BN46/51, a New Nucleolar Protein, Binds to the Basal Body Region in Naegleria Gruberi Flagellates

Journal of Cell Science 103, 167-181 (1992) 167 Printed in Great Britain © The Company of Biologists Limited 1992

BN46/51, a new nucleolar protein, binds to the basal body region in Naegleria gruberi flagellates

GIN A M. TRIMBUR and CHARLES J. WALSH*

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA •Author for correspondence

Summary

Indirect immunofluorescence with the monoclonal antibody region is resistant to extraction, even with 2 M body, BN5.1, labels the nucleolus of amebae of the NaCl. Solubilized BN46/51 exists as a heterogeneous amebo-flagellate Naegleria gruberi. When amebae dif- multimer that elutes on gel filtration with a peak at 400 ferentiate into flagellates, BN5.1 binds to nucleoli and to to 500 kDa and sediments on sucrose gradients at 5.5 S. the basal body region. The BN5.1 antigen is not present The multimers consist of only the 46 kDa and the 51 kDa in basal bodies when basal bodies form at about 60 min subunits in equal amounts as judged by glutaraldehyde after initiation of the differentiation or when flagella cross-linking and by chromatography on BN5.1 affinity form at about 70 min. The BN5.1 antigen is first columns. Nucleolar BN46/51 is associated with the dense detectable in the basal body region at 85 min after fibrillar and granular components of the nucleolus. initiation, a time when the basal body region acts as a However, it does not resemble any previously described microtubule organizing center for the formation of the nucleolar protein. Neither BN5.1, nor three other mAbs microtubule cytoskeleton (MTCS) of flagellates. When that recognize additional epitopes on both the 46 kDa flagellates revert spontaneously to amebae, the BN5.1 and 51 kDa subunits of BN46/51, binds to nucleoli from antigen is lost from the basal body region coincident with Saccharomyces cerevisiae or mammalian cells. BN5.1 the loss of the MTCS. The BN5.1 antigen, composed of does not bind to the nucleoli of Dictyostelium discoideum approximately equal amounts of two subunits of 46 kDa or Euglena gracilis. Thus BN46/51 is an unusual and and 51 kDa, both of which carry the BN5.1 epitope, has perhaps unique nucleolar component whose presence in been named BN46/51. BN46/51 in the basal body region the basal body region presents a challenge to our comigrates with the nucleolar antigen by two-dimen- understanding of the cytoskeleton. sional gel electrophoresis. Approximately 75% of the nucleolar BN46/51 is solubilized by extracton with 0.4 M Key words: nucleolar antigen, basal body, cytoskeleton, NaCl. However, the antigen associated with the basal Naegleria.

Introduction evidence, however, favors the DFC as the site of transcription. rDNA has been localized to the DFC by Nucleoli are the sites of rRNA transcription, rRNA Wachtler et al. (1989, 1992). By pulse-chase labelling, processing and the assembly of ribosomal subunits nascent rRNA transcripts were found first in the DFC (Scheer and Benavente, 1990; Sommerville, 1986; (Deltour and Mosen, 1987; Goessens, 1976). Topo- Warner, 1990). The nucleolus is composed of three isomerase I and RNA polymerase I have both been generally recognized morphological components: the localized in the DFC (Raska et al., 1989; reviewed by fibrillar centers (FC), the dense fibrillar component Jordan, 1991). Fibrillarin (Ochs et al., 1985), a (DFC), and the granular component (GC) (Jordan, component of the U3 snRNP involved in the first 1984; Goessens, 1984; Warner, 1990). However, despite preribosomal RNA processing step (Kass et al., 1990), extensive investigation, the functional significance of is localized to the DFC (Scheer and Benavente, 1990). these morphological components is unclear. Most of the In contrast it is generally agreed that the GC represents discussion has been focused on the function of the FC ribosomal subunits in various stages of assembly (Hiigle and DFC components. For example, the presence of et al., 1985; Warner, 1990). RNA polymerase I (Scheer and Rose, 1984) and rRNA Nuclei of the amebo-flagellate Naegleria gruberi have genes in the FC of nucleoli (Knibiehler et al., 1977; a single large nucleolus (Fulton, 1970; Schuster, 1975). Jordan and Rawlins, 1990) suggests that the FC is the Despite its large size, the Naegleria nucleolus is site of transcription of the rRNA genes. Other composed of the three characteristic morphological 168 G. M. Trimbur and C. J. Walsh components. It is rapidly labeled by RNA precursors vortexed in the presence of non-ionic detergents (Walsh and and it is enriched in 28 S rRNA (Walsh and Fulton, Fulton, 1973). In contrast, nucleoli are quite resistant and 1973) as are nucleoli in other cells. However, the remain intact. The lysate was centrifuged in an IEC Clinical Naegleria nucleolus does differ from most nucleoli in centrifuge at a setting of 6 for 1 min to pellet cysts and unlysed cells. (As noted by Fulton (1970), expression of these brief the organization of the rRNA genes. Instead of a centrifugations as g forces is meaningless because the tandem array of rRNA genes at one or more chromo- centrifuge is accelerating during this time; they are, however, somal locations, the Naegleria rRNA genes are found reproducible.) Nucleoli were pelleted by centrifugation of the exclusively on approximately 4000 copies of a 17 kbp resulting supernatant at 10,400 g for 2 min. Additional plasmid (Clark and Cross, 1987, 1988). Another nucleoli were collected by centrifuging the supematent a unusual feature of the Naegleria nucleolus is that it does second time as above. When flagellates were used, the final not disperse during mitosis (Fulton, 1970; Schuster, supernatant was saved for the isolation of flagellar rootletyfoa- 1975). Mitosis in Naegleria is closed and the nucleolus sal body complexes, described below. divides as the chromosomes separate on the intra- The nucleolar pellets were pooled by resuspension in 4 ml nuclear spindle. of 1 M sucrose-RLB, overlayed on a 1.62 M sucrose-RLB pad and centrifuged for 13 min at 10,400 g. The pellet was This report describes the identification and character- resuspended in 1 ml of SPMG (63 mM sucrose, 25 mM sodium ization of a novel protein, BN46/51, found in the DFC phosphate, pH 7.2, 2.5 mM MgCI, and 0.5 mM EGTA) and and GC of Naegleria nucleoli. BN46/51 is unusual in nucleoli were counted in a hemocytometer. The nucleoli were that it is also associated with the basal body region of pelleted by centrifugation at 15,000g for 1 min. Nucleoli were Naegleria flagellates. Amebae of N. gruberi can solubilized by resuspension in 0.4 M NaCI-SPMG at 4 x 108 synchronously and rapidly differentiate into swimming nucleoli/ml for 30 min on ice. This extract was centrifuged for flagellates when washed free of their food source 10 min at 15,000 g. The supernatant was saved and used as a (Fulton, 1970; Fulton, 1977; Fulton and Dingle, 1967). source of soluble BN46/51. The differentiation results in the de novo assembly of When nucleoli were prepared for immunofluorescence, basal bodies (Dingle and Fulton, 1966; Fulton, 1970; nucleoli were resuspended in SPMG and an equal volume of Fulton and Dingle, 1971), flagellar axonemes (Dingle fixative (MgCF) was added as described below. Nucleolar fractions contained few if any rootlet/basal body complexes and Fulton, 1966), flagellarrootlet s (Dingle and Fulton, when observed by phase-contrast microscopy. 1966; Larson and Dingle, 1981a,b), and a microtubule Rootlet/basal body complexes were prepared from the final cytoskeleton (MTCS) (Walsh, 1984). BN46/51 is associ- nucleolar-depleted supernatant by addition of 1 M KI in RLB ated with the nucleolus in amebae, and appears in the without glycerol to make the extract 0.1 M KI as described by basal body region when the MTCS assembles in Larson and Dingle (1981a). This extract was centrifuged at flagellates. This unexpected observation raises a num- 16,300 g for 10 min. The pellet was resuspended in 1 ml of 0.1 ber of interesting questions about the role of BN46/51 in M KC1-RLB without glycerol, and centrifuged at 15,000 g. the cell. As a first step in examining these questions we The pellet was rinsed with 1 ml of SPMG and resuspended in 9 9 have undertaken a characterization of BN46/51. SPMG at 1 x 10 to 2 x 10 cell equivalents/ml. Rootlet/basal body fractions contained no nucleoli as judged by phase- contrast microscopy. Materials and methods Antigen preparation, monoclonal antibody and ascites production Growth and differentiation of Naegleria gruberi Rootlet/basal body complexes from 2 X 109 flagellates were Naegleria gruberi strain NB-1 (Fulton, 1970; Fulton and solubilized in Laemmli sample buffer (Laemmli, 1970) by Dingle, 1967) was grown on NM or PM agar with Klebsiella heating in boiling water for 2 min and electrophoresed on pneumoniae as previously described (Fulton and Dingle, 7.5% SDS-polyacrylamide gels (Laemmli, 1970). Gels were 1967). Amebae were harvested and washed free of bacteria in stained for 5 min with Coomassie Blue and destained in ice-cold 2 mM Tris-HCl (pH 7.6 at 20°C). Differentiation was deionized water. Proteins in the size range of 46 to 48 kDa initiated in this same buffer at 25°C with 15 ml in 125 ml were cut out of the gel and the gel piece was minced in TBS Erlenmeyer flasks or 25 ml in 250 ml Erlenmeyer flasks on a (50 mM Tris-HCl, pH 7.4, 200 mM NaCl). Complete Fruend's reciprocating shaker as described by Fulton and Dingle adjuvant was added at a 3:1 ratio and the mixture emulsified. (1967). Differentiation was monitered by fixing cells in The emulsion (0.2 ml) was injected intraperitoneally (i.p.) Lugol's iodine. into female Balb/c mice. At 2-week intervals animals were injected with minced gel pieces containing protein emulsified Isolation of nucleoli and flagellar rootlet/basal body with incomplete Fruend's aduvant until they showed antibody complexes production. Animals were boosted i.p. by injection with gel Nucleoli were isolated from either amebae or deflagellated pieces containing protein in TBS 3 days before fusion. This flagellates (Kowit and Fulton, 1974) as described by Larson same procedure was used to obtain the anti-rootlet antiserum, and Dingle (1981a) with minor modifications. Briefly, cells only the 170 kDa rootlet protein, the major protein in the were resuspended at 2 x 107 cells/ml in Rootlet Lyse Buffer rootlet/basal body preparation (Larson and Dingle, 1981a), (RLB): 30 mM Tris-HCl, pH 8.0, 50% glycerol, 3 mM was cut out from gels. Hybridoma production was carried out MgSO4, 1 mM EGTA, 1 mM dithiothreitol (DTT), 10 mM using spleen cells from immunized mice and the myeloma cell epsilon-amino-rc-caproic acid, 0.005% PMSF, and 0.02 mM line P3-X63Ag 8 clone 653 (Dane et al., 1986). Hybridoma leupeptin. Cells were lysed by adding Triton X-100 to a final supernatants were initially screened by immunofluorescence concentration of 0.5% and vortexing for 20 s. The lysate was using flagellates fixed in MgCF as described below. Positive reduced to 25% glycerol by adding RLB without glycerol. cultures were subcloned twice by limiting dilution (Hurrell, Nuclei of Naegleria are very fragile and are broken when 1982). Ascites fluid was obtained by priming 4- to 8-week old BN46/51, a new nudeolar protein 169 female Balb/c mice with 0.2-0.3 ml of pristane and injection then centrifuged at 15,000 g for 10 min. This second with 0.5 x 106 to 1 x 10* hybridomas 10-12 days after priming supernatent is referred to as the NaCl-soluble fraction. (Hoogenraan and Wraight, 1986). Double immunofluorescence of rootlet/basal body Cell fixation and immunofluorescence microscopy complexes Cells were simultaneously fixed and detergent extracted by Rootlet/basal body complexes were isolated as described gently dropping an equal volume of cells in 2 mM Tris-HCI above and incubated in either SPMG or 0.4 M NaCl-SPMG into ice-cold MgCF (50 mM sodium phosphate, pH 7.2, 125 for 30 min on ice. Extracts were centrifuged for 10 min at mM sucrose, 5 mM MgCI, 1 mM EGTA, 0.1% w/v NP-40, 15,000 g. The pellets were fixed with MgCF and prepared for and 0.9% fomaldehyde; Walsh, 1984). After 10 min in ice, immunofluorescence. Slides were incubated with BN5.1 fixed cells were spread on slides, air dried, rinsed twice in ascites at 1: 400 dilution alone, with anti-rootlet serum at TBS, immersed in ice-cold methanol for 10 min, then in ice- 1:100 dilution alone, and with BN5.1 and anti-rootlet together cold acetone for 10 min and air dried. Slides were incubated at the above dilutions. Slides were washed and incubated with with primary antibody, from either hybridoma culture a goat anti-mouse IgG coupled to FITC. supernatents or ascites fluid (diluted with 10% horse serum- TBS), for 60 min at 37°C in a moist chamber. Slides were Partial purification of Triton-soluble BN46/51 washed 4 times with TBS and incubated with a 1:50 dilution of A Triton X-100-soluble extract was adjusted to 50% am- a goat anti-mouse secondary antibody against IgG (whole monium sulfate by addition of ice-cold saturated ammonium molecule, Cappel) conjugated to either FITC or rhodamine, sulfate. The mixture was stirred on ice for 15 min. The for 60 min and washed as above. For DNA localization, the precipitate was removed by centrifugation for 20 min at 16,300 slides were then incubated with DAPI (4',6-diamidino-2- g at 20°C. The supernatent was dialyzed overnight against 20 phenyl indole dihydrochloride) at 0.1 fig/ml in TBS for 15 min mM Tris-HCI, pH 8.0, at 4°C. The dialysate was centrifuged as above. Slides were mounted in 90% glycerol 10% 0.1 M at 16,300 g for 15 min to remove any precipitate. This Na2CC>3, pH 9.6. Cells were examined using incident supernatent was passed over a DEAE column equilibrated in illumination with a Zeiss Universal microscope and photo- 20 mM Tris-HCI, pH 8.0, and the column was washed with graphed using Kodak T-MAX 400, which was developed in T- one column volume of the same buffer. The column was M AX developer according to the manufacturer's instructions. eluted batch-wise with 100 mM, 200 mM and 300 mM NaCl in 20 mM Tris-HCI, pH 8.0. BN46/51 was enriched about 5-fold lmmunoelectron microscopy in fractions which eluted with 300 mM NaCl. This fraction was Cell suspensions were fixed by mixing with an equal volume of dialyzed overnight in SPMG at 4°C and is referred to as a ice-cold 2% glutaraldehyde in 100 mM NaPO4, pH 7.2. After Triton-soluble BN46/51-enriched fraction. 60 min on ice, cells were pelleted by centrifugation for 1 min at a setting of 6 in a Clinical centrifuge. Cell pellets were Glutaraldehyde cross-linking washed twice by overlaying them with 2 ml of ice-cold 50 mM Triton-soluble BN46/51-enriched fractions (330 jig/ml) were NaPO4 and repeating the centrifugation. Washed cells were incubated with 0.08% glutaraldehyde at 25°C. Cross-linking dehydrated through an ethanol series and embedded in LR was stopped at 0, 1, 2, 5 and 10 min with a 25-fold molar White. Gold sections were cut with a diamond knife. Sections excess of glycine. Samples were fractionated by SDS-PAGE were blocked, reacted with BN5.1, washed, and reacted with and visualized by immunoblotting with BN5.1. A solution of anti-mouse antibody complexed to 10 nm colloidal gold ovalbumin at the same protein concentration was treated in (Janssen, Life Sciences Products) as described by Wright and the same way, but protein was visualized with Coomassie Rine (1989). Blue.

Electrophoresis and immunoblots Gel filtration chromatography Polyacrylamide gels, usually 7.5% acrylamide and 0.75 mm Triton-soluble BN46/51-enriched fractions (660 /ig/ml) were thick, were prepared as described by Laemmli (1970). Total fractionated on a 0.7 cm x 22 cm Sephacryl 300 column protein was visualized by Coomassie Blue stain or by silver equilibrated in SPMG. Fractions of 0.3 ml were collected and stain (Morrissey, 1981). Immunoblotting was done as de- analyzed by immunoblotting with BN5.1. Triton-insoluble scribed by Towbin et al. (1979). Proteins were transferred to fractions were solubilized with 0.4 M NaCl-SPMG at 2.7 x 10s nitrocellulose (Millipore) by electroblotting. Blots were cell equivalents/ml and fractionated on the column in the blocked in 10% horse serum-TBS for 1 h and incubated with same way, except that the column was equilibrated with 0.4 M BN5.1 ascites fluid at a 1:400 dilution for 1 h. Blots were then NaCl-SPMG. The column was calibrated using protein washed four times with shaking in 0.5% Tween-20/TBS for 5 standards (SIGMA Chemical Co.) of 669, 443, 200, 150, 66 min, followed by a 5-min TBS wash. Primary antibody was and 29 kDa. located by incubation with a goat anti-mouse IgG coupled to horseradish peroxidase (Cappel) for 1 h, and washed as Affinity chromatography above. The enzyme was visualized with 4-chloro 1-naphthol (Sigma Chemical Co.) and H2O2 as substrate (Hawkes et al., Affinity columns were prepared and run essentially as 1982). described by Schneider et al. (1982). Protein A Sepharose CL- 4B (Pharmacia) (0.5-1 ml swollen) was mixed with 1-2 ml of BN5.1 ascites fluid which had been dialyzed against 0.1 M BN46/51 fractionation boric acid, adjusted to pH 8.2 with NaOH, overnight at 4°C. Triton X-100-soluble extracts were prepared by vortexing 2.5 The mixture was incubated for 2 h at room temperature with x 107cells/ml in SPMG with 0.5% Triton X-100, 0.005% gentle shaking. The Sepharose beads were washed three times PMSF, and 0.02 mM leupeptin. The extract was centrifuged at with 10 volumes of borate buffer, once with 0.2 M tri- 16,300 g for 10 min. The supernatent is referred to as the ethanolamine, pH 8.2, and then resuspended in 20 volumes Triton-soluble extract. The Triton-insoluble pellet was of freshly prepared 20 mM dimethyl pimelimidate dihydro- extracted with 0.4 M NaCl in SPMG on ice for 30 min and chloride (Sigma Chemical Co.) in 0.2 M triethanola- 170 G. M. Trimbur and C. J. Walsh mine with the pH readjusted to 8.2. The mixture was Fig. 1. The distribution of the BN5.1 antigen during the incubated at room temperature with gentle shaking for 1 h to differentiation of Naegleria amebae into flagellates. allow for cross-linking. The reaction was stopped by resus- Amebae were washed free of the bacteria that serve as a pending in an equal volume of 20 mM ethanolamine in 0.2 M food source with ice-cold 2 mM Tris-HCl, pH 7.6 (TRIS). triethanolamine, pH 8.2. After 5 min, the cross-linked BN5.1 Washed amebae were resuspended in 25°C TRIS to initiate Protein A Sepharose beads were washed three times with the differentiation, 0 min. The cell suspension was shaken borate buffer, pH 8.2, and stored in borate buffer with 0.05% on a reciprocating water bath at 25°C. Samples were fixed sodium azide. in a mixture of NP-40/formaldehyde (MgCF) and labelled Triton-soluble BN46/51-enriched fractions were passed with BN5.1 and a FITC-conjugated second antibody. The repeatedly over SPMG equilibrated BN5.1 affinity columns same cells were photographed under epifluorescence (0.5-1 ml) for at least 1 h. The columns were washed with 20 illumination for FITC (left column) and under phase- column volumes of SPMG and bound material was eluted contrast (right column). At 120 min after initiation an with four 0.9 ml samples of 0.1 M sodium carbonate, pH 11, additional sample was fixed and reacted with a mAb into a 1/10 volume of 1 M sodium phoshate, pH 4.3, to against a^tubulin (AA4.3; Walsh, 1984) to visualize the neutralize the pH. microtubule cytoskeleton (MTCS) of flagellates, insert A Triton-insoluble fraction from nucleoli at 4 X 108 between columns. The numbers in the left column indicate nucleoli/ml was solubilized in 0.4 M NaCl-SPMG for 30 min the time in minutes after the initiation of the on ice and centrifuged at 15,000g for 10 min. The supernatant differentiation. Note that at 75 min most cells have visible was passed over a BN5.1 affinity column equilibrated in 0.4 M flagella, and therefore basal bodies, yet there is no BN5.1 NaCl-SPMG as above, but the wash buffer was 0.4 M NaCl- binding in this region. The arrow at 120 min indicates SPMG. Elutions were carried out as above. BN5.1 binding in the basal body region. p-Phenylenediamine at 10 mg/ml was added to the Two-dimensional gel electrophoresis mounting medium to retard photobleaching. Bar, 10 urn. Two-dimensional gel electrophoresis was carried out essentially according to the method of O'Farrell (1975). For the first dimension, ampholytes (BioRad Laboratories) of pH ranges 4-6 and 6-8 were used at a ratio of 3:2. The second evaluate BN5.1 binding, with mixed results. No BN5.1 dimension was 7.5% SDS-PAGE. Gels were blotted to nitrocellulose and probed with BN5.1. Since it was difficult to binding to either nucleoli or the basal body region was solubilize completely the rootlet/basal body complexes in IEF observed when cells were fixed with formaldehyde sample buffer alone, all the samples in Fig. 8, right column, alone, followed by methanol and acetone extraction. were first solubilized in 2% SDS/10% /5-mercaptoethanol for Fixation with glutaraldehyde followed by NP-40 extrac- 15 min at room temperature, then enough Triton X-100 was tion resulted in high levels of background fluorescence added to make an 8:1 ratio of Triton to SDS along with IEF in the cell body, even after reduction of the glutaralde- sample buffer as described by Ames and Nikaido (1976). The hyde with NaBHt and blocking with bovine serum IEF gels had a pH gradient of 4.5 to 5.5 pH units, measured as albumin. The high background was not due to BN5.1, described by O'Farrell (1975). since it was observed in cells incubated with second antibody alone. Despite the high background, BN5.1 binding to nucleoli was apparent in glutaraldehyde- Results fixed cells but BN5.1 binding in the basal body region was not visible. When cells were fixed by suspension in BN5.1 binds to the nucleolus and the basal body ice-cold methanol, BN5.1 bound to the nucleolus, and region in flagellates weakly labeled the surrounding nucleoplasm, but The monoclonal antibody (mAb) BN5.1 was obtained binding to the basal body region was not observed in by immunizing mice with 46-48 kDa proteins from a flagellates. fraction enriched in basal bodies. When hybridomas Since the BN5.1 antigen was partially extracted with were screened by indirect immunofluorescence one non-ionic detergents (see Fig. 4C, below), it was mAb, BN5.1, bound to the basal body region in possible that the presence of the BN5.1 antigen in the flagellates (Fig. 1, 120, 105 and 90 min). BN5.1 also basal body region was due to binding of the antigen bound the nucleolus of both amebae (Fig. 1, 0 min) and released from the nucleolus during fixation in NP- flagellates (Fig. 1,120,105, 90 and 75 min). Cloning and 40/formaldehyde. This possibility was evaluated by recloning the hybridoma line did not separate the fixing a constant number of flagellates in increasing nucleolar binding from basal body region binding. volumes of NP-40/formaldehyde, with rapid mixing. The basal body region fluorescence was not as intense Cells were then concentrated by centrifugation, resus- as nucleolar fluorescence and was sometimes "U"- pended in a small volume of fixative, and processed for shaped or "dot-like" (Fig. 1, 120, 105 and 90 min). indirect immunofluorescence with BN5.1. When flagel- Controls without first antibody showed no fluorescence lates were fixed in volumes up to 50 times greater than (data not shown). The cells used for screening hybrid- standard, there was no decrease of BN5.1 antigen in the oma lines were fixed in MgCF, a mixture of Nonidet- basal body region as judged by indirect immunofluor- P40 (NP-40) and formaldehyde. This technique, which escence (data not shown). Thus either the BN5.1 simultaneously permeabilizes and fixes the cells, was antigen is associated with the basal body region in vivo developed to optimize visualization of the microtubule but is not accessible to the antibody when cells are fixed cytoskeleton (MTCS) in flagellates (Walsh, 1984). in the absence of non-ionic detergent, or the BN5.1 A variety of other fixation techniques were used to antigen must bind during the brief interval after the BN46/51, a new nucleolar protein 171

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cells are permeabilized but before the antigen has been the basal body region was determined by following the diluted. differentiation of amebae to flagellates. Cells were fixed The time of appearance of the the BN5.1 antigen in with NP-40/formaldehyde at intervals during the differ- 172 G. M. Trimbur and C. J. Walsh entiation, and the presence of the BN5.1 antigen was 100 -i visualized by indirect immunofiuorescence. Cells were scored for BN5.1 binding in the basal body region and for the presence of visible flagella (Fig. 2A). The BN5.1 80 - antigen was not detectable at the time when basal bodies form, about 10 min before the flagella emerge (Dingle, 1977; Fulton and Dingle, 1967). BN5.1 antigen was not detected until well after visible flagella were 60 - present in the cells. Because of the synchrony and reproducibility of the differentiation, events are usually described in terms of 40 - their T^, the time when 50% of the population has aquired a given character. The 7^ for the formation of visible flagella is 68 ± 2 min (Dingle, 1977; Dingle and 20 - Fulton, 1966). The BN5.1 antigen appeared with a T^ of 85 min, 17 min after visible flagella are present and approximately 25 min after basal bodies are assembled 0-9 (Dingle, 1977). This is evident in Fig. 1, where cells 20 40 60 80 100 120 fixed at 75 min show visible flagella but no BN5.1 Time after initiation (min) antigen in the basal body region, while BN5.1 binds to the basal body region in cells fixed at 90 min. IUU - B The appearance of the BN5.1 antigen in the basal body region coincides with the assembly of the MTCS in flagellates (Walsh, 1984). As cells proceed from amebae 80 - to flagellates they round up at about the time basal bodies form. Flagella then emerge from these roughly 1 spherical cells. As the flagella elongate a bundle of 60 - r k \ cytoplasmic microtubules grows out from the basal — body region and eventually extends throughout the cell CJ U— i vA (Fig. 1, inset). The assembly of the MTCS coincides O with a change from the spherical shape to a flattened #40 - oval, the flagellate shape. The T^ for the formation of the MTCS, i.e. microtubules extending throughout the cell body, is 87 min, coincident with the appearance of 20 - \\ the BN5.1 antigen in the basal body region. The extent of the correlation between the appearance of BN5.1 antigen at the basal body region and the MTCS was examined by following amebae through the 0 30 60 90 120 150 180 210 240 differentiation into flagellates and reversion back to Time after initiation (min) amebae (Fig. 2B). Flagellates are unstable and spon- Fig. 2. The appearance of flagella, the BN5.1 antigen in taneously revert back to amebae (Dingle, 1970). As the basal body region, and the MTCS during the flagellates revert, the MTCS is disassembled and differentiation of amebae into flagellates. (A) Amebae flagella are absorbed or lost. It is evident that the were washed and differentiated as described for Fig. 1. presence of the BN5.1 antigen in the basal body region Cells were fixed in Lugol's iodine to visualize flagella. The is closely coordinated with the assembly and disassem- percentage of cells with visible flagella was determined by bly of the MTCS. counting 100 cells under phase-contrast (open squares). Cells were also fixed in MgCF and incubated with BN5.1 In nuclei the BN5.1 antigen is confined to the or with AA4.3 as described for Fig. 1. Cells were scored nucleolus. Naegleria nuclei contain one large central for the presence of the BN5.1 antigen in the basal body nucleolus (Fulton, 1970; Schuster, 1975) surrounded by region (open circles) and for the presence of a complete a thin layer of nucleoplasm. Indirect immunofluor- MTCS (filled diamonds). The averaged results of three escence with BN5.1 and DAPI to visualize DNA independent experiments are presented for flagella, BN5.1 demonstrated that the BN5.1 antigen is localized to the binding and MTCS formation during the differentiation. nucleolus (data not shown). The BN5.1 antigen was The bars represent the standard deviation of the mean. (B) The results of a similar experiment in which flagellates restricted to a central ring or circle, depending on the were followed during the spontaneous reversion back to plane of focus, that coincided with the phase-dense amebae. nucleolus. The DAPI staining was concentrated in the thin outer ring of nucleoplasm. The nucleolar localization was more evident when immuno-electron mi- the dense fibrillar and granular components of the croscopy was used to visualize the antigen (Fig. 3). The nucleolus but was not found in the nucleoplasm. BN5.1 antigen was found widely distributed throughout When flagellates were examined by electron mi- BN46/51, a new nudeolar protein 173

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Fig. 3. BN5.1 binding to thin sections of amebae. Amebae were fixed in glutaraldehyde and embedded in LR White. Sections were labelled with BN5.1 and a second antibody conjugated to 10 nm colloidal gold. (A) BN5.1. (B) No first antibody control. Bar, 0.5 /an. croscopy, the nucleolar labelling was similar to that in while extraction with 0.4 M NaCl solubilized almost all Fig. 3 but no labelling was seen around the basal bodies. of the Triton-insoluble antigen (Fig. 4C, lanes 7 and 8). This may simply reflect the small amount of antigen in In an attempt to determine if the 46 kDa polypeptide this region. When NP-40/formaldehyde-fixed cells were arose from proteolytic degradation of the 51 kDa prepared for electron microscopy, no labelling was polypeptide during extraction, whole cells were mixed visible in the basal body region even though these cells directly into sample buffer and immediately heated in label by immunofluorescence (Fig. 1). boiling water. This was expected to minimize proteolysis but it did not change the approximate 1:1 ratio of the 46 and 51 kDa components. All extractions included Characterization of the BN5.1 antigen a mixture of protease inhibitors, which should have When immunoblots of total cell protein were probed minimized degradation. The fact that both the 46 and 51 with BN5.1 peptides of 46 and 51 kDa were identified kDa polypeptides are found in approximate equal (Fig. 4A,B). Thus the BN5.1 antigen has been amounts in all fractions examined, both soluble and designated as BN46/51. As judged by antibody binding, insoluble, and in nucleolar and basal body fractions (see the 46 and 51 kDa bands were present in approximately below) also suggests that the 46 kDa polypeptide is not equal amounts. Subsequent work (see below) demon- a result of proteolysis of the 51 kDa polypeptide during strated that the antigen is composed of a complex of fractionation of the cells. these two polypeptides. Triton X-100 extraction of In flagellates, the Triton-insoluble BN46/51 is associ- amebae solubilized about 75% of BN46/51 (Fig. 4C, ated with nucleoli and with the basal body region. The lane 1). Incubation of the Triton-insoluble fraction with fixation technique used to identify BN5.1 includes NP- 100 jig/ml of DNase I and 100 jug/ml of RNase A for 30 40, a non-ionic detergent similar to Triton X-100, min at 25°C did not solubilize additional BN46/51 (data suggesting that some of both the nucleolar and basal not shown). The Triton-insoluble material was body region-associated BN46/51 is Triton-insoluble. extracted with varying concentrations of NaCl for 30 This was confirmed by extracting cells with Triton- min at 0°C. Extraction with SPMG buffer alone or with containing buffer in the absence of formaldehyde, 0.2 M NaCl solubilized variable amounts of BN46/51, followed by formaldehyde fixation. These Triton- 174 G. M. Trimbur and C. J. Walsh B

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M 1 2 3 4 5 6 7 8 9 10 11 12 Fig. 4. Characterization of the BN5.1 antigen in amebae. (A) Amebae were solubilized in SDS sample buffer at 2 x 107 cells/ml and fractionated by SDS-PAGE. Total protein was visualized by Coomassie blue staining. Lane M, molecular mass markers, values in kDa. (B) Corresponding immunoblot incubated with BN5.1 followed by incubation with a horseradish peroxidase-conjugated second antibody. BN5.1 bound to two polypeptides of 46 kDa and 51 kDa. (C) Amebae were extracted with 0.5% Triton X-100 in SPMG. The Triton-insoluble material was collected by centrifugation and subjected to extraction with various concentrations of NaCl in SPMG followed by centrifugation. The supernatents and pellets were fractionated by SDS-PAGE and the BN5.1 antigen was visualized by immunoblotting with BN5.1. Lanes 1 and 2, the Triton-soluble and -insoluble fraction. Lanes 3 and 4, supernatent and pellet after extraction in SPMG alone. Lanes 5-12, the supernatents and pellets after extraction of the Triton-insoluble fraction with various concentrations of NaCl in SPMG. Lanes 5 and 6, supernatant and pellet in 200 mM NaCl-SPMG. Lanes 7 and 8, supernatent and pellet in 400 mM NaCl- SPMG. Lanes 9 and 10, supernatent and pellet in 600 mM NaCl-SPMG. Lanes 11 and 12, supernatant and pellet in 1 M NaCl-SPMG. Fractions in lanes 3 through 12 were adjusted to contain the same number of cell equivalents. Fractions in lanes 1 and 2 contained one-half the cell equivalents in the other lanes. Virtually all of the Triton-insoluble antigen was solubilized by 400 mM NaCl, lanes 7 and 8. extracted cells contained both nucleolar- and basal the presence of the flagellar rootlet. Basal bodies are body-associated BN46/51. tightly associated with the wider end of the long The NaCl solubility of the Triton-insoluble nucleolar- tapering rootlet. Typically, two basal bodies are and basal body-associated BN46/51 was examined. attached to the rootlet along with a palisade of Nucleoli were separated from basal bodies by differen- microtubules (Schuster, 1963; Dingle and Fulton, 1977; tial centrifugation through glycerol after Triton extrac- Larson and Dingle, 1981a). Inter-basal body linkers are tion. When nucleoli were extracted with 0.4 M NaCl, also thought to hold the rootlet/basal body complex about 80% of BN46/51 was solubilized as judged by together (Dingle and Fulton, 1977; Larson and Dingle, immunoblotting (Fig. 5). In contrast, when basal body 1981a). The basal bodies, the palisade of microtubules, complexes were extracted with 0.4 M NaCl, only about and the interbasal body linkers are all located in a knob- 20% of the BN46/51 was solubilized (Fig. 5). This was like structure visible at the end of the rootlet (Fig. 61). not due to excess protein in the basal body fraction, as When rootlet/basal body complexes were examined up to 8-fold serial dilutions of basal body complexes in by indirect immunofluorescence using BN5.1, numer- NaCl gave the same results (data not shown). ous heterogeneous granular elements were seen (Fig. The distribution of BN46/51 in isolated nucleoli and 6C). In order to determine which granules contained basal body fractions was confirmed by indirect immuno- basal bodies, fractions were also labelled with a mouse fluorescence. After incubation in 0.4 M NaCl, nucleoli polyclonal anti-rootlet antibody. This visualized only were no longer visible by phase-contrast microscopy. the rootlets and not the knob-like structures at the thick Indirect immunofluorescence with BN5.1 showed only end (Fig. 6E). Rootlet/basal body fractions incubated some small clumps of BN46/51, but no nucleolar with both BN5.1 and the anti-rootlet antibody showed structures (Fig. 6B). staining of the rootlets and the knob-like structures at Visualization of basal bodies, isolated from cells in the thicker end of virtually every rootlet (Fig. 6G). The which the fiagella had been removed, was facilitated by granules that bind BN5.1 and are not associated with BN46/51, a new nucleolar protein 175

Rootlet Although there was some heterogeneity in this set of Nucleoli Basal Bodies samples (minor spots are evident with some tailing towards the basic end) the pi of the major 46 kDa spot was 5.0, and the pi of the major 51 kDa spot was 4.9. There were no significant differences in pi between the 46 kDa and 51 kDa polypeptides of nucleoli (NaCl- soluble) and rootlet/basal body (NaCl-insoluble) fractions.

BN46/51 is a complex with an average Mr of 400-500 X 10? Although BN46/51 could be fractionated into Triton- soluble, NaCI-soluble (nucleoli) and NaCl-insoluble (rootlet/basal body) fractions, approximately equal amounts of the 46 and the 51 kDa polypeptides were found in each case. This suggested that the polypeptides may exist as a complex. Glutaraldehyde cross-linking was used to determine the extent of interaction in solution. BN46/51 from a Triton-soluble extract of amebae was partially purified by DEAE column chromatography, this will be referred to as a Triton- soluble BN46/51-enriched fraction. Proteins in this T S P S P fraction were cross-linked with glutaraldehyde. Cross- Fig. 5. Comparison of NaCI solubility of BN46/51 in linking was stopped at intervals, samples were fraction- nucleoli and in flagellar rootlet/basal body complexes. ated by SDS-PAGE and analyzed by immunoblotting Nucleoli and flagellar rootlet/basal body complexes were with BN5.1. As shown in Fig. 8, the two BN46/51 isolated from flagellates and extracted with 0.4 M NaCl- polypeptides were rapidly cross-linked. Intermediates SPMG for 30 min on ice. Extracts were centrifuged, and of about 100 kDa and 150 kDa appeared first. At later supernatents and pellets were adjusted to equivalent times a complex of greater than 200 kDa was formed volumes. Proteins were fractionated by SDS-PAGE and with some material remaining in the stacking gel. analyzed by immunoblotting with BN5.1. The rootlet/basal body fraction contained 5-fold greater cell equivalents/ml Controls with ovalbumin at the same protein concen- than the nucleolar fraction. T, total protein; S, 0.4 M tration showed no cross-linking (data not shown). NaCI-soluble (supernatent); P, 0.4 M NaCl-insoluble Gel filtration chromatography was used to determine (pellet). the MT of the solubilized BN46/51 complex. Proteins from a Triton-soluble BN46/51-enriched fraction were run on a Sephacryl 300 column. Fractions were flagellar rootlets are thought to represent the remnants collected, separated by SDS-PAGE and analyzed by of nucleoli. immunoblotting with BN5.1 (Fig. 9A). Approximately In contrast to nucleoli, the BN46/51 in the basal body equal amounts of the 46 and 51 kDa polypeptides co- complexes was resistant to extraction with 0.4 M NaCI, eluted as a broad peak centered at 400-500 kDa. A as expected from the immunoblottting results. When Triton-insoluble fraction, solubilized with 0.4 M NaCI rootlet/basal body complexes were extracted with 0.4 M was also characterized by gel filtration (Fig. 9 B). Equal NaCI, labelling with BN5.1 and the anti-rootlet anti- amounts of the 46 and 51 kDa polypeptides co-eluted at body showed that all remaining BN46/51-containing a similar position. These results suggest that approxi- granules were associated with the rootlet (Fig. 6H). The mately equal amounts of 46 and 51 kDa polypeptides loss of the non-basal body associated BN46/51 after exist in a heterogeneous complex with an average size NaCI extraction, is consistent with the suggestion that of 400-500 kDa. these are nucleolar fragments. Sucrose gradient sedimentation was also used to BN46/51 was examined by 2-D gel electrophoresis characterize the nature of the BN46/51 complex. A (Ames and Nikaido, 1976; O'Farrell, 1975) followed by NaCl-solubilized nucleolar extract was sedimented immunoblotting with BN5.1. The Triton-soluble and through a 5% to 30% sucrose gradient. Fractions were -insoluble fractions were run separately, and as a 1:1 collected and screened by ELISA assay and SDS- mixture (Fig. 7). The 46 kDa polypeptide focused as PAGE. BN46/51 sedimented as a broad peak centered one spot with a pi of 5.0, while the 51 kDa polypeptide at 5.5 S (data not shown), which would correspond to a focused as one spot with a pi of 4.9. The 46 and 51 kDa globular protein of about 100 kDa (Edsall, 1953). polypeptides from the Triton-insoluble fraction had the Combined with the gel filtration and cross-linking data, same pi as those from the Triton-soluble fraction. The this suggests that BN46/51 is a moderately asymmetric Triton-insoluble fraction was divided into a 0.4 M NaCl- molecule. soluble fraction (nucleoli), and a 0.4 M NaCl-insoluble Because the glutaraldehyde cross-linking and gel fraction (rootlet/basal body complexes). These frac- filtration were done with a complex mixture of proteins, tions were run separately and as a 1:1 mixture (Fig. 7). it was not clear if the 46 and 51 kDa polypeptides were 176 G. M. Trimbur and C. J. Walsh Nucleoli Rootlet/Basal Body Complexes BN5.1 BN5.1 Anti-Rootlet Double

Fig. 6. The NaCl-resistant BN46/51 in rootlel/basal body fractions is concentrated around the basal bodies. Nucleoli and rootlet basal body complexes were isolated and incubated in SPMG with (+), or without (—) 0.4 M NaCl for 30 min on ice. Insoluble material was collected by centrifugation, fixed with MgCF and prepared for indirect v : immunofiuorescence. (A and B) Nucleoli labeled with BN5.1; (C through H) rootlet/basal body complexes; (C and D) labelled with BN5.1; (E and F) labelled with a polyclonal mouse antibody against the rootlet protein; (G and H) double labelled with BN5.1 and the anti-rootlet antibody. Arrows in G and H indicate the knob-like structure labelled by BN5.1. t (I) the composition of a rootlet/basal body fraction as viewed by phase- i — contrast. (A through H) are all at the same magnification. Bar, 10 fim. (I) at a slightly lower magnification, bar, 10 ^m. complexed with other proteins. To resolve this ques- Discussion tion, BN46/51 was purified using a BN5.1 affinity column. A Triton-soluble BN46/51-enriched fraction Our results demonstrate that amebae of Naegleria was passed over the column and the bound material was gruberi contain a novel nucleolar protein, BN46/51. eluted. These fractions contained only the 46 and 51 When amebae differentiate into flagellates, BN46/51 is kDa polypeptides as judged by silver staining (Fig. also found in the basal body region. BN46/51 is a 10A). Immunoblots with BN5.1 confirmed that the multimer of 46 and 51 kDa subunits, both of which polypeptides eluted from the column were recognized share the epitope recognized by mAb BN5.1. The 46 by BN5.1 (data not shown). A 0.4 M NaCl extract of and 51 kDa subunits were present in approximately nucleoli was also passed over the affinity column. Again equal amounts in all cellular fractions examined and the bound material contained only the 46 and 51 kDa under a variety of extraction conditions. This, com- polypeptides (Fig. 10B). Immunoblots incubated with bined with the fact that the ratio of the subunits was not BN5.1 confirmed that the polypeptides eluted from the altered by a variety of treatments that should minimize affinity columns were recognized by BN5.1 (data not proteolysis, suggests that the 46 kDa subunit does not shown). Thus, as judged by binding to a BN5.1 affinity result from partial degradation of the 51 kDa subunit column, only the 46 kDa and 51 kDa subunits are during cell fractionation. However, the 46 and 51 kDa present in the complex. subunits are closely related as demonstrated by the BN46/51, a new nucleolar protein 111 Triton 0.4 M NaCI Soluble vs. Insoluble Soluble vs. Insoluble IEF

Soluble i r

Insoluble

Mixed

Fig. 7. Characterization of BN46/51 by two-dimensional gel electrophoresis. The left column is a comparison of the Triton- soluble and -insoluble fractions. The Triton-soluble and -insoluble fractions were electrophoresed separately, and mixed at a 1:1 ratio and electrophoresed together. Proteins were separated by iso-electric focusing in the first dimension followed by SDS-PAGE in the second dimension. Gels were analyzed by immunoblotting with BN5.1. The right column is a comparison of 0.4 M NaCI-soluble (nucleoli), and 0.4 M NaCI-insoluble (rootle0Dasal body complexes). The IEF gels span one pH unit from about pH 4.5 to pH 5.5 as determined by the method of O'Farrell (1975). The 46 and 51 kDa subunits have pi values of 5.0 and 4.9, respectively. The lane to the far right of each gel is a one-dimensional fractionation of the sample showing the 46 and 51 kDa subunits. observation that they share at least three other mAb (unpublished observations). Two of these additional epitopes in addition to that for BN5.1. These epitopes mAbs show nucleolar and basal body binding similar to were identified by a series of mAbs generated against BN5.1 while the third binds only to the nucleolus. BN46/51 purified on a BN5.1 affinity column. These BN46/51 does not appear to be any of the previously new mAbs are currently being characterized in detail described nucleolar proteins. RNA polymerase I but it is already clear that there are at least three more (Scheer and Rose, 1984), fibrillarin (Ochs et al., 1985; distinct epitopes shared by the two BN46/51 subunits Scheer and Benavente, 1990), nucleolin (Lapeyre et al., 178 G. M. Trimbur and C. J. Walsh &••• 1 ft—

M 1 23456789 10

1 2 3 4 5 6 7 Fig. 8. Glutaraldehyde cross-linking of BN4fy51. A fraction enriched in BN46/51 by DEAE chromatography of Triton- soluble material was cross-linked with 0.08% glutaraldehyde. The cross-linking was stopped with a 25- fold molar excess of glycine. Proteins were fractionated by SDS-PAGE, and analyzed by immunoblotting with BN5.1. Lane 1, starting sample, before addition of glutaraldehyde. Lanes 2, 3, 4, 5, 6 and 7 are at 0, 1, 2, 5, 10 and 15 minutes after the addition of glutaraldehyde, respectively. 43- The * denotes intermediates of about 100 and 150 kDa in lanes 3 and 4. 29-

670 443 200 66 kD I I 1 M1 23456789 10 Fig. 10. (A) Affinity purification of BN46/51 from a Triton soluble fraction enriched in BN46/51 by DEAE chromatography; and (B) a Triton-insoluble fraction from nucleoli solubilized in 0.4 M NaCl-SPMG. Proteins were • fractionated by SDS-PAGE and visualized by silver staining (Morrissey, 1981). Lane 1, starting material; lane B 2, flow through; lanes 3, 4, 5 and 6, column washes with SPMG for (A) and 0.4 M NaCl-SPMG for (B); lanes 7, 8, 9 and 10, elutions with 0.1 M sodium carbonate, pH 11.0. 11 12 13 14 15 16 17 18 19 Lane M indicates molecular mass standards in kDa.

Fig. 9. Fractionation of BN4€/51 by gel filtration chromatography. (A) A fraction enriched in BN46/51 by are 5.0 and 4.9, respectively. Nucleolin differs substan- DEAE chromatography of Triton-soluble material; and (B) tially in molecular mass, ranging from 90 to 100 kDa a Triton-insoluble fraction solubilized with 0.4 M NaCl, and has only been found in higher eukaryotic cells. B23 were fractionated on a Sephacryl 300 column. Fractions (Numatrin) and the 145 kDa protein are not extracted were collected, separated by SDS-PAGE and analyzed by in up to 2 M NaCl, which has led to the suggestion that immunoblotting with BN5.1. The numbers below indicate the fraction number while the numbers above indicate the they are nuclear matrix proteins (Feuerstein et al., elution position of molecular mass standards in kDa. 1988; Krohne et al., 1982). Nucleolar BN46/51 on the other hand is soluble in 0.4 M NaCl. We have been unable to demonstrate any cross- 1987; Warner, 1990), pl80, which is exclusively local- reactivity of BN5.1 with nucleoli of Euglena, Dictyo- ized to the DFC (Schmidt et al., 1984), a 145 kDa stelium, yeast or mammalian cells, by either indirect polypeptide (Krohne et al., 1982) and B23 (Numatrin) immunofluorescence or immunoblotting. This is not (Feuerstein et al., 1988) are all resistant to non-ionic due to to the uniqueness of the BN5.1 epitope as none detergent extraction. In contrast 75% of BN46/51 is of the other three mAbs that recognize separate soluble in 0.5% Triton X-100. Fibrillarin, a 34 kDa BN46/51 epitopes show any cross-reactivity with yeast protein in mammalian cells, has a basic pi of 8.5 (Ochs or mammalian nucleoli. Further work will be needed to et al., 1985), while the pi values of the BN4^/51 subunits determine if this simply represents the selection of BN46/51, a new nucleolar protein 179 unique epitopes by the immune system or if BN46/51 inaccessible to the antibody. When cells were fixed in represents a unique protein not present in most other glutaraldehyde followed by extraction with NP-40, only organisms. nucleoli were seen by immunofluorescence with BN5.1. The association of BN46/51 with the basal body However, due to the high levels of non-specific region in flagellates is interesting and puzzling. There is background fluorescence throughout the cell body, no reason to expect a nucleolar protein to be associated antibody binding to the basal body region may have with the basal bodies or their surrounding structures. been obscured. Nucleoli are involved in the transcription and process- It is possible that BN46/51 is released from the ing of rRNA and the assembly of ribosomal subunits nucleolus and binds to the basal body region during (Scheer and Benavente, 1990; Warner, 1990), while fixation in the presence of detergent. If the basal body basal bodies are templates for axoneme assembly and region association were the result of solubilization of act as microtubule organizing centers (MTOCs) (Brink- BN46/51, then fixation in larger volumes might be ley, 1985; Dingle and Fulton, 1966; Osborn and Weber, expected to reduce the concentration of the antigen 1976). If the presence of BN46/51 in the basal body available to bind to the basal body complex. However, region is a reflection of its affinity for MTOCs, then it is even a 50-fold increase in the volume of the fixative not surprising that it is found only in the nucleolus in failed to reduce the binding of BN5.1. Nonetheless, it is amebae (Fig. 1), since amebae lack centrioles and basal still possible that even at a 50-fold dilution the BN46/51 bodies, even in mitotic cells (Fulton and Dingle, 1971; concentration in the rapidly lysed cells was transiently Schuster, 1975). Furthermore, amebae lack micro- high enough to produce binding to the basal body tubules except in the mitotic spindle, which is intranu- region. A definitive answer to this question will only be clear (Walsh, 1984; Schuster, 1975; Fulton, 1970). possible when we determine why BN5.1 binding to the In Naegleria the basal bodies are assembled de novo basal body complex is not detectable in cells fixed with during the differentiation of amebae to flagellates glutaraldehyde in the absence of non-ionic detergent. (Fulton and Dingle, 1971). The appearance of BN46/51 At present this seems to be a reflection of the limited in the basal body region was initially expected to be amount of antigen. An ultrastructural and biochemical coordinated with basal body assembly. However, investigation of this question is being undertaken. BN46/51 was not found in the basal body region until 85 Regardless of the ultimate resolution of this question, min after initiation of the differentiation, approxi- the presence of BN46/51 in the basal body region mately 25 min after basal bodies form (Dingle, 1977). defines an interesting and potentially instructive tran- Thus BN46/51 is not detected when basal bodies are sition during the development of the flagellar apparatus assembled or when they function as templates for and MTCS. The binding of BN46/51 to the basal body axonemes. Therefore it seems more likely that BN46/51 complex is resistant to extraction with up to 2 M NaCl, is associated with the structures surrounding the basal and yet it is rapidly acquired and lost coincident with bodies, the palisade of tightly bound microtubules, the the polymerization and depolymerization of the micro- flagellar rootlet, or perhaps the inter-basal body linkers tubules that make up the cytoskeleton of flagellates. An (Schuster, 1963). understanding of the biochemical basis for this behavior BN46/51 might be binding to the cytoplasmic micro- may provide some insight into the regulation of MTCS tubules. This possibility is supported by the observation assembly. that BN46/51 can bind to purified rat brain tubulin BN46/51 exists in three solubility states. About 75% bound to nitrocellulose (unpublished observation). The of the BN46/51 is solubilized by 0.5% Triton X-100 recent report that isoforms of the microtubule-associ- (Fig. 4). The detergent-insoluble BN46/51 can be ated protein Tau are localized to the FC of nucleoli further fractionated into a 0.4 M NaCl-soluble and (Loomis et al., 1990) suggests that there may be an -insoluble fraction. The 0.4 M NaCl-soluble fraction is undefined connection between nucleolar proteins and associated with nucleoli, while the 0.4 M NaCl- microtubules. insoluble fraction is associated with the basal body It is also possible that the appearance of BN46/51 in region (Figs 5 and 6). These differences do not appear the basal body region coincident with the assembly of to be due to differences in BN46/51. No differences in the MTCS (Fig. 2) reflects an association with a non- the isoelectric points or Mr of either subunit in the tubulin component of the basal body complex. Unfortu- Triton-soluble or the Triton-insoluble fraction, or in the nately very little is known about the composition and 0.4 M NaCl-soluble and -insoluble fractions were found organization of this complex or about the timing of its by 2-D gel electrophoresis (Fig. 7). Although, the assembly. The appearance of the BN5.1 antigen may possibility that there are multiple modifications which provide a marker for an investigation of the events do not change the overall isoelectric points cannot be surrounding assembly of the MTCS. ruled out. It seems more likely that the differences in BN46/51 is not seen in the basal body region when solubility reflect differences in BN46/51 binding com- flagellates are fixed without the use of non-ionic ponents. BN46/51 binds specifically to two nucleolar detergent. This may be due to inaccessibility or masking proteins, fibrillarin and a previously unidentified com- of the antigen in the basal body region. Formaldehyde ponent, in 0.15 M NaCl but not in 0.4 M NaCl (Trimbur fixation followed by methanol and acetone extraction and Walsh, unpublished data). By this rationale, the resulted in no binding of BN5.1 to either the nucleolus Triton-soluble protein might reflect the solubilization of or the basal body region, suggesting thatBN46/51 was BN46/51, interacting with itself, while the Triton- 180 C. M. Trimbur and C. J. Walsh insoluble and NaCl-insoluble fractions would represent Dingle, A. D. (1970). Control of flagellum number in Naegleria. interactions with non-BN46/51 components. Temperature shock induction of multiflagellate cells. J. Cell Sci. 7, 463-481. Cross-linking, gel filtration chromatography and Dingle, A. (1977). Cell differentiation in Naegleria. In Eukaryotic BN5.1 affinity chromatography suggest that BN46/51 is Microbes as Model Developmental Systems (ed. D. H. O'Day and a multisubunit complex composed of approximately P. A. Horgen), pp. 549-640. New York: Marcel Dekker. equal amounts of the 46 and 51 kDa subunits. The fact Dingle, A. D. and Fulton, C. (1966). Development of the flagellar that the 46 kDa and 51 kDa subunits are lost with about apparatus of Naegleria. J. Cell Biol. 31, 43-54. Edsall, J. T. (1953). The size, shape and hydration of protein the same kinetics during glutaraldehyde cross-linking molecules. In The Proteins. Chemistry, Biological activity, and suggests that the two subunits interact directly (Fig. 8). Methods, vol. I, part B (ed. H. Neurath and K. Bailey), pp. 634- However, these data do not rule out the simultaneous 639. New York: Academic Press Inc. presence of two kinds of multisubunit complexes Feuerstein, N., Chan, P. K. and Mond, J. J. (1988). Identification of composed of only one subunit in each complex. Gel Numatrin, the nuclear matrix protein associated with induction of mitogenesis, as the nucleolar protein B23. J. Biol. Cliem. 263, filtration chromatography demonstrated that the solu- 10,608-10,612. bilized complex was heterogeneous with an average size Fulton, C. (1970). Amebo-flagellates as research partners: The of 400 to 500 kDa whether prepared from a Triton- laboratory biology of Naegleria and Tetramitus. Methods Cell soluble or a 0.4 M NaCl-solubilized Triton-insoluble Physiol. 4, 341-476. fraction (Fig. 9). On the other hand, BN46/51 sedi- Fulton, C. (1977). Cell differentiation in Naegleria gruberi. Annu. Rev. Microbiol. 31, 597-629. ments through sucrose gradients with a peak value of Fulton, C. and Dingle, A. D. (1967). Appearance of the flagellate 5.5 S, which corresponds to a globular protein of about phenotype in populations of Naegleria amebae. Develop. Biol. 15, 100 kDa (Edsall, 1953). Combined, these data suggest 165-191. that solublized BN46/51 is a somewhat asymmetric Fulton, C. and Dingle, A. D. (1971). Basal bodies, but not centrioles, molecule composed of an average of four subunits. in Naegleria. J. Cell Biol. 31, 43-54. Goessens, G. (1984). Nucleolar structure. Int. Rev. Cytol. 87,107-158. Affinity chromatography of solubilized BN46/51 from Hawkes, R., Niday, E. and Gordan, J. (1982). A dot-immunobinding either a Triton-soluble or -insoluble fraction isolated assay for monoclonal and other antibodies. Anal. Biochem. 119, only the 46 and 51 kDa subunits, suggesting that 142-147. BN46/51 is not tightly associated with other components Hoogenraan, N. J. and Wraight, C. J. (1986). The effect of pristane after solubilization (Fig. 10). on ascites tumor formation and monoclonal antibody production. Methods Enzymol. 121, 375-381. The nucleolar localization of BN46/51 to the DFC HUgle, B., Hazen, R., Scheer, U. and Franke, W. W. (1985). and GC (Fig. 3) suggests that it is involved in the Localization of ribosomal protein SI in the granular component of processing of rRNA or the assembly and transport of the interphase nucleolus and its distribution during mitosis. J. Cell Biol. 100, 873-886. ribosomal subunits. An investigation of the association Hurrell, J. G. R. (1982). In Monoclonal Hybridoma Antibodies: of BN46/51 with other nucleolar proteins is under way. Techniques and Applications (ed. J. G. R. Hurrell). pp 33-35. Boca Localization of BN46/51 with the basal body region Raton, Florida: CRC Press, Inc. suggests a novel and unexpected function for a Jordan, E. G. (1984). Nucleolar nomenclature. J. Cell Sci. 67, 217- nucleolar protein. Current experiments are directed at 220. Jordan, E. G. (1991). Interpreting nucleolar structure: Where are the defining the biochemical basis for BN46/51 binding to transcribing genes? J. Cell Sci. 98, 437-442. the basal body region. This information and the cloning Jordan, E. G. and Rawlins, D. J. (1990). Three-dimensional and sequencing of the gene(s) for the subunits are localization of DNA in the nucleolus of Spirogyra by correlated expected to provide the basis for a clearer understand- optical tomogTaphy and serial ultrathin sectioning. J. Cell Sci. 95, ing of the role of this unusual protein in the cell. 343-352. Kass, S., Tyc, K., Steitz, J. A. and Sollner-Webb, B. (1990). The U3 small nucleolar ribonucleoprotein functions in the first step of We thank Susan Hunt and Ya Li Deng for expert technical preribosomal RNA processing. Cell 60, 897-908. assistance. This work was supported by a grant from the Knihiehler, B., Navarro, A., Mirre, C. and Stahl, A. (1977). National Science Foundation DCB: 87-18335. G. M. T. was Localization of ribosomal cistrons in the quail oocyte during partially supported by a fellowship from the University of meiotic prophase I. Exp. Cell Res. 110, 153-157. Pittsburgh Center for Biotechnology and Bioengineering. Kowit, J. D. and Fulton, C. (1974). Purification and properties of flagellar outer doublet tubulin from Naegleria gruberi and a radioimmune assay for tubulin. J. Biol. Chem. 249, 3638-3646. References Krohne, G., Stick, R., Kleinschmidt, J. A., Moll, R., Franke, W. W. and Hausen, P. (1982). Immunological localization of a major Ames, G. F. L. and Nikaido, K. (1976). Two-dimensional gel karyoskeletal protein in nucleoli of oocytes and somatic cells of electrophoresis of membrane proteins. Biochemistry 15, 616-623. Xenopus laevis. J. Cell Biol. 94, 749-754. Brinklcy, B. R. (1985). Microtubule organizing centers. Annu. Rev. Lacmmll, U. K. (1970). Cleavage of structural proteins during the Cell Biol. 1, 145-172. assembly of the head of bacteriophage T4. Nature 227, 680-685. Clark, C. G. and Cross, G. A. M. (1987). rRNA genes of Naegleria Lapeyre, B., Bourbon, H. and Amalric, F. (1987). 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