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Protist, Vol. xx, xxx–xxx, xx 2012 http://www.elsevier.de/protis Published online date xxx

ORIGINAL PAPER

A Centrin3-dependent, Transient, Appendage of the Mother Basal Body Guides the Positioning of the Daughter Basal Body in Paramecium Maria Jerka-Dziadosza,1 , France Kollb,c , Dorota Włogaa,e , Delphine Gogendeaub,c,d , Nicole Garreau de Loubresseb,c , Franc¸oise Ruizb,c , Stanisław Fabczaka , and Janine Beissonb,c aPolish Academy of Sciences, The Nencki Institute of Experimental Biology, Warsaw, bCNRS, Centre de Génétique Moléculaire, UPR404, 91198 Gif-sur-Yvette, France cUniversité Paris-Sud, F-91405 Orsay, France dInstitut Curie - Section Recherche, UMR 144 CNRS, 75005 Paris, France eUniversity of Georgia, Athens, GA 30602, USA

Poland

Submitted June 6, 2012; Accepted November 15, 2012 Monitoring Editor: Michael Melkonian

Basal bodies are tightly controlled not only for their time of duplication but also for their movements, which ensure proper division and morphogenesis. However, the mechanisms underlying these movements only begin to be explored. We describe here a novel basal body appendage in Paramecium, the anterior left filament (ALF), which develops transiently from the mother basal body before duplication and disassembles once the new basal body is docked at the surface. By comparing the ultrastructure of dividing wild type cells to that of cells defective in basal body duplication, either by depletion of conserved proteins required for basal body assembly, or by mutation, we showed 1) that assembly of the ALF requires PtCen3p, one of the two basal body specific centrins and 2) that absence of the ALF correlates with a failure of the newly assembled basal bodies to tilt up to their docking site at the surface. This correlation suggests that the function of the ALF consists in anchoring centrin-containing contractile fibers which pull up the new basal body toward its site of docking. The presence in T. thermophila of an ALF-like appendage suggests the conservation of an ancestral mechanism ensuring the coupling of basal body duplication and cell morphogenesis. © 2012 Elsevier GmbH. All rights reserved. Key words: Anterior-Left-Filament; basal body; centriole; cytotaxis; centrin.

Introduction Centrioles and basal bodies share two remarkable features, their conserved nine-fold symmetry and their mode of duplication. Although de novo assembly of a centriolar structure can be achieved under particular physiological conditions or in particular 1 Corresponding author; e-mail [email protected] (M. Jerka-Dziadosz).

cell types (for reviews: Beisson and Wright 2003; Song et al. 2008), a new basal body or centriole generally arises by the side of the mother organelle according to a precise geometry. In the case of the basal bodies present in unicellular organisms, ciliates or flagellates, in addition to the budding at right angles, the sites of assembly of a new basal body and of its anchoring at the cortex are precisely determined according to the polarity of the cell and of the mother basal body itself (Azimzadeh and

© 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.protis.2012.11.003 Please cite this article in press as: Jerka-Dziadosz M, et al. A Centrin3-dependent, Transient, Appendage of the Mother Basal Body Guides the Positioning of the Daughter Basal Body in Paramecium. Protist (2012), http://dx.doi.org/10.1016/j.protis.2012.11.003

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Marshall 2010; Beisson and Jerka-Dziadosz 1999; Bornens 2008; Dutcher 2003; Feldman et al. 2007; Lacomble et al. 2010; Silflow and Lefebvre 2001). Over the last few years, the molecular dissection of basal body/centriole assembly in a wide range of organisms has led to the characterization of the major conserved constituents and of the basis of the nine-fold symmetry (reviews in: Azimzadeh and Bornens 2007; Azimzadeh and Marshall 2010; Brito et al. 2012; Pearson and Winey 2009; Strnad and Gönczy 2008). In contrast, the nature of the physico-chemical constraints which channel the movements of a new centriolar structure has only recently begun to be considered (Farnum and Wilsman 2011; Vaughan and Dawe 2011), although a specific role of centrin has been demonstrated (Geimer and Melkonian 2005; Koblenz et al. 2003; Ruiz et al. 2005; Stemm-Wolf et al. 2005; Yang et al. 2010). In addition, a breakthrough has recently been obtained by cryo-electron tomography of isolated, duplicating mammalian centrioles: Guichard et al. (2010) described a short stalk connecting a microtubule triplet of the mother centriole and the axis of the cartwheel of the developing procentriole. This is the first evidence of a physical mechanism ensuring the growth at right angle of the pro-centriole. Among unicellular organisms, Paramecium, like other ciliates (Pearson and Winey 2009; Vonderfecht et al. 2011) is a most suitable model to follow the steps of basal body duplication, as their regular organization over the cell surface, the wellknown spatio-temporal pattern of their duplication during division and the homogeneous polarities of the basal body appendages provide unambiguous landmarks in ultra-structural studies (Iftode et al. 1989; Jerka-Dziadosz et al. 1998; Ruiz et al. 1987). In addition, an efficient method of RNAi by feeding ensures a fast targeting of the genes of interest allowing detection of both the early and late effects of gene silencing (Galvani and Sperling 2002). These particularities have permitted us to identify, by standard ultra-structural studies, a previously overlooked transient filamentous appendage, the ALF (Anterior Left Filament), which is assembled at the onset of basal body duplication and develops from the left of the mother basal body, running anterior and along the ciliary row. Although the ultra-structural study of basal body duplication in ciliates seemed to have little new information to reveal since the pioneer studies of Dippell (1968) and Allen (1969), it is by examining dividing cells under depletion of the basal body core proteins PtBald10p or PtSas6p (JerkaDziadosz et al. 2010), that the ALF was pinpointed:

at the empty presumptive docking sites of new basal bodies. We then examined dividing cells in other mutational or physiological conditions affecting basal body duplication and could demonstrate that assembly of the ALF required PtCen3p, a centrin isotype previously shown to localize at the anterior edge of basal bodies and to be involved in new basal body positioning in Paramecium (Ruiz et al. 2005). Finally, we showed that this ALF is not a Paramecium-specific device and that a similar structure exists in Tetrahymena. The general significance and interest of these observations with respect to the physico-chemical constraints at play in the duplication of centriolar structures and guidance of their movements will be discussed.

Results Like centriole duplication (see Azimzadeh and Marshall 2010), basal body duplication in unicellular organisms presents distinct phases, which proceed from initiation, i.e. the canonical budding of the new basal body at right angles to the mother, to elongation of the microtubular shaft, and maturation with assembly of the transition zone. But basal body development presents an additional step: an upward tilting movement of the new basal body leading to its docking at the cell surface (Fig. 1 A) (see also Aubusson-Fleury et al. 2012). As in other unicellular organisms, these movements of the new basal bodies are precisely guided to orchestrate the complex morphogenetic processes of division. For each species, the pattern of basal body distribution on the cell surface is precisely determined and the migration of new basal bodies after budding strictly channeled (Beech et al. 1991; Cavalier-Smith 1974; Lacomble et al. 2010; Tucker 1971). In Paramecium, the ciliature is arranged in parallel longitudinal rows forming an overall pattern faithfully reproduced through binary division and each new basal body aligns within the existing ciliary row, with the same antero-posterior and circumferential polarities (Fig. 2) as the mother. Ciliary rows comprise two types of cortical units that contain either one (1-bb) or two (2-bb) basal bodies (Fig. 1B, C). In all units, the basal bodies are flanked by specific appendages, microtubular ribbons and striated rootlets (Iftode et al. 1989, 1996). In addition, in 2- bb units, the two members of the pair are connected by filamentous linkers, a situation similar to connectors observed between centriole pairs in animal cells (Vladar and Stearns 2007) or basal bodies in flagellates (Geimer and Melkonian 2004).

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Figure 1. Basal body duplication in Paramecium. A. The scheme depicts the stages of duplication, from budding of the new basal body (nbb) at right angle from its mother (obb) to its progressive elongation, maturation and tilting up until its attachment to the cortex (pm), parallel and anterior to the mother organelle. B, C Basal body duplication in 1-bb units (B) and 2-bb units (C) during the first wave of duplication. The distinctive feature concerns the 2-bb units where the linkers (lk) between the two paired basal bodies disassemble and the anterior basal body develops a new ciliary rootlet (ncr) before duplicating. In both cases the preexisting ciliary rootlet (cr) reorganizes. A –P refers to antero-posterior axis of the cell.

Thus, it may represent an ancestral eukaryote condition (Eisler 1992; Lynn 1991). During division, whether in 1-bb or 2-bb units, all pre-existing basal bodies persist and new ones are inserted in the cortex anterior to their mother. In addition to these geometrical aspects, basal body duplication proceeds in two successive waves. At the beginning of mitosis, whether in 1 or 2-bb unit (Fig. 1), the first wave results in addition of one or more new basal bodies anterior to the mother. Duplication is accompanied by a rearrangement of all the appendages of the mother basal body (Iftode et al. 1989, 1997). In particular, in 2-bb units, two striking changes occur (Fig. 1C): 1) the anterior basal body develops a striated ciliary rootlet (ncr), initially present on the sole posterior basal body; 2) the filamentous linkers between the two basal bodies begin to disassemble (Iftode and Aubusson-Fleury 2003), as occurs for centriole pairs at the time of “disengagement” (Tsou and Stearns 2006). This dual change unambiguously

identifies a cortical region, which has been activated in response to duplication signals, and this disengagement individualizes the anterior basal body in a pair, allowing it to duplicate. During the second wave, a new basal body is added to only a subset of old ones, to reconstitute 2-bb units (Iftode et al. 1989, 1996; Iftode and Fleury-Aubusson 2003). The assembly of a new basal body is preceded by the formation, on the old basal body, of a transient filamentous appendage, the ALF. First noticed on thin sections through dividing cells depleted for PtSas6p (Jerka-Dziadosz et al. 2010), a new transient non-microtubular structure appearing at the same time as the new striated ciliary rootlet (cr) in 2-bb units, was then reinvestigated in a collection of sections of wild type cells fixed for control of various mutant cells (JerkaDziadosz et al 1998, 2010) and was in fact regularly observed. This filamentous structure, present in

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Figure 2. Circumferential polarity of basal bodies and numbering of basal body triplets. This schematic 3D representation shows the topographical relationships of the basal body and its anterior appendages: the striated ciliary rootlet (cr, light blue), the ribbon of the transverse microtubules (tmr, blue) and the anterior left filament (ALF, olive). The green circle with red radial spokes represents the new germinative disc with the cartwheel (new cw) structure located under the tmr and between the cr and the ALF. The basal body is viewed from axonemal side of the cell with its anterior side facing the viewer, the basal body triplets are tilted clockwise and the triplet numbering is anti-clockwise. A, B, C depicts the microtubules in the blade. This scheme combines two numbering systems: (1) the color code used in Trypanosoma (Lacomble et al. 2008) where yellow indicates triplet No.1 (anterior most corresponding to triplet No.5 of Paramecium) and red indicates triplet No. 9 (corresponding to Paramecium triplet No.4), and (2) the numbering code (digits on the A microtubule of each triplet) as defined for Ciliates by De Puytorac and Grain (1974), where triplet No. 9 is the one to which the postciliary ribbon (Pc) is attached. The arrows indicate the cell polarities: A – anterior, P- posterior, L – left, R – right, dist – distal, prox –proximal.

left side of the ciliary rootlet (cr) (Fig. 3A - small arrow; compare with Fig. 4C, H, I). This structure that joins the ALF and cr in proliferating cells is also present in mature (interphase) basal bodies while the ALF is transient and disappears after maturation and docking of the new basal bodies. The proximal, upper part of the ALF touches the proximal end of the transverse microtubule ribbon (tmr) (Figs 2, 3A, B, C, E); the distal end is free, not attached to any membranous or filamentous structure. The pro-basal body will develop between the striated ciliary rootlet (cr) and the ALF and underneath the transverse microtubular ribbon (Fig. 2, stars on Fig. 3A, B). While the new basal body elongates, one site of the proximal part of a new basal body (nbb) seems closely connected to the right side of ALF (Fig. 3C, D and E). This association is still visible after the new basal body has tilted upward to the cell surface and anchored to the membrane (Fig. 3F). In contrast to the ciliary rootlet, which is stable until the next division, the ALF can no longer be detected once basal body duplication is completed: in 1-bb units, it disappears, while in the developing 2-bb units, it seems to be incorporated into the forming linker structure (not shown). In fact, ALFs had previously been illustrated (e.g. in fig. 3B in Iftode and Fleury-Aubusson (2003) and fig. 6A in Ruiz et al. (2005)), but never described. Quite prominent ALF structures were illustrated in a previous study (fig. 5B and C in Jerka-Dziadosz et al. (2010) and are shown here in Fig. 3B, D and E). In order to investigate the possible function of this transient appendage accompanying the development of a new basal body, we examined its presence or possible alteration in different previously studied physiological or genetic conditions affecting basal body duplication in Paramecium. Operationally, these conditions fall into two categories: 1) either the assembly of new basal bodies is prevented, or 2) new basal bodies are assembled, but their maturation/positioning and/or the control of their number are altered.

both 1-bb and 2-bb units (Figs 2, 3) runs anterior of the triplet No. 4 toward the anterior left of the cell and accordingly was named Anterior Left Filament (ALF). Figure 2 shows a 3D view of these relationships. As measured on tangential and cross-sections, the ALF is a tapered structure ca 200 nm long, 50 nm high at its base and 10 nm thick. On cross sections it is evident that the ALF is continuous with a fibrous material located anterior of triplets Nos. 4 and 5 and attached to the

Functional analysis of the ALF. I. Inhibition of new basal body assembly does not prevent ALF development. In Paramecium, specific inhibition of basal body assembly had been observed in five cases: upon depletion of PtSas6p, of PtBld10p, of PtSas4p or of gamma-tubulin and in the mutant sm19. Depletion of PtSas6p blocks cartwheel assembly, while depletion of PtBld10p destabilizes PtSas6p in the developing basal body, prevents the assembly of the cartwheel and of the microtubular

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Figure 3. The assembly of a probasal body is preceded by the formation of the anterior left filament (ALF). The orientation of the sections is indicated by arrows pointing to the anterior (A), posterior (P), right (R), left (L) and distal (dist) or proximal (prox) sides of the cell. In B and D, the posterior of the cell is toward the viewer. A, B, C and F: sections through wild type dividing cells; D-E: sections through a dividing cell expressing GFP-PtSas6. A. A tangential section through the surface of a dividing cell at the first wave of basal body proliferation in a 1-bb unit. The ALF runs at the anterior left side of an old basal body (obb), anterior of the microtubule triplet No. 4. A fibrillar structure (small vertical arrow) connects the ALF with the striated ciliary rootlet (cr). The stars on A and B indicate the presumptive site of assembly of a daughter basal body, such as shown in C (nbb). B. A cross-section posterior to a single basal body (cil- cilium of that bb) in a 1-bb unit undergoing the 1st wave of basal body proliferation. The upper part of the ALF is closely apposed to the proximal ends of microtubules from the transverse microtubular ribbon (tmr). C. The new basal body (nbb) anterior to the old one (obb) is located between the striated ciliary rootlet (cr) and the ALF. The new basal body is still oriented perpendicular to the mother. D. A transverse section of a new basal body (nbb) flanked by cross-sections through the ciliary rootlet (cr) and the ALF. Note the connection of the ALF with the transverse microtubular ribbon (tmr). The tilting-up new basal body (nbb) is connected to the ALF and the ciliary rootlet (cr). E. Tangential section near the surface (as on A) of a cell during the second wave of basal body proliferation. The new basal body has tilted up. A prominent ALF touches the left side of the new basal body. F. A young basal body (nbb) already attached to the cell membrane. The section through the ALF is visible adjacent to the proximal part of the new basal body, closely apposed to the new microtubular ribbon (tmr) but does not touch it. pc:–postciliary ribbon, ps: parasomal sac. Bars A-F: 0.2 ␮m.

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Figure 4. Inhibition of new basal body assembly does not affect the ALF formation. The orientation of the sections in A and B is indicated by arrows pointing to the anterior/posterior (A/P), right/left (R/L) and proximal/distal (prox/dist) sides of the cell. A and D show transverse cross-sections anterior to the mother basal body, with the posterior of the cell facing the viewer. All other images show longitudinal tangential sections near

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barrel (Jerka-Dziadosz et al. 2010); depletion of PtSas4p also blocks cartwheel assembly or stability (Gogendeau et al. 2011). We re-investigated the morphology of ciliary units in cells fixed at their first division upon depletion of either Sas6p or Bald10p, focusing on the pre-cartwheel stages. We found that the ALF was formed at stages corresponding to the 1st as well as the 2nd wave of basal body proliferation in PtSas6p depleted cells (Fig. 4A-C). The architecture of the ALF in relation to the mother basal body, the transverse microtubules (tmr) and the striated ciliary rootlet (cr) were the same as in control cells. The formation of the new striated ciliary rootlet (ncr) in the anterior basal body of the 2-bb units (Fig. 4C) occurred normally and correlated with the assembly of the ALF. During the 2nd wave, when the two basal bodies of a pair separate, the ALF was formed but the assembly of the new basal body was inhibited (not shown). Similar observations were made in PtBld10p depleted cells (Fig. 4D-F): although the formation of the cartwheel was perturbed or completely blocked, the ALF was present at both waves of basal body duplication (Fig. 4E, F). We also re-investigated the thermo-sensitive mutant sm19 defective for eta-tubulin (Ruiz et al. 2000): when grown at the non-permissive temperature (35 ◦ C), mutant cells do not assemble new basal bodies. The blockage of basal body duplication does not disturb the cell cycle, as mutant cells continue to divide at the same rate as wild type controls, over 3-5 divisions, after which they die (Ruiz et al. 1987). As in the case of depletion of the core centriolar proteins PtSas6p and PtBld10p, in sm19 cells fixed during the first division at 35 ◦ C, the transient assembly of the ALF takes place despite the absence (Fig. 4G) of a developing basal body. Although less extensively than for the above two situations, we also examined cells undergoing

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their first division upon PtSas4p or gamma-tubulin depletion, which both primarily affects basal body assembly (Gogendeau et al. 2011; Ruiz et al. 1999). In gamma-tubulin depleted cells the ALF is formed in “duplicating units” (Fig. 4H, I) as in SAS4p depleted cells (fig. 4 in Gogendeau et al. 2011). Functional analysis of the ALF. II. Defective positioning of new basal bodies correlates with lack of ALF. We previously showed that depletion of either PtCen2p or PtCen3p caused mis-localization of the newly-formed basal bodies (Ruiz et al. 2005). However, although both are basal body specific, these two centrin isoforms have distinct localizations and affect distinct steps of basal body duplication. GFP-PtCen3p localizes at the proximal part of the basal body, anterior and near the base of the appendages (striated ciliary rootlet, transverse microtubule ribbon) and on the linkers between paired basal bodies in 2bb units (Fig. 5A ). It is also consistently found at the base of the ALF, at the place where the ALF joins the transverse microtubule ribbon adjacent to the basal body (Fig. 5B).This localization of PtCen3p detected here by immunolocalization of the GFP-tag precisely corresponds to that observed in immuno-electron microscopy using the anti-HsCen3 (Ruiz et al. 2005, Supplementary fig. S3). In contrast GFP-PtCen2p is detected within the microtubule barrel and at the transition zone as confirmed by recent studies (Aubusson-Fleury et al. 2012). We found that PtCen3p depletion correlates with ALF deficiency. Although the ciliary rootlet (cr) and the transverse microtubule ribbons (tmr) are normal, the ALF is undetectable even though the pre-cartwheel structures of the new basal body are identifiable (not shown). In 2-bb units, the

➛ the surface, oriented as in B. The triangles in C and E indicate the filamentous linkers between the two old basal bodies of a pair. A-C. Basal bodies during an advanced stage of the 1st wave of basal body proliferation in PtSas6p-depleted cells, after 18 hours under RNAi condition. The star indicates the presumptive site of the missing new basal body. The ALF is present in both 1-bb units (A, B) and in a 2-bb unit (C), anterior to the “activated” anterior basal body with a newly developed striated ciliary rootlet (ncr). D-F. Basal body proliferation after 18 hours of PtBld10p depletion. D, E: The pro-basal body is missing (star) while the ALF is present in an “activated” 2bb unit (E) which has developed a new ciliary rootlet (ncr) at the anterior basal body. F. Formation of a 2-bb unit (2nd wave of basal body proliferation): the ALF is present; the arrowhead points to a group of disorganized microtubules assembled at the place of the nbb. G. Dividing cell of the sm19 mutant affected in eta-tubulin, grown for 1 cell generation at 35 ◦ C (non-permissive temperature). The assembly of a new basal body is inhibited (star in front of old basal body) the ALF is present at the anterior left side of old basal body. H, I. Dividing cells under gamma- tubulin depletion. H. The new basal body is not formed (star) yet the ALF is visible originating near the microtubule triplet No. 4. I. The new basal body is abnormal (arrowhead), yet the ALF is present. Abbreviations as on Figure 3. Bar: 0.2 ␮m.

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pre-cartwheel “activation” of the anterior basal body proceeds normally, the new striated ciliary rootlet is formed, but the ALF is absent (Fig. 6A, B). During the second wave of basal body proliferation, when new 2-bb units are formed, the ALF is not assembled. As in the first wave (Fig. 6B), the new, anterior basal bodies of the pairs form the terminal plate but do not dock at the surface and remain attached to the mother (Fig. 6C arrowheads). Thus, depletion of PtCen3p prevents disengagement of the new basal body and its tilting up. Interestingly, in PtCen2pdepleted cells observed at their first/second division upon inactivation condition, the ALF develops normally and the assembly site of the new basal body is the same as in wild type cells (Fig. 6D, compare with Fig. 3B and E) but it affects the formation of the terminal plate (tp in Fig. 6E), resulting in defective docking (Aubusson-Fleury et al. 2012). We also re-examined the mutant cro1, which displays an erratic burst of basal body duplication during the inter-fission period, in addition to a normal phase of duplication during cell division (Jerka-Dziadosz et al. 1998). We observed that the basal bodies, which duplicate during cell division, develop a normal ALF. In contrast, no ALF was detected in units duplicating during the interfission cells where the newly formed basal bodies often remained parallel to the surface (did not tilt up) and accumulated intra-cytoplasmically (not shown). However, in this case, the defective ALF does not correlate with centrin deficiency, as the wild type PtCEN2 and PtCEN3 genes are present in the cro1 mutant and are expressed in all basal bodies (Ruiz and Koll, unpublished data). Abnormal development and/or positioning of basal bodies had also been observed under depletion of delta-tubulin (Garreau de Loubresse et al. 2001) or epsilon-tubulin (Dupuis-Williams et al. 2002). However, depletion of either delta- or ➛ Figure 5. EM immuno-localization of GFP-PtCen3p in a developing ciliary unit. Sections of dividing cells expressing GFP-PtCen3p and immunolabeled with an anti-GFP antibody followed by a secondary antibody tagged with 5 nm gold grains. A. A tangential section

near the surface. Anterior to the 2-bb unit containg two old basal bodies (obb) connected by linkers (small vertical arrows), a new basal body (nbb) is already formed. The next nbb will be added further anterior between the new ciliary rootlet (ncr) and the ALF. The arrowhead points to gold grains that are located near the triplet No. 4 at the base of the ALF. Gold grains are also present on the linkers in the pair and near the ciliary rootlet (cr) (Compare with Figs 4C and E and Figs 6 A and B-triangles). B. A cross section as on Figure. 3B). The gold grains are visible at the junction of transverse microtubular ribbon (tmr) and ALF, and on the proximal part of the microtubule blade (arrowhead as on A). Bar: 0.2 ␮m.

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Figure 6. Different roles of Cen3p and Cen2p in the formation of ALF. A-E. Sections through an early dividing cells fixed 17 hours upon Cen3p (A-C) and Cen2p (D-E) depletion. A and B. Tangential sections through proliferating 2-bb units under Cep3p depletion The new basal bodies (nbb) anterior to a bb pair do not tilt up. The anterior basal body has already acquired the new ciliary rootlet (ncr) attesting the 1st wave of basal body proliferation. The empty arrow points to the space normally occupied by the ALF (here absent). The triangles in A and B indicate the linkers between the basal bodies of a pair. The posterior basal body from the pair is sectioned through the ciliary rootlet (cr). C. The anterior new and fully grown basal body (nbb) that developed during the formation of a new 2-bb unit is attached by abnormal linkers (arrowheads) to the old basal body (obb), but it did not tilt to dock at the cell membrane (m). D. Cross-section through two adjacent units of an early divider as attested by the presence of the transient, mitotic specific cytospindle microtubules (cs). On the left-hand side, the thin ALF is visible under the transverse microtubular ribbon (tmr) located in front of an old basal body (not seen) below the longitudinally sectioned cilium (cil). Sections of the ciliary rootlet (cr) and parasomal sac (ps) mark the right side of the cell. To the right of the section, a new basal body (nbb), still perpendicular to the axis of the mother basal body and cilium is assembled between the ciliary rootlet and ALF. E. A longitudinal section. The cell anterior is toward the right; the new basal body (nbb) possesses an abnormal terminal plate (tp). The remnant of the ALF persists on the proximal part under the transverse microtubular ribbon (tmr). Bar: 0.2 ␮m.

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epsilon-tubulin led to disorganization not only of the microtubule barrel but also of all the appendages including the ALF. In view of these pleiotropic effects, the published data did not suggest a possible specific relation between the basal body duplication pathway and the assembly of the ALF, as in the case of PtCen3p. Altogether the above observations suggest that the ALF, which develops before and independently of the assembly of the new basal body, is necessary for the movement of new basal bodies towards their docking site at the cortex and therefore provides a structural relay for the perpetuation of the cortical organization and cell morphogenesis. The ALF is present in evolutionary distant ciliate, T. thermophila. In order to ascertain the morphogenetic significance of the ALF, we investigated if this filament is a Paramecium-specific device or a more general ciliate organelle and reexamined the basal body duplication in T. thermophila. Re-examination of Allen’s (1969) classical study on developing T. pyriformis reveals that the ALF could be identified as a dark filamentous material located under the transverse microtubular ribbon, connecting mother and daughter basal bodies (figs 18 and 20 in Allen 1969). Our own studies on the ultrastructure of basal body duplication in T. thermophila revealed a similar structure in front of duplicating basal body units (figs 11 and 12 in Jerka-Dziadosz at al. 2001). Reexamination of previously registered images from wild type and diverse mutant dividing cells with proliferating basal bodies (Jerka-Dziadosz 1981a, b; Jerka-Dziadosz et al. 2001; Sharma et al. 2007; Thazhath et al. 2004; Włoga et al. 2006, 2008a, b, 2009), confirmed the presence of a structure homologous to the Paramecium ALF. The architecture of the basal body unit/territory differs (Fig. 7A) in Tetrahymena and Paramecium by the orientation of the proximal part of the “transverse” microtubular ribbon whose cross-section is aligned in Paramecium, perpendicular to the ciliary row, while in Tetrahymena, its proximal part runs parallel then turns to the left, so that only the most posterior microtubule of the ribbon is apposed to the microtubular blade of the basal body. Also the parasomal sac in Tetrahymena is located anterior to the basal body, while in Paramecium it is to the right of the ciliary rootlet. Similar to Paramecium, a filamentous structure is visible in Tetrahymena (Fig. 7B-D) at the left anterior side of the mother basal body. As in Paramecium it begins close to the microtubule triplet No. 4 (not shown) in early dividing cells

before the assembly of the pre-cartwheel adjacent to the most proximal part of the transverse microtubule ribbon (Fig. 7B and C). It forms a filamentous slightly striated rod-like structure, which underlies the new basal body while it is tilting up (Fig. 7D). The ALF in Tetrahymena is directed anterior but in contrast to Paramecium it is pointing slightly downward (Fig. 7B and C). As in Paramecium, this appendage is transient and precisely accompanies the phase of basal body duplication. Interestingly, the centrin labeling by the monoclonal antibody 20H5 around the basal body in immuno-electron microscopy was detected anterior to the left- proximal side of the basal bodies (Jerka-Dziadosz, unpublished observations) and was studied in detail by Stemm-Wolf et al. (2005) and Vonderfecht et al. (2011). The centrin labeling outside the basal body was identified as the place “where the new basal body is nucleated” and underneath the proximal end of the transverse microtubules (fig. 6b and c in Stemm-Wolf et al. 2005). From these observations it can be concluded that, like in Paramecium, the ALF structure in Tetrahymena originates in close association with centrin located near a specific microtubular triplet (No. 4) to which also the transverse microtubular ribbon is attached. In both ciliates, the ALF structure develops at a similar localization with respect to basal body polarity and appendages, along the antero-posterior cell axis, and is likely to fulfill a similar function.

Discussion Centrosomes and centrioles undergo precisely controlled movements: in the course of cell division towards the mitotic poles, or during cell differentiation, toward the apical cell surface in epithelia or at neuronal and immunological synapses (review: Dawe et al. 2007; Vaughan and Dawe 2011). These movements are likely to rely both on physicochemical and/or structural constraints, and on linkers between centrioles and appendages flanking centrioles whose precise position and function begin to be elucidated (Kim and Rhee 2011; Kobayashi and Dynlacht 2011; Kunimoto et al. 2012; Lim et al. 2009; Lopes et al. 2011; Nigg and Stearns 2011; Stevens et al. 2010; Tsun et al. 2011; Wang et al. 2008). In C. reinhardtii (Cavalier-Smith 1974), in T. brucei (Lacomble et al. 2008) or in P. tetraurelia (Dippell 1968; Beisson and Sonneborn 1965; Sonneborn 1964), the new basal body must move toward a precise final positioning which sustains the reproduction of cell morphology and

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Figure 7. An ALF is present at duplicating basal bodies in Tetrahymena. A. Schematic drawing of the spatial relations of structural elements of 1-bb units in Paramecium and Tetrahymena. Note the difference in location of the parasomal sac (ps) and orientation of the transverse microtubular ribbon (tmr, purple dots). B-D. Sections through early dividing cells of Tetrahymena. B and C. The ALF is visible as a filamentous rod attached to the proximal part of the transverse microtubular ribbon (tmr) at the left anterior side of the old basal body cut obliquely through its cilium (cil) in B. When a new basal body assembles perpendicular to the mother bb (B), the ALF borders the left side of the probasal body (pbb). C. The ALF is directed anterior and slightly downward. Striations in the ALF filaments are indicated by small arrowheads. D. During elongation and tilting up of the new basal body with terminal plate (tp), the ALF touches the proximal part of the new bb (nbb). Note the close proximity of the new basal body to the striated ciliary rootlet (cr); the old basal body (obb) is surrounded by a collar located on the left side of proximal part of the basal body. Bar: 0.2 ␮m.

polarities. The ALF described here provides a new example of an appendage involved in channeling the movement of the new basal body. Like the ciliary rootlet apposed to triplets Nos. 5, 6 and 7 or the transverse microtubule ribbon connected to triplets Nos. 4 and 5 (Beisson and Jerka-Dziadosz 1999), the ALF is nucleated near the basal body triplet No. 4 (Figs. 2, 3A). However, in contrast to the other appendages, the presence of the ALF is restricted to the phase of basal body duplication. This temporal correlation suggested a specific role in the development and/or and positioning of new basal bodies. The identification of a homolog of the ALF in Tetrahymena (Fig. 7) indicates that the function of ALF has been conserved across the ciliate phylum.

Function of the ALF A survey of ultra-structural data previously obtained in a range of conditions affecting basal body

duplication in Paramecium supported two conclusions as to the ALF function. 1) Although coordinated in time, assembly of the new basal body and assembly of the ALF are not interdependent: depletion of basal body core proteins (such as PtSas6p, PtBld10p, PtSas4p, gamma-tubulin), which prevents or blocks assembly of the new basal body or a mutational block of duplication (in the sm19 mutant) does not affect the timely assembly of the ALF. Conversely, no ALF was detected under conditions where basal bodies develop but do not dock at the cell surface or do not do so properly, as observed in PtCen3p-depleted cells, or in the mutant cro1. 2) The correlated absence of the ALF and mis-positioning of newly formed basal bodies observed in two different genetic and physiological backgrounds (PtCen3p depletion and in the cro1 mutant) supports a role of the ALF in the movement of newly formed basal bodies toward their docking site at the cortex.

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By its position within the duplicating cortical unit anchored at the base of the mother basal body and parallel to the striated ciliary rootlet - the ALF frames the space within which the new basal body develops (Fig. 2): Its function might then be to maintain the new basal body along the antero-posterior axis of the basal body row, in conjunction with the ciliary rootlet whose guiding role has previously been pointed out (Iftode and Fleury-Aubusson 2003). In addition, the ALF could also contribute to the tilting up of the developing basal body, since its absence correlates with untilted basal bodies as previously described (Ruiz et al. 2005). The presence of PtCen3p-containing fibrous material, apposed to the proximal end of the basal body, at the base of the ALF and at the site of nucleation of a new basal body (Fig. 5), suggests that contractile fibers linking the ALF to the developing basal body could both lift the growing basal body upward and pull it anterior. The molecular composition of the ALF remains to be ascertained, and the associated centrin-binding protein(s) forming the postulated contractile fibers remain to be identified among the numerous Sfilike proteins detected in the Paramecium genome. In any case, the postulated centrin-binding proteins are distinct from those identified as the backbone of the infraciliary lattice (Gogendeau et al. 2007, 2008), as no defect in basal-body positioning was observed upon inactivation of the latter (Gogendeau, unpublished observations).

Centrins as Ubiquitous Effectors of Organelle Movements The requirement of PtCen3p for ALF assembly suggests that one component of the ALF is a centrin-binding protein. Centrins are ubiquitous effectors in eukaryotes (Dantas et al. 2012) and, since the pioneer work on the green alga Tetraselmis (Salisbury et al. 1984), centrins and centrin-containing contractile fibers have been shown to be essential supports for intracellular motility processes in protists and to drive the movement of the new basal body in Chlamydomonas (Geimer and Melkonian 2005; Koblenz et al. 2003; Taillon et al. 1992), in T. brucei (Selvapandiyan et al. 2007), with centrin deficiency or mutation resulting, as in Paramecium, in defective positioning of neo-formed basal bodies (Ruiz et al. 2005). More generally, the role of centrin, shown in flagellates to form a scaffold throughout the cell (Brugerolle and Mignot 2003; Geimer and Melkonian 2005), is not restricted to basal body movements and also is implicated in the concerted movements of basal bodies with other organelles, including the nucleus

during division. Yeast, where centrin localizes at the spindle pole body nested in the nuclear membrane represents an archetype of this cellular function and its study led to the first characterization of the sfi1 centrin-binding protein (Li et al. 2006) and of the role of its asymmetry in the spindle pole body duplication, a seminal study which provided a model to account for transmission of polarities through cell division (Bornens 2008; Jones and Winey 2006), and put forward the importance of sfi-like centrinbinding proteins to ensure the specificity of centrin action in intracellular movements. We have shown that a transient structure resembling the Paramecium ALF is present in another distant ciliate species, Tetrahymena. In Tetrahymena also, centrin localizes anterior at the proximal side of basal body (Jerka-Dziadosz, unpublished; Stemm-Wolf et al. 2005) and as previously shown (Ruiz et al. 2005), recent studies on basal body phenotypes mutant TtCen1p (corresponding to PtCen2p) showed mis-orientation of newly assembled basal bodies and stability effects (Vonderfecht et al. 2011). It is then likely that functionally similar systems exist in all ciliated cell types (Beech et al. 1991; Dawe et al. 2007; Zhang and He 2011). More generally, the evolutionary conserved association of centrin with basal bodies, centrosomes and spindle pole bodies (Bornens and Azimzadeh 2007) and their role in organelle movements support a general function in the coupling of cell division and organelle duplication. The mechanisms controlling basal body movements and localization are then likely to have a common origin, although they obviously have diversified along with the shape and mode of growth of the organisms.

Basal Body Appendages, as Relays in Polarity Transmission: Cytotaxis in the Move The term cytotaxis was coined by Sonneborn (1964) to designate “the ordering and arranging of new cell structure under the influence of preexisting cell structure”, a concept also referred to as “structural inheritance” or “structural guidance” (Frankel 1974). This concept stemmed from observations on Paramecium, which showed that, in the course of its duplication, a new basal body develops not only a proximo-distal polarity but also a circumferential polarity, ensuring perpetuation along the longitudinal ciliary rows of the different basal body appendages (Beisson and Jerka-Dziadosz 1999) and of the direction of the efficient stroke of ciliary beating (Tamm et al. 1975).

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Evidences for structural differences and/or specializations among triplets have long been recognized in Chlamydomonas (Geimer and Melkonian 2004; Hoops and Whitman 1983; Silflow et al. 2001). More recently, the development of cryo-electron tomography of cilia from different organisms reveals the heterogeneity of doublets in axonemes (Barber et al. 2012; Pigino et al. 2011). Such differentiations among axonemal doublets, with different doublets recruiting different partners, suggest a circumferential anisotropy that is likely to be present in the basal body. A still more direct evidence for basal body circumferential polarity comes from another recent study, focused on the role of Odf2 and the basal foot in the coordination of ciliary beating in ciliated epithelia (Kunimoto et al. 2012). The basal foot - which emanates from the centriole sub-distal appendages and eventually appears as the target of PCP signaling (Bailey and Axelrod 2011; Wallingford and Mitchell 2011) in the final orientation of cilia - develops on specific triplets (Nos. 4 and 5) of the basal body (which correspond to triplets Nos. 8 and 9 in Paramecium (Fig. 2), and in a precise relationship with the position of the central doublet and the future axis of ciliary beating. This strongly suggests that the centriole triplets are not equivalent and that their individuality is defined in relation to the presumptive ciliary function. To focus on the best known basal body systems (Chlamydomonas, Trypanosoma, Paramecium, Tetrahymena (review: Feldman et al. 2007), their circumferential polarity is materialized by their appendages, each nucleated on specific triplets and the problem is how the circumferential polarity is set up. In the case of templated duplication, it may be transmitted by the mother basal body polarity, however the circumferential anisotropy is also present in the basal bodies formed de novo during the differentiation of ciliated epithelia and erratic out of the ciliary rows - basal bodies formed in some Paramecium mutants (Jerka-Dziadosz et al. 1998) develop their appendages at the expected relative position. Assuming now that in these protists a new basalbody acquires its circumferential polarities while developing, this does not suffice for its docking at the right site of the cell surface: it must be guided, through more or less complex movements, depending on the organism. In Paramecium, where the problem is rather simple, that is aligning along the existing ciliary row; the neo-formed basal body needs to tilt up to become parallel to its mother and to anchor precisely along the antero-posterior axis of the ciliary row. Two appendages on the mother basal body, the ciliary rootlet on its right

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and the ALF on its left, frame its movement, while the tilting up toward the docking site would involve PtCen3p-containing contractile fibers. The precise relationships between these contractile fibers and the ALF and the molecular composition of the ALF remain to be ascertained. Finally, it is worth pointing out that transient structural relays set up to guide or control organelle movements, as does the ALF studied here, are not a ciliate peculiarity, only a further example of the importance of precise organelle positioning. The links that tie centrioles over most of the mitotic cycle also pertain of such mechanisms, required to maintain a single centrosome until mitosis is triggered (Mardin and Schiebel 2012). Other examples of transient structures assembled at a specific stage of the cell cycle to accompany duplication processes exist, such as the transient generation of a novel MTOC, which triggers the microtubule rearrangements during meiosis in Schizosaccharomyces. pombe (Funaya et al. 2012), or the setup of a pre-prophase band as a spatial cue for division plane determination in plant somatic cytokinesis (Van Damme 2009).

Methods Strains and culture conditions: For Paramecium, all basic protocols are accessible at http://paramecium.cgm. cnrs-gif.fr/parawiki/. The following strains of Paramecium tetraurelia were used: the wild type stock d4-2 as control and for gene silencing experiments, the mutant nd7-1 (Skouri and Cohen 1997) for microinjection and expression of GFP-tagged proteins, and two mutant strains affected in basal body duplication, sm19 (Ruiz et al. 1987; Ruiz et al. 2000) and cro1 (JerkaDziadosz et al. 1998). Cells were grown at 27 ◦ C in a wheat grass infusion, BHB (L’arbre de Vie) bacterised with K. pneumoniae and supplemented with 0.8 mg/L b-sitosterol (Sonneborn 1970). Depletion of specific proteins - PtSas6p and PtBld10p (Jerka-Dziadosz et al. 2010), PtSas4p (Gogendeau et al. 2011), delta-tubulin (Garreau de Loubresse et al. 2001), epsilontubulin (Dupuis-Williams et al. 2002) - was obtained by the gene silencing technique described by Galvani and Sperling (2002). GFP-tagging of these proteins was carried out as respectively described in the above cited publications. For Tetrahymena thermophila, all cytological protocols were as described by Jerka-Dziadosz (1981a, b). The following strains and/or physiological conditions were examined: wild type and janus mutant for ultrastructural comparison (Strzy˙zewska-Jówko et al. 2003); samples of katanin knockout and overexpression (Sharma et al. 2007), cells overexpressing septin proteins (Włoga et al. 2008a), tagged tubulin (Thazhath et al. 2004), and Nrk2p-K35R-GFP (Włoga et al. 2006) and cells with knocked out TTLL1 and TTLL9 (Włoga et al. 2008b, 2009). Sample preparations for electron microscopy: We detail here important issues pertinent to the orientation of sections and identification of cell cycle stages. Paramecium cells were fixed in a mixture of 1. 5% glutaraldehyde and 1% osmic acid in 0.05 M cacodylate buffer, pH 7.35 (all from Sigma) for 1 h on ice,

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washed three times in the cacodylate buffer, then in H2 O. For agar blocks, random samples from growing cultures and dividing cells of defined stages (early, mid-division, and cytokinesis) were selected into separate samples (5 -10 cells) into small drops on slides previously covered with 2% Agarose (MilesSeravac), spread on microscopic slides and cooled. The cells were then aligned parallel to each other into a small pyramid and covered with hot Agarose from boiling water hot bath. After cooling the agar blocks were cut into small squares and then dehydrated in graded series of ethanol or acetone and embedded in Durcupan (Sigma) on flat longitudinal depressions. The pyramids were arranged in the tip of the longitudinal depression in such a way that when the samples were sectioned by the diamond knife, the cells were cut either transversely or longitudinally. Sectioned samples were contrasted in uranyl acetate and lead citrate (Reynolds). Samples of Tetrahymena cells from growing cultures were centrifuged and the pellet fixed and washed. Small random samples were taken from pellets and embedded in agar blocks and dehydrated followed by embedding in Epon or Durcupan as described (Jerka-Dziadosz 1981a). Samples were first analyzed at low magnification to identify: 1) the characteristics of early vs. late dividers in the oral apparatus, micronucleus, and in cortical structures; 2) the cell polarities indicated by those of striated ciliary rootlets and basal body associated microtubular ribbons (Iftode et al. 1996). Next, the basal body units sectioned at defined angle were photographed at high magnification on an electron microscope JEM 1200 EX, or registered digitally on EM JEM 1400 equipped with Morada 11 Mpx and Olympus item software. Negatives were scanned on an Epson Scanner and processed with the PHOTOSHOP 7.0.1 CE software. Immunoelectron microscopy: Postembeding immunogold methods were basically similar to the described in Klotz et al. (1997). Cell pellets from growing populations were fixed in 2% paraformaldehyde in 0.05 M cacodylate buffer, pH 7.35, at room temperature for 2 h. After washing in the same buffer, cells were dehydrated by passage through a series of ethanol baths (30 -100%) and embedded in LWR (London Resin Ltd) according the manufacturer instructions. Thin sections were collected on nickel grids and saturated and processed with 3% bovine serum albumin (BSA) in phosphate-saline buffer (PBS). The monoclonal anti-GFP antibody (Roche Molecular Biochemicals, Indianapolis IN. USA) were diluted 1:20. After washing the sections were incubated with 5 nm colloidal-gold conjugated anti-mouse immunoglobulin (GAM G5, Amersham International plc, Little Chalfont, England) at 1/50 dilution). After extensive washing the sections were contrasted with ethanolic uranyl acetate end examined in an E.M. Philips 410. Circumferential polarity of basal bodies and numbering of basal body triplets: First defined in relation to the polarity of beating of ciliary axonemes (Afzelius 1959), a circumferential polarity of basal bodies has been defined in diverse unicellular organisms, according to the overall polarities of basal body appendages and of the cell, and subsequently related to structural differentiations in axonemal doublets and to the direction of beating of the cilia or flagella. Circumferential polarity of basal bodies in Paramecium and Chlamydomonas, strictly related to the effective stroke of cilium or flagellum, was previously compared (Beisson and JerkaDziadosz 1999) and the same conventions are used here. Numbering of basal body triplets in Paramecium follows that applied for ciliates by De Puytorac and Grain (1974) and Lynn (1991), where triplet No. 9 is the one to which the post-ciliary ribbon is attached. The numbering in Chlamydomonas and Trypanosoma is different, and based on the pattern of crosssectioned microtubules in the axonemes (Hoops and Witman

1983; Moestrup 2000) and location of the Vfl1p/acorn in the distal part of the basal body (Geimer and Melkonian 2004; Silflow and Lefebvre 2001; Silflow et al. 2001). The colors of the triplet blades on Figure 2 are the same as the code for triplet numbering as used for Trypanosoma (Lacomble et al. 2008). However triplet numbering in Paramecium is different as yellow indicates triplet No. 5 corresponding to No. 1 in Trypanosoma and red color indicates Paramecium triplet No. 4 corresponding to No. 9 in Trypanosoma. The numbering of blades is clockwise when viewed from the proximal side (from inside the cell) or anti-clockwise when viewed from the axonemal site (from outside the cell) (Beisson and Jerka-Dziadosz 1999; Hoops and Whitman 1983; Lacomble et al. 2008; Moestrup 2000). Although the numbering system (allocation of the triplet No. 1) may differ with the organisms, the functional polarity, with respect to the cell’s general polarity, is the same in ciliates, Chlamydomonas and Trypanosoma where No. 1 marks the anterior of the basal body and where the microtubule ribbon attaches and the new basal body is nucleated.

Acknowledgements The authors would like to thank the following colleagues for critical reading and comments on the manuscript: Drs A. Aubusson-Fleury, J. Cohen, J. Frankel, J. Gaertig and A-M Tassin. The investigations performed at the Nencki Institute of Experimental Biology (Warsaw) (MJD, SF and DW) were supported by statute grants from the Ministry of Science and Higher Education (Poland) to the Nencki Institute and EMBO Instalation Grant No. 2331 to DW. Expert technical assistance of H. Bilski from the Laboratory of Electron Microscopy is kindly acknowledged. The work of D. Włoga at the University of Georgia USA was supported by The Kosciuszko Foundation Fellowship (New York, USA) and NSF grants MBC-033965. F. Koll, N. Garreau de Loubresse, F. Ruiz and J. Beisson were supported by the Centre National de la Recherche Scientifique (France) and by a grant from the Agence Nationale de la Recherche, number NT05-2_41522. D. Gogendeau was supported by the Association de la Recherche Contre le Cancer (A06/3). Contribution from the CNRS-supported Research Group « Paramecium Genome Dynamics and Evolution » is kindly acknowledged.

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Please cite this article in press as: Jerka-Dziadosz M, et al. A Centrin3-dependent, Transient, Appendage of the Mother Basal Body Guides the Positioning of the Daughter Basal Body in Paramecium. Protist (2012), http://dx.doi.org/10.1016/j.protis.2012.11.003