the image associated only with the stress fiber asdetermined by the morphology and long recovery time ofpolymerized F-actin structures.

Xenopus embryos

Ovulation was induced by injecting 800 units of human chorionicgonadotropin (Sigma) into the dorsal lymph sac of an adultXenopus laevis. Eggs were expressed and fertilized by addition ofminced testis in 1–2ml of 0.3% MR (100% MR; 100mM NaCl,2mM KCl, 2mM CaCl2, 1mM MgCl2, 2mM CaCl2, 50mg/mlgentamycin, and 5mM HEPES [pH 7.2]). At the early gastrulastage, DAPI (Sigma) was added at a concentration of 1 mg/ml andembryos were imaged in 0.3% MR.

Data analysis

We did not attempt to calculate the actin diffusion rate from theFRAP curves; rather, the qualitative features of the photobleachingrecovery curves were used to identify the degree of actinpolymerization. Diffusion rates have been calculated from FRAPmeasurements (McGrath et al., 1998) and the published monomerdiffusion rate is 5.871.2� 10� 8 cm2/s. For in vivo studies, suchcalculations require a specific model of local cellular geometry andcomposition. The situation is further complicated by the preciseshape of the initial photobleached region; we used a 1.2 NA water

Fig. 1 Nuclear actin. Optical sections through the nucleus of EGFP-actin-expressing cells (A) MDA-MB-231, (B) MCF-7, and (C) MCF-10A. Structures that resemble actin filaments are seen in the nucleus

of live cells examined using confocal microscopy. (D) Anti-actinantibody labeling shows actin in the nucleus of fixed MDA-MB-231(top) and NIH-3T3 (bottom) cells. The scale bar represents 5mm.

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immersion lens for the measurements and a small, 5–10 mmdiameter, circular bleach region. This produced an initial disk-shapedbleach volume on the order of 0.5mm thick. The difficulties associatedwith the detailed analysis of actin diffusion coefficients from FRAPmeasurements in whole cells are well known, and we chose to makequalitative comparisons of our FRAP curves with those found inpublished work to infer the local properties of the actin.Three-dimensional surface rendering of the image shown in

Figure 4 was done using Amira software (TGS Visual Concepts,San Diego, CA).

Results

E-GFP actin reveals the presence of apparentfilaments in the nucleus

To determine the state of actin in the nucleus, wemonitored live NIH-3T3, MCF-10A, and MDA-MB-231 cells that were expressing EGFP-actin. The cellswere incubated for at least 4 hr after electroporationwith pEGFP-actin to allow time for synthesis of protein

levels that generate strong signal-to-noise ratios toenable imaging at low laser intensities. In all cellsexamined, there was a strong actin signal in thecytoplasm that was easily distinguishable from back-ground fluorescence and somewhat lower levels of actinfluorescence emanating from the region of the nucleus.To determine that this actin was unquestionably in thenucleus, we collected 3-D data sets using confocal andmultiphoton microscopy. Diffuse actin was seen inoptical sections 0.5 mm thick collected through thecenter of the nucleus. In addition, many cells demon-strated what appeared to be filamentous actin in theseoptical sections (Figs. 1A–C). We determined that theseapparent filaments were indeed in the nucleus, and notcytoplasmic stress fibers running over or under thenucleus, using two-photon microscopy with a mode-locked Ti-sapphire laser tuned to 900 nm.To assure thatthe actin we observed in the nucleus was not an artifactof overexpression of GFP-actin, we examined cells thatwere fixed and then labeled with anti-actin antibodies,

Fig. 2 FRAP analyses in EGFP-actin-expressing cells. (A) Cyto-plasmic actin stress fibers before photobleaching (0.0 s), bleached(1.6 s), and after partial fluorescence recovery (20.7 s). The scale barrepresents 10mm. (B) Region of nucleus showing a structure thatresembles an actin filament before photobleaching (0.0 s), bleached(1.6 s), and after fluorescence recovery (6.2 s). The fluorescence in

the filamentous structure completely recovered to its originalintensity, whereas the surrounding nuclear actin did not recover by6.2 s post-photobleaching. This suggests that the pool of actincontained within the nuclear envelope was depleted and theapparent filament was filled with fluorescent actin from the large,unbleached cytoplasmic pool.

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and again saw nuclear actin (Fig. 1D). However, cellsthat were fixed and labeled with rhodamine-phalloidin,which is well known to label only filamentous actin, didnot show any of these apparent filaments (data notshown). Therefore, we decided to investigate the nuclearactin using FRAP analyses to determine whether it wasmonomeric or filamentous actin.

FRAP analysis determines that nuclear actinis monomeric

Because filamentous actin has a much longer recoverytime after photobleaching than does monomeric actin,

we used FRAP to examine the nuclear actin todetermine whether it was a form of filamentous thatwas not detectable with rhodamine-phalloidin labelingor an unusual configuration of monomeric actin.Because a large majority of the MDA-MB-231 cellsshowed nuclei with at least one apparent filament, weused these cells for the FRAP studies. Cells wereexamined at least 4 hr after electroporation withpEGFP-actin. We examined the fluorescence recoverycurves for actin in the cytoplasm, both in regionsdemonstrating distinct stress fibers and in regionswithout stress fibers, as well as in the nucleus.

Filamentous actin: To obtain baseline levels for therecovery rate of filamentous actin, we examined anoptical section of a cytoplasmic region of an MDA-MB-231 cell containing three distinct stress fibers. A 33 mm2

area of the section was bleached (Figs. 2A, 3A) thatcontained both filaments and cytoplasmic G-actin; theportions of the filaments that were bleached encom-passed an area of 7.5 mm2. We plotted the recovery offluorescence of the bleached filaments and, after 19 s,the levels of fluorescent actin were only approximately10% of their original intensity (Fig. 3A). The timerequired for complete recovery was significantly longerthan expected for G-actin, based on rates of diffusionfor cytoplasmic monomeric actin (McGrath et al.,1998), and consistent with the extended times requiredfor renewal of stress fibers (Fujiwara et al., 2002).

Cytoplasmic actin: To obtain baseline levels for therecovery rate of unpolymerized actin in the cytoplasm,we examined an optical section of a region of thecytoplasm with no obvious stress fibers. A circlemeasuring a 33 mm2 area was bleached and monitoredfor recovery of fluorescence until it reached a valueclose to its maximal steady-state intensity; this occurredapproximately 2.3 s after the bleach pulse (Fig. 3). Thisfast fluorescence recovery is consistent with the diffu-sion of monomeric actin (McGrath et al., 1998) anddemonstrates that this region of cytoplasm containsG-actin rather than F-actin.

Nuclear actin structures: In an optical section throughthe nucleus, a circular region with an area of 50 mm2

area was bleached and monitored during recovery (Figs.2B, 3). This bleached region contained an apparentactin filament with an area of 3 mm2, and it required 3 sto recover to a steady state (Fig. 3). This recovery timeis significantly faster than that of the cytoplasmic stressfiber we had analyzed (Figs. 2A, 3A), which onlyachieved 10% recovery after 19 s, and is similar to thetime required for recovery of the non-filamentouscytoplasmic actin (Fig. 3). A comparable area of thenucleoplasm that did not contain any filamentous-likestructures returned to close to its steady-state intensity

Fig. 3 Graph of fluorescence recovery after photobleaching (FRAP)of EGFP-actin. (A) Normalized intensity for the monomeric actinin the cytoplasm (blue diamond), nucleus in a region withoutapparent filament (pink square), invagination of cytoplasmic actininto the nucleus (yellow triangle), and stress fiber (blue x) plottedover time. The nuclear actin recovered at the fastest rate (1.3 s), thecytoplasmic actin and nuclear invagination (shown in Fig. 2B)recovered at the same rate (2.3 s), and the stress fiber (shown in Fig.2A) was the slowest to recover, requiring 20 s to return to 10% of itsoriginal intensity. (B) Expanded graph of the fast recovery rates fornuclear, cytoplasmic, and nuclear invagination actin.

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in 1 s (Fig. 3), the fastest recovery rate recorded inthese experiments. These data demonstrate that thediffuse actin in the nucleoplasm is monomeric actinand that the structures resembling filaments mustcontain monomeric actin rather than polymerizedactin. The fact that FRAP analysis shows a turnoverof actin in these structures that is identical to theturnover rate of cytoplasmic actin led us to believe thatthe supposed filaments we see in the nucleus might in

fact be long, thin invaginations of cytoplasmic actininto the nucleus. To further test this, we bleachedthe entire complement of nuclear EGFP-actin, provid-ing no source of fluorescent actin to replenish anystructures in the nucleus without it being transportedfrom the cytoplasm. The apparent filaments wererapidly replenished with actin at a rate between 2 and3 s (data not shown), eliminating the possibility that thesource of actin is nuclear and supporting the idea that

Fig. 4 Three-dimensional reconstruction of the nucleus of anMDA-MB-231 cell. (A) Optical sections were collected throughan EGFP-actin-expressing cell in which the nuclear actin had beencompletely photobleached and the fluorescence of the surrounding

cytoplasmic actin delineated the nuclear surface. (B) Cut away viewof the nucleus showing an invagination of actin-filled cytoplasminto the nucleus. Variations in color represent differences in surfacetopography.

Fig. 5 Immunolocalization of nuclear pore complex proteinsdelineating the nuclear envelope in (A) NIH-3T3, (B) MDA-MB-231, (C) MCF-10A, (D) MCF-7, (E) SW-480, and (F) WI-38 cells.Very few or no invaginations of the nuclear envelope are seen in the

NIH-3T3 (A) and WI-38 (F) cells, several invaginations are seen inthe MDA-MB-231 cells (B), and a small number are seen in theMCF-10A (C), MCF-7 (D), and SW-480 (E) cells. The scale barrepresents 25mm.

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Fig. 6 Ratio of invaginations per cell (percent incidence) as a function of cell type. The largest number of invaginations was seen in highlyde-differentiated cells (MDA-MB-231) and the lowest in differentiated NIH-3T3 cells.

Fig. 7 Time-lapse imaging of interphase nuclei in Xenopus embryo. Five time points of four different nuclei (A–D) that were examined overa 20min time period show the presence of transient invaginations.

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they are a projection of cytoplasmic material into thenucleus.

Completely photobleaching the nuclear actin pro-duces a cell whose nuclear shape is demarcated byfluorescently labeled cytoplasmic actin, making theactin invaginations more prominent. We used thisapproach to collect high-resolution z-series throughthe cell and performed a 3-D reconstruction of thenucleus (Fig. 4). Figure 4A shows a surface view of avolume-rendered nucleus, color coded to accentuatesurface topography, and Figure 4B shows a slicethrough the center of the reconstructed nucleus,displaying an actin invagination identical to that seenin Figure 2B. These data reveal that the surface of thenucleus is far from smooth in the highly de-differen-tiated MDA-MB-231 cells and actually has a veryconvoluted, infolded surface. This nucleus is represen-tative of most nuclei seen in the MDA-MB-231 cells.

Apparent actin filaments in the nucleus areinvaginations of cytoplasmic actin

To demonstrate that these nuclear invaginations occurunder ordinary circumstances and are not artifacts ofoverexpressed actin, we examined the nuclear envelopein fixed cells. We examined MDA-MB-231, MCF-10A,MCF-7, NIH-3T3, WI-38, and SW-480 cells byimmunolabeling with anti-nuclear pore antibodies,which recognize an NPC protein found exclusively onthe cytoplasmic side of the nuclear membrane. Wedetected varying numbers of nuclear invaginations in allcell lines studied (Fig. 5), showing that the actininvaginations into the nucleus are not an artifact ofexpression of GFP-actin and that they do consist ofinfoldings of the nuclear envelope containing nuclearpores. Similar invaginations had been described inseveral cell types using endoplasmic reticulum dyes andNPC proteins (Fricker et al., 1997), so we used thosesame labels to determine whether we were examiningsimilar structures. The invaginations we describe werealso filled with the ER dye DIOC6(3) (data not shown).

Nuclear invaginations correlate with cellde-differentiation

To compare the presence and frequency of nuclearinvaginations in different cell types, we examinedsample fields of normal and tumorigenic epithelial cellsand determined the ratio of the number of invaginationsto the number of cells for each cell type. This ratio wasconverted into a percentage and termed the percentincidence of invaginations. We observed that differentcell types had varying incidents of invaginations(Fig. 6). The human mammary epithelial tumor cellline, MDA-MB-231 cells, had the highest ratio, with a

percent incidence of 90730% invaginations per cell.The non-transformed MCF-10A human mammaryepithelial cells had a 25710% incidence of invagina-tions, the mildly de-differentiated MCF-7 humanmammary epithelial cells had a 972% incidence ofinvaginations, and the colorectal epithelial cell line(SW-480 cells) had a percent incidence of 1472%.Because the MDA-MB-231 cell line is very aggressiveand migrates across a dish much like a fibroblast, unlikethe other mammary epithelial cells, we thought theinvaginations might be associated with cell motility.Therefore, we examined two fibroblast cell lines, NIH-3T3 and WI-38 cells. In contrast to the findings ofFricker et al. (1997), the NIH-3T3 cell line we examineddemonstrated very few invaginations per cell, with apercent incidence of 372%, the lowest incidence of allthe cell lines examined. The WI-38 cells had a percentincidence of invaginations per cell of 975%. These datado not support an association between invaginationsand cell migration. It is interesting to note that thosecell lines with a high incidence of invaginations alsotended to have more invaginations per cell nucleus. Inthose NIH-3T3 cells that had invaginations, there wasonly one per cell, whereas MDA-MB-231 mammarytumor cells had as many as eight in a single nucleus. Theincreased incidence of invaginations also correlatedwith an increase in genotypic and phenotypic modifica-tions of the cell line. MDA-MB-231 breast tumor cellshave numerous genetic mutations and resemble fibro-blasts. To test a possible correlation with degree ofdifferentiation of the cell type, we decided to examineundifferentiated embryonic cells. To determine whetherundifferentiated cells also show the presence of nuclearinvaginations, we examined Xenopus embryonic cells.The nuclei of live embryos, labeled with DAPI, wereexamined at stage 23 using multiphoton microscopy;excitation of DAPI was accomplished with a Ti-sapphire laser at 900 nm to reduce phototoxicity.Time-lapse imaging revealed that the nuclei of allblastomeres were very dynamic and rarely assumed aperfectly round, smooth nuclear envelope (Fig. 7).Numerous transient invaginations were seen in allnuclei throughout the 30min time period examined.Image sequences of four different nuclei are shown hereto demonstrate the presence of invaginations and thechanges in size and shape of these invaginations overtime. In the fields of cells examined, the live cell embryoimages demonstrated a percent incidence of 3574%invaginations per cell.

Discussion

The presence of actin in the nucleus has been anintensely studied and long-disputed topic for more than30 years. Only in the past decade, however, has there

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been significant and reliable evidence to support theobservation that actin exists in the nucleus. We decidedto further examine nuclear actin in several cell types bymonitoring GFP-actin in live cells using confocal andmultiphoton microscopy. Using this approach, weobserved a uniform distribution of actin throughoutthe nucleus of all cells, excluded only by the nucleoli. Insome cell types, using 3-D and 4-D imaging (3-D overtime), we also observed one to five GFP-actin-filledstructures that resembled stress fibers running partiallyor entirely across the nucleus. Although these structuresdid not appear as rigid and immobile as do stress fibers,they were much less dynamic than is the actin inlamellipodia and filopodia.

In order to determine whether the nuclear actin wasmonomeric or filamentous, we conducted FRAPmeasurements, which demonstrate the rate at whichbleached actin is replaced with new fluorescent actin.The majority of actin throughout the nuclei recoveredfrom photobleaching in 1–2 s, a rate compatible withthat of monomeric actin. To our surprise, the ostensibleactin filaments seen in the nuclei recovered fromphotobleaching within 2–3 s. This reveals that therewas a complete turnover of actin in a time framesignificantly shorter than was required for turnover offilamentous actin as reported by others (McKenna andWang, 1986) and for turnover of actin in stress fibersexamined here. Because the photobleaching recoverycurves measured for the filament-like structures wereconsistent with those of monomeric actin (McGrathet al., 1998) and identical to the rate of recovery for G-actin in the cytoplasm, these studies clearly demonstratethat the actin in the filament-like structures seen in thenucleus is monomeric, not filamentous actin. It wasfurther demonstrated that the filamentous appearingstructures are made of cytoplasmic rather than nuclearactin because they were refilled with fluorescent actinwithin seconds after photobleaching, even after theentire pool of nuclear actin had been completelyphotobleached. These studies make it clear that theactin in the filament-like structures seen in the nucleus ismonomeric and that they are actually thin invaginationsof cytoplasmic actin into the nucleus. These structuresappear much brighter than the surrounding nuclearactin because the concentration of cytoplasmic actin issignificantly higher than that of nuclear actin.

Similar invaginations of cytoplasm into the nucleushave been described by Fricker et al. (1997), whoreported the presence of infoldings of dye-labeledendoplasmic reticulum and NPC proteins in several celltypes. These investigators did not examine the distribu-tion of actin, but it is likely that the invaginations theydescribed are the same types of structures that we seefilled with actin. To address this, we examined cellslabeled with the dye DiOC6(3) and the NPC antibodiesused in their study. Our results, like theirs, show thatboth are seen in the actin-filled invaginations.

During the course of these studies, it becameapparent that different cell types tend to have differentnumbers of invaginations. A highly de-differentiated,invasive, mammary epithelial cancer cell line, theMDA-MB-231 cells, had the highest incidence ofinvaginations. Differentiated epithelial cells, such asMCF-10A cells, which are a spontaneously immorta-lized human mammary epithelial cell line, and two othercell lines that have retained characteristics of differ-entiated epithelium, the MCF-7 human mammaryepithelial cells and SW-480 colorectal adenocarcinomacells, demonstrated very few nuclear invaginations.Because the MDA-MB-231 cells morphologically re-semble fibroblasts and vigorously crawl around theculture dish, and the previous work of Fricker et al.(1997) indicated fibroblasts had increased numbers ofinvaginations, we tested the possibility that the invagi-nations correlated with increased cell motility charac-teristic of fibroblasts. But when we compared thenumber of invaginations in the MDA-MB-231 cellswith two other fibroblast cell lines that demonstratedcomparable crawling activity, mouse NIH-3T3 cells andhuman WI-38 cell lines, we obtained unexpected results.The fibroblast cell lines examined here had very lowlevels of actin-filled invaginations. This finding does notagree with the work of Fricker et al. (1997), whoreported a high incidence of dye-filled invaginations inNIH-3T3 cells. In spite of numerous attempts to detectinvaginations in these fibroblast cell lines using GFP-actin and antibody labeling experiments, we repeatedlyobserved very few invaginations; we can only assumethis represents some variability due to different culturesituations.

Because there appears to be no correlation betweenincidence of nuclear invaginations and cell motilitytypical of fibroblasts, we considered the possibility thatthe nuclear invaginations actually correlate with thestate of differentiation of the cell type. To investigatethis possibility, we examined undifferentiated cells indeveloping Xenopus embryos. The embryonic cells weexamined demonstrated moderate levels of nuclearinvaginations that were not as abundant as seen in theaggressive and highly de-differentiated MDA-MB-231cells, but more common than epithelial cell lines thatretained characteristics of differentiated cells, andsignificantly higher than in differentiated fibroblasts.These views of DAPI-labeled nuclei in sheets of cellsfrom a normal, developing embryo demonstrate thatnuclear invaginations are not an artifact of cultureconditions, nor are they an artifact of overexpression ofactin. They appear to be a common manifestation ofnuclei and reveal that the nuclear envelope is a dynamic,fluid structure that undergoes remarkable shapechanges throughout interphase. The function of thesestructures may be revealed by examining the nuclearchanges that accompany the process of cell differentia-tion.

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Conclusion

In recent years, possible functions of nuclear actin havebegun to emerge, one of which is a potential role inreplication. The association of actin with RNA,chromatin, and DNase I imply that it has a regulatoryfunction in nuclear activity. A study researching the roleof BAF (Brg-associated-factor) and actin in the nucleusfound that BAF is a tumor suppressor, and that actin isa functional and necessary subunit of this complex.When the actin subunit is nonfunctional, BAF nolonger acts as a tumor suppressor (Zhao et al., 1998;Rando et al., 2000, 2002; Olave et al., 2002). From thisinformation, one could postulate that cells that arebecoming cancerous and tumorgenic have increasedcellular replication activity. As replication rates in-crease, the need for functional units inside the nucleus,including actin, would increase. Cells such as highly de-differentiated MDA-MB-231 s rapidly proliferate, andthe increased number of invaginations could provideincreased numbers of NPC for transport of proteins andRNA into and out of the nucleus to facilitate nuclearfunctions. Undifferentiated embryonic cells also haveincreased nuclear activity because many genes are beingturned on and off, and have an increased number ofinvaginations. It is also possible that the invaginationssimply reflect a larger nucleus associated with undiffer-entiated and de-differentiated cells, and folding merelyaccommodates the increased surface area of the nuclearenvelope. It is possible that these invaginations could beused as a marker of differentiation state and as a toolfor early detection of cells that are becoming de-differentiated and in turn becoming more cancerous.

Acknowledgments This work was supported by NIH grant no. R01GM63948-01 and by the United States Department of Energy,Office of Biological and Energy Research under contract no. DE-AC03-76SF00098.

References

Berrios, M. and Fisher, P. (1986) A myosin heavy chain-likepolypeptide is associated with the nuclear envelope in highereukaryotic cells. J Cell Biol 103:711–724.

Berrios, M., Fisher, P. and Matz, E. (1991) Localization of amyosin heavy chain-like polypeptide to Drosophila nuclear porecomplexes. Proc Natl Acad Sci USA 88:219–223.

Blikstad, I., Markey, F., Calsson, L., Persson, T. and Lindberg, U.(1978) Selective assay of monomeric and filamentous actin cellextracts, using inhibition of deoxyribonuclease I. Cell 15:935–943.

Clark, T.G. and Merriam, R.W. (1977) Diffusable and bound actinin nuclei of X. laevis oocytes. Cell 2:639–651.

Clark, T.G. and Rosenbaum, J.L. (1979) An actin filament matrixin hand-isolated nuclei of X. laevis oocytes. Cell 18:1101–1108.

Fricker, M., Hollinshead, M., White, N. and Vaux, D. (1997)Interphase nuclei of many mammalian cell types contain deep,dynamic, tubular membrane-bound invaginations of the nuclearenvelope. J Cell Biol 136:531–544.

Fujiwara, I., Takahashi, S., Tadakuma, H., Funatsu, T. andIshiwata, S. (2002) Microscopic analysis of polymerizationdynamics with individual actin filaments. Nat Cell Biol 4:666–673.

Fukui, Y. (1978) Intranuclear actin bundles induced by dimethyl-sulfoxide in interphase nucleus of Dictyostelium. J Cell Biol76:146–157.

Fukui, Y. and Katsumaru, H. (1979) Nuclear actin bundles inAmoeba, Dictyostelium and human HeLa cells induced bydimethyl sulfoxide. Exp Cell Res 120:451–455.

Goldstein, L., Rubin, R. and Ko, C. (1977a) The presence of actinin nuclei: a critical appraisal. Cell 12:601–608.

Goldstein, L., Ko, C. and Errick, J. (1977b) Nuclear actin: anapparent association with condensed chromatin. Cell Biol IntRep 1:511–515.

Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M.and Hinssen, H. (1999) Conformational difference betweennuclear and cytoplasmic actin as detected by a monoclonalantibody. J Cell Sci 112:797–809.

Harkin, D.G. and Hay, E.D. (1996) Effects of electroporation onthe tubulin cytoskeleton and directed migration of cornealfibroblasts cultured within collagen matrices. Cell Motil Cytos-keleton 35:345–357.

Iida, K., Iida, H. and Yahara, I. (1986) Heat shock induction ofintranuclear actin rods in cultured mammalian cells. Exp CellRes 165:207–215.

Kato, M., Sasaki, M., Mizuno, S. and Harata, M. (2001) Novelactin-related proteins in vertebrates: similarities of structure andexpression pattern to Arp6 localized on Drosophila heterochro-matin. Gene 268:133–140.

Kimura, T., Hashimoto, I., Yamamoto, A., Nishikawa, M. andFujisawa, J.I. (2000) Rev-dependent association of the intro-containing HIV-1 gag mRNA with the nuclear actin bundles andthe inhibition of its nucleocytoplasmic transport by latrunculin-B. Genes Cells 5:289–307.

Lazarides, E. and Lindberg, U. (1974) Actin is the naturallyoccurring inhibitor of deoxyribonuclease I. Proc Natl Acad SciUSA 71:4742–4746.

McGrath, J.L., Tardy, Y., Dewey, C.F., Meister, J.J. and Hartwig,J.H. (1998) Simultaneous measurements of actin filament turn-over, filament fraction, and monomer diffusion in endothelialcells. Biophys J 75:2070–2078.

McKenna, N.M. and Wang, Y.L. (1986) Possible translocation ofactin and a-actinin along stress fibers. Exp Cell Res 167:95–105.

Miller, C.A., Cohen, M.D. and Costa, M. III (1991) Complexing ofactin and other nuclear proteins to DNA by cis-diamminedi-chloroplatinum (II) and chromium compounds. Carcinogenesis12:269–276.

Nakayasu, H. and Kiyoshi, U. (1983) Association of actin with thenuclear matrix from bovine lymphocytes. Exp Cell Res 143:55–62.

Nakayasu, H. and Ueda, K. (1985) Association of rapidly-labelledRNA’s with actin filaments in nuclear matrix from mouseL5178Y cells. Exp Cell Res 160:319–330.

Ohnishi, T., Kawamura, H. and Yamamoto, T. (1963) Extraktioneines dem aktin ahnlichen proteins aus DEM zellkern deskalbsthymus. J Biochem 54:298–300.

Ohnishi, T., Kawamura, H. and Tanaka, Y. (1964) Die atkin andmyosin ahnliche proteine in kalbsthymus zellkern. J Biochem56:6–15.

Olave, I.A., Reck-Peterson, S.L. and Crabtree, G.R. (2002) Nuclearactin and actin-related proteins in chromatin remodeling. AnnuRev Biochem 71:755–781.

Ono, S., Hiroshi, A., Nagaoka, R. and Obinata, T. (1993)Colocalization of ADF and cofilin in intranuclear actin rods ofcultured muscle cells. J Muscle Res Cell Motil 14:195–204.

Pederson, T. and Aebi, U. (2002) Actin in the nucleus: what formand what for? J Struct Biol 140:3–9.

Rakowska, A., Danker, T., Schneider, S.W. and Oberleithner, H.(1998) ATP-induced shape change of nuclear pores visualizedwith the atomic force microscope. J Membr Biol 163:129–136.

423

Rando, O.J., Zhao, K. and Crabtree, G.R. (2000) Searching for afunction for nuclear actin. Trends Cell Biol 10:92–97.

Rando, O.J., Zhao, K., Janmey, P. and Crabtree, G.R. (2002)Phosphatidylinositol-dependent actin filament binding by theSWI/SNF-like BAF chromatin remodeling complex. Proc NatlAcad Sci USA 99:2824–2829.

Rungger, D., Rungger-Brandle, E., Chaponnier, C. and Gabbiani,G. (1979) Intranuclear injection of anti-actin antibodies intoXenopus oocytes blocks chromosome condensation. Nature282:320–321.

Sanger, J.W., Sanger, J.M., Kreis, T.E. and Jockusch, B.M. (1980)Reversible translocation of cytoplasmic actin into the nucleuscaused by dimethyl sulfoxide. Proc Natl Acad Sci 77:5268–5272.

Schindler, M. and Jiang, L. (1986) Nuclear actin and myosin ascontrol elements in nucleocytoplasmic transport. J Cell Biol102:859–862.

Tonini, R., Grohovaz, F., Laporta, C.A.M. and Mazzanti, M.(1999) Gating mechanism of the nuclear pore complex channel inisolated neonatal and adult mouse liver nuclei. FASEB J13:1395–1403.

Van Etten, R.A., Jackson, P.K., Baltimore, D., Sanders, M.C.,Matsudaira, P.T. and Janmey, P.A. (1994) The COOH ter-minus of the c-Abl tyrosine kinase contains distinct F- and G-actin binding domains with bundling activity. J Cell Biol124:325–340.

Wada, A., Fukuda, M., Mishima, M. and Nishida, E. (1998)Nuclear export of actin: a novel mechanism regulating thesubcellular localization of a major cytoskeletal protein. EMBO J17:1635–1641.

Welch, W.J. and Suhan, J.P. (1985) Morphological study of themammalian stress response characterization of changes incytoplasmic organelles, cytoskeleton, and nucleoli, and theappearance of intranuclear actin filaments in rat fibroblasts afterheat-shock treatment. J Cell Biol 101:198–211.

Widlak, P., Palyvoda, O., Kumala, S. and Garrard, W.T. (2002)Modeling apoptotic chromatin condensation in normal cellnuclei. J Biochem 277:21683–21690.

Yahara, I., Harada, F., Sekita, S., Yoshihira, K. and Natori, S.(1982) Correlation between effects of 24 different cytochalasinson cellular structures and cellular events and those on actin invitro. J Cell Biol 92:69–78.

Yahara, I., Aizawa, H., Moriyama, K., Iida, K., Yonezawa, N.,Nishida, E., Hatanaka, H. and Inagaki, F. (1996) A role ofcofilin/destrin reorganization of actin cytoskeleton in response tostresses and cell stimuli. Cell Struct Funct 21:421–424.

Zhao, K., Wang, W., Rando, O.J., Xue, Y., Swiderek, K., Kuo, A.and Crabtree, G.R. (1998) Rapid and phosphoinositol-depen-dent binding of the SWI-SNF-like BAF complex to chromatinafter T lymphocyte receptor signaling. Cell 95:625–636.

424