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First published online 4 December 2007
doi: 10.1242/jcs.019414

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Functional diversification of centrins and cell morphological complexity

¹ CNRS, Centre de Génétique Moléculaire, UPR 2167, Gif-sur-Yvette, F-91198, France
² Université Paris-Sud, Orsay, Paris, F-91405, France
³ Université Pierre et Marie Curie, Paris 6, F-75005, France
⁴ Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw Pawinskiego 5a, Poland
⁵ Department of Biology, University of Warsaw, ul. Miecznikowa 3, 02-091 Warsaw, Poland

View larger version (78K): [in this window] [in a new window]   Fig. 1. Comparison of properties of the two families of centrin-binding proteins PtCenBP2/3 and PtCenBP1. (A) Alignment of the primary structures of PtCenBP1p and PtCenBP2/3p shows their regions of homology. R1-R6 correspond to the six identical repeats of 427 amino acids present in PtCenBP1 (Gogendeau et al., 2007). The proteins are highly similar throughout their length, except for the absence of the central repetitions in PtCenBP2/3p. (B) PtCenBP3p displays numerous potential centrin-binding sites that are similar to the centrin-binding consensus defined by Kilmartin (Kilmartin, 2003). The Weblogos of the 30 centrin-binding sites present in PtCenBP3 are compared with those corresponding to the sites present in the human Sfi1p and in PtCenBP1p (Gogendeau et al., 2007). (C) Wild-type cells expressing either GFP-PtCenBP3p (right) or GFP-PtCenBP1p (left) were double-labelled with anti-GFP antibodies (green) and the anti-centrin 1A9 antibody (red). In the GFP-PtCenBP3p expressing cells, the two labels do not colocalise, whereas in the GFP-PtCenBP1p-expressing cells, the two labels colocalise across the network. (D) Wild-type cells were submitted to RNAi targeted either to PtCENBP3 (right) or PtCENBP1 (left) and the organisation of the ICL monitored with the anti-centrin antibody 1A9. A collapse of the ICL is observed in a PtCENBP3-silenced cell after 48 hours. By contrast, in PtCENBP1-silenced cells (after 48 hours of feeding), the ICL is totally disassembled, leaving only small remnants that flank basal bodies. Scale bars: 15 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Phylogenetic tree of ICL centrins. The bootstrap support values from neighbour joining/maximum likelihood analyses are indicated for relevant nodes. /– indicates that some centrins are not grouped by the maximum-likelihood analysis. This tree is supported by the identification of diagnostic amino acid residues specific of each centrin subfamily (supplementary material Fig. S4). Hs-cen2/Cr-VFL2 and Hs-cen3/Cdc31p correspond to the well-defined centriolar centrin subfamilies (Azimzadeh and Bornens, 2004). Cr, Chlamydomonas reinhardtii; Cp, Cryptosporidium parvum; Hs, Homo sapiens; Pf, Plasmodium falciparum; Pv, Plasmodium vivax; Pt, Paramecium tetraurelia; Ta, Theileria annulata; Tt, Tetrahymena thermophila; Tg, Toxoplasma gondii; Vc, Vorticella convallaria; Za, Zoothamnium arbuscula. The accession numbers of Paramecium centrins (named Ptcen_icl or Ptcen) are as in Table 1. Cr-Vfl2=CAA31163; Hs-cen2=AAP35920; Hs-cen3=AAP35334; Pt-cen3=XP_001439003; Pt-cen2=XP_001427485; Tg-cen2=50m03356; Tt-cen1=XP_001019292; Tt-cen2=XP_001470770; Tt-cen3=XP_001026988; Tt-cen4=XP_001023350; Vc-spasmin=AAD00995; Za-spasmin1=BAC43748; Za-spasmin2=BAC43749.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Localisation of GFP-tagged centrins. For a representative of each centrin subfamily, localisation was monitored in GFP-expressing cells, fixed and double-labelled with an anti-GFP polyclonal antibody (green) and either the monoclonal anti-centrin1A9, specific for the ICL (red) or the monoclonal ID5, which labels essentially the basal bodies (red). To the right of each represented cell, the insets show, from top to bottom, GFP, anti-centrin or anti-tubulin labelling and the merged image of a magnified area. (A) In cells expressing ICL1ap-GFP, the two labels precisely colocalise. (B) In cells expressing ICL10ap-GFP, the GFP labelling clearly shows a beaded pattern, whereas the anti-centrin labelling (specific for the ICL1a subfamily) is continuous along the mesh. (C) In cells expressing ICL1ep-GFP, the GFP signal localises not only at the ICL, with a beaded pattern, but also at the contractile vacuole pores (arrowheads), and anteriorly, in close association with basal bodies (arrows). Scale bar: 15 µm.

View larger version (71K): [in this window] [in a new window]   Fig. 4. The effect of centrin depletion is subfamily specific. For all centrins (except ICL8p), RNAi induces a complete disassembly of the ICL observed after a few cell divisions. Although the same terminal phenotype (absence of ICL, small remnants of centrin-containing material near basal bodies) is reached in most cases, the pattern of disassembly, consistently observable throughout the first divisions under RNAi conditions, depends on the targeted subfamily. (A-C) Three of the four recorded modes of disassembly. (A) ICL7p-silenced cells are shown after 24 hours (left) and 48 hours of growth (right): disassembly proceeds progressively and homogeneously. (B) In ICL10ap-silenced cells, thinning of the filament bundles precedes disassembly. (C) In ICL3d-silenced cells, aggregates of ICL material accompany disassembly. The lower panels show enlargements of the above cells. Scale bar: 15 µm.

View larger version (97K): [in this window] [in a new window]   Fig. 5. The pleiotropic effects of ICL1ep depletion. Cells were observed by labelling with either the anti-centrin antibody 1A9 to visualise the ICL (A) or with the anti-tubulin ID5 to visualise microtubular structures and in particular basal bodies (B-C). (A) Disassembly of the ICL after 24 hours of ICL1ep depletion shows a distinctive pattern compared with those in Fig. 4. No terminal phenotype with complete absence of ICL was observed, because cells die after a few divisions under RNAi conditions. (C) Disorganisation of the cortex, observed after 48 hours. Comparison with a control cell (B) shows that ICL1ep depletion leads to fewer and misaligned basal bodies, abnormal and reduced oral apparatus (oa) and post-oral microtubules (pof) and an abnormal number of contractile vacuole pores (cvp). Scale bar: 15 µm.

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