Journal of Cell Science 107, 9-16 (1994) 9 Printed in Great Britain © The Company of Biologists Limited 1994

Cloning of a cDNA encoding human centrin, an EF-hand protein of and mitotic spindle poles

Ramesh Errabolu, Mark A. Sanders and Jeffrey L. Salisbury* Laboratory for Cell Biology, Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, Minnesota 55905, USA *Author for correspondence

SUMMARY

A human cDNA expression library was screened using human centrin is a protein of ~20,000 Mr as predicted from anti-centrin antibodies to obtain a cDNA clone encoding the cDNA clone. Indirect immunofluorescence analysis of human centrin. The cDNA clone contains an open reading HeLa cells demonstrates that centrin is localized at the cen- frame of 516 base pairs and predicts a product of 172 trosome of interphase cells and that it redistributes to the amino acids with a calculated molecular mass of 19,528 and region of the spindle poles during mitosis. When taken a pI of 4.61. Sequence analysis demonstrates that human together with earlier genetic studies, these results demon- centrin and centrins from higher plants, protozoa, algae, strate that centrin is a ubiquitous component of centro- Xenopus and the yeast CDC31 gene product are closely somes and mitotic spindle poles of diverse organisms and related members of a subfamily of the EF-hand superfam- suggest that centrin plays a role in separation ily of calcium-binding proteins. The human centrin at the time of mitosis. sequence has four putative calcium-binding domains as defined by the EF-hand consensus. Immunoprecipitation Key words: centrosome, , centrin, EF-hand protein, and western blot studies from HeLa cells confirm that , mitosis

INTRODUCTION have demonstrated that centrin plays important roles in the determination of centrosome position and segre- The centrosome plays a key role in the organization of cyto- gation, and in the process of microtubule severing (McFadden plasmic , in the determination of cell polarity, and et al., 1987; Salisbury et al., 1988; Sanders and Salisbury, in the establishment of the bipolar mitotic spindle (Kupfer et 1989; Schulze et al., 1987). al., 1982; McIntosh and Koonce, 1989; Mitchison and Kirschner, 1984). During the cell cycle the centrosome dupli- cates, and at prophase the duplicated centrosomes separate into MATERIALS AND METHODS the nascent poles of the forming mitotic spindle (Rieder and Borisy, 1982). Mitotic spindle poles nucleate a larger number Cell culture of microtubules that show relatively rapid turnover when HeLa cells (American Type Culture Collection, Rockville, MD) were compared to the microtubules of interphase centrosomes grown in bicarbonate-buffered modified Eagle’s medium (MEM), pH (Salmon et al., 1988; Saxton et al., 1984; Snyder and McIntosh, 7.3, supplemented with 10% fetal bovine serum at 37ûC and 5% CO2 in air. All culture reagents were purchased from Gibco Laboratories 1975). Centrosome dynamics during the cell cycle are thought (Grand Island, NY). to be influenced by changes in the activity of the principle regulator of cell cycle progression, p34cdc2 kinase (Alfa et al., Isolation of cDNA clones encoding human centrin 1990; Bailly et al., 1989; Riabowol et al., 1989), and through A human testis cDNA expression library in λgt11 (HL 1010b, the action of γ-tubulin, a centrosome-specific tubulin isotype Clontech Laboratories, Palo Alto, CA), was screened with anti-centrin involved in microtubule nucleation (Bass and Joshi, 1992; polyclonal serum 26/14-1 according to the procedures outlined by Joshi et al., 1992; Oakley and Oakley, 1989; Oakley et al., Sambrook and coworkers (1989). λ particles (105 pfu/plate) were r− 1990). Biochemical and genetic studies suggest that the cen- plated with E. coli Y 1090 cells onto 150 mm plates and induced trosome’s protein composition is complex (Bornens et al., with IPTG. The induced plaques were lifted onto nitrocellulose membranes (HATF type HA, Millipore Corp., Bedford, MA) and 1987; Lux and Dutcher, 1991). Here, we identify and sequence screened for reaction against anti-centrin polyclonal serum 26/14-1 a cDNA clone encoding human centrin and demonstrate that using alkaline phospatase-conjugated secondary antibodies and the centrin is a component of centrosomes and mitotic spindle reaction procedures described below for western blots. Screening of poles in cultured human cells. Previous studies in lower fifty filters resulted in the identification of two positive plaques, which 10 R. Errabolu, M. A. Sanders and J. L. Salisbury were plaque-purified through seven additional screenings. These two PWB (three changes, 5 minutes each), and immediately blocked to cDNA clones proved to contain identical inserts and were also eliminate nonspecific binding of antibodies in blocking buffer, which positive for reaction with monoclonal anti-centrin antibody 20H5. consisted of 5% FBS, 5% glycerin, 0.04% sodium azide in PBS (10 Sequence analysis demonstrated an open reading frame encoding the mM KH2PO4 and K2HPO4, 150 mM NaCl), pH 7.2, for 1 hour at complete human centrin sequence. The approximately 1100 bp insert 37ûC. After blocking all specimens were triple-labeled with mono- was transferred from λgt11 into pBluescript SK+ (Stratagene Cloning clonal anti-α-tubulin (Sigma Chem. Co.) antibody, polyclonal anti- Sys., La Jolla, CA) and the recombinant plasmid psk+:333 was used centrin (26/14-1) antiserum, and the DNA-binding fluorochrome 4,6- for sequencing of both strands of the cDNA according to Sanger’s diamidino-2-phenylindole (DAPI; Sigma Chem. Co.). Coverslips dideoxy chain termination method (Sanger et al., 1977) using a were: (1) incubated in rabbit anti-centrin immune serum 26/14-1 Sequenase sequencing kit (United States Biochem. Corp., (1:6000 dilution) overnight at 37ûC, washed with PBS; (2) incubated Cleveland, OH). in mouse monoclonal anti-tubulin ascites fluid (1:500 dilution; Sigma Chem. Co.) for 4 hours at 37ûC, washed with PBS; (3) incubated in Sequence analysis secondary antibodies (goat anti-mouse fluorescein-conjugated IgG DNA and protein sequence analysis was performed using the and goat anti-rabbit rhodamine-conjugated IgG; 1:1000 dilution of sequence analysis software package GCG (University of Wisconsin). each; Caltag Laboratories, San Francisco, CA) for 4 hours at 37ûC, The Motif routine was used to search for consensus patterns defined washed with PBS, rinsed with deionized water to remove salts, and in the Prosite Dictionary of Protein Sites and Patterns. The multiple finally, mounted with Fluoroseal mountant (Microfluor Ltd, Stony sequence alignment routine, Pileup, was used to determine cluster Brook, NY) containing 0.1 µg/ml DAPI to label DNA. relationships among the sequences of interest and to construct den- Micrographs were taken on a Nikon Microphot FXA microscope dograms representing cluster relationships. Sequences for comparison (Nikon Inc., Instrument Div., Garden City, NY) equipped with epi- were obtained from the GenBank sequence data bank (cited in refer- fluorescence illumination using a ×60, 1.4 NA, oil immersion ences) and from Dr T. Stearns (personal communication) for Xenopus objective. Images were recorded on HyperTech film (Microfluor Ltd, centrin and Dr C. Fulton and Y. Levy (personal communication) for Stony Brook, NY) and developed with D-19 for 6 minutes at 20ûC. Naegleria centrin. Immunoprecipitation and western blots A HeLa culture grown in a 100 mm Petri dish was harvested by RESULTS scraping and washing cells in PBS. After centrifugation in a microfuge tube, the cell pellet was lysed in 0.1 ml lysis buffer con- Isolation and sequence of a cDNA clone encoding taining 2.5% SDS, 0.5% deoxycholate, and 0.5% NP40 with brief son- human centrin ication. The cell lysate was diluted with 9 volumes of immunopre- A cDNA clone was obtained by screening 1×106 recombinants cipitation buffer containing 190 mM NaCl, 50 mM Tris HCL (pH from a human testis cDNA λgt11 expression library with anti- 7.4), 6 mM EDTA, and 2.5% NP40 and then clarified by spinning in centrin antibodies raised against bacterially expressed Chlamy- a microfuge for 3 minutes. Anti-centrin serum 26/14-1 (6 µl) was added to the supernatant and allowed to incubate at 4ûC overnight with domonas centrin. Two positive cDNA clones were identified gentle rocking. The preparation was then treated with 30 µl of Protein by reaction with a polyclonal anti-centrin serum (26/14-1). A/G-Sepharose (Pharmacia Biotech Inc, Piscataway, NJ) for one hour These clones were also shown to react with monoclonal anti- at 4ûC, and the immune complexes collected by centrifugation. After centrin antibody (20H5). Sequencing of the cDNA clones SDS-PAGE according to the standard methods described by Laemmli proved them to be identical. An approximately 1170 base pair (1970), the gels were analyzed by western blotting according to Hulen insert, designated Hcen-1, was restriction mapped and and coworkers (1991). The gels were soaked for 15 minutes in 25 mM sequenced, and found to contain an open reading frame of 516 KH2HPO4 buffer, pH 7.0 (KP buffer), followed by transfer to bases beginning with an initiation codon (ATG) starting at bp Immobilon P membranes (Millipore Corp.) in KP buffer at 20 V ′ overnight at 4ûC using a Hoeffer TE Transphor unit (Hoeffer Sci. 49 from the 5 end of the cDNA insert. The complete Instr., San Francisco, CA). After transfer, the membranes were nucleotide sequence of the cDNA insert and the deduced amino incubated for 45 minutes at room temperature in 0.2% (v/v) glu- acid sequence of the encoded protein are shown in Fig. 1. The taraldehyde freshly prepared in KP buffer and then blocked in 5% putative methionine initiation codon is preceded at position, nonfat dry milk (Carnation Co., Los Angeles, CA) in TBS for 1 hour −3 (3 nucleotides upstream of the ATG) by an A residue. A at 37ûC. After three rinses, ten minutes each, with TBS containing purine at this position has been shown to have a dominant 0.05% Tween-20, the membrane was incubated with anti-centrin effect on selection of a functional initiation codon (Kozak, monoclonal ascites diluted 1:1000 in TBS (1 hour, 37ûC), rinsed as 1986). The open reading frame ends with a TGA stop codon above, and incubated in goat anti-mouse IgG (alkaline phosphatase- and would encode a polypeptide of 172 amino acids with a cal- conjugated, Organon Teknika Corp, West Chester, PA) diluted 1:1000 in TBS containing 2% BSA (w/v), 10% normal goat serum (v/v), culated molecular mass of 19,528 and a pI of 4.61. These 0.05% Tween-20 (v/v) for 1 hour at 37ûC. The colored reaction values are in agreement with the estimated molecular mass product was generated using BCIP/NBT tablets (Sigma Chem. Co., (~20,000 Mr) and pI (4.5 to 4.7) of centrin from HeLa cells St. Louis, MO) dissolved in water as the substrate according to the identified by immunoprecipitation and western blot analysis manufacturer. These procedures work especially well for the detection (see below) using the same antibodies used to screen the cDNA of centrin from cell lysates with detection limits in the range of 0.1 library (anti-centrin serum 26/14-1 and monoclonal antibody ng protein. 20H5). The human centrin sequence contains no cysteine, Indirect immunofluorescence histidine, or tryptophan residues, and the only tyrosine is located at carboxy-terminal residue 172. HeLa cells, grown on glass coverslips, were rinsed with PIPES wash Analysis of the predicted amino acid sequence reveals buffer (PWB; 100 mM PIPES, 1 mM CaCl2, 1 mM MgSO4, pH 7.2), fixed in a buffer containing 3% formaldehyde freshly prepared from several consensus sequences that identify important structural paraformaldehyde in PWB for 1 hour, permeabilized with 0.01% motifs in the encoded protein. Most notable among these Triton X-100 in PWB (three changes, 5 minutes each), rinsed with features are four putative calcium ion-binding sites (Fig. 2). cDNA clone encoding human centrin 11

1 gaa ttc ggg ggg cgg tgc cgt tgg gac cac ggc ggc cag agc ggc agg 48 EcoR1 Human Centrin E-F hand Consensus Sequences

49 ATG GCT TCC GGC TTC AAG AAG CCC AGC GCT GCC TCC ACC GGC CAA AAG 96 loop 1 Met Ala Ser Gly Phe Lys Lys Pro Ser Ala Ala Ser Thr Gly Gln Lys 16 ______helix helix 97 AGA AAG GTG GCA CCT AAG CCC GAG CTC ACT GAG GAT CAG AAG CAA GAA 144 17 Arg Lys Val Ala Pro Lys Pro Glu Leu Thr Glu Asp Gln Lys Gln Glu 32 ______1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 145 GTT CGG GAA GCA TTT GAC CTC TTC GAC GTG GAC GGA AGT GGG ACC ATC 192 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 33 Val Arg Glu Ala Phe Asp Leu Phe Asp Val Asp Gly Ser Gly Thr Ile 48 n n n n X Y Z -Y -X -Z n n n n(n)

193 GAC GCG AAG GAG CTG AAG GTG GCC ATG AGA GCG CTG GGC TTC GAA CCC 240 49 Asp Ala Lys Glu Leu Lys Val Ala Met Arg Ala Leu Gly Phe Glu Pro 64 EF-Hand residues 241 AGG AAG GAA GAG ATG AAG AAA ATG ATC TCC GAG GTG GAC AGG GAA GGC 288 65 Arg Lys Glu Glu Met Lys Lys Met Ile Ser Glu Val Asp Arg Glu Gly 80 1 E V R E A F D L F D V D G S G T I D A K E L K V A M R A L 32-60 2 E M K K M I S E V D R E G T G K I S F N D F L A V M T Q K 68-96 289 ACG GGG AAG ATC AGC TTC AAT GAC TTC CTG GCC GTG ATG ACG CAG AAG 336 81 Thr Gly Lys Ile Ser Phe Asn Asp Phe Leu Ala Val Met Thr Gln Lys 96 3 E I L K A F R L F D D D E T G K I S F K N L K R V A N E L 105-133 4 E L Q E M I D E A D R D G D G E V N E E E F L R I M K K T 141-169 337 ATG TCC GAG AAG GAC ACC AAA GAA GAA ATC CTG AAG GCC TTC AGG CTC 384 97 Met Ser Glu Lys Asp Thr Lys Glu Glu Ile Leu Lys Ala Phe Arg Leu 112 Fig. 2. EF-hand domains of human centrin. (A) The canonical domain consists of a helix, loop, helix conformation; the Ca2+, if 385 TTT GAT GAC GAT GAG ACC GGG AAG ATC TCG TTC AAA AAC CTG AAG CGT 432 113 Phe Asp Asp Asp Glu Thr Gly Lys Ile Ser Phe Lys Asn Leu Lys Arg 128 bound, is coordinated by six residues, whose positions are approximated by the vertices of an octahedron (Moncrief et al., 433 GTG GCC AAC GAG CTG GGG GAG AAC CTC ACG GAT GAG GAG CTG CAG GAG 480 1990). Five of these, X, Y, Z, -X, and -Z, usually have oxygen- 129 Val Ala Asn Glu Leu Gly Glu Asn Leu Thr Asp Glu Glu Leu Gln Glu 144 containing side chains, and the calcium ion, when present, is 481 ATG ATC GAC GAA GCT GAT CGG GAT GGG GAC GGC GAA GTG AAC GAG GAG 528 coordinated by an oxygen atom (or by a water molecule bridged to 145 Met Ile Asp Glu Ala Asp Arg Asp Gly Asp Gly Glu Val Asn Glu Glu 160 an oxygen atom) of the side chains of residues 10, 12, 14, 18, and 21; 529 GAG TTC CTT CGG ATC ATG AAG AAG ACC AGC CTT TAC tga agt cgg ttc 576 the carbonyl oxygen of residue 16 also coordinates calcium 161 Glu Phe Leu Arg Ile Met Lys Lys Thr Ser Leu Tyr *** 172 (Moncrief et al., 1990). Amino acids 1-11 comprise the first α-helix; 577 aga agc taa agt gac tct ctg ggt tgc ctg ctt cca ttt tgt gaa acc 624 19-29 the second, and 11-22 the loop. α-Carbons 2, 5, 6, 9, 22, 25, 625 tta gag gac agc ggc tgc ctg tcc ctt ctt cac ccc ctc acc ccc ata 672 26, and 29 (n) usually have hydrophobic side chains (Moncrief et al., 673 att tgt cta gat cta ttt cca tat ctc tag ttc aat aat aga att tga 720 721 aag atg ctt gta atg tga gtt ttg ggt ttt aat tct caa gag cca acc 768 1990). (B) The four EF-hand domains of human centrin are shown 769 tgg agc aca tga ggt taa aca aag ggc cct gaa gtt tga gtg cgc cct 816 beneath the consensus diagram. EF-hands one through four extend 817 cca ttt gcc ctg tgc tga act tgc tgt tca tct gtt gat ctg gag gca 864 865 gga cag ctt ctg gga cac aca aaa atg tgg ttc cct ttg tca ctt ctt 912 from residues 32 to 60, 68 to 96, 105 to 133, and 141 to 169, 913 tgg tgg tct taa att atc ttg ctt cat ata tca ttc ctt aaa ttc cag 960 respectively. 961 tca ttg ttc cag cat aat gag atg gaa tct gcc agt aga ttt gcc tag 1008 1009 cct gtc cac tta gct gaa tac cag ttt gaa gga aaa cag ggt ggc cac 1056 1057 tta caa act tac gga gct cag gac aga tat tct tat aaa gaa tag act 1104 1105 tgc ttg ggt ggt agt acg ttg tgc aat ttt gac tat tca ctg gct tta 1152 ally expressed algal centrin, (S. J. Doig, F. G. Prendergast and 1153 tac ctg caa atg ccc gaa ttc 1174 EcoR1 J. L. Salisbury, unpublished results).

Fig. 1. Human centrin cDNA sequence and deduced amino acid Sequence relatedness of human centrin with sequence. The complete cDNA sequence for clone Hcen-1 is shown, centrins from other eukaryotes bases are numbered from the EcoRI site at the 5′ end to the EcoRI With the complete amino acid sequence for human centrin in site at the 3′ end of the clone. The initiation codon, ATG, at bases hand, the extent of sequence homology to other calcium- 49-51 begins an open reading frame encoding a polypeptide of 172 binding proteins could be evaluated. Not surprisingly, analysis amino acid residues. EcoRI, site of polylinker. Uppercase letters, ′ ′ of the human centrin sequence revealed the highest degree of coding sequence; lowercase letters, noncoding 3 and 5 sequence; identity with the centrin sequence from frogs, followed by single underline, EF-hand loop; ***, stop codon. centrin from algae, protozoa, higher plants, and the yeast CDC31 gene product (Fig. 3). The highest degree of identity is seen for centrins from vertebrate sources; i.e. human centrin, These four sites (residues 32-60, 68-96, 105-133 and 141-169) reported here, compared to the Xenopus centrin sequence (Dr conform to the EF-hand motif originally identified by T. Stearns, personal communication), which shows 81% Kretsinger (1975) as the canonical signature of cytoplasmic sequence identity and 90% similarity. Comparisons between calcium-binding proteins. EF-hand motifs contain a central human centrin and centrins from algae (Chlamydomonas; domain of 12 amino acids, which is predicted to be appropri- Huang et al., 1988), higher plants (Atriplex; Zhu et al., 1992), ately configured in the intact protein to coordinate calcium protozoa (Naegleria; Y. Levy and C. Fulton, personal com- through oxygen-containing side groups, oxygens of the main munication), and the yeast CDC 31 product (Baum et al., 1986, chain, or a water molecule. Each of these central domains is 1988) also show high sequence identity (68%, 65%, 56% and flanked on both sides by an eight to nine residue stretch 52%, respectively). For example, when the amino termini of predicted to form to an α-helix (Moncrief et al., 1990). It is human and algal centrin (a.k.a. caltractin; Huang et al., 1988) likely that at least some of the human centrin EF-hand domains are aligned, and a three amino acid gap is introduced following bind Ca2+ and possibly Mg2+ when present at physiological residue 17 in the algal centrin sequence, the two sequences levels, giving rise to the calcium-modulated behavior of centrin display 68% identity and 85% overall homology, allowing for observed in earlier studies (Salisbury et al., 1984; Salisbury conservative amino acid substitutions, over their entire length. and Floyd, 1978; Sanders and Salisbury, 1989). In this regard, Other calcium-binding proteins in the data bank show lower, we have demonstrated calcium-binding directly by 45Ca2+ gel yet significant, degrees of similarity with the human centrin overlay on centrin immunoprecipitated from vertebrate cells sequence, particularly in EF-hand domains: for example, (Baron et al., 1993) and circular dichroism studies on bacteri- calmodulin (50% identity), and troponin C (34% identity). 12 R. Errabolu, M. A. Sanders and J. L. Salisbury

Cluster relationships, based on sequence similarity, determined of hydrophobic residues. Although the significance of this for representatives of the EF-hand family, clearly demonstrate amino-terminal domain relative to the function of the centrin that the centrins represent a closely related subfamily within molecule is not known, it is tempting to speculate that it may the larger superfamily of calcium-binding proteins (Fig. 3). be involved in centrosome and spindle pole targeting, in This conclusion is supported by the analysis of predicted evo- filament assembly, or in conveying contractile properties to lutionary histories of EF-hand proteins by Nakayama and centrin-based filaments. coworkers (1992). Members of the centrin subfamily of proteins each contain Identification and localization of centrin in HeLa an amino-terminal domain that is not found in calmodulin and cells other related low molecular mass calcium-binding proteins. In Fig. 4 illustrates the identification of human centrin by human centrin, the amino-terminal domain consists of 24 immunoprecipitation followed by western blot analysis of amino acid residues and contains nine hydrophobic and seven extracts from HeLa cells using the same antibodies used to positively charged residues. Although the comparable amino- screen the human cDNA library. Polyclonal anti-centrin serum terminal domains of other centrin molecules bear only modest (26/14-1, lane A) immunoprecipitates a band of approximately sequence homology with each other, they, nonetheless, show 20,000 Mr from HeLa cells that is recognized by the anti- similarity with regard to overall charge density and disposition centrin monoclonal antibody 20H5 by western blot. The HeLa protein co-migrates with centrin immunoprecipitated from Chlamydomonas reinhardtii (lane B). These results demon- MYLC regulatory strate that cultured human cells (HeLa) contain a protein that MYLC essential is of the appropriate molecular mass as the predicted centrin Calmodulin cDNA product. To determine the subcellular distribution of centrin in Troponin C cultured human cells, we performed immunofluorescence Human Centrin experiments using the same anti-centrin polyclonal serum used to screen the human cDNA library. Triple-labeling fluores- Xenopus Centrin cence studies using polyclonal anti-centrin serum 26/14-1, a Chlamydomonas Centrin

Naegleria Centrin

Atriplex Centrin

Yeast CDC 31

Parvalbumin

Calbindin

Aequorin

Vit D CABP

Calpain

Flagellar CABP

Fig. 3. Dendogram illustrating the relationships among centrins from human (this study), Xenopus (T. Stearns, personal communication) Chlamydomonas, (a.k.a calctractin; Huang et al., 1988), Naegleria (Y. Levy and C. Fulton, personal communication), Atriplex (Zhu et al., 1992), and the yeast CDC31 gene product (Baum et al., 1988), and other members of the EF-hand superfamily of calcium-binding proteins (Aoki et al., 1986; Coffee et al., 1974; Engman et al., 1989; Hailstones and Gunning, 1990; Hofmann et al., 1979; Iida 1982; Parmentier et al., 1987; Prasher et al., 1987; Reinach and Karlsson, 1988). These relationships are based on the multiple sequence Fig. 4. Identification of HeLa and Chlamydomonas centrin using the alignment routine, Pileup, from the GCG sequence analysis package same antibodies used to identify the human centrin cDNA clones. (Univ. Wisconsin), which orders sequences using a progressive Whole cell lysate of HeLa (A) and Chlamydomonas (B) cultures alignment method in a paired stepwise comparison. Pairwise were processed for immunoprecipitation using the polyclonal anti- alignments that score the similarity between every possible pair of centrin serum 26/14-1. The precipitates were then run by SDS- sequences are used to create a cluster order that is represented in the PAGE, western transferred to Immobilon P membranes (Millipore) dendogram. The entire sequence of the relevant proteins were used and then immunoblotted using anti-centrin monoclonal antibody for this comparison. Note that the centrins align as a distinct 20H5. A single protein band of 20,000 Mr is identified in lanes A and subfamily and show greater sequence similarity among themselves B. Prestained molecular mass markers (Bio-Rad Laboratories, than with other members within the larger superfamily of EF-hand Richmond, CA) indicated at the left are (top to bottom): 116,500, proteins. 106,000, 80,000, 49,500, 32,500, 27,500, and 18,500. cDNA clone encoding human centrin 13 monoclonal anti-tubulin antibody and DAPI demonstrate that centrin staining spots, often located over the nucleus, at a site centrin is localized at the centrosome, the major microtubule where microtubules originate. Similar staining is typical for all organizing center, in interphase HeLa cells (Fig. 5). The cen- cultured vertebrate interphase cells that we have studied. Fig. trosome of every interphase cell shows an aggregation of anti- 5 also depicts a cell cycle progression: interphase (Fig. 5A-C),

A B C

D E F

G H I

J K L

Fig. 5. Triple-labeling of tubulin, centrin and DNA in HeLa cells. HeLa cells were fixed and stained with a monoclonal anti-tubulin antibody (A,D,G,J), anti-centrin serum 26/14-1 (B,E,H,K), and DAPI, a DNA binding dye (C,F,I,L). Each row shows the same cell viewed to illustrate the different labels. The cell in (A-C) is in interphase, the cell in (D-F) is in early prophase, the cell in (G-I) is in metaphase, and the cell in (J-L) is in anaphase. Anti-centrin antibodies label the centrosome and spindle poles of interphase and mitotic cells, respectively. Bar, 10 µM. 14 R. Errabolu, M. A. Sanders and J. L. Salisbury prophase (Fig. 5D-F), metaphase (Fig. 5G-I), and anaphase 172 amino acid residues and with a calculated Mr of 19,528. (Fig. 5J-L). Centrin staining labels the position of the centro- Sequence analysis of the cDNA clone confirms that human some and mitotic spindle poles throughout the cell cycle in centrin is a member of the EF-hand calcium-binding protein HeLa cells. Careful examination of cells in prophase and family. Four potential calcium-binding domains are present metaphase reveals that the two mitotic poles do not show the spanning residues 32-60, 68-96, 105-133, and 141-169. The same degree of anti-centrin staining; one pole typically has human centrin sequence shows a high degree of sequence more reactive material associated with it than does the other. identity with centrin from Xenopus (81%), Chlamydomonas Also, the images suggest that during anaphase the total amount (70%), Naegleria (65%), Atriplex (56%), and the CDC31 gene of staining diminishes in both spindle poles such that staining product of Saccharomyces cerevisiae (52%). Human centrin of a pair of small spots is observed at each pole. These spots shares lower, yet significant, similarities with other members probably represent the location of the themselves. of the EF-hand family of calcium-binding proteins. Cluster These observations demonstrate that centrin is located at the relationships based on sequence identity demonstrate that the centrosome and spindle poles of HeLa cells where it undergoes centrins represent a closely related subfamily within the EF- a change in organization during mitosis. hand superfamily of calcium-binding proteins. The highest degree of sequence similarity between the human centrin sequence and those of other calcium-binding proteins extends DISCUSSION largely through the four recognized calcium-binding domains. Identification of centrin in human cells Previously, we identified centrin (20,000 Mr) as a component of the flagellar basal body complex in lower flagellated eukary- We have generated a polyclonal antiserum, designated 26/14- otes (Salisbury et al., 1984). In motile unicellular green algae 1, which was raised against a trpE-algal centrin fusion protein such as Chlamydomonas reinhardtii, centrin is a component of expressed in E. coli. This polyclonal antiserum recognizes an calcium-sensitive contractile fibers that link the basal bodies epitope(s) on centrin from diverse organisms including algal, (centrioles) to one another and to the cell nucleus (McFadden protozoan, higher plant, and vertebrate cells. This antiserum et al., 1987; Salisbury et al., 1988; Schulze et al., 1987). In the and other polyclonal and monoclonal anti-centrin antibodies algae, centrin-based fibers play a role in several centrosome- have proven useful for the identification of centrin homologues associated processes, including: (1) segregation of basal bodies in diverse organisms (Baron et al., 1993; Hiraoka et al., 1989; into daughter cells at the time of cell division (Salisbury et al., Salisbury et al., 1986; Schulze et al., 1987; Yu et al., 1993). 1988; Wright et al., 1985, 1989); (2) basal body positioning Using polyclonal antiserum 26/14-1, we have identified ver- tebrate centrin in HeLa cells by immunoprecipitation. HeLa and re-orientation during reversal in swimming direction centrin migrates with an M of approximately 20,000 as deter- (McFadden et al., 1987); and (3) microtubule severing during r mined by SDS-polyacrylamide gel electrophoresis. flagellar excision (Sanders and Salisbury, 1989). Biochemical Indirect immunofluorescence of HeLa cells, using poly- and molecular cloning studies (Huang et al., 1988; Martindale clonal antiserum 26/14-1, demonstrates that vertebrate centrin and Salisbury, 1990; Salisbury et al., 1984) have demonstrated is localized at the centrosome, where interphase cytoplasmic that algal centrin (a.k.a. caltractin; Huang et al., 1988) is a microtubules focus near the cell center. As cells progress phosphoprotein of approximately 20,000 Mr containing four through the cell cycle, centrin remains associated with the putative calcium-binding domains or ‘EF-hands’, as first major MTOC. At prophase, centrin localization marks the described by Kretsinger (1975) for parvalbumin. Mutations in nascent spindle poles as they separate into two distinct entities centrin and a closely related yeast homologue that effect cen- and migrate to opposite sides of the nucleus. At the time of trosome duplication and separation have been identified (Baum metaphase, one of the spindle poles appears to stain more et al., 1986, 1988; Taillon et al., 1992). One of these, the Sac- brightly with anti-centrin antibodies than does the other. charomyces cerevisiae mutant cdc31 (Baum et al., 1986, During anaphase centrin localization is reduced to the area 1988), results in temperature-sensitive cell cycle arrest due to immediately surrounding the centriole pairs of each mitotic a failure of spindle pole bodies to properly duplicate and spindle pole, while the remaining centrin has apparently separate during the cell cycle. The second, a single point dispersed and is no longer detectable above background by mutation in the Chlamydomonas centrin gene, has been iden- immunofluorescence. Thus, centrin localization reflects the tified as the underlying defect in the vfl2 mutant. This mutant dynamic behavior of the centrosome during the cell cycle. fails to assemble the typical centrin-based fiber system, and results in the inappropriate separation of centrosomes (preco- Speculations on centrin function cious separation) that gives rise to the variable flagellar number Functional and genetic studies in lower eukaryotes have phenotype associated with the vfl2 locus (Taillon et al., 1992). demonstrated that centrin plays an important role in basal body (centriole) positioning and orientation, in centrosome segrega- Molecular cloning of a cDNA clone encoding human tion during cell division, and in microtubule severing at the centrin time of flagellar excision (Baum et al., 1986, 1988; McFadden We chose to screen a human testis library because sperm cells et al., 1987; Salisbury et al., 1988; Sanders and Salisbury, show a relatively high content of centrin where it is localized 1989; Wright et al., 1985, 1989). Centrin’s role in centrosome at the base of the flagellar apparatus (Salisbury et al., 1986; dynamics in higher eukaryotes is, as yet, unknown. However, and unpublished results). Here, we have identified and the high degree of conservation of centrin’s molecular structure sequenced a cDNA clone encoding human centrin and demon- across diverse species suggests a functional conservation as strate that human centrin is a low molecular mass protein of well. It is likely that the severing of microtubules anchored at cDNA clone encoding human centrin 15 the centrosome in higher eukaryotes may provide a mechanism centrin encoding genes and suggest the possibility that there for releasing a subset of microtubules, as is the case during may be unique tissue- or cell type-specific expression of their flagellar excsion in Chlamydomonas. Microtubule severing products. The development of immunological and molecular activity has been observed in mitotically activated Xenopus egg probes for vertebrate centrin will allow for the further dissec- extracts (Vale, 1991), and individual microtubules have been tion of the human centrin genes and the functional properties demonstrated to detach from centrosomes in cultured epithe- of this important centrosome protein. lial cells and neurons and to subsequently depolymerize (McBeath and Fujiwara, 1990) or be transported to distal We thank Dr T. Stearns (University of California, San Francisco, regions within axons (Baas et al., 1992; Yu et al., 1993). Newly CA), Dr P. Hodges (Univ. Edinburgh, Edinburgh, Scotland) and Y. generated microtubule fragments may be predisposed to Levy and Dr C. Fulton (Brandeis University, Boston, MA) for com- unique behavior, including rapid disassembly through the municating their unpublished sequence data, and Drs E. Stone and B. dynamic instability pathway first described by Mitchison and Nichols (Univ. Iowa, Iowa City, IA) for relating their chromosomal Kirschner (1984). Microtubule release would also allow for panel analysis to us. Also, thanks to Drs A. Baron, W. Lingle, N. Maihle, and W. Lutz for comments on the manuscript. The nucleotide new polymerization from nucleation sites within the centro- sequence data reported in this paper have been submitted to GenBank some, and/or allow for microtubule transport to distal sites in and assigned the accession number U03270. This work was supported the cytoplasm. The process of microtubule severing may also by NIH grants GM 35258 and HD 29366. contribute to the increased turnover and decreased half-life observed for microtubules at the onset of mitosis (Salmon et al., 1988; Saxton et al., 1984; Snyder and McIntosh, 1975). REFERENCES Furthermore, centrosome separation and the alterations in microtubule dynamics seen at mitosis are likely to not only be Alfa, C., Ducommun, B., Beach, D. and Hyams, J. (1990). Distinct nuclear coincident but also dependent events; that is, it may be and spindle pole body populations of cyclin-cdc2 in fission yeast. Nature necessary to disassemble and reassemble new microtubules of 347, 680-682. Aoki, K., Imajoh, S., Ohno, S., Emori, Y., Koike, M., Kosaki, G. and a nascent spindle in order to allow for spindle pole migration Suzuki, K. (1986). Complete amino acid sequence of the large subunit of the toward opposite sides of the nucleus. Centrosome and mitotic low-Ca2+-requiring form of human Ca2+-activated neutral protease spindle pole shape appears to be variable or ‘flexible’ (Mazia, (mCANP) deduced from its cDNA sequence. FEBS Lett. 205, 313-317. 1984; Paintrand et al., 1992). A flexible centrosome would Baas, P. and Joshi, H. (1992). γ-tubulin distribution in the neuron: effect the spatial orientation of microtubule nucleating sites implications for the origins of neuritic microtubules. J. Cell Biol. 119, 171- 178. and could result in changes in the ‘casting’ direction for new Bailly, E., Dorée, M., Nurse, P. and Bornens, M. (1989). p34cdc2 is located in microtubules (Rieder and Alexander, 1990; Waters et al., both the nucleus and cytoplasm: part is centrosomally associated at G2/M 1993). Thereby, alterations in the shape of the centrosome may and enters vesicles at anaphase. EMBO J. 8, 3985-3995. effect small changes in the pattern and frequency of micro- Baron, A., Greenwood, T., Bazinet, C. and Salisbury, J. (1993). Centrin is a tubule interdigitation within the nascent forming spindle. Cen- component of the pericentriolar lattice. Biol. Cell. 76, 383-388. Baum, P., Furlong, C. and Byers, B. (1986). Yeast gene required for spindle trosome separation, which appears to be dependent on the pole body duplication: Homology of its product with Ca2+-binding proteins. activity of motor proteins of the kinesin family (Bloom, 1992; Proc. Nat. Acad. Sci. USA 83, 5512-5516. Enos and Morris, 1990; Rose et al., 1993), may also require Baum, P., Yip, C., Goetsch, L. and Byers, B. (1988). A yeast gene essential both the local severing of microtubules and subtle changes in for regulation of spindle pole duplication. Mol. Cell. Biol. 8, 53-86-5397. the shape of the MTOC itself. Centrin’s role in microtubule Bloom, G. S. (1992). Motor proteins for cytoplasmic microtubules. Curr. Opin. Cell Biol. 4, 66-73. severing and centriole orientation in lower eukaryotes is well Bornens, M., Paintrand, M., Berges, J., Marty, M.-C. and Karsenti, E. documented (McFadden et al., 1987; Sanders and Salisbury, (1987). Structural and chemical characterization of isolated centrosomes. 1989). Based on the demonstrated conservation of molecular Cell Motil. Cytoskel. 8, 238-249. features of centrins from all sequences currently available, and Coffee, C., Bradshaw, R. and Kretsinger, R. (1974). The coordination of the centrosomal localization of centrin, we suggest that the calcium ions by carp muscle calcium binding proteins A, B, and C. Adv. Exp. Med. Biol. 48, 211-233. functional role of centrin in microtubule severing and centro- Engman, D., Krause, K.-H., Blumin, J., Kim, K., Kirchhoff, L. and some orientation may also be a common feature of this protein. Donelson, J. (1989). A novel flagellar Ca2+-binding protein in Finally, as we were completing the studies reported here, Trypanosomes. J. Biol. Chem. 264, 18627-18631. Drs T. Stearns (Univ. CA, San Fransisco, CA) and P. Hodges Enos, A. P. and Morris, N. R. (1990). Mutation of a gene that encodes a (Univ. Edinburgh, Edinburgh, Scotland) communicated to us kinesin-like protein blocks nuclear division in A. nidulans. Cell 60, 1019- 1027. sequence data for cDNA clones encoding a distinct human Hailstones, D. and Gunning, P. (1990). Characterization of human myosin partial product that was homologous to our Hcen-1 cDNA light chains 1sa and 3nm: implications for isoform evolution and function. clone. These communicated cDNA clones were identical to Mol. Cell. Biol. 10, 1095-1104. one another and 90% identical to Hcen-1 and certainly encode Hiraoka, L., Golden, W. and Magnuson, T. (1989). Spindle-pole a unique yet closely related human centrin product (Hcen-2, organization during early mouse development. Dev. Biol. 133, 24-36. ′ Hofmann, T., Kawakami, M., Hitchman, A., Harrison, J. and Dorrington, our designation). Using primers based on unique 3 untrans- K. (1979). The amino acid sequence of porcine intestinal calcium-binding lated sequence from Hcen-1 and Hcen-2, Drs E. Stone and B. protein. Can. J. Biochem. 57, 737-748. Nichols (Univ. Iowa, Iowa City, IA) mapped the chromosome Huang, B., Mengersen, A. and Lee, V. (1988). Molecular cloning of cDNA location of the respective genes by hybridization analysis of for caltractin, a basal body-associated Ca2+-binding protein: Homology in its their PCR products with members of a human chromo- protein sequence with calmodulin and the yeast CDC31 gene product. J. Cell Biol. 107, 133-140. some/mouse hybrid library panel. Hcen-1 and Hcen-2 mapped Hulen, D., Baron, A., Salisbury, J. and Clarke, M. (1991). Production and to human chromosomes 18 and X, respectively. These obser- specificty of monoclonal antibodies against calmodulin from Dictyostelium vations suggest that there are at least two distinct human discoideum. Cell Motil. Cytoskel. 18, 113-122. 16 R. Errabolu, M. A. Sanders and J. L. Salisbury

Iida, Y. (1982). Molecular evolution of protein: internal homology in the amino Rieder, C. L. and Alexander, S. P. (1990). Kinetochores are transported acid sequence of calmodulin. J. Mol. Biol. 159, 167-177. poleward along a single microtubule during chromosome attachment to the Joshi, H., Palacios, M., McNamara, L. and Cleveland, D. (1992). γ-tubulin is spindle in newt lung cells. J. Cell Biol. 110, 81-96. a centrosomal protein required for cell cycle-dependent microtubule Rose, M. D., Biggins, S. and Satterwhite, L. L. (1993). Unraveling the tangled nucleation. Nature 356, 80-83. web at the microtubule-organizing center. Curr. Opin. Cell Biol. 5, 105-115. Kozak, M. (1986). Point mutations define a sequence flanking the AUG Salisbury, J. and Floyd, G. (1978). Calcium-induced contraction of the initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, rhizoplast of a quadriflagellate green alga. Science 202, 975-977. 283-292. Salisbury, J., Baron, A., Surek, B. and Melkonian, M. (1984). Striated Kretsinger, R. (1975). Hypothesis: calcium-modulated proteins contain EF- flagellar roots: Isolation and partial characterization of a calcium-modulated hands. In Calcium Transport in Contraction and Secretion (ed. Carafoli), p. contractile protein. J. Cell Biol. 99, 962-970. 469. North Holland Publishing, Amsterdam. Salisbury, J., Baron, A., Coling, D., Martindale, V. and Sanders, M. (1986). Kupfer, A., Luovard, D. and Singer, S. (1982). The polarization of the Golgi Calcium-modulated contractile proteins associated with the eucaryotic apparatus and microtubule organizing center in cultured fibroblasts at the centrosome. Cell Motil. Cytoskel. 6, 193-197. edge of an experimental wound. Proc. Nat. Acad. Sci. USA 79, 2603-2607. Salisbury, J., Baron, A. and Sanders, M. (1988). The centrin-based Laemmli, U. (1970). Cleavage of structural proteins during the assembly of the cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and head of bacteriophage T4. Nature 277, 680-685. mitotic cells. J. Cell Biol. 107, 635-641. Lux, F. G. and Dutcher, S. K. (1991). Genetic interactions at the FLA10 locus: Salmon, E., Saxton, W., Leslie, R. J., Karow, M. and McIntosh, J. (1988). suppressors and synthetic phenotypes that affect the cell cycle and flagellar Spindle microtubule dynamics in sea urchin embryos: Analysis using a function in Chlamydomonas reinhardtii. Genetics 128, 549-561. fluorescein-labeled tubulin and measurements of fluorescence redistribution Martindale, V. and Salisbury, J. (1990). Phosphorylation of algal centrin is after laser photobleaching. J. Cell Biol. 99, 2165-2174. rapidly responsive to changes in the external milieu. J. Cell Sci. 96, 395-402. Sambrook, J., Fritsch, E. and Maniatis, T. (1989). Molecular Cloning: A Mazia, D. (1984). Centrosomes and mitotic poles. Exp. Cell Res. 153, 1-15. Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor McBeath, E. and Fujiwara, K. (1990). Microtubule detachment from the Laboratory Press. microtubule-organizing center as a key event in the complete turnover of Sanders, M. and Salisbury, J. (1989). Centrin-mediated microtubule severing microtubules in cells. Euro. J. Cell Biol. 52, 1-16. during flagellar excision in Chlamydomonas reinhardtii. J. Cell Biol. 108, McFadden, G., Schulze, D., Surek, B., Salisbury, J. and Melkonian, M. 1751-1760. (1987). Basal body reorientation mediated by a Ca2+-modulated contractile Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNA sequencing with protein. J. Cell Biol. 105, 903-912. chain-terminating inhibitors. Proc. Nat. Acad. Sci. USA 75, 5463-5467. McIntosh, J. and Koonce, M. (1989). Mitosis. Science 246, 622-628. Saxton, W., Stemple, D., Leslie, R., Salmon, E., Zavortink, M. and Mitchison, T. and Kirschner, M. (1984). Microtubule assembly nucleated by McIntosh, J. (1984). Tubulin dynamics in cultured mammalian cells. J. Cell isolated centrosomes. Nature 312, 232-237. Biol. 99, 2175-2186. Moncrief, N., Kretsinger, R. and Goodman, M. (1990). Evolution of EF- Schulze, D., Robenek, H., McFadden, G. and Melkonian, M. (1987). hand calcium-modulated proteins. I. Relationships based on amino acid Immunolocalization of a Ca2+-modulated contractile protein in the flagellar sequences. J. Mol. Evol. 30, 522-562. apparatus of green algae: the nucleus-basal body connector. Eur. J. Cell Biol. Nakayama, S., Moncrief, N., and Kretsinger, R. (1992). Evolution of EF- 45, 51-61. hand calcium-modulated proteins. II. Domains of several subfamilies have Snyder, J. and McIntosh, J. (1975). Initiation and growth of microtubules diverse evolutionary histories. J. Mol. Evol. 34, 416-448. from mitotic centers in lysed mammalian cells. J. Cell Biol. 67, 744-760. Oakley, C. and Oakley, B. (1989). Identification of γ-tubulin, a new member Taillon, B., Adler, S., Suhan, J. and Jarvik, J. (1992). Mutational analysis of of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans. centrin: an EF-hand protein associated with three distinct contractile fibers in Nature 338, 662-664. the basal body apparatus of Chlamydomonas. J. Cell Biol. 119, 1613-1624. Oakley, B., Oakley, C., Yoon, Y. and Jung, M. (1990). γ-Tubulin is a Vale, R. (1991). Severing of stable microtubules by a mitotically activated component of the spindle pole body that is essential for microtubule function protein in Xenopus egg extracts. Cell 64, 827-839. in Aspergillus nidulans. Cell 61, 1289-1301. Waters, J. C., Cole, R. W. and Rieder, C. L. (1993). The force-producing Paintrand, M., Moudjou, M., Delacroix, H. and Bornens, M. (1992). mechanism for centrosome separation during spindle formation in Centrosome organization and centriole architecture: their sensitivity to vertebrates is intrinsic to each aster. J. Cell Biol. 122, 361-372. divalent cations. J. Struct. Biol. 108, 107-128. Wright, R., Salisbury, J. and Jarvik, J. (1985). A nucleus-basal body Parmentier, M., Lawson, D. and Vassart, G. (1987). Human 27-kDa connector in Chlamydomonas reinhardtii that may function in basal body calbindin complementary DNA sequence. Evolutionary and functional localization or segregation. J. Cell Biol. 101, 1903-1912. implications. Eur. J. Biochem. 170, 207-215. Wright, R., Adler, S., Spanier, J. and Jarvik, J. (1989). Nucleus-basal body Prasher, D., McCann, R., Longiaru, M. and Cormier, M. (1987). Sequence connector in Chlamydomonas: Evidence for a role in basal body segregation comparisons of complementary DNAs encoding aequorin isotypes. and against essential roles in mitosis or in determining cell polarity. Cell Biochemistry 26, 1326-1332. Motil. Cytoskel. 14, 516-526. Reinach, F. and Karlsson, R. (1988). Cloning, expression, and site-directed Yu, W., Centonze, E., Ahmad, F. J. and Baas, P. W. (1993). Microtubule mutagenesis of chicken skeletal muscle troponin C. J. Biol. Chem. 263, 2371- nucleation and release from the neuronal centrosome. J. Cell Biol. 122, 349- 2376. 359. Riabowol, K., Draetta, G., Brizuela, L., Vandre, D. and Beach, D. (1989). Zhu, J.-K., Bressan, R. A. and Hasegawa, P. M. (1992). An Atriplex The cdc2 kinase is a nuclear protein that is essential for mitosis in nummularia cDNA with sequence relatedness to the algal caltractin gene. mammalian cells. Cell 57, 393-401. Plant Physiol. 99, 1734-1735. Rieder, C. and Borisy, G. (1982). The centrosome cycle in PtK2 cells: Asymetric distribution and structural changes in . Biol. Cell 44, 117-132. (Received 6 August 1993 - Accepted 15 October 1993)