An Sfi1p-Like Centrin-Binding Protein Mediates Centrin-Based Ca2+-Dependent Contractility in Paramecium tetraurelia

The previous characterization and structural analyses of Sfi1p,a Saccharomyces cerevisiae centrin-binding protein essentialfor spindle pole body duplication, have suggested molecularmodels to account for centrin-mediated, Ca²⁺-dependent contractilityprocesses (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson,R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877,2006). Such processes can be analyzed by using Paramecium tetraurelia,which harbors a large Ca²⁺-dependent contractile cytoskeletalnetwork, the infraciliary lattice (ICL). Previous biochemicaland genetic studies have shown that the ICL is composed of diversecentrin isoforms and a high-molecular-mass centrin-associatedprotein, whose reduced size in the démaillé (dem1)mutant correlates with defective organization of the ICL. Usingsequences derived from the high-molecular-mass protein to probethe Paramecium genome sequence, we characterized the PtCenBP1gene, which encodes a 460-kDa protein. PtCenBP1p displays sixalmost perfect repeats of ca. 427 amino acids (aa) and harbors89 potential centrin-binding sites with the consensus motifLLX₁₁F/LX₂WK/R, similar to the centrin-binding sites of ScSfi1p.The smaller (260-kDa) protein encoded by the dem1 mutant PtCenBP1allele comprises only two repeats of 427 aa and 46 centrin-bindingsites. By using RNA interference and green fluorescent proteinfusion experiments, we showed that PtCenBP1p forms the backboneof the ICL and plays an essential role in its assembly and contractility.This study provides the first in vivo demonstration of the roleof Sfi1p-like proteins in centrin-mediated Ca²⁺-dependent contractileprocesses.

INTRODUCTION

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INTRODUCTION

Centrins are EF-hand calcium-binding proteins conserved in alleukaryotes, where they localize as a discrete component at microtubule-organizingcenters. Their implication in the duplication of the spindlepole body (SPB) in Saccharomyces cerevisiae (6, 41), of thecentrosome in mammalian cells (6, 26, 37), and of basal bodiesin ciliates and flagellates is well documented (21, 31, 39,43, 45). In unicellular organisms, centrins are also associatedwith different organelles, like cilia (14, 15) and contractilevacuole pores (43), and are major constituents of diverse contractileorganelles, such as the striated flagellar roots of Tetraselmisstriata, where centrin was first characterized (34); the nucleus-basalbody connector and the striated fibers of Chlamydomonas reinhardtii(35); the spasmoneme of Vorticella convallaria (2); and theinfraciliary lattice (ICL) of Paramecium tetraurelia (11). Likecalmodulin, centrins act as effectors of different Ca²⁺-mediatedprocesses, and understanding their mode of action requires theidentification of their partner(s).

Among the proteins associated with centrins, Sfi1p previouslyidentified in S. cerevisiae (19) is of particular interest.Sfi1p interacts with the yeast centrin Cdc-31p and localizesto the half-bridge of the SPB. The study of different mutantalleles of SFI1 showed that this gene plays an essential rolein SPB duplication both in the formation of the new half-bridge(19, 22) and in the processes allowing the separation of thenew SPB from the old one (3, 44). Sfi1p, a 120-kDa polypeptide,presents 21 motifs with the consensus sequence AX₇LLX₃F/LX₂WK/R,presently known as centrin-binding sites, and each repeat iscapable of binding to centrin at a 1:1 molar ratio (19, 22).The coexpression of 15 repeats and Cdc-31p leads to the in vitroformation of filaments that can be observed by electron microscopy.However, in this in vitro system, Ca²⁺ has no effect on thelength of the filaments (22).

By BLAST and motif searching for proteins with Sfi1 motifs,at least 23 Sfi1-like proteins in diverse organisms have beenidentified (22). The human homolog, HsSfi1p, contains 23 repeats,some of which have previously been shown to bind Cdc-31p aswell as mammalian centrins (19). The HsSfi1p-green fluorescentprotein (GFP) fusion localizes at the centrosome, suggestingthe conservation of function between yeast and human proteincentrin-binding sites (19).

The discovery of Sfi1p provides new clues to understand howcentrins intervene in Ca²⁺-mediated contractility. An attractivemodel proposes that the association of Sfi1p and centrins formscontractile fibers (19, 33): upon Ca²⁺ influx, centrins wouldundergo a conformational change leading to the torsion of theSfi1 molecule, to which they would remain attached, and, asa consequence, to the shortening of the fiber. This hypothesisfits the in vitro observation of the Ca²⁺-sensitive behaviorof the centrosome and its filamentous associated matrix: calciuminduces a decrease in the centriole diameter and a shorteningof the intercentriolar link that connects centrioles (27). Similarly,Ca²⁺-induced basal body reorientation on an isolated basal apparatusof C. reinhardtii was observed previously (16).

While the validity of this model implying the contractilityor the elasticity of Sfi1p-centrin complexes remains to be demonstratedfor SPB or centrosome duplication, it can be experimentallytested in the case of the cytoskeletal contractile arrays presentin unicellular organisms. The ICL of Paramecium is a Ca²⁺-associatedcontractile cytoskeletal network made of bundles of 4-nm filamentssubtending the whole cell surface (1). In previous biochemicaland genetic studies, we have identified, in addition to at least10 centrin isoforms (12, 24), a large centrin-associated protein(L-CAP; >350 kDa) as a major component of the ICL (20). Weshow here that L-CAP, hereinafter referred to as PtCenBP1p (forP. tetraurelia centrin-binding protein 1), is indeed an Sfi1p-likeprotein which presents the same remarkable succession of centrin-bindingsites over the whole length of the molecule. By using RNA interference(RNAi) and GFP fusion experiments, we demonstrated that PtCenBP1pis an essential constituent of the filamentous bundles thatform the ICL and, thus, provide the first experimental demonstrationof the models proposed by Salisbury (33) and Kilmartin (19)for the direct involvement of Sfi1p-like proteins in centrin-mediatedCa²⁺-dependent contractility.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Strains and culture conditions. Stock d4-2 of P. tetraurelia, the wild-type reference strain,was used in all experiments. Two mutants derived from the wild-typestock were also used: the démaillé (dem1) mutant,which contains a monogenic nuclear mutation affecting the organizationand/or the dynamics of the ICL (20), and the ${Delta}$ -PtCenBP1 cellline, described herein, which carries an epigenetically maintaineddeletion of the macronuclear PtCenBP1 gene (see below). Cellswere grown at 27°C in a wheatgrass infusion (BHB; l'Arbrede Vie, Luçay Le Male, France) or wheatgrass powder (PinesInternational, Lawrence, KS) bacterized with Klebsiella pneumoniaeand supplemented with 0.4 µg of ß-sitosterol/mlaccording to standard procedures (42).

In vivo contractility assay. Aminoethyldextran (AED) is a vital secretagogue that inducesthe exocytosis of trichocysts, the secretory vesicles dockedbeneath the plasma membrane (28). Exocytosis is accompaniedby a transient intracellular Ca²⁺ influx (18), which provokesthe contraction of the ICL, resulting in cell contraction. Forin vivo observations, cells were transferred into a mixtureof 10 mM Tris buffer, pH 7.3, and 40 µM CaCl₂ containing3 µM AED. Cell contraction, which persists for ca. 30s, is easily monitored under a dissecting microscope.

Peptide sequencing. One hundred micrograms of an ICL-enriched fraction (11) wassubjected to limited trypsin proteolysis in a solution of 0.1M ammonium carbonate, pH 8.0, and 5 mM CaCl₂ (with 0.17-mg/mltrypsin from Boehringer) for 5 min at 25°C. Proteolysiswas stopped by the addition of 0.05 mg of phenylmethylsulfonylfluoride (Sigma)/ml. The digested fragments were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE),and the two heaviest major bands (45 and 40 kDa), generatedby the proteolysis of the heaviest protein, were electroblottedonto Problott membranes (Applied Biosystems, CA) and sequencedon a 470A protein sequencer (ABI; Applied Biosystems, CA) usinga Problott cartridge. Phenylthiohydantoin amino acids were identifiedusing an online analyzer (ABI 120 A). For the determinationof internal sequences, gel bands were digested by endoproteinaseAspN according to the method described in reference 29. Peptideswere purified by reverse-phase high-performance liquid chromatographyand sequenced.

Gene silencing by feeding. The gene sequences of interest were amplified by PCR and clonedinto the L4440 feeding vector between two T7 promoters (46).For the CenBP1 gene, we amplified the region encompassing positions2627 to 4768 with the following primers: 5'-ATTTGGAATCCATTATACGA-3'(sense) and 5'-TGTGTGTATTCTTATGGTAT-3' (antisense). The resultingplasmid was used for the transformation of HT115, an RNase III-deficientstrain of Escherichia coli with an isopropyl-ß-D-thiogalactopyranoside(IPTG)-inducible T7 polymerase (38). Wild-type paramecia wereincubated in double-stranded RNA-expressing bacteria, as previouslydescribed (9), and observed over successive days through dailytransfer into freshly induced feeding bacteria. For negativecontrols, Paramecium cells were fed with HT115 carrying theL4440 plasmid without the insertion.

Expression vector for GFP fusion proteins and transformation. The GFP synthetic gene sequence designed by E. Meyer and J.Cohen (unpublished data) was cloned at its 3' extremity intothe plasmid pPXV (32). The part of the PtCenBP1 gene encodingamino acids (aa) 59 to 1344 was amplified using primers 5'-TTAAGGTACCGGAGGAATGATCGAGAAAAAAATAGATGTCTCand 3'-TTTTTGGTACCTCAAATAATAGCTCTCTTCTTCAAAGC and cloned intothe KpnI site (underlined) of pPXV-GFP. Plasmid DNA was preparedwith a plasmid midi kit according to the protocol of the manufacturer(QIAGEN, Courtaboeuf, France). Microinjection of the DNA wascarried out as previously described (13).

Fluorescence microscopy. The immunostaining of cells was carried out as previously described(20). The monoclonal antibody 1A9, raised against the ParameciumICL (7), was used at a dilution of 1:200; the polyclonal anticentrinantibody 26/14-1 (9), the monoclonal antitubulin antibody ID5(47), and the secondary anti-mouse and anti-rabbit antibodiesfrom Jackson ImmunoResearch Labs (West Grove, PA) were usedat a dilution of 1:500. For the recording of GFP expressionin living cells, cells were washed twice in Dryl's buffer (46)containing 0.2% bovine serum albumin and then transferred ina small drop onto a coverslip and overlaid with paraffin oil.Alternatively, GFP-labeled paramecia were fixed in 2.5% formaldehyde.Cells were observed under an Axioskop 2-plus fluorescence microscopeequipped with a Roper Coolsnap-CF intensifying camera (Zeiss,Oberkochen, Germany) with GFP filters. Images were processedusing the Metamorph software (Universal Imaging, Downington,PA).

Electron microscopy. Cell pellets were fixed in 2% glutaraldehyde in 0.05 M cacodylatebuffer for 90 min at 4°C. Fixed cells were washed threetimes in the same buffer and postfixed in 1% (wt/vol) osmiumtetroxide in 0.05 M cacodylate buffer for 1 h at 4°C. Afterdehydratation, thin sections were contrasted with saturatedethanolic uranyl acetate and lead citrate and examined witha Philips CM10 microscope.

Computational sequence analysis. Sequences were compared by ClustalW alignment (http://www.ch.embnet.org/software/ClustalW.html).Dot plots were established by Self-Matrix from DNA Strider (CEA,France). Sequence logos (8) was developed using WebLogo (http://weblogo.berkeley.edu/logo.cgi).The two-dimensional structure analysis was performed with PredictProtein(http://www.predictprotein.org). PROFsec or PHDsec (30) analyzesthe ${alpha}$ -helicoidal content of a protein. The probability of a coiled-coilstructure was calculated according to the prediction programsPepcoil and COILS, version 2.2, using the MTIDK matrix withdifferent weights for positions a through g, as follows: a andd, 2.5, and b, c, e, f, and g, 1.0. The prediction was madeusing a scanning window of 28 residues (23).

Nucleotide sequence accession number. The PtCenBP1 gene sequence was deposited in the EMBL databaseunder accession number CR932274.

RESULTS

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RESULTS

The ICL of P. tetraurelia forms a conspicuous cytoskeletal networkwhose Ca²⁺-dependent contractility can be observed in vivo uponCa²⁺ influx (Fig. 1). Biochemical analysis of purified ICL fractionshad previously established that the building blocks of the networkare complexes of different centrin isoforms with a large, >350-kDa,centrin-associated protein and that these complexes are stablein the absence of Ca²⁺ (20). In vivo evidence for the role ofL-CAP in the organization of the ICL was provided by the propertiesof the dem1 mutant, which displays a shorter L-CAP polypeptideand looser organization of the ICL (Fig. 2). The characterizationof the corresponding gene led us to rename the L-CAP gene PtCenBP1(for P. tetraurelia centrin-binding protein 1).

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FIG. 1. The Ca²⁺-dependent contractility of the ICL. Cells were immunolabeled with the monoclonal anticentrin antibody 1A9 (7). (Left panel) Ventral side of a wild-type resting cell. (Right panel) Cell labeled during the Ca²⁺ influx induced by AED treatment (see Materials and Methods), which triggers the contraction of the ICL, resulting in a general contraction of the cell. Scale bar, 10 µm.

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FIG. 2. Phenotypical and biochemical features of the dem1 mutant. (Left panel) Wild-type (wt; left) and dem1 mutant (right) cells were immunolabeled with the monoclonal anticentrin antibody 1A9. Scale bar, 10 µm. The ICL of the dem1 mutant is characterized by a reduced number of meshes and an apparent deficit in branching. This phenotype is exhibited only by cells in growing populations, while cells in stationary phase reach a wild-type phenotype. (Right panel) Sodium dodecyl sulfate-PAGE pattern of purified ICL from wild-type and dem1 mutant cells. The gel is a 6 to 15% acrylamide gradient stained with Coomassie blue. A striking difference in the bands corresponding to the high-molecular-mass protein of wild-type and dem1 mutant cells, at 450 and 250 kDa (stars), respectively, is observed, whereas the same pattern for the low-molecular-mass bands (bracket), which correspond to centrins, is seen for both cell types. The presence of a doublet at the PtCenBP1-dem1p position may be due to proteolysis or to an unknown posttranslational modification.

Characterization of the PtCenBP1 genes from the wild type and the dem1 mutant. To identify the gene encoding PtCenBP1p, the Paramecium genomesequence (4, 5) was probed by using BLAST with four sequencesgenerated by tryptic proteolysis of the protein (Fig. 1 andMaterials and Methods). As the BLAST search targeted two adjacentgene models (GSPATG00034434001 and GSPATG00034433001) correspondingto products whose sizes (714 and 1,233 aa, respectively) werenot compatible with the observed size of the high-molecular-massprotein of more than 350 kDa, the genomic region was reassessed.The steps to the characterization of the wild-type gene organizationand the characterization of the mutation present in the dem1mutant PtCenBP1 allele are presented in the supplemental material.The wild-type PtCenBP1 gene turned out to be 11,862 bp longand to encode a 3,893-aa protein with a theoretical molecularmass of ca. 460 kDa. This protein contains six almost perfectrepeats (P1 to P6), each with 427 aa, except for P1, with 428aa, and P6, with 419 aa (Fig. 3). PtCenBP1p has a pI of 10.1owing to a high content (18.5%) of the basic amino acids K andR. According to secondary-structure predictions, PtCenBP1p isan all-alpha protein that is ca. 86% ${alpha}$ -helical. Coiled-coil arrangementswere detected in the N-terminal part, between residues 210 and245, and in the C-terminal part, between residues 3814 and 3867,with a high probability (>90%). These characteristics arecompatible with a filamentous protein able to form homopolymers.

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FIG. 3. Analysis of the PtCenBP1p protein in the wild-type and dem1 mutant cells. The wild-type (Wt) PtCenBP1p sequence (orange rectangles) is composed of six repeats (P1 to P6) plus a divergent repeat (P0) and an incomplete repeat (P7). The mutant PtCenBP1-dem1p (CenBP1p-dem1) sequence derives from the wild-type sequence by the deletion of repeats P3 through P5. For both sequences, the positions of the sequenced peptides are indicated by colored boxes, red, black, blue, and green for the peptides KFERTLDILFRV/S(K)LKVSFDPLKE(I)YMXALNIK(T)M(G)LKKLF, DXAQALKKRAIXLMIKLQ, ETQLNKFTLXI, and DSNLRYFFMK, respectively. Vertical bars represent the centrin-binding motifs as defined by Kilmartin (19) and indicate whether the diagnostic hydrophobic amino acid is a tryptophan, a leucine, a tyrosine, a phenylalanine, or a methionine or valine.

We first looked for 23-aa centrin-binding motifs by using theconsensus previously defined by Kilmartin, with a diagnostictryptophan in position 22 (19). PtCenBP1p contains 44 such sites,with the consensus sequence KX₄QXG/SX₄A/TFX₂WK/R (Fig. 4). Thisconsensus is clearly similar to that of the Sfi1 proteins ofS. cerevisiae, Schizosaccharomyces pombe, and H. sapiens previouslydescribed as being a centrin-binding domain and further demonstratedto act as a centrin-binding site in vitro (19). We then enlargedthe search by allowing L, Y, F, M, or V in addition to W atthe diagnostic position 22. This allowance led to the recognitionof a total of 89 centrin-binding sites, which share a shorterconsensus sequence, LLX₁₁F/LX₂WK/R (Fig. 4). Comparison of thePtCenBP1p centrin-binding sites with those of yeast and of ahomologous protein found in the annotation of the Tetrahymenathermophila genome (corresponding to gene XM_001030914.1) showsthat the most conserved residues are the C-terminal ones, F/LX₂WK/R,which appear to be the true signature of the consensus. Thisfinding is consistent with the crystal structures showing contactsbetween these conserved residues and helices VI and VII of theC-terminal centrin domain (22). Since the ICL is built up froma diverse multigene family of centrins, it is possible thatdifferent subclasses of these sites are specific for given ICLcentrin subfamilies.

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FIG. 4. Sequence logos of the Sfi1 repeats in S. cerevisiae and homolog in P. tetraurelia. Among the potential centrin-binding sites identified in PtCenBP1p, 44 present a diagnostic W at position 22 in the 23-aa sequences defined as centrin-binding sites by Kilmartin (19). A less stringent definition of the consensus sequence, allowing L, Y, F, M, or V in addition to W at the diagnostic position 22, led to the identification of a total of 89 potential centrin-binding sites. The two sets of centrin-binding sites (44 and 89 consensus sites, respectively) were submitted to WebLogo. For comparision, logos corresponding to two sets of consensus sequences from S. cerevisiae are shown: a first set of 17, each with a W or F at position 22 (19), and a second set including all 21 centrin-binding sites recognized in Sfi1p (22) and showing more variability at position 22.

The mutation present in the dem1 strain was found to correspondto a large deletion of ca. 5,000 bp (see Fig. S1 in the supplementalmaterial). The conceptual translation of the 6,738-bp dem1 mutantPtCenBP1 gene gives a 2,186-aa protein with a theoretical molecularmass of ca. 260 kDa and a pI of 10.1. The dem1 mutant proteinPtCenBP1-dem1p lacks the central repeats (P3 through P5), retaining,however, 46 potential centrin-binding sites. Previous analyses(20) had shown that the patterns of centrin isoforms presentin the ICL were the same in dem1 mutant and wild-type cells,consistent with the finding that only the number of each bindingsite, and not the specificity of the binding sites, is modifiedin the mutant.

Functional analysis of PtCenBP1p: RNAi experiments. RNAi targeting the ICL1a gene, which codes for one of the constitutivecentrins, was previously shown to cause the total disassemblyof the ICL, without any other effect on cell growth or morphology(7). This dispensability of the ICL provides a favorable situationfor its functional analysis. To investigate the role of PtCenBP1p,we silenced the corresponding gene in both wild-type and dem1mutant cells by an RNAi approach using feeding (9). PtCenBP1-silencedcells grew normally without visible defects in swimming behavior,shape, or generation time. However, as early as two divisionsafter inoculation into the feeding medium, cells were no longerable to contract upon AED-induced Ca²⁺ influx (Fig. 1 and Materialsand Methods), suggesting the disappearance of the ICL. Immunolabelingwith anticentrin antibodies allowed us to monitor the disassemblyof the network (Fig. 5). Although both wild-type and mutantcells continued to grow normally, disassembly was complete for100% of the cells examined (i.e., 80 to 120 cells in each offive independent RNAi experiments) after three to four divisionsfor the wild type and slightly sooner for dem1 mutant cells.As shown in Fig. 5C, silenced cells displayed only ICL remnants,in the form of anticentrin-reactive spots close to each basalbody, morphologically identical to the previously describedICL-organizing centers (ICLOCs) observed after RNAi in the ICL1agene (7).

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FIG. 5. Disassembly of the ICL upon PtCenBP1 silencing by RNAi as visualized by using immunofluorescence. Disassembly triggered by PtCenBP1p depletion was monitored using the anticentrin antibody 1A9. (A) Ventral side of a wild-type cell before the start of the experiment. (B) Ventral side of a cell after 24 h (ca. three cell divisions) of gene silencing. The cell presents a disrupted ICL in which only the longitudinal part of the meshes is still present and the transversal branches are missing. (C) Ventral side of a cell after 48 h (ca. six to seven fissions) of CenBP1p depletion. The disassembly is complete. Double labeling with a polyclonal anticentrin antibody (26/14-1) and a monoclonal antitubulin antibody (ID5) reveals both basal bodies (red) and the remnants of the ICL (green), which form discrete masses of amorphous material, shown to be reactive to anticentrin antibodies and to be the sites of ICL reassembly, the ICLOCs (7). Enlargements of representative areas selected from panels A, B, and C are presented in the lower panels. Scale bar, 10 µm.

The RNAi effect could be reversed by transferring the cellsback to normal growth medium. The reassembly of the networkstarted from the ICLOCs, as previously described (7). Aftera few divisions, cells reacquired a complete contractile ICL.Conversely, a stable ICL-less condition can be established.When cells are subjected to RNAi during the nuclear reorganizationaccompanying sexual processes, some clones develop a new macronucleus(somatic nucleus) in which at least part of the target geneis deleted, although their micronulei (germinal nuclei) stillretain the wild-type gene (10). Such clones carrying a deletionof PtCenBP1 in the macronucleus were obtained (data not shown)and were observed to be devoid of the ICL. These clones, designated ${Delta}$ -PtCenBP1, could be maintained under normal culture conditionsthrough cellular continuity over hundreds of cell divisions,and the phenotype was inherited maternally in crosses. Observationsof a ${Delta}$ -PtCenBP1 cell line by electron microscopy (Fig. 6B) confirmedthat the meshes constitutive of the network were absent andthat small masses of amorphous material corresponding to theICLOC, as observed by anticentrin immunolabeling, were leftnear the basal bodies. Some small electron-dense triangularstructures, the posts (1), normally located at the branchingpoints of the meshes and possibly serving as anchoring pointsfor the filament bundles in the native network, were observedwithin the ICL remnants (Fig. 6B). Spontaneous reversions toa contractile phenotype can occur during nuclear reorganization.Reversions always correlate with the restoration of wild-typecopies of the PtCenBP1 gene within the maronucleus (data notshown). These experiments demonstrated that the assembly ofthe ICL, i.e., the organization of the residual masses of centrin-containingmaterial into filaments, was directly dependent upon the availabilityof the PtCenBP1 gene product.

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FIG. 6. Disassembly of the ICL upon PtCenBP1 silencing by RNAi as shown by electron microscopy. Tangential thin sections of the cortex show the differences between the organization of the ICL in a control cell (A) and that in a cell after the inactivation of the PtCenBP1 gene (B). (A) The polygonal meshes surrounding basal bodies (bb) with their attached ciliary rootlet (cr) and secretory vesicle tip (t) are composed of branched filamentous bundles. At the branching points, note the presence of one or two dense structures (arrows) called posts (1). (B) In the inactivated cells, the meshes of the network are no longer present. Only small pieces of amorphous material (stars) are visible to the right of each basal body: the enclosed posts (arrows) ensure that they are genuine ICL remnants. Note the mitochondria (m), which are always found at the level of the proximal part of basal bodies in the absence of the ICL, while they are found deeper in the cytoplasm in wild-type cells when the ICL is present (our unpublished observations). Scale bar, 0.30 µm.

In vivo localization of GFP-PtCenBP1p. In order to determine the localization of PtCenBP1p, a sequenceencoding a GFP tag was fused to the part of the PtCenBP1 geneextending from positions 226 to 4160. The gene segment encodeda ca.-150-kDa polypeptide spanning aa 59 to 1344 and containingthe presumed coiled-coil region of the N terminus and 30 consensuscentrin-binding sites. The pPXV vector containing this constructionwas used to transform, by microinjection, both wild-type and ${Delta}$ -PtCenBP1 cells. In the transformed wild-type cells, the GFPsignal homogeneously localized to the preexisting network fibersas observed on living cells. GFP labeling of the ICL was detectedas early as 3 h after microinjection (Fig. 7A). In living cells,in addition to the network labeling, strong background fluorescencethroughout the cytoplasm was visible, most likely due to excessmolecules in the cytosol and possibly also to the fact thatthe truncated GFP construct may have had lowered affinity forthe network. Interestingly, when we processed the cells forlabeling with anti-GFP antibodies in order to enhance the GFPsignal, this signal was lost. However, if 1 mM Ca₂Cl was addedto the permeabilization-fixation buffers, the GFP signal waspreserved and could be revealed by the anti-GFP antibodies.The Ca²⁺-dependent rescue of the GFP signal showed that theincorporation of the GFP fragment into the network was Ca²⁺sensitive since the GFP signal was detected in vivo, destabilizedin the standard EGTA-containing permeabilization-fixation buffer,and stabilized if the calcium concentration was raised. Underthe latter conditions, as in vivo, the GFP label localized preciselywith the centrin label along the extant ICL as shown in Fig.7B to D.

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FIG. 7. GFP-tagged PtCenBP1p localizes at the ICL. Transformants expressing a GFP-tagged fragment of PtCenBP1p comprising aa 59 to 1344 were examined in vivo and after immunolabeling with anti-GFP antibodies. (A) Ventral and dorsal faces of a living cell showing the homogeneous fluorescence of the ICL. (B to D) Transformants after permeabilization and fixation in the presence of 1 mM Ca²⁺ and double labeling with a polyclonal anti-GFP antibody and the monoclonal anticentrin antibody 1A9. (B) Anti-GFP staining. (C) Anticentrin staining. (D) Merged images. Scale bar, 10 µm.

In ${Delta}$ -PtCenBP1 cells transformed with pPXV, expressing GFP fusedto the product of positions 226 to 4160 of the PtCenBP1 gene,the GFP signal specifically localized near basal bodies at thesites of the ICLOCs (data not shown), where ICL centrins localizeeven in the absence of the network. No rescue of the phenotypewas observed, probably owing to the small size of the PtCenBP1gene fragment used and/or the absence of the N- and C-terminalparts of the gene product.

DISCUSSION

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DISCUSSION

We have used the ICL of P. tetraurelia to study the molecularmechanisms of Ca²⁺-mediated centrin-based contractility. Wepreviously demonstrated that the building block of the ICL wasa complex of different centrin isotypes and a high-molecular-massprotein, L-CAP (20). We report here the characterization ofthe L-CAP gene and its functional analysis. We showed that L-CAP,now renamed PtCenBP1p, is a 460-kDa, Sfi1p-like protein featuring89 motifs of 23 aa, previously described by Kilmartin (19) ascentrin-binding consensus sites. We demonstrated that PtCenBP1pforms the backbone of the contractile ICL by three convergingexperimental approaches and in vivo observations: (i) the monogenicrecessive dem1 mutation, which causes a looser organizationof the network (Fig. 2), corresponded to the deletion of 43consensus centrin-binding sites; (ii) in cells depleted of PtCenBP1pfollowing RNAi treatment, the ICL was totally disassembled butreassembled upon return to normal growth conditions; and (iii)a GFP-tagged fragment of PtCenBP1p comprising aa 59 to 1344localized with the whole network in a Ca²⁺-dependent manner.

We will first examine to what extent the present findings accountfor all previous data on the biochemical composition, cytologicalorganization, and contractile properties of the ICL. Then wewill discuss how these results fit present models concerningthe structure and function of Sfi1p-centrin complexes (19, 25).

PtCenBP1p as the backbone of the ICL. In Paramecium, different centrins are present in different organellesand ensure different functions, not only in the ICL but alsoin basal body positioning (31) and in ciliary Ca²⁺ channels(14). While these diverse functions are also likely to involvecentrin-binding proteins, our data show that the protein characterizedhere, PtCenBP1p, is specific to the contractile ICL, since itsdepletion results only in ICL disassembly without any effecton cell growth, morphology, or behavior. Conversely, most ofthe centrins of the ICL are specific to this structure: we havepreviously shown that ICL organization is not affected by thesilencing of the basal body-specific centrins (31) and thatdifferent monoclonal antibodies against ICL centrins do notcross-react with basal bodies (7). Compared to the half-bridgeof the SPB, the ICL stands out by virtue of its dimensions,which parallel the huge size of PtCenBP1p and the diversityof its constitutive centrins (20, 31). As first described byAllen (1), the ICL forms a subcortical, transcellular reticulumof polygonal meshes running around each basal body or pair ofbasal bodies and around the tips of trichocysts, the secretoryvesicles docked at the cell surface (Fig. 6A). Each mesh correspondsto a bundle of 3- to 4-nm-diameter microfilaments. Filamentbundles merge at one apex of the polygons, marked by a singulartriangular structure, the post (12), which can be viewed asthe branching or anchoring point of the network (Fig. 6A). Thelength of filament bundles varies from 0.3 to ca. 2 µm.In the images of the contracted network (as in Fig. 2), themeshes are 20 to 25% shorter. The molecular data presented hereare in good agreement with these cytological data. The characterizationof PtCenBP1p as a 3,893-aa protein of an essentially ${alpha}$ -helicalstructure makes it reasonable to postulate that each moleculeis ca. 600 nm long (a maximal value, calculated for a fullyextended helix), and assuming that the numerous centrin isoformsknown to colocalize to the ICL (7; D. Gogendeau et al., unpublishedresults) occupy their potential binding sites, the width ofthe PtCenBP1p molecule would be ca. 4 nm, as shown by the crystalstructures of Sfi1p-centrin complexes (22). Furthermore, thepredicted coiled-coil arrangement of the C and N termini ofPtCenBP1p may be compatible with end-to-end interactions asshown for Sfi1p in the duplicating SPB (22). Clearly, the observed(3- to 4-nm) width of the unit filament within the ICL meshesin electron micrographs corresponds to the estimated width ofthe PtCenBP1p helix with its bound centrins, while the estimatedlength of the complex, 600 nm, falls within the size range ofthe shortest meshes in the resting network.

Biochemically, the unit complex (one PtCenBP1p molecule plus89 centrin molecules) would have an estimated molecular massof ca. 2,700 kDa. The size previously measured by the classicalmethods (liquid chromatography, ultracentrifugation, and PAGE)had led to a size estimate of more than 1,000 kDa for the elementaryprotein complex obtained by the dissociation of the purifiedICL in the absence of Ca²⁺ (20).

PtCenBP1p would then form the backbone of the ICL, in agreementwith the continuous and homogeneous GFP labeling of the network(Fig. 7). Finally, it is of interest to examine how the characteristicsof the ICL in dem1 mutant cells (Fig. 2) can be accounted forby the reduced size of the mutant protein. With the length ofmutant building blocks being half of that found in the wildtype, the deletion of the central repeats may affect the flexibilityof the bundles of filaments and lead to reduced or delayed branchingactivity. However, the question remains open as to the mechanismof formation of the branches and the nature of the posts.

Molecular mechanisms of centrin-based contractility. Different models for the organization of Sfi1p and centrin havebeen proposed previously (19, 22, 25, 33, 41). Centrins boundto Sfi1p by their C-terminal moiety, but also by their freeN-terminal moiety (41), are able to interact with other centrinslocated on the same Sfi1p molecule (22). Centrins may also interactwith other centrins bound to a different Sf1p molecule (25),owing to their strong tendency to self-assemble (48). In allcases, the binding of Ca²⁺ onto centrins would induce conformationalchanges triggering or enhancing the interactions between centrinsand resulting in the shortening of the filament. Moreover, becausethese filaments are highly ${alpha}$ -helicoidal, it is possible thatinteractions between adjacent centrins bound along Sfi1p filaments(22) induce the supercoiling of the filament. This scenariocorresponds to the idea first proposed in the case of the contractionof the rhizoplast of Platymonas subcordiformis, that centrin-basedmotility involves filament supercoiling (36).

In this conceptual framework, what information does the ICLcontribute? For the general consensus that the association betweenthe Sf1p-like protein and centrins is not Ca²⁺ dependent (17,19, 25), the ICL provides experimental confirmation: the completedissociation of the isolated network in the absence of Ca²⁺(Ca²⁺ concentration, 10^–8 M) could not dissociate centrinsfrom the high-molecular-mass partner CenBP1p, as establishedby PAGE of high-speed pellets or exclusion chromatography. TheCenBP1p-centrin association was quite strong, as a partial dissociationof centrins from the complex could be achieved only in the presenceof 2 M urea (20).

Concerning the Ca²⁺ dependence of the interactions between centrins,both our in vitro and our in vivo observations indicate interactionsbetween centrins bound to adjacent Sfi1p-like proteins. Whenthe purified ICL has been completely dissociated in the absenceof Ca²⁺, raising the Ca²⁺ concentration to 10^–7 M (comparableto the resting-cell Ca²⁺ concentration) provokes the self-associationof the elementary complexes into filamentous aggregates (12).However, association into ordered bundles of filaments was notobserved in vitro. In vivo, the total disassembly of the networkupon the depletion of the ICL1a centrin (7) shows that centrinscontribute to the cohesion of the network and/or to the bundlingof the filaments. Furthermore, we have shown here that the integrationof GFP-tagged fragments of PtCenBP1p into the ICL filament bundlesrequired a Ca²⁺ concentration at least equal to that in restingliving cells (ca. 10^–7 M). The range of calcium dependenceobserved for the ICL proteins, both in vitro (20) and in vivoas shown here, is similar to the values obtained previouslyby others measuring interactions between Sfi1p (one centrinbinding site) and C. reinhardtii centrin at different Ca²⁺ concentrations(40). Evidence for interactions between centrins bound to differentSf1p-like proteins may not have general relevance, since theICL architecture involves filament bundling and may have specificrequirements for such lateral interactions. It is also possiblethat of the numerous centrin isotypes present in the ICL, someare more reactive than others for establishing intercomplexinteractions. Our observations show, nevertheless, that suchlateral interactions do occur and are likely to occur also inother systems since, in unicellular organisms, contractile organellessuch as the distal connecting fibers in C. reinhardtii all consistof bundles of filaments.

As depicted in Fig. 8, we propose that in unstimulated Parameciumcells, PtCenBP1p-centrin molecules are assembled longitudinallyin filaments with lengths that are multiples of 600 nm: onlythe high-affinity Ca²⁺-binding sites of the centrins are saturated,and there is no contraction. When the intracellular Ca²⁺ levelrises, the lower-affinity sites are recruited and their bindingprovokes conformational changes leading to the supercoilingand thickening of the filaments and to the shortening of thebundles. This idea is in contrast to the model proposed forthe contraction of the distal connecting fibers of Chlamydomonas(25), in which the contraction is proposed to occur perpendicularto the axis of the filaments. The large number of differentcentrins found in Paramecium as well as their diversity withrespect to the numbers and positions of the functional Ca²⁺-bindingsites (Gogendeau et al., unpublished) are likely to play a rolein the fine regulation of the contractile strength of the ICLand its adjustment to the mosaic organization and dynamics ofthe cortex.

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FIG. 8. Role of CenBP1p in the Ca²⁺-dependent contractility of the ICL. (A) Schematic representation, based on Fig. 1, of the ICL. The geometry of the polygonal ICL meshwork at resting (10^–7 M) and physiologically high (10^–6 M) Ca²⁺ concentrations is shown. The filament bundles that constitute the edges of the polygons are on average 25% shorter in the presence of high levels of Ca²⁺. (B) Schematic representation of one ICL polygon. Under resting conditions, the unit filament consisting of one PtCenBP1p molecule with associated centrins is likely to be $~$ 0.3 to 0.5 µm in length (depending on whether or not the Sfi-like backbone is a completely extended ${alpha}$ -helix). This is the distance between posts (red triangles) along the shortest polygon edges. The lateral association of unit filaments builds up the bundles. The N- and/or C-terminal coiled-coil domains may be involved in tethering the filament bundles to the posts. At high Ca²⁺ concentrations, the filament bundles shorten and the tension is relayed through the posts to yield a global reduction in the size of the ICL, and hence of the whole cell, with very little distortion of global cell shape (Fig. 1). (C) Schematic representation of a possible arrangement of PtCenBP1p and centrins. At resting Ca²⁺ levels, the ICL unit filament is probably a disordered helical array of $~$ 4-nm centrins (pink) wrapped around an essentially extended ${alpha}$ -helical PtCenBP1p molecule (gray), as originally proposed by Kilmartin for yeast Sfi1p-Cdc31p complexes (19). The binding of Ca²⁺ to the centrin high-affinity sites at in vivo Ca²⁺ concentrations favors the lateral interactions necessary for filament bundling. At high Ca²⁺ levels, the occupation of centrin low-affinity Ca²⁺-binding sites leads to interactions between adjacent centrins along the length of the filament, as seen in the yeast Sfi1p-Cdc31p crystal structures (22). The consequence is the shortening of the filaments, probably owing to reduced distances between adjacent centrins and the consequent bending, twisting, or supercoiling of the Sfi1-like PtCenBP1p backbone.

The ICL still raises a number of intriguing questions concerningits elaborate cellular organization and the specific roles ofits diverse centrins. However, the interactions between centrinsand Sfi1p-like proteins, presumed to mediate Ca²⁺-dependentcontractile processes in the SPB and centrosomes and in a varietyof contractile organelles in unicellular eukaryotes, are likelyto involve similar molecular mechanisms. The demonstration thatCenBP1p forms the backbone of the ICL, a contractile networkof Paramecium, provides the first direct evidence of the functionalassociation of centrins and centrin-binding proteins to formcontractile fibers, as proposed previously for the Sfi1p protein(22, 33).

ACKNOWLEDGMENTS

We are particularly grateful to Michel Bornens for first drawingour attention to Kilmartin's publication on the properties ofSfi1p (19) and for critical reading of the manuscript. We aremuch indebted to Maud Sylvain for DNA sequencing, to Jean-PierreDenizot (Institut A. Fessard, Gif-sur-Yvette, France) for electronmicroscopy facilities, and to R. Le Guyader for her participationin the artwork. We thank J. Wehland and J. Salisbury for thegenerous supply of antibodies.

This work was supported by the Centre National de la RechercheScientifique and by grant ACI:IMPBIO 2004 no. 14 (L.S.). D.G.was supported by the Association de la Recherche Contre le Cancer(A06/3).

FOOTNOTES

* Corresponding author. Mailing address: Centre de Génétique Moléculaire, UPR 2167, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France. Phone: 33 1 6982 4392. Fax: 33 1 6982 3181. E-mail: klotz{at}cgm.cnrs-gif.fr

Published ahead of print on 3 August 2007.

Supplemental material for this article may be found at http://ec.asm.org/.

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