A Novel 95-kD Protein Is Located in a Linker between Cytoplasmic Microtubules and Basal Bodies in a Green Flagellate and Forms Striated Filaments In Vitro Stefan Geimer, Judith Clees, Michael Melkonian, and Karl-Ferdinand Lechtreck Botanisches Institut, Universität zu Köln, D-50931 Köln, Germany

Abstract. The flagellar basal apparatus comprises the 95-kD protein exclusively decorated the striated, basal bodies and the attached fibrous structures, which wedge-shaped fibers, termed sinister fibers (sf-fibers), together form the organizing center for the cytoskele- attached to the basal bodies of S. similis. Striated fibers ton in many flagellated cells. Basal apparatus were iso- with a periodicity of 98 nm were assembled in vitro

lated from the naked green flagellate Spermatozopsis from the purified protein expressed from the cloned Downloaded from similis and shown to be composed of several dozens of cDNA indicating that the 95-kD protein could be a ma- different polypeptides including a protein band of 95 kD. jor component of the sf-fibers. This structure intercon- Screening of a cDNA library of S. similis with a poly- nects specific triplets of the basal bodies with the micro- clonal antibody raised against the 95-kD band resulted tubular bundles that emerge from the basal apparatus. in a full-length clone coding for a novel protein of 834 The sf-fibers and similar structures, e.g., basal feet or

amino acids (90.3 kD). Sequence analysis identified satellites, described in various eukaryotes including jcb.rupress.org nonhelical NH2- and COOH-terminal domains flanking vertebrates, may be representative for cytoskeletal el- -residues, which was predicted ements involved in positioning of basal bodies/centri 650ف a central domain of to form a series of coiled-coils interrupted by short oles with respect to cytoskeletal microtubules and spacer segments. Immunogold labeling using a poly- vice versa.

clonal antibody raised against the bacterially expressed on September 9, 2017

he base of eukaryotic flagella is formed by special- central structures of the basal apparatus—a functional ho- ized microtubular structures, basal bodies. The struc- mologue of the centrosomal complex of animal cells Tture of the basal bodies, consisting of nine microtu- (Baron and Salisbury, 1992). The basal apparatus consists bular triplets, is highly conserved and also characteristic for of the basal bodies and a diverse array of filaments at- centrioles, which are part of the centrosome in many eu- tached to them. Sets of flagellar root microtubules usually karyotic cells. During sexual and asexual reproduction, basal radiate from the basal apparatus and form the major part bodies are often converted into centrioles, indicating their of the microtubular cytoskeleton (which is involved in the functional similarity (Kellogg et al., 1994). It is generally determination of polarity, shape, and internal organization accepted that basal bodies/centrioles function as a template of flagellated cells) (Moestrup, 1982; Melkonian, 1984; for the structure of the axoneme (Lange and Gull, 1996b). Holmes and Dutcher, 1989). Thus, analysis of the flagellar An open question is whether or not centrioles contribute basal apparatus could provide information about the basal to the centrosome of animal cells, for example, by organiz- bodies/centrioles as centrosomal organizers. ing its microtubule nucleation sites or by controlling its du- In green flagellates like Chlamydomonas reinhardtii, two plication. types of the filaments present in the basal apparatus have The basal bodies of many flagellated cells not only serve been analyzed in detail (Lechtreck and Melkonian, 1991a). as assembly sites for the axoneme but also represent the One is characterized by the presence of centrin, a 20-kD protein belonging to the EF-hand family of calcium-bind- ing proteins (Huang et al., 1988). Centrin-based filaments Address all correspondence to Karl-F. Lechtreck, Botanisches Institut, (system II fibers; Melkonian, 1980) form a connection be- Gyrhofstrasse 15, Universität zu Köln, D-50931 Köln, Germany. Tel.: 49-221- tween the basal bodies, interconnect the basal bodies and 470-3795. Fax: 49-221-470-5181. E-mail: [email protected] J. Clees’ present address is Universitätskrankenhaus Eppendorf, Zen- the cell nucleus, and are part of the flagellar transitional trum für Molekulare Neurobiologie, Institut für Biosynthese Neuraler region; they undergo calcium-induced contractions and Strukturen, Martinistrasse 85, D-20251 Hamburg, Germany. are involved in various motile functions of the basal appa-

 The Rockefeller University Press, 0021-9525/98/03/1149/10 $2.00 The Journal of Cell Biology, Volume 140, Number 5, March 9, 1998 1149–1158 http://www.jcb.org 1149

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ratus (Melkonian et al., 1992; Salisbury, 1995). The other Tris-HCl, pH 7.4, 2 mM EDTA), disintegrated by homogenization, well-characterized filaments of the basal apparatus are washed with TE buffer/0.5 % Triton X-100 (800 g, 30 min), and then the suspension was loaded onto a discontinuous sucrose gradient (6 ml 60%, the noncontractile, striated microtubule-associated fibers 9 ml 50%, 9 ml 40%, and 6 ml 20% in TE buffer/0.1% Triton X-100) and 1 (SMAFs, or system I fibers) (Melkonian, 1980) that are centrifuged for 1 h at 12,500 g in a swing-out rotor (HB-4; Sorvall, Bad composed of striated fiber (SF)-assemblin, a highly ␣-heli- Homburg, Germany). Highly enriched basal apparatus were collected cal 30-kD protein (Lechtreck and Melkonian, 1991b; We- from a milky band below the 20–40% sucrose interphase, and the proteins ber et al., 1993). These are attached to the four bundles of were separated by preparative SDS-PAGE. A band of 95 kD was excised from Coomassie blue–stained gels, concentrated by electrophoresis, and ␮g 10ف flagellar root microtubules characteristic for Chlamydo- then blotted onto nitrocellulose membrane. The membrane with monas-like flagellates and are thought to function as stabi- of the 95-kD band was dissolved in dimethyl sulfoxide, mixed with com- lizing elements in the basal apparatus (Lechtreck and Sil- plete Freunds adjuvant, and subcutanously injected at several sites into a flow, 1997). Electron microscopical studies have revealed young, male rabbit. For subsequent booster injections on day 15 and 53, ,␮g antigen, homogenized in PBS 10ف we used acrylamide gel pieces with additional filamentous structures attached to the basal and then mixed with incomplete Freunds adjuvant. An IgG fraction (anti- bodies in the green algal basal apparatus (Ringo, 1967; p95) was purified from the serum taken 10 d after the second injection us- Beech and Melkonian, 1993; Geimer et al., 1997a): transi- ing a fast protein liquid chromatography (FPLC) system (Pharmacia LKB, tional fibers (connectives between the basal bodies and Uppsala, Sweden) and a protein A column (Pharmacia LKB). the plasma membrane), proximal plates or connecting fi- bers, and linkers between basal bodies and flagellar root Cloning and Sequencing of a cDNA Coding for the microtubules. The latter may establish the spatial relation- 95-kD Protein ship between the basal bodies and the flagellar root micro- Total RNA from S. similis was isolated by the phenol/SDS method basi- tubules, as well as direct the microtubular roots to specific cally following the protocol of Palmiter (1974). To purify mRNA, a self- locations in the cell. In contrast to centrin- and SF-assemb- packed oligo-dT column (Boehringer Mannheim GmbH, Mannheim, lin–based fibers, these other fibers and linkers are rela- Germany) was used. A Uni-Zap XR cDNA library was constructed using

a cDNA synthesis and cloning kit (Stratagene, Heidelberg, Germany) ac- Downloaded from tively small, making it more difficult to identify their bio- cording to the manufacturer’s instructions with the following modifica- chemical composition. tions: first-strand synthesis was done for 15 min, 45ЊC; 20 min, 50ЊC; and We have recently developed a method to obtain homo- 20 min, 55ЊC, with reverse transcriptase from GIBCO BRL (Eggenstein, geneous preparations of basal apparatus from the naked, Germany). The library was screened with anti-p95, and positive clones were analyzed by restriction mapping and partial sequencing after in vivo biflagellate green alga Spermatozopsis similis (Geimer et al., excision of the pBluescript phagemid. In addition, bacterial lysates ob- 1997b). The complex, filamentous system surrounding the tained from isopropyl-␤-d-thiogalactopyranoside (IPTG)-induced cul-

two basal bodies in the isolated state has been studied in tures were analyzed with anti-p95 in Western blots. A 3.1-kb clone (clone jcb.rupress.org detail by serial thin section electron microscopy (Geimer 7/1) produced an immunoreactive band of 95 kD. The insert was se- a quenced completely in both directions (Abiprism, 310 Gene Analyzer; et al., 1997 ). Besides the major proteins of the basal appa- Perkin Elmer Applied Biosystems, Weiterstadt, Germany). The AutoAs- ratus (tubulin, centrin, and SF-assemblin) more than two sembler 1.4.0 (Perkin Elmer Applied Biosystems) program was used for dozen different polypeptides were present in our prepara- gene assembly and the sequence was analyzed using programs (COILS, tions. Here we identify a novel 95-kD protein as a compo- FASTA, BLAST) provided by European Bioinformatics Institute (EBI; nent of the sinister fibers (sf-fibers), which are linkers be- http://www.ebi.ac.uk/services/services.html) and Baylor College of Medi- on September 9, 2017 cine (BCM; http://dot.imgen.bcm.tmc.edu:9331/seq-search/struc-predict.html) tween the basal bodies and two of the four microtubular via the internet (Altschul et al., 1990; Lupas, 1996). flagellar roots in the basal apparatus of S. similis. The pro- tein contains extended coiled-coil domains and assembles Purification of the Recombinant 95-kD Protein and into striated fibers in vitro. The green algal sf-fibers resem- Antibody Production ble the basal feet that have been described in various eu- karyotes (Gibbons, 1961), and may be representative of a For induction of gene expression, Escherichia coli XL-1Blue cells contain- ing clone 7/1 were grown at 37ЊC to a density of OD600 0.8–1.0 and induced particular type of basal body–microtubular flagellar root with 2 mM IPTG. After 4 h the cells were harvested (1,000 g, 15 min, 4ЊC), linker that is a common component of the cytoskeleton. resuspended in 50 mM NaH2PO4, 300 mM NaCl, pH 7.8, and frozen at Ϫ80ЊC for 20 min. After thawing, the bacteria were sonicated five times for 10 s, and the insoluble material was pelleted by centrifugation (10,000 g, Materials and Methods 10 min, 4ЊC). The pellet was washed three times (10,000 g, 10 min, 4ЊC) in washing buffer (50 mM Tris-HCl, 1 mM EDTA, 3% Triton X-100, 0.05% Strains and Culture Conditions ␤-mercaptoethanol, pH 7.5) and finally twice in 50 mM Tris-HCl, pH 7.5. Then, the pellet was resuspended in 20 mM Tris-HCl, 2 mM EDTA, 1 mM Spermatozopsis similis (Sammlung von Algenkulturen Göttingen [SAG] DTT, pH 8.0, containing 8 M urea and extracted for 2 h at room tempera- B 1.85; Schlösser, 1994) and Chlamydomonas reinhardtii cw 15ϩ (SAG ture. To remove insoluble material the suspension was centrifuged at -liter ϫ minϪ1) at 15ЊC with 18,000 g for 30 min and filtered (0.22 ␮m, Millex-GV; Millipore, Esch 1ف) were maintained in aerated cultures (83.81 a light-dark cycle of 14:10 h and a photon flux density of 20 ␮Einstein (E) born, Germany). The proteins were separated on a MonoQ column (HR ϫ mϪ2 ϫ sϪ1 (Osram 40W/25 universal white) in Waris medium (McFad- 5/5, Pharmacia LKB) equlibrated with the urea buffer using a FPLC sys- den and Melkonian, 1986). tem (Pharmacia LKB). The column was eluted with a linear salt gradient (0–50 mM KCl) in the same solvent and fractions containing the 95-kD Isolation of a 95-kD Protein Band from the protein were identified by SDS-PAGE. MonoQ fractions of interest were Basal Apparatus applied to preparative SDS-PAGE. For internal or NH2-terminal amino acid sequencing, the protein was transferred to polyvinylidene difluoride The isolation and purification of basal apparatus of Spermatozopsis similis membrane. For antibody production the recombinant 95-kD protein was has been described (Snell et al., 1974; Geimer et al., 1997b). Briefly, con- cut out, reelectrophorized, and electroeluted. The protein concentration centrated cells were lysed with 1.5% Triton X-100 in TE buffer (10 mM was determined using the method of Neuhoff et al. (1979), and four injec- tions of 75 ␮g of homogenous 95-kD protein each were used to generate 1. Abbreviations used in this paper: IPTG, isopropyl-␤-d-thiogalactopyra- polyclonal antibodies (anti-rec95) in rabbit (as above or by BioGenes, noside; sf-fibers, sinister fibers; SMAF, striated microtubule-associated Berlin, Germany) (day 1, intralymphatic; day 7, intramuscular; day 14, fibers. subcutan; day 29, intramuscular).

The Journal of Cell Biology, Volume 140, 1998 1150

Reassembly of Striated Fibers from the Recombinant and Melkonian, 1991b). The anti-p95 and anti-rec95 sera were used in a 95-kD Protein 1:100 dilution; monoclonal anti–␣-tubulin was applied simultaneously. Sec- ondary antibodies (anti–rabbit IgG or anti–mouse IgG conjugated to FITC ␮g/␮l) or TRITC, Sigma Chemical Co.) were diluted 1:50. Immunofluorescence 0.1ف ,MonoQ fractions containing the 95-kD protein (Ͼ95% pure were dialyzed using a floating membrane filter (type VS, pore size 0.025 images were observed using a Zeiss IM35 equiped with a 1,3/100ϫ oil ␮m; Millipore) against an excess amount of 10 mM Tris-HCl, 1 mM immersion objective (Carl Zeiss, Oberkochen, Germany), documented on EDTA, 150 mM KCl, pH 7.0, with decreasing concentrations of urea (4 M, HP5 (Ilford Ltd., Knutsford, UK), digitized, labeled, and assembled using 2 M, 0.8 M, and 0 M) for 120 min at each urea concentration. An alterna- Photoshop (Adobe Systems Inc., Mountain View, CA) and Powerpoint tive protocol used dialysis tubes (12,000–14,000 D; Medicell, London, UK) (Microsoft, Redmond, WA). and prolonged dialysis times (4 h) for each concentration. Reassembled fi- bers were analyzed by whole mount electron microscopy. The fibers were Preembedding Immunogold Electron Microscopy harvested by centrifugation (100,000 g, 30 min), the proteins in the super- natant were precipitated by the addition of acetone (1:9), and both frac- Cells of S. similis were washed by centrifugation in MT (30 mM Hepes, 5 tions analyzed by SDS-PAGE. mM EDTA, 15 mM KCl, pH 7) supplemented with 5 mM MgSO4 (MT- Mg2ϩ) and lysed by the addition of an equal volume MT-Mg2ϩ/2% Triton Immunoblot Analysis X-100, immediately followed by fixation with 1% paraformaldehyde (final concentration in MT-Mg2ϩ) for 5 min. The cytoskeletons were washed Whole cells, isolated cytoskeletons, or enriched basal apparatus of S. simi- twice in the presence of the fixative (80 g, 10 min, 15ЊC). All subsequent lis were separated by SDS-PAGE and transferred to PVDF membrane steps were performed as described previously (McFadden et al., 1987). (Millipore Corp., Bedford, MA). After extensive blocking with PBS-T The anti-rec95 was used at a 1:200 final dilution and detected with protein (PBS with 0.05% Tween 20) containing 3% gelatin from cold water fish A conjugated to 15-nm gold particles. Samples were transferred to agar, (Sigma Chemical Co., St. Louis, MO) the membrane strips were incubated with the primary antibody (anti-p95, 1:500 or anti-rec95, 1:1,000 in PBS-T/3% gelatin) for 60 min. The membrane was washed five times for 15 min with PBS-T, and then blocked again for 30 min, before incubation with anti– rabbit IgG peroxidase conjugate (Sigma Chemical Co.; 1:1,000 in PBS-T/3%

gelatin) for 60 min. Membrane strips were washed five times for 15 min in Downloaded from PBS and developed using 4-chloro-1-naphthol as the substrate.

Indirect Immunofluorescence The preparation of isolated cytoskeletons of S. similis for indirect immuno- fluorescence and antibody staining has been described previously (Lechtreck jcb.rupress.org on September 9, 2017

Figure 1. (a) SDS-PAGE analysis (11%) of isolated basal appa- ratus of S. similis. Protein pattern characteristic for basal appara- tus of S. similis purified by sucrose gradient centrifugation. Cen- trin (C), SF-assemblin (A), tubulin (T), and the 95-kD band (arrowhead) are marked. A polyclonal antibody (anti-p95) was raised against the electrophoretically purified 95-kD band. (b) Western blot analysis of S. similis with anti-p95 and anti-rec95. Membrane strips were stained with amido black (lanes 1–3), anti- p95 (lanes 4–6), or anti-rec95 (lanes 7–9), an antibody directed against bacterial expressed 95-kD protein (see also Fig. 4). 10 ␮g Figure 2. Sequence of the 95-kD protein. The cDNA sequence of of whole cells (1, 4, and 7), isolated cytoskeletons (2, 5, and 8), or clone 7/1 and the deduced amino acid sequence of the longest enriched basal apparatus (3, 6, and 9) were loaded per lane. Anti- open reading frame are shown. The start codon is preceded by a p95 labeled two closely spaced bands, but only the upper one also stop codon (᭛). Peptide sequences determined by NH2-terminal cross-reacts with anti-rec95. The antibody staining reveals that or internal sequencing of the recombinant 95-kD protein are un- the 95-kD proteins were highly enriched in the isolated basal ap- derlined. The SPS(R/K) motifs present in the NH2- and COOH- paratus of S. similis. Preimmune controls are shown in Fig. 4 b. terminal domains are marked by bold letters. These sequence The positions of standard proteins are indicated on the left (from data are available from GenBank/EMBL/DDBJ under accession top for a/b: 205, 116, 97, 66, 45, and 29 kD). number AJ001438.

Geimer et al. Green Algal Basal Feet 1151

Figure 4. (a) Purification of the recombinant 95-kD protein (SDS-PAGE 11%). Lane 1, bacteria without plasmid; lane 2, bac- teria induced with IPTG for 2 h. Note the additional band of 95 kD. Lane 3, inclusion bodies enriched in the 95-kD protein; lane 4, MonoQ fraction containing the 95-kD protein; and lane 5, 95-kD protein purified by preparative electrophoresis from fractions as shown in lane 4. MonoQ-purified 95-kD protein was used for re- assembly experiments, SDS-PAGE–purified protein was used for antibody (anti-rec95) production. (b) Western blot analysis of re- combinant 95-kD protein (1, 3, 6, 7, and 10) and isolated basal ap- Figure 3. Structure of the 95-kD protein. (a) Coiled-coil–forming paratus of S. similis (2, 4, 5, 8, and 9). Membrane strips were probability (P) of the 95-kD protein as calculated with the pro- stained with amido black (1 and 2), anti-p95 (3 and 4), anti-rec95 gram COILS (parameters: MTIDK matrix, window ϭ 28, weight- (5 and 6), and preimmune sera of both animals (7 and 8, and 9 ing of positions a and d ϭ 2.5; Lupas, 1996). The central part of and 10), respectively. To allow an exact comparison of the anti- Downloaded from the 95-kD protein is formed by a series of predicted coiled-coils genic bands recognized by anti-p95 and anti-rec95, “double slots” interrupted by short spacers. (b) Schematic presentation of the were loaded with basal apparatus proteins (4/5 and 8/9), and the 95-kD protein. Boxes, coiled coil regions; arrowheads, position of blot cut before antibody staining. Only the upper one of the two SPS(R/K) motifs. bands labeled with anti-p95 is also stained with anti-rec95 (com- pare lanes 4 and 5), demonstrating that anti-rec95 is monospe- cific. The additional bands visible in lanes 3 and 6 are caused by the presence of degradation products of the 95-kD protein (see dehydrated, and then embedded in Epon 812 (Serva, Heidelberg, Ger- jcb.rupress.org also lane 1). (c) SDS-PAGE of in vitro reassembly experiments many) as previously described (McFadden and Melkonian, 1986). with recombinant 95-kD protein. Lane 1, MonoQ fraction; lane 2, pellet (100,000 g) after stepwise dialysis against urea-free buffer; Standard and Whole Mount Electron Microscopy and lane 3, supernatant corresponding to lane 2. Similar amounts For whole mount electron microscopy, 5 ␮l of suspended particles were of the samples were loaded in each lane. Almost the entire applied to copper grids coated with pioloform (Plano GmbH, Marburg, amount of the 95-kD protein is found in the pellet. on September 9, 2017 Germany) and allowed to adhere for 10–60 min. The grids were stained with 1% aqueous uranyl acetate for 1–2 min. Utrathin sections were cut with a MT-6000 microtome (RMC, Tucson, AZ) and stained with uranyl acetate and lead citrate (Reynolds, 1963). Micrographs were taken with a body No. 1), or 2s and 2d if associated with basal body No. transmission electron microscope (CM 10; Philips Electron Optics, Mah- 2. The fibrous structures that connect the basal bodies to wah, NJ) using Scientia EM Film (Agfa, Leverkusen, Germany). the microtubular roots are called 1sf, 1df, 2sf, and 2df, re- spectively (Beech and Melkonian, 1993). The dominant Results proteins in preparations of isolated basal apparatus of S. similis were tubulin, centrin, and SF-assemblin (Geimer et Identification of 95-kD Components in the al., 1997b; Fig. 1 a). Several proteins with molecular Basal Apparatus weights Ͼ50 kD were enriched in fractions of basal appa- Spermatozopsis similis is a naked, biflagellate green alga ratus purified by sucrose gradient centrifugation in com- that resembles Chlamydomonas reinhardtii in its major ul- parison to crude fractions. Most of these proteins consti- trastructural features. Detailed descriptions of the cells, tute only 1% or less of the total protein; however, a band isolated cytoskeletons, and basal apparatus are given in of 95 kD was present comprising 2–4% of the proteins in Preisig and Melkonian (1984), Lechtreck et al. (1989), and purified basal apparatus (Fig. 1 a). The 95-kD band was Geimer et al. (1997a). A uniform terminology for the purified by SDS-PAGE and used to raise a polyclonal an- structures in the basal apparatus of green flagellates has tibody (anti-p95). When isolated cytoskeletons of S. similis now been established (Moestrup and Hori, 1989; Beech et al., were stained with anti-p95 for indirect immunofluores- 1991), and the developmentally oldest basal body is desig- cence, the region of the basal apparatus was labeled (Fig. nated as basal body No. 1, and the suffixes “s” (for sinister 6, a and b). In Western blotting experiments, this antibody (Latin) ϭ left) and “d” (for dexter ϭ right) are used for labeled two closely spaced bands of 95 kD, which were no- the associated structures according to their position rela- tably enriched in the basal apparatus in comparison to tive to basal body No. 1 (see Fig. 9). The four microtubular whole cells or isolated cytoskeletons (Fig. 1 b). roots of the cruciate x-2-x-2 flagellar root system (where x ϭ 3–6 microtubules), which are characteristic for Chlamydo- Cloning of the 95-kD Protein monas-like flagellates, are called 1s (x-root of basal body SDS-PAGE analysis, indirect immunofluorescence and No. 1) and 1d (two-stranded microtubular root of basal Western blotting analysis indicated that the antigens rec-

The Journal of Cell Biology, Volume 140, 1998 1152 Figure 5. Negatively stained preparations of fi- bers assembled in vitro from the recombinant 95-kD protein. (a–e) Fibers from reassembly ex- periment using floating microdialysis membrane. (a) Overview. Note the tapering ends of several filaments (arrowheads). (b) Detail revealing the cross-striation pattern with a periodicity of 98 nm (indicated by arrows). (c and d) Fibers with alter- nating filamentous (arrow) and condensed re- gions. Similar filamentous parts are also present in the sf-fiber (compare Fig. 8, a and c). (e) Rod- like particles (arrowheads) and small filaments. These structures were observed frequently in re- assembly experiments in addition to the fibers shown in a–d. Overview (f) and detail (g) of fi- Downloaded from bers formed by the 95-kD protein at high protein concentrations (Ͼ0.4 ␮g/␮l) or using dialysis tubes. These spindle-shaped fibers often exhibit a 16-nm cross-striation. Bars: (a, d, and f) 500 nm; (b, c, e, and g) 200 nm. jcb.rupress.org ognized by anti-p95 were components of the basal appara- SPS(K/R) motifs (four in the COOH-terminal domain), tus of S. similis (Figs. 1 and 6). Screening of a S. similis which resemble the known consensus sequences for p34cdc2 cDNA expression library with anti-p95 resulted in three kinase (Moreno and Nurse, 1990; Figs. 2 and 3 b). Data- clones with inserts of 3.1 kb and two shorter variants (1.9 base searches using the complete amino acid sequence of and 2.3 kb), all with identical 3Ј sequences. Both strands of the 95-kD protein detected only low (Ͻ25%) homologies on September 9, 2017 one of the 3.1-kb clones (clone 7/1) were completely se- to other proteins with large coiled-coil domains (e.g., myo- quenced. The sequence contained a large open reading sin, paramyosin, and intermediate filament proteins). In frame of 2,505 bp that had a GC content of 75%; the puta- comparison with those proteins, the 95-kD protein dis- tive initiation codon was preceded by an in-frame stop plays a high content of alanine (19.7%), whereas isoleu- codon (Fig. 2). An immunoreactive 95-kD protein was cine (1.4%) is underrepresented. This might reflect the present in bacteria containing clone 7/1 after induction high GC-content of the cDNA of the 95-kD protein (e.g., with IPTG (see Fig. 4, a, lane 2, and b), demonstrating that isoleucine has the codons AT[A/T/C]; 161 alanines are en- the clone is full length. Southern blot analysis using the in- coded by GC[C/G], but only three by GC[A/T]). sert of clone 7/1 as a probe indicated that the protein is en- coded by a single copy gene in S. similis (data not shown). Purification of Recombinant 95-kD Protein and Antibody Production Sequence Analysis Electrophoretic analysis of bacterial lysates showed that The ORF of clone 7/1 encodes a protein of 834 amino ac- large quantities of recombinant 95-kD protein were ids that has a predicted isoelectric point of 7.4 and a mass present in the insoluble fraction (Fig. 4 a, lanes 2 and 3). of 90.3 kD (Fig. 2). Sequence analysis using the algorithm The 95-kD protein was further purified by anion exchange of Garnier et al. (1978) identified a central domain of chromatography and used for amino acid sequencing and reassembly experiments (Fig. 4 a, lane 4). NH2-terminal ,(%88ف) residues that is predominantly ␣-helical 650ف flanked by nonhelical NH2- and COOH-terminal domains sequencing of the 95-kD protein identified two staggered -and 120 residues, respectively. The sequences starting with methionine 1 or 4 (Fig. 2). Recom 65ف with lengths of program COILS (Lupas, 1996) predicted a high probabil- binant 95-kD protein (Fig. 4 a, lane 5) was used to raise a ity of the central domain forming coiled-coil structures polyclonal antibody (anti-rec95). In Western blotting ex- separated by short spacers around residues 140, 270, 450, periments this antibody recognized only the upper of the and 620 (Fig. 3 a). Because of the presence of heptad re- two bands labeled by the original anti-p95 (Fig. 4 b, lanes 4 peats, two leucine zipper motifs (L-6X-L-6X-L-6X-L in and 5). In the meantime, immunoscreening of a cDNA li- position 392–413 and 572–593) were identified by PRO- brary with anti-p95 yielded a clone different from clone 7/1 SITE. The head and tail domains contained all prolines as revealed by partial sequences. Bacteria transformed (14 of 17 located in the COOH-terminal domain) and five with this clone expressed a protein of 95-kD that cross-

Geimer et al. Green Algal Basal Feet 1153 reacts with anti-p95 but not with anti-rec95 (data not shown). We conclude that anti-p95 was heterospecific and that clone 7/1 encodes one of the two antigens of anti-p95.

Reassembly Experiments with Recombinant 95-kD Protein Purified recombinant 95-kD protein (Ͼ95%) dissolved in 8 M urea was dialyzed successively against buffers with 4, 2, 0.8, and 0 M urea. EM analysis of the final dialysate showed the formation of striated fibers with a periodicity of 98.2 Ϯ 5.6 nm (n ϭ 20; Fig. 5, a and b). Correspond- ingly, the post-centrifugation pellet was almost entirely composed of 95-kD protein (Fig. 4 c, lane 2). Structurally, the reassembled fibers showed repeats consisting of a nm) divided by an 60ف) broad electron-translucent band nm and an electron-opaque 6ف electron-opaque line of ,nm; Fig. 5 b). The 95-kD protein formed thick 40ف) band short fibers (length 643 Ϯ 358 nm, n ϭ 16; width 106 Ϯ 61 nm, n ϭ 15) often with tapered ends (Fig. 5 a). We some- times observed ladder-like fibers with a periodicity of nm, consisting of alternating loose regions formed 100ف by thin filaments and condensed regions (Fig. 5, c and d); Downloaded from these may represent incompletely assembled intermedi- ates. We also observed clusters of rod-like particles (aver- age length of 81 nm [Ϯ 18 nm, n ϭ 16] and a diameter of 2–3 nm; Fig. 5 e), which may represent oligomeric precursors Figure 6. Indirect immunofluorescence of S. similis. (a) Isolated of the striated fibers and also reflect the elongated shape cytoskeletons of S. similis stained with monoclonal anti–␣-tubu- of the 95-kD molecules. Reassembly experiments using di- lin. (b) Anti-p95 immunofluorescence corresponding to a. In ad- alysis tubing instead of microdialysis membrane (see Ma- dition to the prominent dot-like staining in the basal apparatus, jcb.rupress.org we often observed a faint labeling at the posterior end of the cell, terials and Methods) or higher protein concentrations where the microtubules are interconnected by a cap-like struc- (Ͼ0.4 ␮g/␮l instead of 0.1 ␮g/␮l) resulted in the formation ture. (c and d) Isolated cytoskeletons of S. similis stained with of electron-dense, spindle-shaped protein aggregates (length anti-rec95, which labels two dots of unequal size. (e) Anti-tubulin 557 Ϯ 55 nm, n ϭ 15; width 243 Ϯ 77 nm, n ϭ 12), which staining of isolated cytoskeletons of S. similis. The corresponding were sometimes cross-striated with a periodicity of 16 nm signal obtained by anti-rec95 (f) consists of two dots in the region on September 9, 2017 (n ϭ 3; Fig. 5, f and g). of the basal apparatus. Bars, 10 ␮m.

Localization of the 95-kD Protein of the sf-fibers or any other parts of the cytoskeleton (Fig. Immunofluorescence staining of isolated cytoskeletons of 7, b and g). The sf-fibers were also decorated by immu- S. similis with anti-rec95 resulted in comparatively weak nogold labeling using anti-p95 (data not shown). We con- labeling of two dots, frequently of unequal size, in the re- clude that the sf-fibers are the primary location of the 95-kD gion of the basal apparatus (Fig. 6, c, d, and f). To localize protein in the basal apparatus of S. similis. the 95-kD protein more precisely, we performed immu- nogold electron microscopy (preembedding technique) on Structure of the sf-Fibers isolated cytoskeletons from S. similis (Fig. 7). In samples treated with anti-rec95, cone-like structures associated The sf-fibers connect each of the two basal bodies with the with the basal bodies were decorated (Fig. 7, a and f). The corresponding bundle of five microtubules. The latter are immunoreactive structures were identified as the sf-fibers part of the cruciate microtubular flagellar root system that link the basal bodies with the five-stranded microtu- characteristic for Chlamydomonas-like flagellates, which bular roots (s-roots). The intense labeling of the exposed in the case of S. similis, consist of two two-stranded (d-type) surface was most clearly seen in cross-sections through the and two five-stranded (s-type) microtubular bundles (Prei- sf-fibers (Fig. 7, h and i). Previously, we have shown that sig and Melkonian, 1984). The 1sf-fiber is attached to basal the two sf-fibers present in a basal apparatus differ in size body No. 1 in a subdistal position and extends about 200 nm depending on their association with the developmentally towards the s-type root (Fig. 8, a and b). The connection to older (basal body No. 1) or younger (No. 2) basal body the basal body involves 1–3 triplets depending on the level (Geimer et al., 1997a; Fig. 8). Immunogold labeling of the of section (Fig. 7, e and f; Fig. 8 b), and the junctions with small 2sf-fiber attached to basal body No. 2 is depicted in at least some of the basal body triplets are formed by sev- Fig. 7, c and d; sections through the prominent 1sf-fiber eral thin filaments (Geimer et al., 1997a). Similar filaments are shown in Fig. 7, a, b, and e–i. Extensive analyses of im- are present between the part of the sf-fiber associated with munolabeled cytoskeletons revealed no specific reaction the basal body and its distal part which overlies the flagel- of anti-rec95 with any other component of the basal appa- lar root microtubules, suggesting a flexible junction (Fig. 8 ratus and preimmune controls showed no specific labeling a). The 1sf-fiber exhibits a cross-striation consisting of broad

The Journal of Cell Biology, Volume 140, 1998 1154 Figure 7. Ultrastructural lo- calization of the 95-kD pro- tein in S. similis by immu- nogold labeling of isolated cytoskeletons using the preem- bedding technique. Cytoskel- etons treated with anti-rec95 (a, c–f, h, and i) or preim- mune serum (b and g). (a and b) Longitudinal section through No. 1 basal bodies with attached 1sf-fiber (ar- rowheads). The sf-fiber in a is decorated with 15-nm gold particles, no labeling is visi- ble in b (preimmune control). Open arrow in a, distal con- necting fiber. (c and d) the 2sf-fiber (arrowheads) in lon- gitudinal section (c) or cross- section (d). (e and f) Two con- secutive cross-sections through the basal body No. 1 and the 1sf-fiber. Arrow, five-stranded root. Note that the sf-fiber is Downloaded from attached to only one triplet in the more distal section (e). (g) Similar section as in f, but the preimmune control. (h and i) Cross-sections through the 1sf-fiber revealing an intense labeling of the exposed surface. Arrow in h, five-stranded roots with characteristic four over one-pattern of the microtubules. Bar, 200 nm. jcb.rupress.org electron-translucent and electron-opaque bands (Fig. 8, a protein decorate two dots in the basal apparatus of C. rein- and b). The 2sf-fiber resides in a proximal position on hardtii in indirect immunofluorescence (not shown). A nm long (Fig. 8 c). For a possible explanation for the low species cross-reactivity of-80ف basal body No. 2 and is only detailed analysis of the structure of the sf-fibers by serial the antibodies to the 95-kD protein could be the low se- ultrathin sections, see Geimer et al. (1997a). After intensive quence conservation characteristic for many coiled-coil on September 9, 2017 mechanical disintegration of basal apparatus, we still ob- proteins. This is also true for SF-assemblin, the major pro- served complexes of basal bodies and attached sf-fibers, tein of the noncontractile striated roots in green flagel- indicating that the 1sf-fibers are linked firmly to the basal lates, which has only 54% identity of the amino acid se- bodies (Fig. 8, d and e). quence between S. similis and C. reinhardtii (Lechtreck and Silflow, 1997), but obviously has homologues in or- Discussion ganisms as phylogenetically distant as Giardia lamblia (Weber et al., 1993). We have identified a 95-kD protein as a component of the The sf-fibers are the third nontubulin filament system basal apparatus of the green flagellate Spermatozopsis si- identified in the green algal basal apparatus (Fig. 9). They milis. Analysis of the deduced sequence of the 95-kD pro- are distinct from the previously characterized centrin- and tein showed a high probability for the formation of ex- SF-assemblin filaments in their biochemical composition tended coiled-coils. The 95-kD protein forms rod-shaped and putative function (Salisbury et al., 1984; Lechtreck particles in vitro and is located in the sf-fibers, which con- and Melkonian, 1991a). Centrin fibers maintain the spatial nect the basal bodies with the two five-stranded microtu- orientation of the basal bodies to each other and to the nu- bular roots. Furthermore, fibers reconstituted in vitro cleus. Further, centrin may be involved in controlling basal from the recombinant 95-kD protein resembled the sf-fibers body replication (Taillon et al., 1992). Because centrin fi- in that they had a cross-striation pattern consisting of bers are contractile, they also serve to reorientate the broad electron-translucent and electron-opaque bands. basal bodies (e.g., during reversal of swimming direction), We conclude that the 95-kD protein is the major structural to decrease the distance between the basal apparatus and component of the sf-fibers in S. similis. the nucleus, or to sever microtubules during flagellar auto- Until now there are only very limited data about the dis- tomy (McFadden et al., 1987; Salisbury et al., 1987; Sand- tribution in other organisms of proteins homologous to the ers and Salisbury, 1994). SMAFs run parallel to the micro- 95-kD protein from S. similis: no specific labeling was ob- tubular roots and may stabilize the flagellar root microtubules served in various green algal (Dunaliella bioculata, Poly- and absorb any mechanical stress caused by the flagellar tomella agilis), protist (Tetrahymena), or animal cells (sea beat (Lechtreck and Melkonian, 1991b). The sf-fibers, fi- urchin sperm, 3T3 cells, rod cells of bovine retina, mouse nally, link the basal bodies to the five-stranded microtubu- trachea). However, the antisera raised against the 95-kD lar roots (s-roots; Beech and Melkonian, 1993). Functionally,

Geimer et al. Green Algal Basal Feet 1155 Figure 9. Cartoon of the basal apparatus of S. similis in top view. 1, older basal body; 2, younger basal body; 1d, 1s, 2d, and 2s, mi- crotubular roots with attached SMAFs. dcf, distal connecting fi- ber indicated by a dotted line. ey, eyespot apparatus. Three bio-

chemically distinct filament systems (centrin, SF-assemblin, and Downloaded from 95-kD protein) have been identified in the basal apparatus of flagellate green algae.

most flagellate cells that is thought to be maintained by the microtubular flagellar roots (Melkonian, 1984). Previ-

ous studies in green algae have shown that the 2s-root con- jcb.rupress.org nects the eyespot apparatus with the corresponding basal body (Holmes and Dutcher, 1989; Lechtreck et al., 1997). Thus, the eyespot is in a defined position with respect to the plane of flagellar beat, a situation important for photo- taxis (Kreimer, 1994). Further, the two s-roots often per- sist during mitosis and form a specialized structure, which on September 9, 2017 in Chlamydomonas is known as the metaphase band (Johnson and Porter, 1968). The s-root microtubules inter- connect the dividing basal apparatus in an antiparallel fashion and arc over the mitotic spindle (Gaffal and el- Gammal, 1990). Thus, the metaphase band and homolo- Figure 8. Structure of the sf-fibers. (a) Longitudinal section gous structures present in other green flagellates (Segaar through basal body No. 1 with 1sf-fiber. Arrow, filamentous part and Gerritsen, 1989; Lechtreck et al., 1997) may be involved of the 1sf-fiber. (b) Basal body and 1sf-fiber in cross-section. Note that the 1sf-fiber is attached to three triplets. Arrowhead, in the separation of the duplicated basal apparatus and the attachment sites of the df-fiber that links the basal body to the positioning of the spindle apparatus and the cleavage fur- two-stranded microtubular root. (c) Cross-section through the row (Ehler et al., 1995). Another important feature of the proximal parts of the basal bodies in antiparallel configuration sf-fibers is the difference in size between the two fibers (the left basal body [No. 2] is pointing towards the observer, the present in the basal apparatus of S. similis. The 1sf-fiber is No. 1 basal body away from the observer). The 2sf-fiber (arrow- much more prominent than its counterpart at the younger head), the 2s-root (arrowhead with 2), the 1sf-fiber (large arrow), basal body resulting in a rather unequal distribution of the one of the proximal plates (arrow with PP), and the cartwheels 95-kD protein to the two basal bodies. A similar distribu- (arrows with 1 and 2, respectively) are marked. (d and e) Isolated tion has been described for cenexin, a 96-kD protein of un- basal bodies obtained by mechanical disintegration of basal appa- known primary structure, that is located exclusively in the ratus with a French press at 300, 250, and 200 bar, consecutively (French Pressure Cell Press, 40,000 Psi Pressure cell; SLM Instru- pericentriolar material surrounding one of the two centri- ments Inc., Urbana, IL). The distal end of the basal bodies con- oles in the centrosome of mammalian cells (Lange and tains remnants of the axonemal microtubules. The 1sf-fibers are Gull, 1995). Interestingly, as described for cenexin, the labeled with arrowheads. Bar, 200 nm. prominent sf-fiber is a marker for the older basal body/ centriole. Furthermore, monoclonal antibodies directed against Spc110p from the yeast spindle pole body identi- the sf-fibers could determine the position of the microtu- fied an immunologically related 100-kD protein at the bular roots with respect to the basal bodies and guide the proximal end of centrioles of mammalian cells, sometimes s-roots into a specific direction. This seems important with more intensely decorating the parental centriole (Tassin et respect to the stereotyped internal order characteristic for al., 1997). In summary, the link formed by the sf-fibers be-

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