The Cell-Surface-Expressed Nucleolin Is Associated with the Actin Cytoskeleton

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Experimental Cell Research 261, 312–328 (2000)doi:10.1006/excr.2000.5071, available online at http://www.idealibrary.com on

The Cell-Surface-Expressed Nucleolin Is Associatedwith the Actin Cytoskeleton

Ara G. Hovanessian,*,‡,1 Francine Puvion-Dutilleul,† Sebastien Nisole,* Josette Svab,*Emmanuelle Perret,‡ Jau-Shyong Deng,§ and Bernard Krust*

*Unite de Virologie et Immunologie Cellulaire and ‡URA 1930 CNRS, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15,France; †Laboratoire Organisation Fonctionnelle du Noyau de l’UPR 1983 CNRS, BP 8, F-94801 Villejuif Cedex, France;

and §Department of Veterans Affairs, Medical Center, Pittsburgh, Pennsylvania 15240-1001

Nucleolin is a RNA- and protein-binding multifunc-tional protein. Mainly characterized as a nucleolarprotein, nucleolin is continuously expressed on thesurface of different types of cells along with its intra-cellular pool within the nucleus and cytoplasm. Byconfocal and electron microscopy using specific anti-bodies against nucleolin, we show that cytoplasmicnucleolin is found in small vesicles that appear totranslocate nucleolin to the cell surface. Translocationof nucleolin is markedly reduced at low temperatureor in serum-free medium, whereas conventional inhib-itors of intracellular glycoprotein transport have noeffect. Thus, translocation of nucleolin is the conse-quence of an active transport by a pathway which isindependent of the endoplasmic reticulum–Golgi com-plex. The cell-surface-expressed nucleolin becomesclustered at the external side of the plasma membranewhen cross-linked by the nucleolin-specific monoclo-nal antibody mAb D3. This clustering, occurring at20°C and in a well-organized pattern, is dependent onthe existence of an intact actin cytoskeleton. At 37°C,mAb D3 becomes internalized, thus illustrating thatsurface nucleolin can mediate intracellular import ofspecific ligands. Our results point out that nucleolinshould also be considered a component of the cell sur-face where it could be functional as a cell surfacereceptor for various ligands reported before. © 2000

cademic Press

Key Words: nucleolin; actin; intracellular import; ac-tive transport; cell surface; electron microscopy; con-focal microscopy.

INTRODUCTION

Nucleolin has been described as an abundant nucle-olar protein that plays an important role in ribosomebiogenesis [1, 2]. Because of its reported function as ashuttle between the nucleus and the cytoplasm [3], it

1 To whom reprint requests should be addressed. Fax: (33-1) 4061-
3012. E-mail: [email protected].
3120014-4827/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

has been suggested that nucleolin could be involved inthe import of cytoplasmic or export of nuclear compo-nents. The subcellular localization of nucleolin, ini-tially called C23 [4], has primarily been characterizedin relation to the nucleolus despite several reportspointing out the presence of nucleolin on the surface ofdifferent types of cells [5–13].

Nucleolin is a ubiquitous protein which is encoded bya gene localized on chromosome 12 with the character-istic GC-rich promoter sequences found in housekeep-ing genes [14]. As it is directly involved in the regula-tion of ribosome biogenesis and maturation [15–17],nucleolin is one of the key elements regulating cellgrowth. Throughout the years, numerous reports haveshown that nucleolin could be implicated in processessuch as chromatin decondensation, cytoplasmic nu-clear transport of ribosomal components and preribo-somal particles, cytokinesis, replication, embryogene-sis, and nucleogenesis [1, 2]. In addition, nucleolin hasbeen shown to be a component of B-cell-specific tran-scription factor LR1 [18], a transcriptional repressor[19], and to manifest a helicase activity capable ofunwinding RNA–RNA duplexes, as well as DNA–DNAand DNA–RNA duplexes [20]. Nucleolin appears tobind specifically several nuclear proteins, such as nu-cleophosmin or B23 [21], topoisomerase 1 [22], and thegrowth factor midkine [23, 24], and such associationmay represent a mechanism for nuclear localization ofcellular proteins. As a posttranscriptional regulator,nucleolin binds the 39-untranslated region of amyloidprecursor protein mRNA and stabilizes it [25]. In po-liovirus infection, nucleolin binds poliovirus RNA andcould be implicated in the viral RNA replication pro-cess [26]. In the case of infection by hepatitis deltavirus, nucleolin binding activity of the hepatitis deltaantigen appears to be required for nucleolus targetingand virus replication [27]. The different reports pointout that nucleolin is a multifunctional protein which,in addition to biosynthesis of ribosomes, could directlyor indirectly regulate mechanisms essential for cell
growth and virus infection. The function of nucleolin

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313CELL-SURFACE EXPRESSION OF NUCLEOLIN

could be regulated by partial proteolytic cleavage [28],phosphorylation [29], methylation [30], and ADP-ribo-sylation [31].

Analysis of the amino acid sequence of nucleolin [32,33] and its molecular characterization have revealedthe existence of three main structural domains innucleolin which are involved in the organization ofnucleolar chromatin, packaging of pre-RNA, rDNAtranscription, and ribosome assembly [1, 2]. These do-mains are: (1) the amino-terminal domain controllingrRNA transcription, (2) the central globular domaincontrolling pre-RNA processing, and (3) the carboxyl-terminal domain controlling nucleolar localization. TheN-terminal domain contains a-helical structures com-prising four acidic stretches interspersed with motifs ofbasic residues. The acidic stretches have been proposedto bind histone H1 [34], while the basic residues mightbe implicated in the capacity of nucleolin to modulateDNA condensation in chromatin [35]. This domain alsocontains phosphorylation sites for casein kinase II(CKII) [36] and p34cdc2 [37, 38]. Interestingly, CKII-

ediated serine phosphorylation of nucleolin occursuring interphase, whereas p34cdc2-mediated threonine

phosphorylation occurs during mitosis, thus suggest-ing that the functioning of nucleolin could be regulateddifferentially by phosphorylation during the cell cycle[2]. The basic residues in the N-terminal domain arealso the site for the proteolytic cleavage of nucleolinwhich appears to be correlated with its phosphoryla-tion state [39]. The central domain of nucleolin con-tains four RNA-binding domains that confer RNA-binding specificity to nucleolin [40]. Accordingly,nucleolin could be associated with nascent preriboso-mal RNA and bind with high affinity and specificitywith rRNA fragments [17]. The C-terminal domain isrich in motifs containing glycine and is interspersedwith dimethylarginine and phenylalanine residues[30]. Structural studies have pointed out that the C-terminal domain can adopt repeated b-turns [41] andmediate protein/protein [42] and protein/nucleic acid[40, 43, 44] interactions. The mechanism of nucleolaraccumulation of nucleolin appears to be complex, im-plicating a classical bipartite nuclear localization sig-nal situated between the N-terminal domain and theRNA binding domain and the affinity of RNA bindingdomains and the C-terminal domain for different nu-cleolar components including rRNA [1]. By virtue of itsdistinct RNA and protein binding, nucleolin couldbring together both ribosomal proteins and RNA dur-ing assembly of ribosomal subunits. Finally, throughits nucleocytoplasmic shuttling property [3], nucleolincould act as a chaperone during either the nuclearimport of cytoplasmic [45] or the export of nuclear [46]cell components.

The cell-surface expression of nucleolin in cells of
different species was first suggested by its isolation as n
a phosphoprotein from the surface [5, 47], the phos-phorylation of which is catalyzed by an ectoproteinkinase [7]. Since then nucleolin has been recoveredfrom the cell surface as a binding protein for differentligands, including virus particles, and consequently ithas been suggested that it could serve as a cell surfacereceptor. By studies using electron and confocal laserimmunofluorescence microscopy, here we confirm thatnucleolin is expressed at the cell surface where it existsin close association with the intracellular actin cy-toskeleton. Furthermore, disruption of actin microfila-ments by cytochalasin D leads to the collapse of clus-tered nucleolin along the condensed bundles of actin,thus pointing out the tight association of intracellularactin with surface nucleolin. We also demonstrate thatthe translocation of nucleolin to the cell surface impliesan alternative secretion pathway independent of theclassical pathway of secretion through the endoplasmicreticulum (ER) and Golgi apparatus.

MATERIALS AND METHODS

Materials. The monoclonal antibody (mAb) D3 specific for humannucleolin is known to react with the cell-surface-expressed nucleolin[10]. Other mAbs against human nucleolin, CC98 and 3G482, wereprovided by Dr. N. H. Yeh (National Yang-Ming Medical College,Shih-Pai, Taiwan, Republic of China) and Dr. R. Foisner (Universityof Vienna, Austria), respectively [28, 48]. Rabbit polyclonal antibod-ies against human nucleolin were provided by Dr. M. Erard, Tou-louse, France. Rabbit polyclonal antibody against a synthetic peptidecorresponding to the first 26 amino acid residues was as described[12]. The histone H2B-specific mAb LGG2-2 was provided by Dr. S.Muller, IBMC, Strasbourg, France. Anti-CXCR4 mAbs 12G5, 44708,and 44717 were from R&D Systems. The anti-CD4 mAb CBT4 wasfrom Dr. E. Bosmans (Eurogenetics, Tessenderlo, Belgium), whilemAbs OKT4 and OKT4A were purchased from Ortho DiagnosticsSystems. FITC-labeled goat anti-mouse IgG was from Sigma (St.Louis, MO), whereas Texas red dye-conjugated horse anti-mouse IgGwas from Valbiotech, Paris, France. FITC-labeled phalloidin waspurchased from Sigma. The biotin-labeled HB-19 (5[Kc(CH2N)PR]-

ASP)2 was as described before [12]. Cytochalasin D, brefeldin A,onensin, methylamine, actinomycin D, cycloheximide, and the cal-

ium ionophore A23187 were from Sigma.Cells and extracts. Human Daudi (a Burkitt’s lymphoma) and937 (a promonocytic leukemia) cells and CD41 lymphoblastoidEM (clone 13), MOLT4-T4 (clone 8), and MT-4 cell lines and theurine hybridoma T-cell line T54 [49] were cultured in the suspen-

ion medium RPMI 1640 (Bio-Whittaker, Verviers, Belgium). Hu-an HeLa (cervix carcinoma) and RD (ATCC; rhabdomyosarcoma)

ells and murine L929 cells (fibroblast-like cells derived from normaldipose tissue from C3H mouse) were grown as monolayers in Dul-ecco’s medium. Human peripheral blood mononuclear cells (PBMC)rom an healthy donor were stimulated by phytohemagglutininPHA) and cultured in RPMI 1640 medium [12]. All cells were cul-ured with 10% (v/v) heat-inactivated (56°C, 30 min) fetal calf serumFCS).

For the preparation of cytoplasmic extracts, cells were first washedxtensively in phosphate-buffered saline (PBS) before lysis in buffer

2 Abbreviations used: PFA, paraformaldehyde; HB-19, the nucleolinbinding pseudopeptide 5[Kc(CH2N)PR]-TASP; PHA, phytohemaggluti-

in; NRP, nucleolin-related protein from the surface of rat cells.

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E (20 mM Tris–HCl, pH 7.6, 150 mM NaCl, 5 mM MgCl2, 0.2 mMPMSF, 5 mM b-mercaptoethanol, aprotinin (1000 U/ml) and 0.5%Triton X-100) and the nuclei were pelleted by centrifugation (1000gfor 5 min). For the preparation of nuclear extracts, the nuclear pelletwas first washed in buffer E and the nuclear pellet was disrupted inbuffer I (20 mM Tris–HCl, pH 7.6, 50 mM KCl, 400 mM NaCl, 1 mMEDTA, 0.2 mM PMSF, 5 mM b-mercaptoethanol, aprotinin (1000

/ml), 1% Triton X-100, and 20% glycerol). The nucleus-free super-atant and the nuclear extract were then centrifuged at 12,000g for0 min, and the supernatants were stored at 280°C.Purification of the cell-surface-expressed nucleolin. Cells were in-

ubated for 45 min at 5°C with the biotin-labeled HB-19 (5 mM) inPBS containing 1% bovine serum albumin (BSA) and 0.02% sodiumazide. Cells were then washed with PBS containing 1 mM EDTA andcytoplasmic extracts were prepared using buffer E but containingunlabeled HB-19 (50 mM). The complex formed between the cell-urface-expressed nucleolin and the biotin-labeled HB-19 was recov-red by affinity chromatography using avidin-agarose (ImmunoPuremmobilized avidin from Pierce Chemical Company, U.S.A.). Theurified nucleolin was eluted in the electrophoresis sample bufferontaining SDS and analyzed by SDS–polyacrylamide gel electro-horesis (PAGE). The presence of nucleolin was then revealed bymmunoblotting using polyclonal and monoclonal antibodies againstucleolin. The detailed experimental procedure was as describedefore [12, 13].Two-dimensional gel isoelectric focusing. Experimental condi-

ions were as described previously [50]. The concentration of ampho-ine (Pharmacia Biotech, Sweden) was at 2% of pH range 3–10. TheH gradient obtained by isoelectric focusing (first dimension) wasrom 4 to 7. The proteins were resolved in the second dimension on.5% SDS–PAGE and the nucleolin spots were revealed by immuno-lotting using mAb D3.Confocal microscopy. For laser scanning confocal microscopy

Leica TCS4D) studies, HeLa cells were plated 24 h before thexperiment in eight-well glass slides (Lab-Tek Brand; Nalge Nuncnternational, Naperville, IL). Cells were fixed with either parafor-aldehyde (PFA; 3.7%) to monitor the clustered surface nucleolin orFA/Triton X-100 solution (PFA/Triton) for intracellular staining asreviously described [51]. Consistently in different types of cells,xation of cells with 3.7% PFA resulted in the permeabilization ofhe plasma but not the nuclear membrane. Consequently, specificell surface labeling was investigated by incubation of cells directlyith the antibody at room temperature before PFA fixation. On thether hand, staining of cytoplasmic actin was performed by theddition of the FITC-labeled phalloidin after PFA fixation. For im-unofluorescence analysis of cells in suspension, cells were added to

lides which were precoated with poly-L-lysine at 30 mg/ml (Sigma)nd left for 15 min before the attached cells were washed with PBSnd the experimental protocol was continued.Fixation and embedding for electron microscopy. For studying

he distribution of nucleolin within the cells, the postembeddingmmunogold procedure was used as follows. CEM or HeLa cells werexed for 1 h at 5°C, with either 1.6% glutaraldehyde (Taab Labora-ory Equipment Ltd., Reading, UK) or 4% formaldehyde (Merck,armstadt, Germany) in 0.1 M Sorensen phosphate buffer, pH 7.3–.4. Following dehydration in increasing concentrations of methanol,he cells were embedded in Lowacryl K4M (Polysciences EuropembH, Eppelheim, Germany) as described before [52]. Polymeriza-

ion was carried out at 230°C under long wave-length UV lightPhilips TL 6-W fluorescent tubes) for 5 days. Ultrathin sections wereollected on Formvar carbon-coated gold grids (mesh 200) and usedor immunogold labeling prior to observation with a Philips 400ransmission electron microscope, at 80 kV, at 13,000 and 22,000agnification.For the exclusive detection of nucleolin expressed at the cell sur-

ace, a preembedding immunogold procedure was used. HeLa cells
reviously fixed with 0.2% formaldehyde (in PBS containing 1% BSA
nd 0.02% sodium azide) at 5°C for 30 min before further incubation30 min, 5°C) with 25 mg/ml mAb D3 specific for nucleolin. After this

incubation, cells were washed and further incubated in PBS (30 minat 5°C) containing gold-labeled (10 nm in diameter) anti-mouse IgGat 1:25 dilution (British Biocell International Ltd., Cardiff, UK). Thisprocedure was adopted in order to avoid internalization of antibodiesduring the surface labeling of cells. The cells were postfixed withglutaraldehyde and subsequently with osmium tetroxide, dehy-drated in ethanol, and embedded in Epon. Sections were collected onFormvar carbon-coated copper grids and observed without stainingor following uranyl acetate staining with or without lead citratecounterstaining.

Assay for cell surface translocation of cytoplasmic nucleolin.HeLa cells passaged in eight-well glass slides were incubated infresh culture medium (containing 10% FCS) in the absence (thecontrol) or presence of different agents: brefeldin A (0.5 mg/ml),

onensin (10 mM), methylamine (10 mM), and the calcium iono-hore A23187 (5 mM). After incubation for 5 h at 37°C, the slides

were left at room temperature for 15 min before addition of mAb D3(15 mg/ml) to each culture and further incubation for 1 h at roomtemperature (to avoid mAb D3 entry). Cells were then fixed withPFA and the bound mAb D3 was revealed by the addition of FITC-labeled anti-mouse IgG. The samples were processed for confocalimmunofluorescence laser microscopy. The degree of clustering of thecell-surface-expressed nucleolin was used as a measure to estimatetranslocation of cytoplasmic nucleolin. In each experiment, HeLacells were also incubated in serum-free medium in order to showinhibition of cytoplasmic nucleolin translocation to the cell surface. Afurther control was obtained by incubation of serum-stimulated cellsat 5°C.

RESULTS

Cell-Surface-Expressed Nucleolin Could BeDifferentiated from That of the Nucleus

The anti-HIV pseudopeptide HB-19 binds cells spe-cifically and forms a stable complex with the cell-sur-face-expressed nucleolin, as demonstrated previouslyin CEM and HeLa cell lines and in primary bloodT-lymphocytes and macrophages [12, 13, 53]. By usingthe biotin-labeled HB-19, here we show that the cell-surface-expressed nucleolin could be efficiently recov-ered from different human (HeLa, RD, Daudi, MOLT4,CEM, U937, and Jurkat) and murine (L929 and T54)cell lines. Nucleolin was revealed by immunoblottingusing rabbit polyclonal antibodies raised against hu-man nucleolin which cross-react with nucleolin fromdifferent species (Fig. 1A). It is of interest to note thatmurine nucleolin migrated slightly faster than the hu-man nucleolin. The 80-, 60-, and 50-kDa bands in somesamples represent partial degradation products ofnucleolin as has been reported before [28]. Previously,the RD cell line was reported not to express surfacenucleolin [54]. However, by our experimental proce-dure we could demonstrate that these cells do expresscell-surface nucleolin.

By immunoblotting using subcellular fractions fromPHA-activated blood lymphocytes and different celllines, nucleolin was recovered as was expected from the
nuclear fraction but also always in the nucleus-free

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fraction corresponding to the cytoplasm and from thecell surface. In different cell types, the surface-ex-pressed nucleolin represented less than 20% of thecytoplasmic proportion (not shown). Further character-ization of nucleolin found in the cytoplasm and nuclearcompartment was carried out by two-dimensional gelisoelectric focusing experiments along with the nucleo-lin sample purified from the cell surface. The immuno-blotting was carried out with the monoclonal antibodymAb D3 specific for human nucleolin (Fig. 1B). Thecytoplasmic and the cell-surface-expressed nucleolinhad similar isoelectric points with pI values at about.5, whereas nuclear nucleolin was resolved as several

FIG. 1. The cell-surface-expressed nucleolin in different types ofcells. (A) The surface-expressed nucleolin was isolated by incubationof different human (HeLa, RD, Daudi, MOLT4, CEM, U937, andJurkat) and murine (T54 and 929L) cell lines with the biotin-labeledHB-19. The samples (material recovered from 15 3 106 cells) wereanalyzed by immunoblotting using rabbit polyclonal antibodiesagainst the purified human nucleolin. (This antibody also cross-reacts with nucleolin from different species.) On the left is the posi-tion of protein markers in kilodaltons. On the right are the positionsof nucleolin (p95) and its partial degradation products (p60, p50). (B)The cell-surface-expressed nucleolin could be differentiated by its pI.

he cell-surface-expressed nucleolin recovered as in A (Surface; ma-erial recovered from 10 3 106 CEM cells) and crude nuclear and

cytoplasmic extracts (each corresponding to that from 2 3 106 CEMcells) were analyzed by two-dimensional gel isoelectric focusing fol-lowed by immunoblotting using mAb D3 (Materials and Methods).The pH gradient obtained by isoelectric focusing (first dimension)was from 4 to 7. On the left of each gel is the profile of proteinmarkers in the second dimension.

elated species with pI values between pH 4 and 6. The s

ifference between the nuclear and the cytoplasmic/ell surface nucleolin might be the consequence of post-ranslational modifications, which could determine theargeting of the newly synthesized nucleolin towardhe nucleus or the plasma membrane. Both nuclearnd cell surface nucleolin have been reported to behosphorylated [7, 37], thus other posttranslationalodifications might account for their distinct pI val-es.

ntibody-Dependent Clustering of the Cell-Surface-Expressed Nucleolin Revealed by ConfocalImmunofluorescence Microscopy

Previously, monoclonal antibodies against nucleolinave been employed to demonstrate the surface expres-ion of nucleolin by both immunofluorescence and elec-ron microscopy [10, 55] or by fluorescence-activatedell sorter (FACS) analysis [13, 53] in different types ofells. Here, the subcellular localization of nucleolin wasnvestigated by using mAb D3 and confocal immuno-uorescence microscopy, by fixing cells either withFA/Triton for intracellular staining or with PFAlone for staining the cell-surface-expressed nucleolin.As membrane proteins could cluster into patcheshen they are cross-linked by antibodies, HeLa cellsere incubated with the mAb D3 at room temperature

or 1 h before PFA fixing and analysis for confocalicroscopy. This latter incubation was carried out at

oom temperature in order to avoid entry of mAb D3hat occurs at 37°C [10]. Consistently, incubation ofells with mAb D3 resulted in the condensed stainingf cell-surface nucleolin into large patches. A crossection of such cells is shown in Fig. 2B demonstratinghe clustering of surface nucleolin which was revealedy distinct staining at the periphery of the HeLa cellshat grow as monolayers. Under these experimentalonditions, Triton X-100 treatment of PFA-fixed cellsesulted in the destruction of the clustered nucleolinonsistent with detergent-mediated solubilization ofhe cell-surface-expressed nucleolin (not shown). Ithould also be noted that mAb D3 is specific to humanucleolin since in immunoblotting experiments it re-cts with nucleolin of human origin only (not shown).onsequently, under similar experimental conditionssed for the clustering of surface nucleolin in humanells (Fig. 2B), no cell-surface signal was observed inonhuman cells that were tested, such as in hamsterHO or mouse L929 cells.In PFA/Triton-permeabilized cells, mAb D3 stained

ntracellular nucleolin as expected in the nucleolus,hereas in the cytoplasm it revealed nucleolin in dis-

inct vesicle-like structures (Fig. 2A). It should beoted that the detection of such nucleolin-containingytoplasmic vesicles by confocal microscopy required
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saturated signal in the nucleolus. Furthermore, thesecytoplasmic vesicles were readily lost due to their sol-ubility during detergent treatment used for the pur-pose of permeabilization of plasma and nuclear mem-branes. In the nucleus, mAb D3 stained primarily theperiphery of the nucleolus (Fig. 2A), at a region whichis reported to have most of the rRNA accumulation incells at interphase [56]. No cell surface staining wasobserved in HeLa cells incubated with mAb anti-his-tone H2B, which in permeabilized cells stained the

FIG. 2. Detection of intracellular and membrane-associated nuclpermeabilized cells (A), HeLa cells cultured in fresh medium for 5 h atemperature) containing mAb D3 (5 mg/ml). For the detection of thefresh medium) were further incubated for 1 h at room temperature in thextensively, and the bound anti-nucleolin antibody was revealed byorresponding to a cross section toward the middle of the cell monolayehe immunofluorescence scanning was 745 and 716 V in A and B, respeytoplasm. Note. In PFA/Triton-fixed cells, mAb LG2-2 specific for hi

cytoplasm, whereas in cells preincubated with mAb LG2-2 before PFAFIG. 3. Clustering of the cell-surface-expressed nucleolin in cells

medium were incubated (1 h at room temperature) in the presence ofand Methods). The scans of cells toward the middle cell layer are pr

nucleoplasm but not the nucleolus (not shown). The

clustering of the cell-surface-expressed nucleolin bymAb D3 occurred in different types of cells. A typicalexample is shown in Fig. 3 in which we used PBMCand CD41 T cell lines CEM and MT-4. In these cells theclustering of the cell-surface nucleolin occurred as cap-ping, which is typically observed in cells in suspensionwhen a membrane-anchored antigen is cross-linkedwith a specific antibody.

By immunoblotting using crude cytoplasmic and nu-clear extracts from HeLa cells, we demonstrated that

in by confocal immunofluorescence laser microscopy. For staining of7°C were fixed with PFA/Triton before incubation in PBS (1 h, roomll-surface-clustered nucleolin (B), HeLa cells (after 5 h of culture inesence of 15 mg/ml mAb D3 before PFA fixation. Cells were then washedTC-labeled goat anti-mouse antibodies. For each condition, a scanshown along with the respective phase contrast. The voltage used forely. A higher voltage was used in A in order to pick up the spots in thee H2B stained primarily the nucleus and not the nucleolus nor the

ation, no surface staining was apparent (not shown).suspension. CEM, MT-4, and PHA-activated PBMC in fresh cultureb D3 (15 mg/ml) before processing for confocal microscopy (Materialsnted with the respective phase contrasts.

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and its partial degradation products of 60 and 50 kDa.The pattern of nucleolin and the related nucleolinbands (in both cytoplasmic and nuclear extracts) re-vealed with mAb D3 was similar to that obtained withother monoclonal antibodies against human nucleolin,mAbs CC98 and 3G482, or with polyclonal antibodiesraised against a purified preparation of human nucleo-lin (Fig. 4). It is interesting to note that the rabbitpolyclonal antibody raised against a synthetic peptidecontaining the first 26 amino acids of human nucleolinreacted only with the nondegraded nucleolin (Fig. 4B),thus suggesting that the 60- and 50-kDa bands corre-spond to cleavage products toward the COOH-terminalportion of nucleolin; i.e., the partial cleavage of nucleo-lin starts at the amino-terminal end [12, 13]. The par-tial degradation of nucleolin which most probably ismediated by membrane-associated proteases [12, 13,53] has been well documented in the literature [28].The fact that the 60- and 50-kDa nucleolin by-productsare recognized by each of the three mAbs indicates thattheir respective epitopes should be located in theCOOH-terminal portion of nucleolin.

FIG. 5. Organized clustering of the cell-surface-expressed nucleo37°C) in the absence or presence of cytochalasin D (5 mg/ml) were fumg/ml). Cells were then washed in PBS and incubated at room temfixation. (B) HeLa cells preincubated in the absence or presence of cytmAb D3 (5 mg/ml). The bound mAb D3 was then revealed by the FITa cross section toward the middle of the cell monolayer is presented. TV in A and B, respectively. At an increased voltage during scanninvisible; however, the nucleolin signal in the nucleolus becomes overstreatment affected the clustering of the chemokine receptor CXCR4

FIG. 6. Association of the cell-surface-expressed nucleolin with(A) or presence (B) of cytochalasin D (5 mg/ml) were further incubate

ere then washed in PBS and further incubated at room temperatugG. Finally, cells were fixed with 3.7% PFA (to permeabilize the pla

FIG. 4. Detection of nucleolin in crude cytoplasmic and nuclearHeLa cell extracts by different monoclonal and polyclonal antibodies.Cytoplasmic and nuclear extracts (lanes C and N, respectively) wereprepared from proliferating HeLa cells as described under Materialsand Methods. The samples (material recovered from 3 3 106 cells)were analyzed by immunoblotting using rabbit polyclonal antibodiesraised against a purified preparation of human nucleolin (A) oragainst a synthetic peptide corresponding to the first 26 amino acidsof human nucleolin (B) and mAbs D3 (C), CC98 (D), and 3G482 (E).On the right are the positions of nucleolin (p95) and its partialdegradation products (p60, p50).

orresponding to a middle cross section of cell monolayers show nucleo

Despite the fact that the three different monoclonalantibodies (mAb D3, CC98, and 3G482) react with thedenatured nucleolin preparations, only mAb D3 ap-peared to react with the cell-surface-expressed nucleo-lin in its native state. This differential effect is mostprobably a consequence of the respective epitope in theCOOH-terminal portion of nucleolin that is recognizedby these antibodies. The mAb D3 was raised againstthe nucleolin preparation from the whole-cell lysates[10], whereas mAb CC98 and 3G482 were raisedagainst preparations from the nuclear fraction of cells[28, 48]. Consequently, mAb D3 might be against anepitope which is unmasked in the cell-surface-ex-pressed nucleolin. Whatever is the case, it should benoted that the capacity to cluster nucleolin by mAb D3and not mAb CC98 and 3G482 is not unique. Indeed,several antibodies specific to transmembrane proteins,such as CXCR4 and CD4, also failed to cluster therespective cell-surface-expressed antigen. For exam-ple, among mAbs 12G5, 44708, and 44717, only mAb12G5 [57] clustered CXCR4 in HeLa and CEM cells(not shown). On the other hand, different antibodiesagainst the CD4 antigen (mAb CBT4, OKT4, andOKT4A) which react efficiently with surface CD4 inFACS analysis [58], failed to trigger clustering of CD4.

Organized Clustering of the Cell-Surface-ExpressedNucleolin Is Dependent on the Actin Cytoskeleton

The clustering of membrane proteins has beenshown to be dependent on the actin cytoskeleton. Forthis purpose, we used cytochalasin D, a specific inhib-itor of actin polymerization in the cytoskeleton, whichcauses the collapse of actin microfilaments into con-densed bundles of monomeric actin [59, 60]. Interest-ingly, treatment of cells with cytochalasin D resultedin the aggregation of cell-surface-expressed nucleolin,whereas no effect was observed on the nuclear nucleo-lin (Fig. 5). In contrast, either demecolcine or nocoda-zole (each at 10 mM), known to cause perinuclear ag-gregation of intermediate filaments or inhibit tubulinpolymerization into microtubules [61, 62], had no ap-

is dependent on actin microfilaments. (A) HeLa cells incubated (3 h,er incubated (1 h, room temperature) in the presence of mAb D3 (15ature (1 h) with the FITC-labeled anti-mouse antibody before PFAalasin D (as in A) were fixed with PFA/Triton before incubation withabeled mouse antibody. For each condition, a scan corresponding tovoltage used for the immunofluorescence scanning was 725 and 680B (for example, like in Fig. 2A), the intracellular vesicles become

rated. Note. Under similar experimental conditions, cytochalasin DmAb 12G5 (not shown).actin cytoskeleton. HeLa cells incubated (3 h, 37°C) in the absence1 h, room temperature) in the presence of mAb D3 (15 mg/ml). Cells(1 h) with the secondary Texas red (TR) dye-conjugated anti-mousea membrane) to stain actin with the FITC-labeled phalloidin. Scans

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FIG. 8. Detection of nucleolin by immunogold electron microscopy in CEM and HeLa. Post- and preembedding immunogold procedures weresed to reveal the presence of nucleolin in the cytoplasm and associated with the cell membrane (Materials and Methods). Bars represent 0.2 mm.

a to c) Postembedding procedure on CEM cells using the rabbit polyclonal antibody raised against the N-terminal nucleolin peptide (a, b) or mAb3 (c). Cells were fixed with 4% formaldehyde. The arrows point to gold particles located in a clear closed vesicle adjacent to the plasma membranend at the cell surface (a), at the cell surface in front of an opened vesicle (b), and in the cytoplasm within a clear closed vesicle or clustered amonghe ribosomes (c). (d to i) The pericellular distribution of nucleolin in Epon sections of glutaraldehyde- and osmium tetroxide-fixed HeLa cells bysing a preembedding immunogold labeling performed on 0.2% formaldehyde-fixed cells, either before addition of mAb D3 (d to f) or after

ncubation of cells with mAb D3 (g to i). The sections were stained with uranyl–lead. (d to f) Different aspects of the association of gold particlesith opened vesicles which might correspond to exocytosis or endocytosis of nucleolin. (g to i) The clustered nucleolin at the external surface of thelasma membrane (only limited portions of the cell surface are labeled due to antibody-induced clustering of nucleolin). (i) A tangential section ofhe cell surface showing the accumulation of gold particles in front of the opened vesicle. Note. No gold particles were detectable at the cell surface
r within vesicles when control rabbit or mouse immunoglobulin preparations were used instead of the primary antibodies (not shown).
FIG. 7. The effect of cytochalasin D on the clustered nucleolin. (A and B) HeLa cells were incubated in culture medium containing mAbD3 (15 mg/ml) for 2 h at room temperature. The antibody-containing medium was then removed and cells were incubated at 37°C for 2 h inhe absence (A) or presence (B) of cytochalasin D (5 mg/ml). Cells were then fixed with 7% PFA and the bound antibody was revealed by the

addition of FITC-labeled goat anti-mouse IgG. A scan corresponding to a middle cross section of cell monolayers is shown. (C and D) HeLacells preincubated with mAb D3 and treated with cytochalasin D (as above) were fixed with 7% PFA. The bound antibody was revealed usingas the secondary antibody the Texas red dye-conjugated anti-mouse IgG. Cells were then stained with the FITC-labeled phalloidin. (C) A scancorresponding to the superimposed images of nucleolin (red) and actin (green). (D) An enlargement of the large actin–nucleolin bundle in C.

FIG. 9. Increased surface nucleolin expression in proliferating cells upon serum stimulation. HeLa cells passaged 24 h earlier wereincubated at 37°C as such (A) or in fresh medium in the absence (B) or presence of 5 (C) and 10% (D) FCS or in 10% FCS containing 50 mg/mlcycloheximide (E). In parallel, cells stimulated with 10% FCS were incubated at 5°C (F). At 5 h, the clustering of the surface nucleolin wasmonitored in all samples by incubation of all cells with mAb D3 at room temperature for 1 h. Finally, cells were fixed with PFA and processed
for immunofluorescence confocal microscopy.

ip


parent effect on the clustering of surface nucleolin (notshown). Efficient nucleolin clustering therefore is de-pendent on actin microfilaments, but not vimentin,which is a constituent of intermediate filaments, ortubulin, which is a constituent of microtubules.

As phalloidin is a highly poisonous alkaloid thatbinds and stabilizes actin filaments, the FITC-labeledphalloidin could be used to stain actin filaments insidecells. No labeling occurred in unfixed HeLa cells con-sistent with the inaccessibility of the FITC-labeledphalloidin in nonpermeabilized cells. On the otherhand, a classical pattern of actin staining was observedin HeLa cells permeabilized by treatment with highconcentrations of PFA (3.7%). The association of thecell-surface-expressed nucleolin with the actin cy-toskeleton was further investigated by double stainingof HeLa cells for nucleolin and actin in the absence orpresence of cytochalasin D treatment [59]. In the ab-sence of cytochalasin treatment, typical patterns of theclustered surface nucleolin and actin cytoskeleton wereobserved. When the nucleolin- and actin-specific stainswere superimposed, then the colocalization of both an-tigens was emphasized by the yellow color in the merge ofnucleolin red and actin green stains (Fig. 6A). Cytocha-lasin treatment triggered the expected collapse of actinmicrofilaments into condensed bundles which were ran-domly distributed. Interestingly, the distribution of thecell-surface-expressed nucleolin was also modified in cy-tochalasin-treated cells, but once again it colocalized withthe condensed actin bundles (Fig. 6B). These results sug-gest that clustered nucleolin at the cell surface should beassociated directly or indirectly with the intracellularactin. In this respect, it is worthwhile to mention herethat a proportion of the extranuclear nucleolin has re-cently been isolated as a detergent-soluble phosphopro-tein extractable from the cytoskeleton [63].

In order to investigate the effect of actin disruptionon the cell-surface-clustered nucleolin, cells preincu-bated with mAb D3 were washed and further incu-bated in the absence or presence of cytochalasin D. Thesurface clustered nucleolin was revealed as a well-organized and concentrated pattern at the cell periph-ery (Fig. 7A), whereas in cytochalasin D-treated cells itappeared to be disintegrated, as the nucleolin signal atthe cell periphery became dispersed and disrupted

FIG. 10. Induction of surface nucleolin in growth-arrested cells. H16 h (A, Growth-Arrested). Cells were then further incubated in fresh mactinomycin D (C) or 50 mg/ml cycloheximide (D). After 5 h at 37°C, th

FIG. 11. Entry of nucleolin-specific mAb D3 into HeLa cells is inhn fresh culture medium containing 10% FCS for 2 h at 37°C under dresence of mAb D3 (15 mg/ml) and sodium azide (NaZ3, 25 mM), an

Cells were then washed and fixed with 7% PFA and permeabilized wiwith the cell surface or having entered the cell (intracellular mAbantibodies. For each condition, a scan corresponding to a cross sec
respective phase contrast. The voltage used for the immunofluorescenc
(Fig. 7B). Double staining of cytochalasin-treated cellsdemonstrated the coexistence of the clustered nucleo-lin with actin in aggregates at the cell periphery (Fig.7C). Interestingly, the enlargement of the large con-densed actin–nucleolin bundle indicates the coexist-ence of a distinct layer of actin alongside a layer of theclustered surface nucleolin, with the middle yellowstaining representing the superimposed actin-FITCand nucleolin-Texas red layers (Fig. 7D). It should benoted that the disruption of clustered nucleolin wasobserved with cytochalasin D [59, 60] but not demecol-cine and nocodazole treatment [61, 62] (data notshown).

The Detection of Cell-Surface-Expressed Nucleolin byImmunogold Electron Microscopy

The intracellular distribution of nucleolin was inves-tigated by the use of rabbit polyclonal antibodies raisedagainst the synthetic peptide corresponding to theNH2-terminal 26 amino acids of nucleolin and themonoclonal antibody mAb D3, on Lowacryl sections offormaldehyde-fixed cells. The bound primary antibod-ies were revealed by gold-labeled secondary antibodies.Gold particles were found to be scattered over thenucleoplasm and as expected they were numerous overthe nucleolus (not shown). Few gold particles werepresent at the cell surface, sometimes in associationwith opened vesicles (Figs. 8a and 8b). Regarding thecytoplasm, gold particles were found over the ribo-some-rich areas and additionally over the electron-translucent lumen of smooth vesicles (Figs. 8a and 8c).

In order to confirm the presence of nucleolin on thecell surface, we used a preembedding immunogold la-beling procedure in which cells were first fixed par-tially with 0.2% formaldehyde before incubation withmAb D3, to block the potential internalization of anti-bodies that might occur in unfixed cells. The boundmAb D3 was then revealed by the addition of gold-labeled secondary antibodies (10 nm in diameter)which can be accessible only to antigens exposed at thecell surface. Consistent with this, gold particles wererestricted to the cell surface and often associated withopened vesicles either in the interior (Figs. 8e and 8f)or at their junction with the plasma membrane (Fig.

cells passaged 24 h earlier were incubated in serum-free medium forium containing 10% FCS in the absence (B) or presence of either 5 mMustering of the surface nucleolin was monitored as for Fig. 9.ed by sodium azide. HeLa cells passaged 24 h earlier were incubatedrent conditions: (A) in the presence of mAb D3 (15 mg/ml), (B) in theC) in the presence of mAb LG2 (15 mg/ml) specific for histone H2B.Triton X-100. The anti-nucleolin antibody mAb D3, either associated3), was revealed by the addition of FITC-labeled goat anti-mouse

toward the middle of the cell monolayer is shown along with the

eLaed

e clibitiffed (th

Dtion

e scanning was 720 V.


8d). The different aspects of the association of goldparticles with the opened vesicles might correspond tosuccessive steps of either endocytosis or exocytosis ofnucleolin. The interior of cells was entirely devoid ofgold particles, demonstrating the total absence of in-ternalization of the anti-nucleolin antibody. Furtherexperiments by electron microscopy were carried out todemonstrate the antibody-dependent clustering ofnucleolin at the cell surface, by first incubation of cellswith mAb D3 before fixation with 0.2% formaldehydeand addition of the secondary antibodies. By this pro-cedure, nucleolin associated with the plasma mem-brane became abundant in accord with the results ob-tained by the confocal microscopy (Fig. 2B), andconsistently the clustered nucleolin molecules werepresent on the external side of the plasma membrane(Figs. 8g, 8h, and 8i).

Expression of the Cell-Surface-Expressed Nucleolin inResponse to Serum Stimulation of Proliferating andGrowth-Arrested Cell Cultures

Nucleolin was constantly detectable on the surface ofproliferating cells. However, removal of FCS from theculture of proliferating cells (Figs. 9A and 9B) or trans-fer of cells from 37 to 5°C for 5 h (not shown) resultedin an almost complete disappearance of surface nucleo-lin. Therefore nucleolin appears to be constantly trans-located to the surface by an active process, as has beenreported to be the case for other secreted proteins [64–66]. When cultures of proliferating cells were furtherstimulated for 5 h by the addition of fresh culturemedium containing 10% FCS, then there was a signif-icant enhancement in the level of surface nucleolin at37°C but not at 5°C (Figs. 9D and 9F). At a reducedtemperature, therefore, the translocation of nucleolintoward the surface is blocked even in the presence ofserum stimulation. The enhancement of surfacenucleolin in serum-stimulated proliferating cells wasnot affected by de novo RNA and protein synthesisinhibitors actinomycin D (not shown) and cyclohexi-mide (Fig. 9E), respectively, consistent with a translo-cation process for the preexisting cytoplasmic nucleolintoward the plasma membrane. It should be noted thatno apparent effect was detectable in the level of nucle-olar nucleolin in cell cultures incubated in the absenceor presence of serum (not shown), thus pointing outthat the expression of surface nucleolin is regulatedindependent from that of the nucleolus.

We next investigated the expression of surfacenucleolin in serum-starved cells in which, due to inhi-bition of metabolic processes such as RNA and proteinsynthesis, the cytoplasmic nucleolin stores become ex-hausted. For this purpose, HeLa cells were growtharrested by culturing 16 h in serum-free medium be-
fore serum stimulation in the absence or presence of
actinomycin D and cycloheximide. As was expected, theexpression of surface nucleolin was almost not detect-able in serum-starved cells (Fig. 10A), but a strongsignal was induced upon addition of fresh culture me-dium containing 10% FCS (Fig. 10B). Both inhibitors,actinomycin D and cycloheximide, blocked significantlythe induction of surface nucleolin in response to serumstimulation (Figs. 10C and 10D), thus demonstratingthat surface expression of nucleolin in such growth-arrested cells requires both transcriptional and trans-lational events. These results also point out that sur-face expression of nucleolin is highly associated withcell growth conditions.

The Translocation of Nucleolin to the PlasmaMembrane

During serum stimulation of proliferating cells, thesecretion of a small amount of nucleolin was detectablein the culture medium at 1 h with maximum levels at2 h, whereas at 3 h poststimulation of cells nucleolinsecretion was no longer detectable (not shown). Thus,nucleolin appears to be released in the culture me-dium, but this effect is an early and a transient eventin response to serum stimulation. The role, if any, ofthe secreted nucleolin remains to be investigated. Itmight simply be the consequence of an increased rateof nucleolin translocation toward the plasma mem-brane. The increased levels of surface nucleolin re-mained highly stable, since shedding of surface nucleo-lin did not occur even after extensive washing of cellsin solutions containing EDTA, EGTA (each at 5 mM),or heparin (100 mg/ml; not shown).

The results shown in Fig. 9 demonstrate that trans-location of nucleolin toward the surface is a continualprocess which is increased rapidly in serum-triggeredcells. This latter observation was used here to evaluatethe effect of inhibitors of the classical pathway forsecretion through the ER and Golgi apparatus (Table1). These inhibitors were brefeldin A and monensin,which block protein transport within the ER–Golgicomplex [67, 68], and methylamine, which is an inhib-itor of the endocytotic and exocytotic pathways [69].None of these inhibitors exerted an apparent effect onnucleolin translocation, thus indicating that the trans-port of nucleolin occurs by an alternative secretorypathway independent of ER–Golgi, as has been previ-ously shown to be the case for a few other secretedproteins [64]. In addition, no significant effect onnucleolin translocation was observed in the presence ofthe calcium ionophore A23187, known to stimulatecalcium-dependent exocytosis of proteins by a mecha-nism independent of the ER–Golgi complex pathway[70]. In contrast to classical inhibitors of glycoproteintransport, surface nucleolin expression was reduced
when serum-stimulated cells were incubated at room

cpposM


temperature or 5°C and when cells were incubated inthe absence of serum (Table 1). The enhancement ofsurface nucleolin upon serum stimulation at 37°C ap-pears to be a specific event since it occurs in the ab-sence of any apparent effect on the level of surfacetransmembrane glycoprotein-receptors, such asCXCR4 or CD4 (not shown).

Internalization of the Anti-nucleolin mAb D3 Boundto the Surface of Human Cells at 37°C

Previously, mAb D3 was reported to be internalizedin human cells following prolonged incubation of cellcultures at 37°C [10]. Under experimental conditions ofTriton X-100 permeabilization of 7% PFA-fixed cells,intracellular mAb D3 was clearly detectable in cellspreincubated with mAb D3 for 15–30 min. This entry ofmAb D3 was progressive since it was increased mark-edly with time. Figure 11A shows the presence of mAbD3 in the cytoplasm as well as at the membrane ofHeLa cells preincubated with mAb D3 for 2 h. Thislatter was not an artifact of the experimental proce-dure, since no entry was observed in HeLa cells prein-cubated with the anti-histone H2B antibody mAb LG2(Fig. 11C), which does not bind to the surface althoughit reacts strongly with histone H2B in the nucleoplasm.The internalization of mAb D3 did not occur at 5°C,and even at room temperature there was very littleentry (not shown), thus indicating that mAb D3 entryoccurs by an active process. Furthermore, internaliza-tion requires metabolic energy as it was drasticallyreduced in the presence of high concentrations of so-dium azide (Fig. 11B). Consistent with its specificity tonucleolin of human origin, mAb D3 does not bind ham-ster CHO cells although they express surface nucleolin.

TABLE 1

Translocation of Cytoplasmic Nucleolin to the Surface

Serum Temperature InhibitorNucleoein surface

expression

10% 37°C — Enhancement10% RT — Reduction10% 5°C — Reduction0% 37°C — Reduction

10% 37°C Brefeldin A (0.5 mg/ml) No effect10% 37°C Monensin (10 mM) No effect10% 37°C Methylamin (10 mM) No effect10% 37°C Ca21 ionophore (5 mM) No effect

Note. HeLa cells passaged 48 h earlier were incubated in freshulture medium containing 10 or 0% FCS and in the absence orresence of different protein transport inhibitors as indicated. Inarallel, cells stimulated with 10% FCS were incubated either at 5°Cr at RT (room temperature, 21°C). At 5 h, the clustering of theurface nucleolin was monitored as described for Fig. 9 and underaterials and Methods.

Furthermore, mAb D3 does not become internalized in

CHO cells, even after prolonged incubation at 37°C(not shown). Consequently, the entry of mAb D3 inhuman cells is a specific event as a consequence of theantibody binding to the surface expressed nucleolin.

DISCUSSION

Previously, we had isolated a 95-kDa protein fromthe surface of different types of human cells andshowed that it is recognized by different monoclonalantibodies specific for human nucleolin and by rabbitpolyclonal antibodies raised against either a purifiedpreparation of human nucleolin or a synthetic peptidecorresponding to the first NH2-terminal 26 amino acidsof human nucleolin. Surface iodination of intact cellsresulted in the iodine labeling of the 95-kDa protein,whereas protease digestion of surface proteins resultedin the loss of this protein among other surface proteins.Finally, the amino acid sequence obtained by microse-quencing of the NH2-terminal end and of several inter-nal peptides obtained by partial proteolytic digestion ofthe 95-kDa protein was found to correspond to theamino acid sequence reported for human nucleolin[12]. The cell-surface presence of nucleolin was con-firmed here by immunoelectron microscopy using therabbit polyclonal antibodies raised against the NH2-terminal synthetic peptide (Figs. 8a and 8b) and themonoclonal antibody mAb D3 (Figs. 8c to 8i). In addi-tion, incubation of cells with mAb D3 resulted in theclustering of the cell-surface-expressed nucleolin, as isgenerally observed with transmembrane proteins uponincubation of unfixed cells with their respective anti-bodies. The surface nucleolin therefore could behave asa well-characterized membrane-anchored proteinwhen cross-linked with a specific antibody [71]. Like-wise, the clustering of surface nucleolin by mAb D3also occurred at low temperature, thus indicating thatit is via an energy-independent process as is the casefor transmembrane antigens.

By immunoblotting using crude cytoplasmic and nu-clear extracts, we show that mAb D3 reacts withnucleolin and its partial degradation products, a reac-tivity pattern identical to that obtained by two othermonoclonal antibodies and by a polyclonal antibodyraised against a purified preparation of human nucleo-lin (Fig. 4). In view of these observations, it is veryunlikely that the reactivity of mAb D3 with the cell-surface-expressed 95-kDa protein (Fig. 1B) is simplydue to recognition of an epitope common to nucleolinand a 95-kDa protein which is not related to nucleolin.It is worthwhile to note that in rat cells, a calcium-binding surface protein has been isolated and shown tobe 83% homologous to rat nucleolin [72]. Interestingly,by aligning the amino acid sequence of this nucleolin-related protein (referred to as NRP) with that of hu-
man nucleolin, we found that the homology is much

te1mAitho

imvcscsk6tpstsndaiucmnenbawcatw

asasi1a


more significant between NRP and human nucleolincompared to NRP with rat nucleolin. Indeed, if oneeliminates the NH2-terminal domain containing theacidic stretches, the last 437 amino acids at the COOH-terminal end of NRP share 97.5 and 89.2% sequenceidentity with the corresponding sequence in humanand rat nucleolin, respectively. Moreover, rat NRPcould be differentiated from the rat nucleolin by theexistence of a distinct motif containing 5 amino acids atresidues 10 to 14: AsnGlnSerAspPro for rat NRP com-pared to ThrHisGlyGluSer for rat nuclear nucleolin[72] (Fig. 12). Intriguingly, this AsnGlnSerAspPro mo-tif in rat NRP is quite similar to the correspondingmotif AsnGlnGlyAspPro in the sequence reported forhuman nucleolin [33]. It should be noted that the NH2-erminal microsequence of the 95-kDa protein recov-red from the surface of human cells was found to be00% identical with the NH2-terminal sequence of hu-an “nuclear” nucleolin [12], i.e., containing thesnGlnGlyAspPro motif (Fig. 12). Consequently, if an

soform of nucleolin exists as a surface-expressed-pro-ein, it should be highly homologous to the sequence ofuman “nuclear” nucleolin which was reported previ-usly.In the cytoplasm, nucleolin appears to be packaged

n intracytoplasmic vesicles (Fig. 2) that at the plasmaembrane seem to fuse (Fig. 8). This latter could pro-

ide a pathway for the translocation of nucleolin to theell surface by an active process, since the level ofurface nucleolin is markedly reduced by incubation ofells in serum-free medium or incubation of serum-timulated cells at 5°C (Fig. 9), i.e., under conditionsnown to block endo- and exocytosis of proteins [64–6].Classically, exocytosis involves fusion of small in-racellular vesicles with the inner surface of thelasma membrane and is of fundamental importanceince it represents the route by which many neuro-ransmitters, hormones, and enzymes, packaged inuch vesicles, are secreted [70]. The detection ofucleolin inside intracytoplasmic vesicles, in vesiclesuring the fusion process with the plasma membrane,nd in vesicles opened at the cell surface, the increasen the amount of clustered nucleolin upon serum stim-lation at 37°C but not at 5°C and the existence of thelustered nucleolin at the external side of the plasmaembrane (Figs. 8, 9, and 10) favor the suggestion thatucleolin becomes transported to the cell surface by anxocytosis mechanism. The translocation of nucleolinot being affected by methylamine and monensin (Ta-le 1), our results suggest that nucleolin is exported bypathway independent of the classical secretion path-ay for glycosylated proteins through the ER–Golgi

omplex. Furthermore, as nucleolin does not possessn N-terminal leader sequence [32, 33], its transloca-ion should occur through a leaderless secretion path-
ay. In this respect, the secretion of nucleolin is simi-
lar to the release of other proteins devoid of a signalsequence, such as IL-1b [64], the lactose-binding lectin[73], thioredoxin [74], FGF-1 [75], FGF-2 [65], and theHIV-1 Tat transactivator protein [66]. It should also benoted that there might be some differences in themechanism of nucleolin secretion compared to the se-cretion of other leaderless proteins, since methylamineinhibits the release of IL-b and FGF-1 [64, 65] but notthat of nucleolin.

The close association of nucleolin with actin in theabsence or presence of cytochalasin D treatment (Figs.6 and 7) indicates the close association of surfacenucleolin with the intracellular actin cytoskeleton.These results also point out that the association ofnucleolin with actin is not dependent on the filamen-tous and polymerized state of actin, but it is an intrin-sic property of nucleolin to interact directly or indi-rectly with globular actin. The mechanism responsiblefor the association of nucleolin with the actin cytoskel-eton remains to be elucidated. However, by analogywith other transmembrane receptors, cytoskeleton as-sociation could be mediated by intermediary proteinsthat bind both nucleolin and actin filaments [71, 76].As the reported nucleolin sequence does not predict ahydrophobic domain to account for potential integra-tion into the plasma membrane, by virtue of its affinityto heparin, nucleolin could bind syndecans, known fortheir association with actin. Syndecans are type Itransmembrane proteoglycans that could function ascell-surface receptors of extracellular matrix mole-cules, mediating the effects of matrix components tothe cytoskeleton and acting as coreceptors of heparinbinding proteins [76]. The potential association ofnucleolin with syndecans is enforced by experimentalevidence demonstrating the strong affinity of purifiednucleolin to bind heparin (unpublished results). TheC-terminal domain in nucleolin rich in arginine resi-dues could represent a potential syndecan binding site.

FIG. 12. The homology between the NH2-terminal amino acidsequence of rat NRP and the corresponding sequence in humannucleolin. The amino acid sequence of the first 16 NH2-terminalmino acid residues of the rat nuclear nucleolin and that of raturface NRP [72] are aligned with the first 16 NH2-terminal aminocid residues of human nucleolin [33] and those of the amino acidequence obtained by microsequencing of the 95-kDa protein whichs recovered from the surface of human cells [12]. Amino acids 10 to4 containing the motif which is not conserved between rat nucleolinnd rat NRP are presented in bold letters.

Interestingly, this domain has been shown to be impli-

n

1

1

1

1

1

1


cated in the binding of soluble nucleolin to polyanioniccompounds such as the biotin-labeled G-G-paired DNAETS-1 [43]. On the other hand, however, ETS-1 DNAdoes not bind the cell surface, thus suggesting thatwhen nucleolin is expressed on the cell surface itsC-terminal domain is not accessible (unpublished re-sults).

In accord with several previous reports on the sur-face expression of nucleolin [2, 55, 77], our results pointout that along with its nucleolar localization, nucleolinshould also be considered a surface antigen. The closeassociation of surface nucleolin with intracellular actinand its clustering by the anti-nucleolin antibody pointout that surface nucleolin exists and behaves as clas-sical surface antigens with well-defined transmem-brane domains. In view of these and by virtue of itsRNA and protein binding domains, the cell-surface-expressed nucleolin could function as a cell surfacereceptor by interacting with specific ligands. Severalligands of the cell-surface-expressed nucleolin havebeen reported in the literature: lipoproteins in HepG2cells [78], laminin in neural cells [6], fructosyllysine inthe human monocytic cell line U937 [8], Factor J inseveral cell lines and in primary lymphocytes [11], thecytokine midkine [23, 24], the coxsackie B virus inHeLa cells [9], and the human immunodeficiency virusin different types of cells as well as in primary T-lymphocytes and macrophages [12, 13, 53]. Consistentwith the observation that the anti-nucleolin mAbbound to human cells becomes internalized at 37°C(Fig. 11), the binding of these different ligands withsurface nucleolin could represent the first step neces-sary for chaperoning these ligands into the cytoplasm[2, 55, 77]. In view of this, and the increase of surfacenucleolin in serum-stimulated cells (the results herein)or during activation of PBMC [79], it is plausible tosuggest that the cell-surface-expressed nucleolin has arole in the chain of events necessary for cell growth andproliferation.

This work was supported by grants from the Institut Pasteur, theCentre National de la Recherche Scientifique (CNRS), and the As-sociation pour la Recherche sur le Cancer. F.P.-D. and B.K. aremembers of the Institut National de la Sante et de la RechercheMedicale (INSERM). A.G.H. and J.S. are members of CNRS. Wethank E. Pichard, S. Souquere-Besse, and N. Robert for their tech-

ical assistance.

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Received July 3, 2000Revised version received August 30, 2000

The Cell-Surface-Expressed Nucleolin Is Associated with the Actin Cytoskeleton

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