effects of anti-ﬁbrillarin antibodies on building of ... · during mitosis and then reintegrated...

359Journal of Cell Science 111, 359-372 (1998)Printed in Great Britain © The Company of Biologists Limited 1998JCS9682

Effects of anti-fibrillarin antibodies on building of functional nucleoli at the

end of mitosis

N. Fomproix, J. Gébrane-Younès and D. Hernandez-Verdun*

Institut Jacques Monod, Paris, France*Author for correspondence (e-mail: [email protected])

Accepted 17 November 1997: published on WWW 15 January 1998

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During mitosis some nuclear complexes are relocalized atthe chromosome periphery and are then reintegrated intothe re-forming nuclei in late telophase. To addressquestions concerning translocation from the chromosomeperiphery to nuclei, the dynamics of one nucleolarperichromosomal protein which is involved in theribosomal RNA processing machinery, fibrillarin, wasfollowed. In the same cells, the onset of the RNApolymerase I (RNA pol I) activity and translocation offibrillarin were simultaneously investigated. In PtK1 cells,RNA pol I transcription was first detected at anaphase B.At the same mitotic stage, fibrillarin formed foci ofincreasing size around the chromosomes, these foci thengathered into prenucleolar bodies (PNBs) and later PNBswere targeted into the newly formed nucleoli. Electronmicroscopy studies enabled the visualization of the PNBsforming the dense fibrillar component (DFC) of newnucleoli. Anti-fibrillarin antibodies microinjected at

different periods of mitosis blocked fibrillarin translocationat different steps, i.e. the formation of large foci, focigathering in PNBs or PNB targeting into nucleoli, andthereby modified the ultrastructural organization of thenucleoli as well as of the PNBs. In addition, antibody-boundfibrillarin seemed localized with blocks of condensedchromatin in early G1 nuclei. It has been found thatblocking fibrillarin translocation reduced or inhibited RNApol I transcription. It is postulated that when translocationof proteins belonging to the processing machinery isinhibited or diminished, a negative feed-back effect isinduced on nucleolar reassembly and transcriptionalactivity.

Key words: Perichromosomal protein, Prenucleolar body, NucleoluMitosis, Transcription, Nucleologenesis, PtK1 cell, Microinjection,Organelle inheritance

SUMMARY

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INTRODUCTION

During mitosis, different partitioning mechanisms ensuorganelle inheritance in the two daughter cells (for a review Warren and Wickner, 1996). One such mechanism involvemitotic disorganization of the organelles and therelocalization in the cytoplasm or in the mitotic spindle. addition to the membrane-bordered organelles, some molecmachineries and supramolecular complexes have also bfound to be equally partitioned during mitosis. This illustrated by the nuclear machineries that can be relocaliduring mitosis and then reintegrated in the re-forming nucin late telophase. One of the major sites of relocalizationnuclear complexes during mitosis is the perichromosomal laat the chromosome periphery, a region also designatedsurface domain of the chromosomes (for reviews sHernandez-Verdun and Gautier, 1994; Rattner, 1992).

In mitotic cells the perichromosomal layer constitutesspecific domain of compartmentation for nucleolar protesuch as nucleolin (Weisenberger and Scheer, 1995), proB23 (Schmidt-Zachmann et al., 1987; Zatsepina et al., 199fibrillarin (Dundr et al., 1997; Gautier et al., 1994; Yasuda a

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Maul, 1990), and the small nucleolar RNAs (snoRNAs) U(Azum-Gélade et al., 1994; Gautier et al., 1994) and U1(Beven et al., 1996) suggesting the presence of nucleolar Rcomplexes. The presence of nuclear proteins related to the cycle has also been described (Willingham and Bhalla, 199as well as highly phosphorylated nuclear proteins (Dilworth1991; Starborg et al., 1996; Verheijen et al., 1989) and smribonuclear proteins (snRNP) (Leser et al., 1989). It has befound that the proliferating marker Ki-67 forms a reticulatestructure surrounding metaphase chromosomes (Verheijenal., 1989). This perichromosomal compartmentation advantageous for equal partitioning between daugther cells amay have an important role in nuclear reconstruction. Taddress these questions, the dynamics of one nucleoperichromosomal (NoPC) protein (for a review see Gautier al., 1992a), fibrillarin, was followed in relationship with theonset of ribosomal RNA (rRNA) synthesis and chromatidecondensation, as well as the effect of immunodepletion this protein on mitotic partition and nuclear reconstruction.

Fibrillarin (Ochs et al., 1985b), a highly conserved nucleolaprotein (Aris and Blobel, 1991) was chosen for thiinvestigation because its compartmentation in th

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N. Fomproix, J. Gébrane-Younès and D. Hernandez-Verdun

perichromosomal layer was observed in a variety of animal aplant cells (Beven et al., 1996; Dundr et al., 1997; Gautieral., 1994; Yasuda and Maul, 1990). This nucleolar proteininvolved in all major post-transcriptional activities in ribosomsynthesis, the first steps of rRNA processing, pre-rRNmodification and ribosome assembly (Tollervey et al., 199Fibrillarin is associated with several snoRNAs required for prrRNA cleavage (U3, U8, U14 and U22) and methylation rRNA (U14, U18, U24-U63) (Cavaillé et al., 1996; Smith anSteitz, 1997; Tollervey, 1996). Sequence analyses indicate human fibrillarin has potential RNA binding domain in itcentral part, a glycine-arginine-rich (GAR) domain at thamino-terminal end and an α-helical domain at the carboxy-terminal end (Aris and Blobel, 1991). These three main regioare evolutionarily conserved in yeast, Physarum polycephalum,Xenopus laevis, and mammalian species (for a review seKasturi et al., 1995). However, a different organization of tfibrillarin GAR-domain has recently been described Tetrahymena thermophila(David et al., 1997).

During interphase, fibrillarin localized in the dense fibrillacomponent (DFC) of the nucleoli (Ochs et al., 1985b). In laG2, several nucleolar proteins including fibrillarin, migratefrom the nucleoli towards the periphery of the nucleus (Gautet al., 1992b). This cordon-like pattern formed a network nucleolar proteins around the condensing chromosom(Hernandez-Verdun and Gautier, 1994). As mitosis progressthe condensed chromosomes were surrounded by perichromosomal layer at the end of prophase.

At the end of mitosis, fibrillarin was detected in prenucleolbodies (PNBs) scattered in telophase nuclei (Azum-Géladeal., 1994; Benavente et al., 1987; Beven et al., 1996; JiménGarcia et al., 1989; Ochs and Smetana, 1991; Scheer Weisenberger, 1994). It has been reported that these Pfused at the nucleolar organizer regions (NORs), when rRNsynthesis resumed (Jiménez-Garcia et al., 1994). U3 and snoRNAs as well as other nucleolar proteins were aassociated with PNBs (Azum-Gélade et al., 1994; Beven et1996; Gautier et al., 1994; Jiménez-Garcia et al., 199Therefore, the PNBs appeared as pre-package nuclecomplexes mainly involved in processing steps of the prRNAs that associated to the re-forming nucleolus.

In nucleolus formation (for a review see Thiry and Goesse1996), the NORs, sites of ribosomal gene (rDNA) clusterinare indeed the sites that ‘organize’ preexisting nucleomaterial. Several ways to deliver preexisting components toforming nucleoli during mitosis have been proposed (Dundral., 1997). Nevertheless, the PNBs constitute the main pathwfollowed to reach the nucleolus because they correspondtargeting of the major nucleolar proteins such as nucleoprotein B23 and fibrillarin. It has been proposed that PNtargeting to the nucleolus is in some way dependant on Nactivity, i.e. active rDNA transcription (Bell et al., 1992)However, there is presently no information concerning the rof PNB targeting on rDNA transcription and nucleolaformation as well as on the fate of perichromosomal proteat stages preceding the formation of the PNBs.

In the present work, translocation of fibrillarin at the end mitosis was investigated and correlated with the kinetics activation of RNA pol I transcription. Microinjection of anti-fibrillarin antibodies was used to check the effects of blockiof fibrillarin translocation on the events leading to nucleol

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MATERIALS AND METHODS

CellsPotorous tridactyliskidney cells (PtK1 cells) were cultured withoutantibiotic in Eagle’s minimum essential medium (EMEM) with 0.8g/l NaHCO3, 10% fetal calf serum and 2% glutamine in 5% CO2 at37°C. The cell monolayers were grown on glass slides as previoudescribed (Robert-Fortel et al., 1993) or on microgrid coversl(CELLocate, Eppendorf). The alphanumeric labeling of the grifacilitated the location of microinjected cells.

HeLa cells were cultured in MEM supplemented with 10% fetcalf serum. Cells were seeded three times a week and extracts prepared 24 hours after seeding from exponentially growing cells

AntibodiesThe following antibodies were used: the monoclonal antibody 72(kindly provided by M. Pollard, La Jolla, CA, USA), produced ancharacterized as an anti-fibrillarin antibody by Reimer et al. (198two human auto-antibodies directed against fibrillarin, S4 describby Ochs et al. (1985b) (kindly provided by R. Ochs, La Jolla, CUSA), and GM4 described by Gautier et al. (1994). Both human simmunoprecipitated U3 snoRNAs and cross-reacted with the yefibrillarin equivalent (Gautier et al., 1994; Schimmang et al., 1989

Run-on transcription assayIn permeabilized cellsRun-on transcription was performed as previously describ(Wansink et al., 1993) except that glycerol was omitted from tmedia and the reactions were performed at room temperature (Briefly, cultured cells were washed rapidly in PBS, pH 7.4, and wpermeabilization buffer (20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.5mM EGTA, 0.5 mM PMSF). The cells were permeabilized in the sabuffer containing 0.05% Triton X-100 (IBI, USA) for 5 minutes at Rand permeabilization was stopped by extensive washing in the sbuffer without Triton X-100. They were incubated in run-on buffe(Wansink et al., 1993) containing 0.5 mM of ATP, CTP, GTP and 0mM Br-UTP (Sigma) for 20 minutes. The cultured cells were thwashed twice with PBS containing 5 units/ml of RNase inhibit(Boehringer), fixed immediately in 2% paraformaldehyde for 4minutes at 4°C, and permeabilized with 0.1% Triton X-10(PBS/Triton). For control experiments, either 10 µM dideoxyTTP(Boehringer) plus 5 µg/ml aphidicolin (Sigma), either actinomycin D(1 µg/ml) or α-amanitin (100 µg/ml), were added in the run-on bufferEnzymatic digestion with RNase A (50 µg/ml in PBS) was performedfor 10 minutes after run-on transcription.

In ‘weakly fixed’ cellsRun-on transcription was performed as previously described Moore and Ringertz (1973). The cells were fixed for exactly 5 minuin absolute ethanol/acetone (1:1, v/v) at 4°C, washed 3 minutereaction mixture containing 100 mM Tris-HCl, pH 7.9, 12 mM 2mercaptoethanol, 150 mM sucrose and 12 mM MgCl2, and incubatedin run-on buffer containing 0.5 mM of ATP, CTP, GTP and 0.2 mBr-UTP for 15 minutes. They were then washed 3 minutes in reaction mixture, postfixed immediately in 2% paraformaldehyde 40 minutes at 4°C and permeabilized with 0.1% Triton X-100 in PBfor 5 minutes at RT.

To detect transcription, the cells were incubated for 60 minutes wa monoclonal anti-Br-dU antibody (Sigma) diluted 1/100 in PBS, thfor 30 minutes with goat anti-mouse FITC-conjugated antibo(Jackson Immunoresearch Laboratories) and for 5 minutes in DA

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(1/10,000). Control of the anti-Br-dU antibody specificity waperformed by inclusion of UTP instead of Br-UTP in the run-obuffer.

Microinjection in mitotic cellsInjections were performed using a micromanipulator/inject(Eppendorf) with glass capillaries (Eppendorf). All microinjectioexperiments were carried out in 3.5 cm diameter Petri discontaining 3 ml of Hepes MEM in order to avoid a decrease in pH of the medium during the injection. The cells were grown alphanumeric microgrids to provide the cell coordinates. GM4 serdiluted 1/10 in PBS was microinjected into the cytoplasm of mitocells or into the nuclei of interphasic cells including late G2 cells. Insome cases, Dextran Texas Red was also added to the microinjbuffer because cells injected with the control sera presented eithelabeling or a weak fluorescence labeling. The control sera were a of 10 non-autoimmune sera. Fixation was performed in some cajust after the injection. In most cases, following injection, cells weleft in the culture medium at 37°C for 2 hours and were either fix1 hour at 4°C with 3% formaldehyde and 0.1% Triton X-100 or usfor run-on transcription. The injected antibodies were revealed witgoat anti-human FITC conjugated antibody (1/100) or with a ganti-human rhodamine conjugated antibody (1/50).

Immunofluorescence microscopyThe cells grown as monolayers on glass slides were fixed in onthe following fixatives: acetone at −20°C for 5 minutes, ethanol at −20°C for 5 minutes or 3% formaldehyde (BDH) for 20 minutes. the latter case, cells were permeabilized with 0.5% Triton X-1during fixation, or following fixation with methanol or acetone for minutes at −20°C. The antibodies GM4 (1/100), 72B9 (1/10) and annucleolin (1/10) were incubated for 1 hour and revealed by a goat ahuman FITC-conjugated antibody (1/100), by a goat anti-mouFITC-conjugated antibody (1/50) or by a goat anti-rabbit FITconjugated antibody (1/100) for 35 minutes in PBS. Five minubefore the end of the incubation, the DNA-specific dye DAPI wadded. All samples were mounted with Citifluor and photographusing a TMAX 400 Kodak film in a Leica microscope or directobserved on a Video Microscope Control. The scanned imagesthe video microscope images were assembled on a Macincomputer equipped with an Adobe Photoshop 4.0 software progrImages were printed directly from the computer on colour paper usa dye sublimation printer (Colorease, Eastman-Kodak).

Western blottingThe procedure followed for preparing nuclear HeLa extracts wdescribed earlier (Gautier et al., 1992a). PtK1 cell extracts wereprepared as follows. The cells were washed twice in PBS, lysePBS containing 1% Triton X-100 for 10 minutes on ice and pelleby centrifugation for 10 minutes at 10,000 g. The pellets weresolubilized in Laemmli buffer (Laemmli, 1970) or 2-dimensional (D) O’Farrell lysis buffer (O’Farrell, 1975) and were sonicated for seconds in ice using a Soniprep apparatus at a setting of 6.

The cell extracts were electrophoresed on a 2-D gel with isoelecfocusing in the first dimension and an 8% polyacrylamide-SDS gethe second dimension.

Polymerization of the gel for the first dimension was performedglass capillaries (Bio-Rad) according to the O’Farrell procedure. Tampholines were between pH 3.5 and 10. Briefly, a pre-run wperformed 15 minutes at 200 V, 20 minutes at 300 V and 20 minuat 400 V with electrophoresis buffer 0.01 M H3PO4 (anode), 0.75 Methylenediamine (cathode). Separation of the proteins requiredminutes at 500 V followed by 3 hours 30 minutes at 750 V.

For one-dimensional (1-D) electrophoresis, proteins were separby 8% or 10% SDS-PAGE according to the method of Laemmli. Sstandards from 14 to 200 kDa (Bio-Rad) were also loaded onto egel. The polypeptides were electrotransferred to reinforced cellul

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nitrate membrane (BA S83; Scleicher and Schuell). The nitrocellulomembranes were stained with Ponceau Red to visualize transferred proteins and cut into strips for 1-D immunoblotting. Tnon-specific sites were blocked by incubating the membrane forminutes in PBS containing 5% dried milk and 0.5% Tween-2Incubation with the sera (dilution 1/10,000) was performed overnigin the same buffer. The membranes were washed three times with containing 5% dried milk and 1% Tween-20, and incubated in the fibuffer for 1 hour in the presence of peroxidase-labeled secoantibodies (dilution 1/5,000). After several rounds of washing, tchemiluminescence substrate (Super Signal, Pierce) for the enzwas added and blots were exposed to X-ray films for 30 seconds minute.

Electron microscopyStandard electron microscopyThe cells were treated in situ on the microgrid coverslips. After brrinsing in PBS, the cells were immersed in fixative solution (2paraformaldehyde (w/v) and 2.5% glutaraldehyde (v/v) in 0.1 sodium cacodylate buffer, pH 7.2) at 4°C. After 1 hour, they wewashed in 0.1 M cacodylate buffer, postfixed in 1% (w/v) osmiutetroxide in the same buffer for 30 minutes, dehydrated throughethanol series and immersed in an Epon/ethanol mixture (1:1, vfollowed by pure Epon 812. Finally, an Epon-filled gelatin capsuwas inverted over the microgrid coverslip.

After resin polymerization, the embedded cells were separated frthe microgrid coverslips by brief immersion of the Epon-filled capsuin liquid nitrogen. As a consequence, the surface of the block carthe imprint of the alphanumeric labeling. The cells previously locatby light microscopy were serially sectioned parallel to the planethe growing cells and collected on Formvar-coated grids with a sinslot. Ultrathin sections were conventionally contrasted with uranacetate (10 minutes at RT) followed by lead citrate (5 minutes at R

Microinjected cellsAfter incubation in complete medium for 2 hours at 37°C, thmicroinjected antibodies were visualized as described above, exthat 4% (w/v) paraformaldehyde in PBS was used as fixative solutand 0.1% saponine was added to PBS, the buffer used to dilutesecondary antibodies. Photographs of microinjected cells were tawith a Leica microscope equipped with epifluorescence optics andappropriate filter sets. Then the coverslips were dismounted, rinsePBS, fixed with 1% (v/v) glutaraldehyde in 0.1 M cacodylate buffepH 7.4, overnight at 4°C, postfixed with 1% (w/v) osmium tetroxidand processed for electron microscopy as described above.

RESULTS

Fibrillarin in PtK 1 cellsFibrillarin has been characterized by western blot in sevespecies, from human to yeast. However, no informationavailable concerning the Potorous tridactylis species,especially PtK cells, which are frequently used to localifibrillarin in nucleoli.

Fibrillarin in PtK1 cell extracts was identified by S4 andGM4 sera on 1-D immunoblots. Fibrillarin recognized by bosera migrated at 36 kDa, that is, slighly slower than othmammalian fibrillarins found at 34 kDa. To ascertain thdifference in migration between PtK and human fibrillarinextracts from PtK1 and HeLa cells were compared and probein parallel (half strips) with S4 and GM4 sera (Fig. 1a). Thsame polypeptides were recognized by both sera but migration of the fibrillarins differed. PtK1 fibrillarin migratedat 36 kDa and HeLa fibrillarin at 34 kDa (Fig. 1a).

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Fig. 1. Identification of fibrillarin in PtK1 cells with GM4 serum (1/10,000) and S4 serum (1/10,000). (a) 1-D immunoblot of transferredproteins from HeLa (right) and PtK1 (left) cell extracts. Each strip was cut into two parts for separate incubations, respectively, with S4 andGM4 sera. S4 and GM4 sera bound to a protein (fibrillarin) with the same molecular mass: 36 kDa in PtK1 extracts and 34 kDa in HeLaextracts. (b) 2-D electrophoresis pattern of PtK1 extracts showing a single spot (pI approximatively 9.5) with the GM4 serum as well as with theS4 serum. The pH range was from 7 to 9.7; basic peptides migrate to the right. The 1-D gels show the migration of fibrillarin in PtK1 cellextracts in one dimension. In PtK1 cells, fibrillarin has a molecular mass of 36 kDa and a pI of about 9.5.

To more precisely characterize PtK1 fibrillarin, 2-Dimmunoblots were performed using GM4 and S4 sera. Thdecorated indistinguishably the same protein with the sa

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molecular mass and the same isoelectric point (Fig. 1b). Itnoticeable that the isoelectric point of PtK1 fibrillarin is higher(9.5) than has been reported for fibrillarin of other mamma

Fig. 2.Distribution of fibrillarin during the laststeps of mitosis in PtK1 cells. Bar, 5 µm. The cellsimmunolabeled with the monoclonal antibody72B9 (1/10) (a), or the auto-immune serum GM4(1/100) (b,c,d) presented the same labelingpatterns. DNA was stained with DAPI (a′,b′,c′,d′),allowing identification of the mitotic stages. (a) Atearly anaphase A, fibrillarin detected with themonoclonal antibodies 72B9 is observed inassociation with the moving chromosomes. (b) Atanaphase A, fibrillarin was detected as a layeraround the chromosomes. (c) At anaphase B,recruitment of fibrillarin was observed. The sheathof fibrillarin around the chromosomes consisted infoci of different sizes, and accumulation offibrillarin began in the NOR areas. (d) Attelophase, fibrillarin accumulated in newly formednucleoli and remained in the PNBs associatedwith the condensed parts of the chromosomes.


Fig. 3. Electron micrographs of nuclei of untreated cells. (A) Late telophase: the chromatin (c) is still decondensing and two PNBs (arrows) areseen in the vicinity of the re-forming nucleolus. Bar, 0.5 µm. (A′) Detail of the nucleolar compartment showing one PNB (single arrow) lyingbetween the fibrillar components and the condensed chromatin (c), whereas the second PNB (double arrow) is still included in the condensedchromatin. Dense fibrillar component (df), fibrillar centers (*). Bar, 0.2 µm. (B) Early G1 phase with the chromatin almost completelydecondensed. Two nucleoli are seen: one nucleolus shows a PNB (large arrow) in the process of fusion with the tangentially sectioned densefibrillar component, whereas the other, loosely organized nucleolus shows three PNBs (small arrows). Bar, 1 µm. (B′) Detail of the latternucleolus: three dense spherical PNBs (arrowheads) assemble at the site where the nucleolus is being re-formed. The df forms two massesadjacent to the fibrillar center (*). Granules 15 nm in diameter (arrows) can be identified. Nucleolus associated chromatin (c). Bar, 0.2 µm.

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Fig. 4.Schematic diagram of the behavior of fibrillarin during the laststeps of mitosis, and nucleolus re-formation related to rDNAtranscription. In step 1, anaphase A, the condensed chromosomes aresurrounded by a homogeneous sheath of fibrillarin. At this stage, norDNA transcription can be detected (−). In step 2, anaphase B, as rDNAtranscription is triggered in the NOR areas, recruitment of fibrillarin isobserved at the chromosome periphery, fibrillarin consists in foci ofdifferent sizes. In step 3, telophase, the chromatin is less condensed andthe fibrillarin gathers in PNBs. At this stage, the nuclear envelope is nottotally re-formed. Most of the PNBs have been targeted to the nucleoliand become the fibrillar elements of the nucleolus. In step 4, early G1,rDNA transcription is amplified and fibrillarin accumulates in newlyformed nucleoli, some PNBs remain asssociated with the condensedchromatin in the periphery of the nucleus. In step 5, G1 phase, no PNBscan be observed associated with the interphasic chromatin.Nucleologenesis is complete. Plus signs indicate level of transcription:(+) weak spot; (++) several weak spots; (+++) several bright spots;(++++) numerous bright spots with extended distribution.

(Ochs et al., 1985b). However, the differences detected PtK1 fibrillarin did not alter its recognition by the antifibrillarin sera used.

The localization of fibrillarin during the cell cycle waexamined after microinjection of antibodies into living celor after in situ immunolabeling using several fixatioprocedures. In vivo, as in fixed cells, a similar distributiopattern of fibrillarin was observed with each serum. Durimitosis as already reported for fixed cells, fibrillarin walocated at the mitotic chromosomal surface surrounding echromosome (Fig. 2a,b,c). To investigate precisely the kineof fibrillarin translocation at the time of nucleologenesis, tlocalization of fibrillarin during the last mitotic steps wainvestigated.

During anaphase A, fibrillarin was associated wichromosomes as small dots uniformely and reguladistributed around all chromosomes (Fig. 2a,b). In anaphasfibrillarin was observed in the NOR areas and remainassociated with the perichromosomal regions, forming focidifferent sizes (Fig. 2c). These foci probably resulted from trecruitment of fibrillarin either dispersed in the cytoplasbelow the level of detection, or by regrouping of fibrillarifrom the chromosome periphery. At telophase, fibrillarin wassociated with the condensed parts of chromosomes (Figand accumulated into newly formed structures that resemPNBs by phase contrast microscopy. Later, fibrillarin wentirely associated with the newly formed nucleoli.

To more precisely characterize PNB genesis, colocalizatof fibrillarin with nucleolin and Ag-NOR staining wasperformed. At anaphase B and telophase, PNBs couldvisualized simultaneously by Ag-NOR staining and bimmunofluorescence with anti-fibrillarin antibodies (data nshown). In addition, during anaphase B and telophafibrillarin and nucleolin were colocalized (data not shownTherefore, we propose that the NoPC proteins participate information of PNBs in telophase.

At the resolution of electron microscopy, re-formation of thnucleolus, which begins in late anaphase, became eaidentified in telophase when the PNBs, which appearedsmall dense fibrillar structures, started fusing with the NORAt late telophase (Fig. 3A,A′), as the chromosomes were stidecondensing, the PNBs could be seen either adjacenchromatin or closely apposed to the DFC of the re-formatnucleolus. At this time, the PNBs appeared more electdense than the DFC.

At early G1 (Fig. 3B,B′), as the PNBs unwound, the DFCbecame more extended but some PNBs were still presaround the fibrillar centers (FCs) that form clear areasdifferent sizes. Even if the granular component did not formvisible entity, a few granules 15 nm in diameter could observed within the nucleolar structures.

These observations are schematized in Fig. 4. Three sfor fibrillarin translocation at the end of mitosis in PtK1 cellscan be proposed: first, a recruitment of fibrillarin iheterogeneous foci at the chromosome periphery, secondgathering of these foci in PNBs still in association with thcondensed chromatin and third the targeting of the PNBs tonucleolus. It is a dynamic process illustrated by a gradienfibrillarin regrouping depending on the mitotic stageTherefore, the kinetics proposed are indicative of the mafibrillarin pool detected.

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Different approaches were used to label nascent rRNAs (Materials and Methods). The data using permeabilized ‘weakly fixed’ cells were similar. However, visualization omitosis was optimized with ‘weakly fixed’ cells. Moreover, th‘weakly fixed’ cell technique made it possible to fix precisewhen the rDNA transcription started at the end of mitosis. cells were fixed, they could not progress through the cell cyand the time when precursor incorporation began could exactly determined.

Double labeled cells for nascent rRNA and fibrillarirevealed association between the site containing nascent Rpol I transcripts and NOR areas stained with fibrillarin. Ianaphase B, RNA pol I transcription was reinitiated wherepart of chromosomes were still condensed and surroundedfibrillarin (Figs 4, 5a,a′). Transcription sites started forming adoublet. Two signals were observed in each group of separachromatids, i.e. one signal at each secondary constriction. Ltranscription was observed at the NOR areas whereas fibrillawas still at the chromosome periphery and progressivtargeted to transcription sites (Figs 4, 5b,b′). As the cycleprogressed, PNBs were still observed in the nucleoplasm wtranscription was amplified as well as the nucleolar domacontaining fibrillarin (Figs 4, 5c,c′).

Microinjection of anti-fibrillarin antibodies in mitoticPtK1 cellsTo determine whether fibrillarin translocation has a criticaction on nucleolar genesis, anti-fibrillarin antibodies wemicroinjected into living PtK1 cells at each stage of mitosis. Tochoose the antibodies with the highest potential efficienimmuno-competitions were performed. GM4 and 72B9 sewere successively incubated or vice versa on fixed interphPtK1 cells. Complete extinction of the nucleolar signal usinthe 72B9 serum was observed when GM4 was applied fiwhereas only a decrease of the GM4 nucleolar signal wobserved when 72B9 was applied first (data not shown). It cbe postulated that GM4 recognized larger epitopes than 72which is in agreement with the fact that auto-immunantibodies are known to recognize several epitopes of the sprotein. In addition, GM4 presented a higher titer than 72Bantibodies, an advantage given the very small volume that be microinjected. Therefore, GM4 serum was selected further microinjections as it potentially neutralized a largnumber of fibrillarin epitopes.

Fig. 5. Comparison of the fibrillarin pathway and activation of rDNAtranscription at the end of mitosis in PtK1 cells. Bar, 5 µm. Fibrillarinwas labeled with GM4 serum (1/100) (a′,b′,c′). Run-on transcriptionwas performed in ‘weakly fixed’ cells, under conditions favoringrRNA pol I. rRNA synthesis could be detected by incorporation ofBr-UTP revealed by immunofluorescence (a,b,c). DNA was stainedwith DAPI (a′′ ,b′′ ,c′′ ). In anaphase B, as rDNA transcription started(a), fibrillarin was still localized at the chromosome periphery (a′).Transcription started as a doublet at each NORs. Concomittantlywith the onset of transcription, fibrillarin was recruited and appeareas small dots at the chromosome periphery (b′). At telophase,increase of rDNA transcription (c) could be noted as well asamplification of the nucleolar surface labeled with fibrillarin (c′).Some fibrillarin remained in the PNBs, still associated with thecondensed parts of chromatin.

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Control microinjection experimentsTo set up the protocol for microinjection into mitotic cellscontrol sera (see Materials and Methods) were first testMicrogrids with alphanumeric labeling helped to identify thmicroinjected cells as well as the fluorescent Dextramicroinjected together with the sera. After microinjection, thcells were left to progress into the cell cycle for differenperiods (1-5 hours). It has been found that two hours is the ti

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necessary to complete mitosis and to observe two daugcells in early G1. Therefore, this time of recovery was used fall the subsequent observations.

The microinjected samples became homogeneoudistributed throughout the cytoplasm two hours aftmicroinjection. These control sera had no discernible effeon the formation of early G1 nuclei in the 126 cells injected adifferent stages of mitosis. Nucleolar re-formation was aunaffected in cells microinjected in this way. Taken togeththese results demonstrate that the experimental conditionsnot interfere with nuclear and nucleolar re-formation.

Fig. 6. Abnormal nuclear phenotypes obtained 2 hours after anti-fiµm. In the subsequent interphase, 40% of the microinjected cellschromatin remained in a telophase-like configuration (a′,b′,c′). The locamade it possible to classify cells in 3 categories. In pattern 1 (27%nucleoplasm (a) and no nucleolar structure could be detected by fibrillarin in the nucleoli (weakly stained) and also dispersed as smlocated in the nucleoli (c). Run-on transcription in conditions favortranscription was weaker in these nuclei than in the adjacent cont

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Microinjection of anti-fibrillarin antibodiesGM4 anti-fibrillarin serum was injected into the cells: 58 inlate G2, 38 in prophase, 30 in metaphase, 64 in anaphase. Tfate of each microinjected cell for each mitotic stage waexamined. Two hours after microinjection, the distribution othe fibrillarin antibodies was observed as well as the DNA anthe general organization of the cells. Cells that generated twdaughter cells or made abnormal mitoses were classified different categories depending on the fibrillarin localizationthe aspect of the DNA and the structures observed by phacontrast microscopy.

brillarin antibody microinjection (GM4 1/10) in PtK1 mitotic cells. Bar, 5 presented abnormal chromatin condensation. DAPI staining showed thatlization of anti-fibrillarin antibodies by indirect immunofluorescence), the abnormal cells contained fibrillarin distributed as small dots in the

phase contrast microscopy. In pattern 2 (27%), the abnormal cells containedall dots in the nucleoplasm (b). In pattern 3 (46%), fibrillarin was only

ising RNA pol I was performed on this cells. It can be noted that rDNArol cell (c′′ , cell on left).


Fig. 7.Effects of fibrillarin antibodies two hours after microinjection into mitotic cells. (A) Two postmitotic daughter cells, each showing apolylobate nucleus with two nucleoli. Chromatin that is still condensed is mostly associated with the nuclear envelope. Bar, 5 µm. (B) Part ofthe nucleus with a nucleolus (Nu) consisting mainly of fibrillar components that are less contrasted than the chromatin (c, arrowheads). Thenumerous fuzzy dense aggregates (arrows) of irregular size scattered throughout the nucleoplasm are identified as PNBs. These structures arenever seen fusing with the nucleolus. (C) High magnification of a pseudo-nucleolus completely depleted of granules and showing two fibrillarcenters (*) surrounded by dense fibrillar material. Note the presence of a small PNB in the vicinity (arrow), and condensed chromatin (c).(D) Typical PNB similar to those observed at telophase of normal cells. (E) The three prenucleolar bodies with irregular limits are composed ofdensely packed fibrils. (F,G) The rounded spherical masses with a loose fibrillar aspect (arrows) are usually seen in close contact with non-decondensed chromatin (c). Bars: (B-G) 0.2 µm.

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Fig. 8. rRNA transcription, 2 hours aftermicroinjection of anti-fibrillarinantibodies in the nucleus of an interphasiccell. The nucleoli are labeled with anti-fibrillarin antibodies 2 hours aftermicroinjection (a). rRNA transcriptionwas detected in conditions favoring RNApol I activity. The level of BrUTPincorporation was comparable in themicroinjected cell and in the adjacentcontrol cell (a′). DNA was stained withDAPI (a′′ ). Bar, 5 µm.

To find out if the differences observed between the conand anti-fibrillarin sera were significant, a χ2 analysis wasperformed to calculate the probability for each cell to obtathe observed distribution by chance. The results indicated the effects of microinjection of anti-fibrillarin antibodies wersignificant as compared to control antibodies. These effedescribed below were specifically due to the injection of anfibrillarin antibodies and are presented as percentages.

Effects on nuclear and nucleolar reconstructionThe effects were first analyzed with respect to the stagemicroinjection. There was no obvious difference betweinjections performed in late G2, prophase, metaphase oanaphase. Consequently, the results were compiled analyzed independently of the injection time. Microinjectioof anti-fibrillarin antibodies did not block mitosis, bunevertheless induced mitotic abnormalities in a few casIndeed, 10% of the cells injected at any time displayed nuthat were fragmented into micronuclei of variable sizes anumbers (data not shown) whereas in control microinjeccells only 5% cells with micronuclei were observed.

Of the anti-fibrillarin antibody microinjected cells, 70%generated two daughter cells. By comparison with the consera, these cells were expected to be in G1, 2 hours aftermicroinjection. However, significant changes in the nuclemorphology were observed, since 40% of the cells presenchromatin condensation reminiscent of a telophase-lconfiguration. These cells were apparently unable to progrinto G1 and their chromatin remained condensed. Thereforeseems that microinjection of anti-fibrillarin antibodies causapparent modifications of interphasic chromatdecondensation.

The distribution of the anti-fibrillarin antibodies waanalyzed in the 40% of cells presenting abnormal chromacondensation. The cells could be classified into thrcategories. In the first category, pattern 1 (27% of the ceno typical interphasic nucleolar structure could be seen phase contrast microscopy suggesting that nucleolar formation was inhibited. The antibody-bound fibrillarin wascattered over the nucleus appearing as dense structurephase contrast microscopy (Fig. 6a,a′). In the second categorypattern 2 (27% of the cells), antibody-bound fibrillarin wapresent in the re-formed nucleoli but also in PNBs in t

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nucleoplasm (Fig. 6b,b′). In the third category, pattern 3 (46%of the cells), the cells had apparently regained a normnucleoplasmic compartmentalization, because nucleoli weobserved by phase contrast microscopy and antibody-boufibrillarin was able to fuse in the new nucleoli (Fig. 6c,c′).

Nuclear organization of the microinjected cellsA careful examination of serial ultrathin sections of postmitotidaughter cells blocked at pattern 2 revealed disorganized apolylobate nuclei (Fig. 7A). Even two hours aftermicroinjection, the chromatin was still much more condensethan at late telophase (compare with Fig. 3A) and formeblocks mainly associated with the nuclear envelope (Fi7A,B,F,G). The nucleoli which were still visible by phasecontrast microscopy, presented an abnormal organization atultrastructural level and were smaller. However, their numbdid not change. There were always two nucleoli, alwayadjacent to non-decondensed chromatin. The nucleconsisted of material apparently identical to the fibrillar centand associated with more electron dense fibrillar structur(Fig. 7B,C), which were, however, less contrasted than tDFC of an interphase nucleolus. We have designated thonucleoli, which are completely depleted of morphologicallidentifiable granular component, pseudo-nucleoli.

In addition, unravelled structures were also visible, scatterthroughout the nucleoplasm, and never associated with pseudo-nucleoli (Fig. 7B,C,E). They exhibited irregular limitand were frequently smaller than the PNBs observed nucleologenesis of normal cells. However, based on thfibrillar morphology and heavy staining property, they weridentified as PNBs. Besides these structures and the rare PNof normal shape (Fig. 7D), small fibrillar structures about 0µm in diameter and of low electron density were systematicaseen in close association with the non-decondensed chrom(Fig. 7F,G). They may result from the segregation of the PNcontent, following fibrillarin depletion.

Transcription in microinjected cellsThese abnormal nuclear phenotypes could be related inhibition of fibrillarin translocation from its perichromosomallocation to the newly formed nucleoli. To determine whethesteps in the early post-mitotic phase of nucleolar re-formatiwere correctly accomplished, RNA pol I transcription wa


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studied 2 hours after anti-fibrillarin antibody microinjection. decrease in transcription was observed in nucleoli microinjected cells blocked in pattern 3 compared to contadjacent cells and early G1 cells (Fig. 6c). Extinction oftranscription was observed in nuclei of cells blocked in patt2 containing pseudo-nucleolar structures and several type

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Fig. 9. Schematic diagram indicating the effects of anti-fibrillarinantibody microinjection in mitotic PtK1 cells after 2 hours. Theseeffects are related to the normal pathway of fibrillarin. When anti-fibrillarin antibodies were injected, 40% of the cells failed to re-fora normal nucleus in the subsequent interphase. Chromatin remaiin a telophase-like configuration. Depending on the localization othe antibody-bound fibrillarin, 3 steps at which the fibrillarinpathway could be blocked were proposed. In pattern 1 (27%),fibrillarin was dispersed as little dots around the nucleoplasm andthese cells failed to re-form nucleoli and no rDNA transcription waobserved. In pattern 2 (27%), the newly re-formed nucleoli wereabnormal because no granular component was observed by elecmicroscopy. No rDNA transcription was detected in these nucleoland fibrillarin was localized in these pseudo-nucleoli as well as inPNBs that remained attached to condensed chromatin. In pattern(46%), only nucleoli were labeled with fibrillarin and rDNAtranscription was weaker than in control cells. The broken arrowsindicate the putative steps of blocking produced by themicroinjection of anti-fibrillarin antibodies. Plus signs indicate leveof transcription: (+) weak spot.

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PNBs. This was in agreement with the electron microscoobservations which showed that these microinjected cefailed to re-form normal nucleoli. In cells without visiblenucleolar structure (pattern 1), no rDNA transcription wadetected, even in small areas that could correspond to NO

To determine whether microinjection of fibrillarin antibodiehad a direct effect on the level of rDNA transcription ifunctional nucleoli, microinjections were performed ininterphasic nuclei. Two hours after microinjection thantibody-bound fibrillarin was detected in the nucleoli (Fig8a). Indeed, the antibodies did not induce the releasefibrillarin from the nucleoli nor PNB formation. A comparisonof the level of rDNA transcription in control microinjectedcells and in anti-fibrillarin microinjected cells, did not reveaany significant difference (Fig. 8a′).

Therefore it seems that the effect on rDNA transcriptioinduced by microinjection of anti-fibrillarin antibodies isprobably related to depletion of fibrillarin during nucleolar reformation.

Interpretation of the effects of the anti-fibrillarinantibodies on nucleolar re-formationConsidering the distribution of the antibody-bound fibrillariin cells 2 hours after microinjection during mitosis, it seempossible that some steps of the normal fibrillarin translocatipathway (Fig. 4) might have been disturbed. We propose tinterphasic nuclei without nucleolus and antibody-bounfibrillarin appearing in small foci in the nucleoplasm (patter1) were generated by blocking of the early stages of fibrillartranslocation (Fig. 9). Interphasic nuclei containing antibodbound fibrillarin in small incomplete nucleoli and in PNB(pattern 2) could derive from a blocking in telophase (Fig. 9The same blocking step is postulated for interphasic nuclepattern 3 because chromatin condensation is similar telophase cells and is not comparable to early G1 nuclei. Theeffects on transcription (inhibition or decrease) support thmodel as well as the abnormal chromatin condensationinterphasic nuclei (Fig. 9).

DISCUSSION

NoPC proteins participate in PNB formationVarious nuclear complexes have been detected in perichromosomal domain, among which nucleolar NoPproteins and nucleolar RNP complexes are largely represen(Beven et al., 1996; Dundr et al., 1997; Gautier et al., 199Medina et al., 1995; Stracke and Martin, 1991; Weisenbergand Scheer, 1995). Fibrillarin is characteristic of thredistribution of the NoPC proteins at the end of G2 (Gautieret al., 1992b), but other major nucleolar proteins such nucleolin and protein B23 (Zatsepina et al., 1997b) are ainvolved, indicating that this common location has a biologicsignificance.

As anaphase progresses, the fibrillarin of thperichromosomal layer is recruited in foci of heterogeneosizes located at the periphery of the chromosomes which still condensed. Therefore in telophase, fibrillarin is found the PNBs colocalized with nucleolin and Ag-NOR protein(Jiménez-Garcia et al., 1994; Ochs et al., 1985a,b). Here it wshown that fibrillarin localized on the chromosome periphe

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up to anaphase as well as nucleolin and Ag-NOR proteins (unpublished data), and was recruited in a number heterogeneous foci. Therefore it seems that the PNBs formduring telophase by gathering the material surrounding chromosomes during mitosis. The ultrastructural organizatof the PNBs (for a review see Zatsepina et al., 1997a)compatible with this hypothesis. Consequently it can proposed that in late anaphase, the first step of PNB formais the recruitment of NoPC proteins in foci at the peripherychromosomes. The second step of PNB genesis would begathering of these heterogeneous foci as proposed in Fig.

The microinjected antibodies could block gathering fibrillarin in PNBs, since only small scattered structurappeared in some newly formed nuclei and no nucleolus visible (pattern 1). However, it cannot be simply concluded thfibrillarin is essential for the formation of PNBs, since in nuclassembled in Xenopus egg extracts, PNBs depleted ofibrillarin can be formed, albeit in reduced numbers (Bell aScheer, 1997). It is noticeable that the pool of PNB proteother than fibrillarin (mainly nucleolin and protein B23) icertainly higher in Xenopus egg extracts than in PtK1 cells andthis difference may affect the efficiency of PNB formation sub-optimal conditions, i.e. depletion of fibrillarin.

The effects of antibodies on fibrillarin translocation depeon the epitopes recognized by the serum. It is known that afibrillarin auto-antibodies present in systemic sclerosis (as GM4 and S4) interact with epitopes of the amino- and carboterminal domains of fibrillarin but less frequently with thcentral RNA binding domain (Kasturi et al., 1995). It reasonable to predict that the same epitopes are also recogby GM4. The fact that the amino-terminal domain is involvein recognition of fibrillarin is also supported by the absencereduced cross-reaction with S4 serum in species (archebacand tetrahymena) in which the amino-terminal part fibrillarin differs from that of fibrillarin in higher eukaryotes(Agha Amiri, 1994; David et al., 1997). The amino-termindomain of fibrillarin is characterized by the GAR-domain,sequence also present in nucleolin and in other nuclesnoRNPs as well as in hnRNP-A proteins (for a review sBurd and Dreyfuss, 1994). This GAR-domain must be presfor nucleolin to associate with nucleoli in vivo (Heine et a1993; Schmidt-Zachmann et al., 1993; Schmidt-Zachmann Nigg, 1993). In the case of inhibition of fibrillarin translocatioto PNBs, the GAR-domain could also be involved in inhibitioof PNB targeting to nucleoli in nuclei with pattern 1 (Fig. 9

Pathway of fibrillarin to the nucleoliPNBs containing fibrillarin were still present around thcondensed part of the chromosomes in telophase, and fibrillwas simultaneously targeted to the nucleoli as visualized byaccumulation in nucleoli. This process continued in early G1,and later PNBs were generally not detected. This dynaprocess should be studied in living cells to be completunderstood. Electron microscopy studies demonstrated thaPNBs corresponding to dense fibrillar structures associatenucleoli in sites close to the DFC. Differences in electrdensity of the two contiguous components was obvious, was no longer visible after fusion.

The antibody-bound fibrillarin can be blocked along imigration pathway to nucleoli. Electron microscopy studirevealed different kinds of PNB structures that can

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generated by dissociation of PNB components. In addition tnucleoli exhibited an unusual organization of fibrillacomponents: the components were less electron dense anddeveloped than the DFC in PtK1 cells at G1 phase. Thepresence of modified DFC is compatible with the absence reduction of PNB fusion in these nucleoli as well as with thabsence of active rDNA transcription. It can be proposed thDFC depleted of fibrillarin nevertheless possesses nucleocomponents not delivered by PNBs (Dundr et al., 1997).

Dynamics of nucleolar assembly at the end ofmitosisThe first event that should unambiguously define nefunctional nucleoli is the resumption of the RNA pol I activityIt is well known that transcription is stopped during mitosi(Prescott, 1964). Because the level of transcription will initiallbe low, a very sensitive method is needed to detect it. Thisthe case for RNA transcription detected in vivo by modifieRNA precursors introduced in permeabilized cells (Jacksonal., 1993; Wansink et al., 1993), in conditions favouring RNApol I activity (Masson et al., 1996). The same procedure walso adapted on ‘weakly fixed’ cells, adapting a protocdescribed for the incorporation of tritiated uridine (Moore anRingertz, 1973). In this case, the cells did not progress in tcell cycle during precursor incorporation; this is particularladvantageous when trying to precisely determine the mitostage at which RNA pol I activity is resumed. In PtK1 cells,RNA pol I activity was first detected in anaphase B, earlier thain HeLa mitotic cells examined using the same approa(Roussel et al., 1996). This can be due to differences betwecell lines or to the fact that the mitotic phase is more easdetermined in PtK1 cells. It is noticeable that activation ofrDNA transcription is an early event in the formation of newinterphase nuclei.

In the present study, it was shown that transcription by RNpol I starts at the end of anaphase concommittantly with trecruitment of fibrillarin in foci on the surface of thechromosomes. Thus, the beginning of PNB formation occusimultaneously with the onset of rDNA transcriptionPresently, we do not know if these two events are linked, orthey are regulated by the same mechanism, such as possmitotic regulation during anaphase.

In addition, it was demonstrated by immunodepletion ofibrillarin that the translocation of PNB to NORs not onlydepends on rDNA transcription as generally reported (forreview see Scheer et al., 1993; Bell et al., 1992; Benaventeal., 1987). An hypothesis of the capturing of nucleolar proteiby transcribing nucleolar RNAs has been proposed. Howevit is noticeable that antibody-bound fibrillarin was noefficiently translocated into the nucleoli at stages where RNpol I is active. Because it was observed that antibody-boufibrillarin was still associated with condensed chromatin, wpostulate that fibrillarin was not translocated, but rather thinteraction of fibrillarin with rRNAs was impaired.

It has been shown in yeast that fibrillarin may organize thnucleolar structure (Jansen et al., 1991). Fibrillarin is believto play a role in the nuclear matrix (Ochs and Smetana, 199On the other hand, yeast depleted of NOP1 can complemented by human fibrillarin, but this results inmodifications of nuclear morphology and in aberrant nucleolmorphology (Jansen et al., 1991). This indicates that fibrillar


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could be involved in nucleolar structure. After microinjectioof anti-fibrillarin antibodies that prevented normatranslocation of fibrillarin from the chromosome periphery the nucleoli, nucleolar function was disturbed. This wdemonstrated by inactivation of transcription and by abnormnucleolar organization. It is tempting to postulate that when assembly of the processing machinery is inhibited diminished, it induces a negative feed-back effect on nucleoreassembly and, further, on transcription activity.

The authors are grateful to A. Lepage for help in cell culture, ato M. Barre and R. Schwartzmann for photographic work. We particularly grateful to A.-L. Haenni for critical reading of thmanuscript. This work was supported in part by grants from the CeNational de la Recherche Scientifique, the European Biomed conBM H4-Ct95-1139 and the Association pour la Recherche surCancer (ARC, contract no. 6703). N.F. was the recipient ofFellowship from ‘la Ligue Nationale contre le Cancer’, anacknowledges the ARC for financial support.

REFERENCES

Agha Amiri, K. (1994). Fibrillarin-like proteins occur in the domain archaeJ. Bacteriol.176, 2124-2127.

Aris, J. P. and Blobel, G. (1991). cDNA cloning and sequencing of humafibrillarin, a conserved nucleolar protein recognized by autoimmuantisera. Proc. Nat. Acad. Sci. USA88, 931-935.

Azum-Gélade, M.-C., Noaillac-Depeyre, J., Caizergues-Ferrer, M. andGas, N. (1994). Cell cycle redistribution of U3 snRNA and fibrillarinPresence in the cytoplasmic nucleolus remnant and in the prenuclebodies at telophase. J. Cell Sci.107, 463-475.

Bell, P., Dabauvalle, M. C. and Scheer, U. (1992). In vitro assembly ofprenucleolar bodies in Xenopusegg extract. J. Cell Biol.118, 1297-1304.

Bell, P. and Scheer, U.(1997). Prenucleolar bodies contain coilin and aassembled in Xenopusegg extract depleted of specific nucleolar proteins aU3 RNA. J. Cell Sci.110, 43-54.

Benavente, R., Rose, K. M., Reimer, G., Hügle-Dörr, B. and Scheer, U(1987). Inhibition of nucleolar reformation after microinjection oantibodies to RNA polymerase I into mitotic cells. J. Cell Biol.105, 1483-1491.

Beven, A. F., Lee, R., Razaz, M., Leader, D. J., Brown, J. W. S. and ShawP. J. (1996). The organization of ribosomal RNA processing correlates wthe distribution of nucleolar snRNAs. J. Cell Sci.109, 1241-1251.

Burd, C. G. and Dreyfuss, G.(1994). Conserved structures and diversity ofunctions of RNA-binding proteins. Science265, 615-621.

Cavaillé, J., Nicoloso , M. and Bachellerie, J. P. (1996). Targeted ribosemethylation of RNA in vivo directed by tailored antisense RNA guideNature 383, 732-735.

David, E., McNeil, J. B., Basile, V. and Pearlman, R. E. (1997). An unusualfibrillarin gene and protein: structure and functional implications. Mol. Biol.Cell 8, 1051-1061.

Dilworth, S. M. (1991). A perichromosomal region contains proteinphosphorylated during mitosis in Xenopus laeviscells. J. Cell Sci. 98, 309-315.

Dundr, M., Meier, U. T., Lewis, N., Rekosh, D., Hammarskjöld, M.-L. andOlson, M. O. J. (1997). A class of nonribosomal nucleolar componentslocated in chromosome periphery and in nucleolus-derived foci duranaphase and telophase. Chromosoma 105, 407-417.

Gautier, T., Dauphin-Villemant, C., André, C., Masson, C., Arnoult, J. andHernandez-Verdun, D. (1992a). Identification and characterization of new set of nucleolar ribonucleoproteins which line the chromosomes dumitosis. Exp. Cell Res. 200, 5-15.

Gautier, T., Robert-Nicoud, M., Guilly, M.-N. and Hernandez-Verdun, D.(1992b). Relocation of nucleolar proteins around chromosomes at mitoA study by confocal laser scanning microscopy. J. Cell Sci. 102, 729-737.

Gautier, T., Fomproix, N., Masson, C., Azum-Gelade, M.-C., Gas, N. andHernandez-Verdun, D. (1994). Fate of specific nucleolar perichromosomproteins during mitosis: cellular distribution and association with UsnoRNA. Biol. Cell 82, 81-93.

nltoas

altheorlar

ndareentretract le ad

a.

nne

.olar

rend

.f

,ith

f

s.

s

ising

aring

sis.

al3

Heine, M. A., Rankin, M. L. and DiMario, P. J. (1993). The gly/arg-rich(GAR) domain of Xenopusnucleolin facilitates in vitro nucleic acid bindingand in vivo nucleolar localization. Mol. Biol. Cell 4, 1189-1204.

Hernandez-Verdun, D. and Gautier, T. (1994). The chromosome peripheryduring mitosis. BioEssays 16, 179-185.

Jackson, D. A., Hassan, A. B., Errington, R. J. and Cook, P. R. (1993).Visualization of focal sites of transcription within human nuclei. EMBO J.12, 1059-1065.

Jansen, R. P., Hurt, E. C., Kern, H., Lehtonen, H., Carmo-Fonseca, M.,Lapeyre, B. and Tollervey, D.(1991). Evolutionary conservation of thehuman nucleolar protein fibrillarin and its functional expression in yeast. J.Cell Biol. 113, 715-729.

Jiménez-Garcia, L. F., Rothblum, L. I., Busch, H. and Ochs, R. L. (1989).Nucleologenesis: use of non-isotopic in situ hybridization animmunocytochemistry to compare the localization of rDNA and nucleolaproteins during mitosis. Biol. Cell 65, 239-246.

Jiménez-Garcia, L. F., Segura-Valdez, M. d. L., Ochs, R. L., Rothblum, L.I., Hannan, R. and Spector, D. L. (1994). Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNtranscription after mitosis. Mol. Biol. Cell5, 955-966.

Kasturi, K. N., Hatakeyama, A., Spiera, H. and Bona, C. A. (1995).Antifibrillarin autoantibodies present in systemic sclerosis and otheconnective tissue diseases interact with similar epitopes. J. Exp. Med. 181,1027-1036.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembof the head of bacteriophage T4. Nature 227, 680-685.

Leser, G. P., Fakan, S. and Martin, T. E.(1989). Ultrastructural distributionof ribonucleoprotein complexes during mitosis. snRNP antigens acontained in mitotic granule clusters. Eur. J. Cell Biol. 50, 376-389.

Masson, C., Bouniol, C., Fomproix, N., Szöllösi, M. S., Debey, P. andHernandez-Verdun (1996). Conditions favoring the RNA polymerase Itranscription in permeabilized cells. Exp. Cell Res. 226, 114-125.

Medina, F. J., Cerdido, A. and Fernandez-Gomez, M. E.(1995).Components of the nucleolar processing complex (pre-rRNA, fibrillarin, annucleolin) colocalize during mitosis and are incorporated to daughter cnucleoli. Exp. Cell Res.221, 111-125.

Moore, G. P. M. and Ringertz, N. R. (1973). Localization of DNA-dependentRNA polymerase activities in fixed human fibroblasts by autoradiographExp. Cell Res. 76, 223-228.

O’Farrell, P. H. (1975). High resolution two-dimensional electrophoresis oproteins. J. Biol. Chem. 250, 4007-4021.

Ochs, R. L., Lischwe, M. A., Shen, E., Caroll, R. E. and Busch, H. (1985a).Nucleologenesis: composition and fate of prenucleolar bodies. Chromosoma92, 330-336.

Ochs, R. L., Lischwe, M. A., Spohn, W. H. and Busch, H. (1985b).Fibrillarin: a new protein of the nucleolus identified by autoimmune serBiol. Cell 54, 123-134.

Ochs, R. L. and Smetana, K. (1991). Detection of fibrillarin in nucleolarremnants and the nucleolar matrix. Exp. Cell Res. 197, 183-190.

Prescott, D. M. (1964). Cellular sites of RNA synthesis. Prog. Nucl. AcidsRes. 3, 33-57.

Rattner, J. B. (1992). Integrating chromosome structure with functionChromosoma 101, 259-264.

Reimer, G., Pollard, K. M., Penning, C. A., Ochs, R. L., Lischwe, M. A.,Busch, H. and Tan, E. M.(1987). Monoclonal autoantibody from a (NewZealand black × New Zealand white) F1 mouse and some humascleroderma sera target an Mr 34,000 nucleolar protein of the U3 RNparticle. Arthr. Rheum.30, 793-780.

Robert-Fortel, I., Junéra, H. R., Géraud, G. and Hernandez-Verdun, D.(1993). Three-dimensional organization of the ribosomal genes and ANOR proteins during interphase and mitosis in PtK1 cells studied byconfocal microscopy. Chromosoma 102, 146-157.

Roussel, P., André, C., Comai, L. and Hernandez-Verdun, D. (1996). TherDNA transcription machinery is assembled during mitosis in active NORand absent in inactive NORs. J. Cell Biol. 133, 235-246.

Scheer, U., Thiry, M. and Goessens, G. (1993). Structure, function andassembly of the nucleolus. Trends Cell Biol. 3, 236-241.

Scheer, U. and Weisenberger, D. (1994). The nucleolus. Curr. Opin. Cell Biol.6, 354-359.

Schimmang, T., Tollervey, D., Kern, H., Frank, R. and Hurt, E. C. (1989).A yeast nucleolar protein related to mammalian fibrillarin is associatewith small nucleolar RNA and is essential for viability. EMBO J. 8, 4015-4024.

Schmidt-Zachmann, M. S., Dargemont, C., Kühn, L. C. and Nigg, E. A.

372

II.

se

s

arin

e


(1993). Nuclear export of proteins: the role of nuclear retention. Cell 74,493-504.

Schmidt-Zachmann, M. S., Hügle-Dörr, B. and Franke, W. W.(1987). Aconstitutive nucleolar protein identified as a member of the nucleoplasmfamily. EMBO J. 6, 1881-1890.

Schmidt-Zachmann, M. S. and Nigg, E. A. (1993). Protein localization tothe nucleolus: a search for targeting domains in nucleolin. J. Cell Sci.105,799-806.

Smith, C. M. and Steitz, J. A. (1997). Sno storm in the nucleolus: new rolefor myriad small RNPs. Cell 89, 669-672.

Starborg, M., Gell, K., Brundell, E. and Höög, C. (1996). The murine Ki-67 cell proliferation antigen accumulates in the nucleolar aheterochromatic regions of interphase cells and at the periphery of mitotic chromosomes in a process essential for cell cycle progression. J. CellSci. 109, 143-153.

Stracke, S. and Martin, R. (1991). Postmitotic reassembly of the cell nucleuin whole cells: an electron-spectroscopic study. J. Cell Sci. 100, 541-550.

Thiry, M. and Goessens, G. (1996). The Nucleolus During the Cell Cycle.Springer-Verlag, Heidelberg.

Tollervey, D. (1996). Trans-acting factors in ribosome synthesis. Exp. CellRes. 229, 226-232.

Tollervey, D., Lehtonen, H., Jansen, R., Kern, H. and Hurt, E. (1993).Temperature-sensitive mutations demonstrate roles for yeast fibrillarinpre-rRNA processing, pre-rRNA methylation, and ribosome assembly. Cell72, 443-457.

Verheijen, R., Kuijpers, H. J. H., van Driel, R., Beck, J. L. M., vanDierendonck, J. H., Brakenhoff, G. J. and Ramaekers, F. C. S. (1989).

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s

ndthe

s

in

Ki-67 detects a nuclear matrix-associated proliferation-related antigen Localization in mitotic cells and association with chromosomes. J. Cell Sci.92, 531-540.

Wansink, D. G., Schul, W., van der Kraan, I., van Steensel, B., van Driel,R. and de Jong, L. (1993). Fluorescent labeling of nascent RNA revealtranscription by RNA polymerase II in domains scattered throughout thnucleus. J. Cell Biol. 122, 283-293.

Warren, G. and Wickner, W. (1996). Organelle inheritance. Cell 84, 395-400.

Weisenberger, D. and Scheer, U. (1995). A possible mechanism for theinhibition of ribosomal RNA gene transcription during mitosis. J. Cell Biol.129, 561-575.

Willingham, M. C. and Bhalla, K. (1994). Transient mitotic phaselocalization of Bcl-2 oncoprotein in human carcinoma cells and itpossible role in prevention of apoptosis. J. Histochem. Cytochem. 42, 441-450.

Yasuda, Y. and Maul, G. G.(1990). A nucleolar auto-antigen is part of amajor chromosomal surface component. Chromosoma99, 152-160.

Zatsepina, O. V., Dudnic, O. A., Todorov, I. T., Thiry, M., Spring, H. andTrendelenburg, M. F. (1997a). Experimental induction of prenucleolarbodies (PNBs) in interphase cells: interphase PNBs show similcharacteristics as those typically observed at telophase of mitosis untreated cells. Chromosoma 105, 418-430.

Zatsepina, O. V., Todorov, I. T., Philipova, R. N., Krachmarov, C. P.,Trendelenburg, M. F. and Jordan, E. G. (1997b). Cell cycle-dependanttranslocations of a major nucleolar phosphoprotein, B23, and somcharacteristics of its variants. Eur. J. Cell Biol.73, 58-70.

effects of anti-ﬁbrillarin antibodies on building of ... · during mitosis and then reintegrated...

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