EXPERIMENTAL CELL RESEARCH 200, 5-15 (1992)

Identification and Characterization of a New Set of Nucleolar Ribonucleoproteins Which Line the Chromosomes during Mitosis

THIERRY GAUTIER, *'1 CHANTAL DAUPHIN-VILLEMANT,* CHANTAL ANDR]~,t CLAUDE MASSON,*

JACQUELINE ARNOULT,* AND DANII~LE HERNANDEZ-VERDUN*

*Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05 France; and tService Central d'Hdmatologie-Immunologie, Hdpital Henri Mondor, CHU Crdteil France

W e i n v e s t i g a t e d the p e r i c h r o m o s o m a l a r c h i t e c t u r e e s t a b l i s h e d d u r i n g m i t o s i s . E n t r y in to m i t o s i s b r i n g s about a d r a m a t i c r e o r g a n i z a t i o n o f bo th n u c l e a r and cy - t o p l a s m i c s t r u c t u r e s in p r e p a r a t i o n for ce l l d i v i s i o n . W h i l e the n u c l e a r e n v e l o p e b r e a k s d o w n , n u c l e a r pro- t e i n s a r e r e d i s t r i b u t e d d u r i n g c h r o m o s o m e c o n d e n s a - t ion . S o m e o f t h e s e p r o t e i n s a r e f o u n d a r o u n d t h e chro- m o s o m e s , but l i t t l e is k n o w n c o n c e r n i n g t h e i r n a t u r e and func t ion . T e n a u t o i m m u n e s e r a w e r e u s e d to s t u d y the m i c r o e n v i r o n m e n t o f c h r o m o s o m e s and, in p a r t i c u - lar , the c h r o m o s o m e p e r i p h e r y . T h e y w e r e s e l e c t e d for t h e i r a n t i - n u c l e o l a r s p e c i f i c i t y and w e r e f o u n d to r e c o g - n i z e t h r e e n u c l e o l a r p r o t e i n s that coa t the c h r o m o - s o m e s d u r i n g m i t o s i s . T h e d i s t r i b u t i o n o f t h e s e a n t i g e n s w a s f o l l o w e d t h r o u g h the ce l l c y c l e b y c o n f o c a l l a s e r s c a n n i n g m i c r o s c o p y . T h e a n t i g e n s d i s p e r s e d v e r y e a r l y d u r i n g p r o p h a s e and s i m u l t a n e o u s l y w i t h the c h r o m o s o m e c o n d e n s a t i o n s u g g e s t i n g a c o r r e l a t i o n be- t w e e n t h e s e t w o p r o c e s s e s . T h e a n t i g e n s h a v e a p p a r e n t m o l e c u l a r w e i g h t s o f 5 3 , 6 6 , and 1 0 3 k D a on S D S - P A G E m i g r a t i o n . E l u t i o n o f the a n t i b o d i e s and i m m u - n o p u r i f i c a t i o n s h o w e d that t h e s e p r o t e i n s a r e the s i t e s of t h e p e r i c h r o m o s o m a l and n u c l e o l a r l a b e l i n g ob- s e r v e d by i m m u n o f l u o r e s c e n c e . C h a r a c t e r i z a t i o n o f the a n t i g e n s s h o w e d that t h e y a r e R N A - a s s o c i a t e d pro- t e ins . T h e c o i m m u n o p r e c i p i t a t i n g R N A m o i e t y in- v o l v e d in t h e s e R N P s a p p e a r e d to b e U 3 , but the ant i - g e n s are not r e l a t e d to the f ibr i l lar in f a m i l y . T h e r e f o r e , s m a l l n u c l e o l a r R N P s f o l l o w the s a m e d i s t r i b u t i o n dur- ing m i t o s i s as that d e s c r i b e d for s m a l l n u c l e a r R N P s . P o s s i b l e f u n c t i o n s for t h e s e a n t i g e n s a r e d i s c u s s e d . �9 1992 Academic Press, Inc.

INTRODUCTION

Interphase is characterized by the presence of a com- plex architecture separating the cytoplasm and the ge- nome. This structure is destabilized and disappears when the cell enters mitosis. The nuclear envelope

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breaks down into small vesicles and the lamina is solubi- lized by lamin depolymerization [1-4]. Simultaneously, the chromosomes condensed and the nucleolar struc- tures desassemble [5]. The required modifications are mainly regulated by specific phosphorylation of some proteins as extensively reviewed by several authors [6- 8]. The breakdown of the envelope allows chromatin and cytoplasmic constituents to come into contact with each other. It is possible therefore that a particular orga- nization forms at the chromosome periphery in mitotic cells.

During mitosis, nuclear proteins become distributed throughout the mitotic cells and adopt a totally differ- ent organization. The great majority of them disperse throughout the cytoplasm without any particular local- ization during the different phases of mitosis. Some spe- cific proteins remain associated with one particular re- gion of chromosomes, where rDNA is clustered as nu- cleolar organizer regions (NORs) [9]. Other nuclear proteins wrap around the chromosomes and remain as- sociated with the surface of the chromosome or the per- ichromosomal region. These proteins are believed to play a role in chromosome organization throughout the cell cycle [10, 11]. A mouse monoclonal antibody, Ki-67 specific for proliferative cells, exclusively recognizes the nucleoli in interphase cells and is distributed in a reticu- lated structure surrounding the condensed chromo- somes at metaphase. Although it is extensively used to determine the growth fraction of human tumors in situ and has been shown to be of value for medical prognosis, its antigen has not been characterized [12, 13]. Per- ichromonucleolin is the name for nucleolar antigens that decorate the chromosome surface during mitosis. Preliminary study suggests that these antigens have mo- lecular weights of 30,000 and 36,000 Da, and in some experiments are sensitive to RNase, but not to DNase I digestions [14]. Furthermore, earlier cytochemical staining of proteins indicated that RNPs are accumu- lated at the chromosome periphery [15], and by EDTA differentiation authors pointed out a layer of perichro- mosomal RNP fibrils and isolated granules on the pe- riphery of bleached chromosomes [16].

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GAUTIER ET AL.

T A B L E 1

Characterist ics of the Antigens

Immunofluorescence

HeLa cells Western Extraction procedures blot Antibody

Sera lnterphase Mitosis Lymphocytes (mol wt) class NaC1 2 M RNase DNase

A05 Nu PC Nu 53 IgG S S NS G08 Nu PC Nu 53-51 IgG S S NS E07 Nu PC Nu 53 IgG S S NS C12 Nu PC Nu 53 IgG * * * D09 Nu PC Nu 53-51 IgG S S + / - G04 Nu PC Nu 66 IgG S + / - NS B03 Nu PC Nu 66-62 * * * * B06 Nu PC Nu 66 IgG S + / - NS B01 Nu PC Nu 103-66 IgG S + / - NS L02 Nu PC Nu 103 IgG S S NS

Note. The behaviors of the antigens were tested onto different cell lines both during mitosis and interphase. Results of the various treat- ments exhibit common features for the antigens. Nu, nucleolar location; PC, perichromosomal location; S, sensitive to extractions; NS, not sensitive to extractions; *not determined.

L i t t l e is k n o w n t h e r e f o r e a b o u t t h e m i c r o e n v i r o n - m e n t o f t h e c h r o m o s o m e s d u r i n g m i t o s i s a n d o n l y f rag- m e n t e d i n f o r m a t i o n c o n c e r n i n g v a r i o u s a s s o c i a t e d an- t i gens is ava i lab le . H o w e v e r , t h e d a t a sugges t t h a t t h e c h r o m o s o m e p e r i p h e r y is c o m p o s e d o f R N P s o f n u c l e a r o r n u c l e o l a r o r ig in [15, 17] w h i c h cou ld p l a y a ro le in t h e

c h r o m o s o m e o r g a n i z a t i o n . T h e a v a i l a b i l i t y o f a u t o i m - m u n e se ra specif ic fo r n u c l e o l a r c o m p o n e n t s a l lows fur - t h e r i n v e s t i g a t i o n s o f t h e n u c l e o l a r o r g a n i z a t i o n a n d o f c o m p o n e n t s o f t h e c h r o m o s o m e p e r i p h e r y d u r i n g m i t o - sis. M o r e o v e r , s e l e c t i o n of a n t i b o d i e s is a p o w e r f u l m e t h o d to so r t p r o b e s a g a i n s t n e w n u c l e a r a n t i g e n s ( for r e v i e w see [18]). A l t h o u g h n u c l e o l a r spec i f i c i ty is less f r e q u e n t , s e v e r a l a u t o i m m u n e se ra h a v e a l r e a d y b e e n s e l e c t e d a n d u s e d as specif ic p r o b e s (for r e v i e w see [ 19]).

In th i s pape r , we desc r ibe t h e i d e n t i f i c a t i o n a n d t h e c h a r a c t e r i z a t i o n o f a s e t o f n u c l e o l a r p r o t e i n s f o r m i n g a s h e a t h a t t h e c h r o m o s o m e p e r i p h e r y . T h e s e p r o t e i n s w e r e s t u d i e d wi th 10 d i f f e r e n t a u t o i m m u n e se ra con- t a i n i n g a n t i b o d i e s o f a n t i - n u c l e o l a r speci f ic i ty . T h e re- su l t s i n d i c a t e t h a t t h e s e a n t i g e n s f o r m a h i t h e r t o u n d e - s c r ibed c lass o f p r o t e i n s a n d a re a s s o c i a t e d w i t h a s m a l l n u c l e o l a r R N A . T h e i r d i s t r i b u t i o n t h r o u g h o u t m i t o s i s i n d i c a t e s t h a t b o t h n u c l e o l a r a n d n u c l e a r R N P s p r e f e r - e n t i a l l y a c c u m u l a t e a t t h e c h r o m o s o m e p e r i p h e r y sug- ge s t i ng t h a t t h e y h a v e a d y n a m i c ro le in c h r o m o s o m e o r g a n i z a t i o n a n d / o r r e f l ec t i ng a m e c h a n i s m n e c e s s a r y for t h e e q u a l d iv i s ion o f t h e n u c l e o l a r m a t e r i a l b e t w e e n t h e d a u g h t e r cells.

MATERIALS AND METHODS

Cell culture. Human established ceil lines, (HeLa, HEp-2, TG), were grown in MEM-Eagle medium containing 10% fetal calf serum and 1% glutamine, CHO ceils were grown in ~-MEM-Eagle medium containing 10% fetal calf serum and 1% glutamine. PtKa cells were cultured in 0.85 g/liter NaHCOs, 2% glutamine, and 10% fetal calf serum.

Serum selection. Sera were screened on unfixed rat liver cryosec- tions and human HEp-2 cells, by indirect immunofluorescence. Of 40,000 sera tested, 380 contained antibodies against mammalian nu- cleolus. Antisera which reacted with one or more extranucleolar anti- gens were eliminated, leaving 36 sera. Among the 36 anti-nucleolar sera selected, 10 bound to the chromosome periphery during mitosis. These 10 sera were tested by several techniques to verify that they do not contain other anti-nuclear antibodies associated to the anti-nu- cleolar antibodies. Anti-double-stranded DNA antibodies were de- tected by indirect immunofluorescence using Crithidia luciliae. Precip- itating antibodies against extractable nuclear antigens were revealed by Ouchterlony double diffusion using a rabbit thymus extract (rabbit acetone powder, Pel-Freeze Biologicals, Rogers AR) containing nu- clear RNP (nRNP), Sm, La/SS-B, Scl 70, Jol, and PCNA antigens [18]. Ribosomal RNP (rRNP) antigens were also present in this ex- tract. Precipitating antibodies against Ro/SS-A antigens were re- vealed by counterimmunoelectrophoresis using human spleen extract as the antigen source. All 10 selected sera scored negative in all the above tests andwere presumed therefore not to contain specific anti- bodies for DNA, soluble nRNPs, RNA-protein complexes containing small nuclear RNAs designated U1, U2, U4, and U6 snRNAs, RNA polymerase III transcripts including 5S ribosomal RNA, DNA topo- isomerase I, or "cyclin."

The patients from whom these sera were obtained suffered of chronic graft versus host disease after allogenic bone marrow trans- plantation (D09, C12), scleroderma (B01, G04, and A05), undifferen-

FIG. 1. Immunolabeling of PtK~ cells cultured on coverslips. Antibodies were revealed by an anti-human IgG coupled to FITC. (a) Early prophase (P) and interphase (I) observed after labeling with B01 serum and in phase contrast. (b) The two nucleoli are labeled and antigens begin to diffuse around the chromosomes in the late prophase (P). (c-d) Pseudometaphase (PM) and interphase (I) labeled with G04 serum. (e-f) metaphase (M) and interphase (I) labeled with L02 serum. Chromosomes were stained with Hoechst 33342; the immunofluorescence signal is visible around the chromosomes forming a thin layer. Bar = 10 ttm.

PERICHROMOSOMAL DISTRIBUTION OF NUCLEOLAR RNPS 7

8 GAUTIER ET AL.

tiated connective tissue disease (E07), and nonclassified disease (L02, B03, B06, and G08). Nine of these 10 anti-nucleolar antibodies were immunoglobulin G.

Localization of antigens. For immunofluorescence microscopy, Human HEp-2 cells and P tK 1 cells seeded on coverslips, and rat liver cryosections were used. The serum dilutions ranged from 1:40 to 1:640 for use with fixed cells and from 1:100 to 1:5000 for use with unfixed rat liver sections. DNA was stained with Hoechst 33342 (5 ~g/ml) or DAPI (1:10000). The mounting medium was citifluor LTD (City University, London). CLSM was performed using a Bio-Rad MRC-600 mounted on an Optiphot II Nikon equipped with a 60x objective (plan apo; NA 1.4). An Argon-ion laser adjusted at 488 nm wave length was used for fluorescein excitation.

For extraction procedures unfixed rat liver sections were floated on one of the following solutions: DNase-free 0.4% RNase I (A) (Worth- ington) in PBS for 1 h; 0.4% DNase I (Worthington) in PBS contain- ing 0.4 mM MgC12 for i h; 0.05% Pronase in PBS for 20 min at 4~ or 2 M NaC1 in 10 mM Tris-HC1, pH 7.6, containing 0.2 mM MgC12. Controls were carried out by incubating sections in the buffers with- out enzymes.

Electron microscopy. For cryoultramicrotomy, HeLa cells were treated with colchicine, and then fixed for 1 h as a pellet with phos- phate-buffered 2% paraformaldehyde containing 0.1% glutaralde- hyde. Free aldehyde groups were blocked for 2 h by incubation in 0.5 M NH4C1 in phosphate buffer. The samples were soaked overnight at 4~ in 1.6 M sucrose as a cryoprotectant and then for 2 h in 2.1 M sucrose. Samples were then rapidly plunged into liquid nitrogen. Ul- trathin sections were cut with a Reichert-FC 4D microtome cooled to -110~ [20], stained with uranyl acetate for 1 min, and observed with a Philips 410 electron microscope.

For immunolabeling, HEp-2 cells were fixed with 3% formaldehyde in PBS for 15 min and with methanol for 6 min at -20~ then per- meabilized with 0.25% Triton X-100 for 2 min. Cells were incubated with the autoimmune sera followed by peroxidase-conjugated sheep anti-human immunoglobulins (Amersham, France) and treated as previously described [21]. The samples were dehydrated by immer- sion in a series of ethanol solutions of increasing concentration, and embedded in Epon. The sections were contrasted with uranyl acetate for 1 min. Nonautoimmune human serum was used for controls.

Characterization of antigenic proteins. Nuclear proteins from ac- tively growing HeLa cells were used as a source of antigens. Cells were suspended in TKM buffer (10 mM Tris-HC1, pH 7.4, 10 mM KC1, 3 mM MgC12) , containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 #g/ml pepstatin, 10 #g/ml aprotinin, and 1 #g/ml leupep- tin. Cells are swollen by hypotonic shocks in the TKM buffer. The procedure was performed at 4~ Triton X-100 (1%) was added to obtain very pure nuclei. Isolated nuclei were suspended in buffer con- taining sodium-dodecyl-sulphate (SDS) according to Laemmli [22] and stored at -80~ until use. The nuclear protein concentrations were measured by the BCA protein assay [23].

Nuclear proteins were separated by electrophoresis in SDS-10% polyacrylamide slab gels. Molecular weight standards were obtained from Bio-Rad Laboratories (Ivry sur Seine, France). Samples were loaded to obtain 10 ~g of protein per lane. Proteins were then trans- ferred to a nitrocellulose sheet, as previously described [24]. The ni- trocellulose sheets were stained with 0.2% Ponceau red in 0.3% tri- chloracetic acid (TCA) and then destained with 0.3% TCA. The blots were saturated for 1 h with 5% dried milk in Tris buffer solution (TBST: 10 mM Tris-HC1 pH 8.0, 150 mM NaC1, 0.5% Tween 20). Cut strips were incubated with a different human serum in TBST for 2 h overnight at room temperature. Alkaline phosphatase-conjugated goat anti-human IgG (H + L) was used as the second antibody and was detected with nitro-blue-tetrazolium substrate.

Immunopurification of antibodies. Monospecific antibodies were purified following the strategy of Olmsted [25]. The selected serum was incubated for 12 h with a blot to bind antibodies specific for nuclear extract antigens. The position to which antibodies bound was performed on one control strip by immunodetection and horizontal bands of nitrocellulose were cut. Antibodies were eluted from the hori- zontal bands by incubation in 3 M KSCN for 5 min. The suspension was immediately diluted in PBS and dialyzed under vacuum to a final volume of 200 ~1 as described by Krohne [26]. The antibodies immun- opurified by this method were tested by immunofluorescence on HEp-2 cells and used on Western Blot to verify elution and to deter- mine the possible cross-reactions.

CharacterizationofassociatedRNAs. The procedure for ultrastruc- tural autoradiography has been reported and extensively described elsewhere [27]. TG cell monolayers were labeled with tritiated uridine at a final concentratio n of 100 #Ci/ml for 3 h. The cells were washed three times in PBS containing nonradioactive uridine at a final con- centration of 1 x 104 mM. Classical Epon embedding was performed. Ilford L4 emulsion was applied onto single grids using the gold inter- ference colored zone of the emulsion film. After exposure the signals were amplified by gold latensification and then developed using phen- idon [28].

Analysis of immunoprecipitated RNAs. The procedure was based on the technique described by Steitz [29]. For RNA analysis, 6 >< 107 HeLa or CHO cells were labeled for 16 h with [32P]orthophosphate at a final concentration of 10 ~Ci/ml (Amersham, France) in phosphate- free MEM (or a-MEM for CHO cells). Cells were harvested and washed in 15 ml of cold TBS (40 mM Tris-HC1, pH 7.4, 150 mM NaC1), suspended in 1.2 ml NET-2 (50 mM Tris-HCl, pH 7.4, 150 mM NaC1, 0.05% Nonidet P40) containing the same anti-proteases as described above and broken by sonication for 10 s by a soniprep 150 (MSE) at a setting of 14. The samples were centrifuged at 10,000 rpm for 10 min in an Eppendorf microfuge and the clarified cell superna- tant was collected and used immediately for immunoprecipitation as- says. Protein A-Sepharose CL-4B (Pharmacia, France) 2.5 mg, pres- wollen in 500 #l of PBS, pH 8.0, was incubated with 10 ~1 of crude serum by end-over-end rotation for 16 h at 4~ and then washed five times with NET-2. The antibody-bound beads were incubated with 200 #l of the [32P]-labeled HeLa or CHO cell extract and 300 #1 of NET-2 for 2 h at 4~ The samples were centrifuged for 15 s and the supernatant was collected and stored for subsequent RNA extraction. The pelleted beads were washed five times in NET-2 and then sus- pended in 300 ul of NET-2; 30 #1 of 10% SDS and 2 ug of yeast tRNA (Boehringer) as carrier was added. RNAs were extracted with phenol- chloroforme-isoamyl alcohol (25:24:1) containing 0.1% hydroxyquin- oline and precipitated with ethanol. They were separated by electro- phoresis on 8 M urea-10% polyacrylamide gels, 100 mM Tris-borate, pH 8.3, and 2 mM EDTA and identified by autoradiography with X-ray films (Fuji, Euromedica, France). Nonautoimmune and anti- Sm sera were used simultaneously as negative and positive controls for immunoprecipitation. The anti-Sm serum that we used is specific for U1, U2, U4, and U5 snRNAs. Its specificity has been established by Tan's group (LaJolla, CA) by comparison with their anti-Sin au- toimmune sera.

RESULTS

Selection of Anti-nucleolus Antibodies Labeling the Chromosome Periphery

T h e 36 an t i -nuc leo lus sera were each i n d e p e n d e n t l y used to label fixed PtK1 cells. This cell line was used

FIG. 2. Confocal laser scanning microscopy of human HEp-2 cells after labeling with D09 serum. Metaphase (M) and anaphase (A) stages are observed at three different levels obtained at z = 1, 2.5, and 4.5 #m. Analysis of the images shows that the labeling is present in close contact with the mass of the compacted chromosomes. No signal can be found in the cytoplasm of the two cells. Bar = 10 ~m.

PERICHROMOSOMAL DISTRIBUTION OF NUCLEOLAR RNPS 9

10 GAUTIER ET AL.

because it has only six ch romosome pairs, which simpli- fies the s tudy of mitosis. T e n sera labeled the per iphery of the chromosomes of cells undergoing mitosis. Only these 10 sera (Table 1) were used. T h e pa t t e rns of label- ing of each serum, at each stage of the cell cycle, were examined and found to be 'similar. At the beginning of the prophase , the nucleolar ant igens migra ted f rom the nucleolar body to form a ne twork in the nucleoplasm at the t ime of the ch romat in condensa t ion (Figs. l a - l b ) . T h e nex t step of the process revealed t ha t each chromo- some was su r rounded by nucleolar ant igens in p rometa - phase (Figs. l c - l d ) . During metaphase , the chromo- some per iphery exhibi ted a positive labeling. T h e signal was found at the border of Hoechs t s ta ined chromo- somes (Figs. l e - l f ) . During anaphase, per ichromoso- mal mater ia l followed the chromosomes dur ing thei r migrat ion to each pole. At late te lophase, this s taining fused to the presumpt ive NORs to form the in te rphase nucleolus. The in te rphase cells visibly in the different fields show the labeling of nucleoli for the th ree sera i l lustrated (Figs. la , lc , and le) .

The labeling of human lymphocytes was examined to investigate whether these nucleolar ant igens were cell cycle related. The sera bound to the nucleolus of the lymphocytes as found by indirect immunofluorescence , indicat ing tha t the ant igens were still p resen t in these noncycl ing cells (Table 1).

On confocal sections, in te rconnec ted s t rands forming an intr icate ne twork a round metaphase ch romosomes were found on H e L a cells. T h e images obta ined with D09 serum indicated t ha t the labeling is not only at the per iphery of the plate in me taphase cells bu t also be- tween the chromosomes. Th ree levels of 0.5 um in thick- ness and separa ted of 1, 2.5, 4.5 #m from the cell a t tach- men t level are shown (Figs. 2a, 2b and 2c). T h e pa t t e rn of labeling of late anaphase suggests t ha t the binding is s t rongly associated with the ch romosome per iphery during cell division (Figs. 2a, 2b, and 2c).

T o exclude any ar t i factual localization within the cell volume, c ryou l t ra th in sections of h u m a n H e L a cells were observed under the e lect ron microscope. A dense mater ia l was visible associated with chromosomes (Fig. 3a) and even between two chromat ids of one chromo- some (Fig. 3b). This technique minimizes fixative modi- fications, and a layer of mater ia l was clear at the chro- mosome per iphery wi thout preferent ia l staining. Im- munolabel ing with G04 serum on HEp-2 cells was carr ied out and revealed by peroxidase-conjugated anti-

bodies. Cells were embedded in Ep o n and th in sections were per formed. T h e p a t t e rn of b inding was very simi- lar to t h a t observed af te r immunof luorescence localiza- t ion (Fig. 3d). A similar pe r ichromosomal accumula t ion was obta ined when immunolabel ing was p e r fo rmed with the D09 and the L02 sera (data not shown). No such peroxidase accumula t ion was visible in sect ions t r ea t ed by the same protocol incubated with a nonau- t o im m u n e serum (Fig. 3c).

Extractive Properties of the Antigens Recognized by the Sera

Th e labeling d isappeared af ter ex t rac t ion of the rat l iver sections with e i ther p ronase or pro te in solubiliza- t ion with 2 M NaC1 indicat ing tha t the epi topes belong to prote ins not res is tant to high salt extract ion. Nucleo- lar labeling was sensit ive to RNase digestion, disap- pear ing to ta l ly in mos t cases. This indicated t ha t the nucleolar ant igens are probably associated with RNA. In contras t , DNase digestion did not decrease the inten- sity of f luorescence staining. There fore , it is infer red tha t the ant igens are mos t probably nucleolar R N P s with no binding to nucleolar mat r ix and do no t a t t ach to ch romat in in nucleoli. These results are summar ized in Table 1.

Antigen Identification by Western Blotting

T h e 10 sera selected for the i r nucleolar and per ichro- mosomal affinity, as shown by the indirect immunof luo- rescence assay on HEp-2 cells, were tes ted on immuno- blot using H e L a nuclei as the source of ant igens (Fig. 4). A to ta l of three major bands of prote ins with appa ren t molecular weights of 53 kDa (Figs. 4a-4e), 66 kDa (Figs. 4f-4i), and 103 kDa (Figs. 4i and 4j) were de tec ted with the 10 sera. These bands were complete ly absent when the immunolabe l ing was pe r fo rmed with a pool of non- au to immune sera (Fig. 4k). A minor band at 51 kDa was also detec ted associated with the 53-kDa pro te in with two sera (Figs. 4b and 4e).

To de te rmine if all the revealed ant igens corre- sponded to polypept ides located bo th in the nucleolus and at the ch romosome per iphery, ant ibodies bound to each ant igen were eluted. After immunopur i f ica t ion , only the ant ibodies bound to the 53-kDa pro te ins gave a clear nucleolar and pe r ichromosomal p a t t e rn of fluores- cence on HEp-2 cells. Th e antibodies f rom the L02 serum bound to the 103-kDa pro te in gave a weak nu-

FIG. 3. Electron microscopy of human mitotic cells. (a-b) Cryoultramicrotomy of HeLa cells reveals the fibrillar and granular structure of the layer (arrowheads) that is darker than the chromatin. The perichromosomal material can be seen between chromatids of one chromosome. Bar = 0.5 ttm. (c) HEp-2 cells treated with nonautoimmune serum and peroxidase-conjugated antibodies. No antibody binding can be ob- served. (d) As(c) with G04 serum. The perichromosomal material is revealed by the peroxidase reaction (arrowheads) around the chromo- somes. Only the periphery of chromosomes binds the autoimmune serum. Bar = 1 ttm. (e-f) In situ radioactive labeling of total RNAs of TG cells with tritiated uridine. Bar = 2 ttm. At higher magnification, accumulation of silver particles is visible at the chromosome periphery. Bar 0.5 ~m.

PERICHROMOSOMAL DISTRIBUTION OF NUCLEOLAR RNPS 11

12 GAUTIER ET AL.

a b c d e f g h i ] k

200 --

116--

9 7 -

66,.~

4 5 ~

31 ,-

, , 1 0 3

~,66

~,53

FIG. 4. Immunoblot t ing of nuclear extracts of HeLa cells with the panel of 10 sera revealed by ant i -human IgG coupled with peroxi- dase. Lane a, A05; lane b, G08; lane c, E07; lane d, C12; lane e, D09; lane f, G04; lane g, B03; lane h, B06; lane i, B01; lane j, L02; lane k, nonautoimmune serum, and lane l, nuclear proteins stained with Ponceau red. Three antigens of 53, 66, and 103 kDa are revealed. Some sera are specific of one protein (A05, E07, C12, G04, B06, B03, and L02) and the other recognized two proteins (G08, D09, and B01).

cleolar fluorescence and a perichromosomal signal. We could not obtain such patterns after elution of the anti- bodies bound to the 66-kDa protein. To determine if the different antibodies of each serum recognized the same antigens, immunoblots were performed after the im- munopurification. We verified that the antibodies recog- nizing the 53-kDa proteins did not cross-react with the two other proteins and that the antibodies of the L02 serum bound to 103-kDa polypeptides only recognized these polypeptides (data not shown). Moreover, none of the two types of antibodies of the B01 serum eluted from one protein reacted with the other one (Figs. 5A, lanes c and d).

The effect of the inhibition of rRNA transcription on the presence of the 53-kDa antigens was tested. The patterns of binding of each of the 10 antisera on HeLa cells after actinomycin D t rea tment were compared with those of control HeLa cells. Binding to the 53-kDa antigens was identical in both cases (Fig. 5B, lanes a and b). Similar results were found with the sera recognizing the 66- and the 103-kDa proteins (data not shown).

RNA Characterization

TG cells were labeled with tri t iated uridine, embed- ded in Epon, and autoradiography was performed. RNAs were thereby detected in the cytoplasm and around some chromosomes (Fig. 3e). In favorable sec- tions including a large chromosome part a preferential accumulation of the silver particles was visible lining the chromosome arms even between the two chromatids

(Fig. 3f). These observations indicate an association of RNAs with chromosomes during mitosis. This localiza- tion is consistent with a RNP sheath lining the chromo- somes in mitosis.

To characterize the RNAs in this putative sheath some sera were used to immunoprecipitate antigens, and any RNA contained therein was extracted and sepa- rated on gels. Figure 6 shows the pat tern of HeLa and CHO cell RNAs obtained after immunoprecipitation by a nonautoimmune serum, and G04 and anti-Sm sera (Fig. 6A, lanes c, d, and f). In each case, the RNAs re- maining in the supernatant were each characterized (Fig. 6A, lanes b and e). They exhibited a pat tern char- acteristic of total RNAs (Fig. 6A, lanes a and g). G04 sera precipitated three bands, and the two lower bands present the same electrophoretic mobility as the U1 and U2 specifically immunoprecipitated by the anti-Sm sera (Fig. 6A, lane d). These bands were also precipitated with the nonautoimmune serum in a very low amount and were not specific of the G04 serum. This can be explained by the large copy number of these two RNAs in the cells as visible in Fig. 6A, lanes f and g; and 6B, lane a. In contrast, only the G04 serum coprecipitated significantly a RNA having the same molecular weight tha t U3 snRNA. This finding is corroborated by the fact that the same pat tern of RNA was observed when these sera were used to immunoprecipitate total extract of CHO cells (Fig. 6B). The efficiency was clear when we compared the amount of this RNA in the pellet with

a b c d

a b c

103 ,.- ~

66,.~ 5 3 , - ~

' i

?;G

%

;~; ;~ B

FIG. 5. (A) Immunopurif icat ion and elution of antibodies. Lane a, Western blott ing of HeLa nuclear proteins stained with Ponceau red; lane b, B01; lane c, eluted anti-103 kDa antibodies; lane d, eluted anti-66 kDa antibodies. No cross-labeling between the antibodies was detected. (B) Effects of actinomycin D inactivation. Lane a, immuno- blott ing with C12 serum; lane b, HeLa cells incubated with actinomy- cin D (2 ttg/ml for 5 h) prior to protein extraction and immunoblot- t ing with C12 serum; lane c, Western blott ing of HeLa nuclear ex- t racts stained with Ponceau red.

PERICHROMOSOMAL DISTRIBUTION OF NUCLEOLAR RNPS 13

M a b c d e f g

U3,.-

M

B

a b c d e

-.,U2 -.,U1 -,U4

-.U5

. .U6

A

FIG. 6. (A) Immunoprecipitation of HeLa RNAs separated on urea 8 M-10% polyacrylamide gel electrophoresis. The supernatants and the pellets of immunoprecipitation were compared. Lane M, pBR 322 digested with MspI (622-570-404-309-242-217-190-180-160-147-122-110- 90-76-67 nucleotides). Lanes a, g, total RNAs of the extract at two different concentrations; lane b, supernatant of nonautoimmune precipita- tion; lane c, nonautoimmune serum; lane d, G04 serum; lane e, supernatant of G04 immunoprecipitation; lane f, anti-Sm serum. (B) Immuno- precipitation of CHO RNAs as described above. Lane M, pBR 322 cut by MspI; lane a, anti-Sm serum; lane b, G04 serum; lane c, supernatant of G04 precipitation; lane d, nonautoimmune serum; lane e, total RNAs of the extract. The position of U3 is indicated.

t h a t of the supe rna t an t , i.e., to the to ta l a m o u n t of th is R N A in the cells (Fig. 6A, lanes d and e; and 6B, lanes b and c). T h e G04 se rum coprec ip i t a ted a R N A t h a t mi- gra tes to a pos i t ion of 210 to 225 nucleot ides in bo th cell lines. Th is is ve ry close to the length of U3 (217 nucleo- tides). U3 is the re fo re a good candida te for the s n R N A assoc ia ted with the 53- and 66-kDa prote ins .

DISCUSSION

Prev ious resul ts have indica ted t h a t dur ing mitosis nuc lear p ro te ins m a y be d i s t r ibu ted a t the c h r o m o s o m e pe r iphery a f te r the nuclear envelope d isappears . T h e subsequen t changes in cell o rganiza t ion showed the ap- pea r ance of a layer a t the c h r o m o s o m e surface where nuclear p ro te ins belonging to the ma t r ix and to the nu- cleolus accumula t ed [10, 11, 14]. However , there have been few studies address ing nuclear an t igens p re sen t ing

a p e r i c h r o m o s o m a l locat ion and the da ta avai lable for the different an t igens are l imited.

T h e major i ty of prev ious ly descr ibed ant igens , shar- ing bo th a nucleolar locat ion and a d is t r ibut ion over the c h r o m o s o m a l surfaces, have a molecu la r weight in the range of 25 to 40 k D a [14, 19, 30]. T h e p ro te ins de- scr ibed here have molecu la r weights of 53, 66, and 103 kDa. T h e solubil izat ion of these ant igens by high sal t concen t r a t ion t r e a t m e n t showed t h a t they can not be p a r t of the so-cal led nuc lear matr ix . Moreover , we suc- ceeded in label ing lymphocytes , which are noncycl ing cells. Th i s resul t excludes the possibi l i ty t h a t sera con- ta in Ki-67 ant ibodies , a l though the molecu la r weight of the co r respond ing an t igens r e m a i n u n k n o w n [12]. T h e p ro te in b a n d of 53 k D a recognized by five sera (A05, E07, C12, B06, and G04) is the m o s t r ep resen ta t ive t h a t we found. I t could not be re la ted to a 52-kDa ant igen coprec ip i ta t ing with t r ansc r ip t ion complexes [31]. T h e finding t h a t the level of synthes is of the th ree an t igens

14 GAUTIER ET AL.

was not modified by the act ion of ac t inomycin D sug- gests t ha t antigens are not direct ly dependen t on nu- cleolar t ranscr ip t ion activity. These resul ts are not in favor of the presence in the sera of ant ibodies against known per ichromosomal proteins , Ki-67, or RNA poly- merase I t ranscr ip t ion complexes.

T he sensit ivity of the ant igens to RNase digestion and i n v i v o RNA labeling exper iments suggested tha t ant igens were RNPs . Th is idea was suppor ted by pre- vious cytochemical observat ions t ha t de tec ted the pres- ence of R N P s at the ch romosome per iphery [15, 16, 32] and by pulse-chase exper iments with [3H]-uridine [17]. However , we were able to observe the pe r i ch romosomal layer in cryosect ions wi thout preferent ia l or regressive staining. We charac ter ized by immunoprec ip i t a t ion with the G04 serum a specific RNA associated with the ant igens tha t has a molecular weight very close to tha t described for U3. Th is snRNA is known to b ind to fibril- larin (36 kDa) and to five prote ins of 12.5, 13, 30, 59, and 74 kDa. However , none of these molecular weights cor respond to those of the ant igens we identified (for review see [33]). T h e molecular weights of the ant igens tha t we charac ter ized indicate t ha t the serum does not conta in anti-f ibri l larin antibodies. Moreover anti-fibril- larin ant ibodies immunoprec ip i ta te snoRNAs including U8, U13, and U14 [33, 34] and the pe r ichromosomal distr ibution of fibrillarin is still controvers ia l [30, 35, 36]. U1 and U2 RNAs were coprecipi ta ted with the anti- gens recognized by the G04 serum. However they are not considered to be specifically immunoprec ip i t a t ed RNAs because they were also found af ter immunoprec i - p i ta t ion with the cont ro l serum and because they are only presen t in nucleoplasm of in te rphase cells (for re- view see [37]). Moreover , they seem to be sensit ive to ac t inomycin D inact ivat ion bu t the th ree ant igens we identified were not [38].

T he behavior of the prote ins we identified is consis- t en t with a specific funct ion necessary to the mitot ic process. The different character is t ics we found for these nucleolar prote ins suPport a role in spatial order ing of the chromosomes. We observed t ha t the ant igens were released f rom the nucleolus at the beginning of pro- phase and remained associated with the ch romosome per iphery unti l late telophase. Th is suggests t ha t the ant igens could play a role during the condensa t ion and / or decondensa t ion of the chromosomes . Th is funct ion has been proposed for pro te ins t ha t do not cover the ch romosome surface including H1 and H3 [39, 40], nu- cleolin [41], topoisomerase II [42, 43], and per ichromo- somal pro te ins [14]. T h e ant igens may have a s t ruc tura l role in ch romosome organizat ion, possibly in associa- t ion with o ther nuclear proteins.

The dispersion of the ant igens in the cells was con- comi tan t with the depolymer iza t ion of the lamins. T h e breakdown of the nuclear envelope at the end of pro- phase exposes the chromat in to cytoplasmic const i tu- ents. Thus , a s t ruc ture specific for the mitotic stage may

be e labora ted with pro te ins of nuclear origin to replace the nuclear envelope when it breaks down af ter the de- polymer iza t ion of the lamins [6, 8]. T h e layer covering the ch romosome may the re fore be composed of various nuclear pro te ins tha t specifically in te rac t with the chro- mosome surface at a def ined m o m e n t of the mitot ic pro- cess.

Third , the dis t r ibut ion of the ant igens on the chromo- some surface may be a more passive storage role, as was described for nucleoplasmin and proposed for p400+ [44]. Th is region was also demons t r a t ed to be a t ran- s ient accumula t ion site for various pro te ins including r ibosomal pro te in $1 [45], r ibochar in [45, 46], and U- s n R N P s recognized by ant i -Sin and anti-f ibri l larin sera [30, 47]. In this case, l ining the ch romosome per iphery could be an efficient mechan ism to dis tr ibute nuclear and nucleolar prote ins equally be tween daughte r cells.

T h e pe r ichromosomal region appears to be a very complex s t ruc ture involving a great n u m b er of nuclear proteins. T h e mechan ism by which these pro te ins are t rans loca ted and addressed to the ch romosome surface and how they are regula ted are unknown. I t is necessary to s tudy the s t ruc tures establ ished by nuclear and nu- cleolar prote ins to u n d e r s t an d clearly the sequent ia l events tak ing place at the t ime of nuclear breakdown. T h e presence of a s t ruc ture at the ch romosome periph- ery seems to be the resul t of very coord ina ted move- ments in space and t ime during mitosis in which phos- phory la t ion cycles may play a cent ra l role. T h e charac- ter izat ion of all the pro te ins involved in s t ruc tur ing the mic roenv i ronmen t of ch romosomes is the re fore neces- sary to u n d e r s t an d the different funct ions of the peri- ch romosomal a rch i tec ture tha t seems to be essential for complete mitosis.

The authors are very grateful to Dr. N, Rottman for helpful discus- sions, and to F. Devienne for artwork. We thank Professor J. Revuz and Professor J. P. Vernant for the provision of the patients clinical data and Mrs. S. Bouralha, A. Charnay, and M. Assouvie for technical assistance. This study was supported by grants of Le Centre National de la Recherche Scientifique, of Association pour la Recherche contre le Cancer 6703 and of Assistance Publique.

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Received September 19, 1991 Revised version received December 27, 1991