the conserved centrosomal protein for20 is required for … · 2012. 11. 7. · journal of cell...

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The conserved centrosomal protein FOR20 is requiredfor assembly of the transition zone and basal bodydocking at the cell surface

Anne Aubusson-Fleury1,2,*, Michel Lemullois1,2, Nicole Garreau de Loubresse1,2, Chloé Laligné1,2,Jean Cohen1,2, Olivier Rosnet3, Maria Jerka-Dziadosz4, Janine Beisson1,2 and France Koll1,2

1CNRS, Centre de Génétique Moléculaire, UPR3404, 91198 Gif-sur-Yvette, France2Université Paris-Sud, 91405 Orsay, France3Centre de Recherche en Cancérologie de Marseille, INSERM UMR1068, CNRS UMR7258, Institut Paoli-Calmettes, 13273 Marseille Cedex 09,France4Polish Academy of Sciences, M. Nencki Institute of Experimental Biology, Department of Cell Biology, 3 Pasteur Street, 02-093 Warsaw, Poland

*Author for correspondence ([email protected])

Accepted 14 May 2012Journal of Cell Science 125, 4395–4404� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.108639

SummaryWithin the FOP family of centrosomal proteins, the conserved FOR20 protein has been implicated in the control of primary ciliumassembly in human cells. To ascertain its role in ciliogenesis, we have investigated the function of its ortholog, PtFOR20p, in themulticiliated unicellular organism Paramecium. Using combined functional and cytological analyses, we found that PtFOR20p

specifically localises at basal bodies and is required to build the transition zone, a prerequisite to their maturation and docking at the cellsurface and hence to ciliogenesis. We also found that PtCen2p (one of the two basal body specific centrins, an ortholog of HsCen2) isrequired to recruit PtFOR20p at the developing basal body and to control its length. By contrast, the other basal-body-specific centrin

PtCen3p is not needed for assembly of the transition zone, but is required downstream, for basal body docking. Comparison of thestructural defects induced by depletion of PtFOR20p, PtCen2p or PtCen3p, respectively, illustrates the dual role of the transition zone inthe biogenesis of the basal body and in cilium assembly. The multiple potential roles of the transition zone during basal body biogenesis

and the evolutionary conserved function of the FOP proteins in microtubule membrane interactions are discussed.

Key words: FOR20, FOP, Centrin, Cilium, Paramecium, Ciliogenesis

IntroductionCilia are highly conserved eukaryotic cell appendages present as

either motile or non motile organelles (Carvalho-Santos et al.,

2011; Bornens, 2012). They constitute cell compartments

involved in reception and signal transduction (Bloodgood,

2010). Any defect in their functions leads to dramatic

consequences at the level of the organism, as illustrated by

developmental disorders and/or ciliopathies in human

(Hildebrandt et al., 2011). Cilia are generally assembled from a

nine fold-symmetrical microtubular template, the basal body

which is docked at the cell surface, either transiently as in most

metazoan tissues where the older centriole docks at the cell

surface to generate a primary cilium as the cell enters the G0

phase (Sorokin, 1968; Kobayashi and Dynlacht, 2011), or

permanently. In this second case, basal bodies may be

assembled de novo, during epithelium differentiation (Sorokin,

1968; Lemullois et al., 1988; Laoukili et al., 2000), in flagellates

like Naegleria (Fulton and Dingle, 1971), in ciliate cystic cycle

(Grimes, 1973), in male gametes as in Marsilea vestita (Hart and

Wolniak, 1998), or be assembled close to parental ones, as during

the division in ciliates (Dippell, 1968; Allen, 1969). In all cases,

ciliogenesis is a multi-step process: basal body assembly involves

nucleation, elongation, maturation of the microtubular scaffold,

docking at the cell surface and growth of a cilium.

Recently it was found that FOR20, a protein conserved in most

ciliated organisms, is involved in the assembly of the primary

cilium (Sedjaı̈ et al., 2010). FOR20 belongs to the family of FOP-

related proteins (Azimzadeh et al., 2008), which is defined by the

presence of two N-terminal domains, the LisH domain, necessary

for the dimerisation yielding the functional form of the protein, and

the TOF domain, required in addition to LisH for centrosomal

localisation, and a Pro-Leu-Leu (PLL) domain of unknown

function (Sedjaı̈ et al., 2010). In this family, four subgroups can

be recognised: (1) the FOP proteins, identified as oncogene

partners associated to the centrosome (Andersen et al., 2003;

Delaval et al., 2005; Popovici et al., 1999), required for

microtubule anchoring (Yan et al., 2006) and cell cycle

progression (Acquaviva et al., 2009); (2) the centrosomal OFD1

proteins, whose mutations cause oral facial digital diseases in

humans (Ferrante et al., 2001); (3) the land plant TONNEAU 1

protein, required for the normal cortical cytoskeleton of

microtubules and pre-prophase band formation at the division

plane (Azimzadeh et al., 2008); (4) a subgroup of shorter proteins

corresponding to FOR20. Proteins of this subfamily are the most

evolutionarily conserved. In RPE1 cell lines, HsFOR20p localises

at the centrioles and the pericentriolar satellites, close to the

centrosome and its depletion has been shown to affect ciliogenesis.

However, the primary effect of HsFOR20p deficiency remains

Research Article 4395

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unclear. In order to obtain information about the function of theHsFOR20p, we investigated the role of its ortholog, PtFOR20p, in

Paramecium, a multi-ciliated unicellular organism that offers thepossibility to follow the lineage of each basal body over cellgenerations (Iftode et al., 1989), hence to detect and analyse any

defects in the mechanisms of basal body assembly andciliogenesis. We found that, in Paramecium, GFP-PtFOR20localises at the transition zone of all basal bodies, whetherciliated or not, and that its depletion induces defects in basal body

maturation and docking. Although the protein localised at thedistal part of docked basal bodies, as previously noted inTetrahymena (Kilburn et al., 2007), its presence could be

detected very early during basal body assembly. Furthermore,PtCen2p, the ortholog of the human centriolar centrin 2 (Ruiz et al.,2005), is required for the recruitment and/or stabilisation of

PtFOR20p at the basal body and both proteins are essential forbuilding the transition zone. In contrast, PtCen3p, the ortholog ofthe mammalian centrin 3, is only required for docking the basalbody at the cell membrane. Our findings shed new light on the

assembly of the transition zone as well as on the docking of basalbodies in Paramecium and explain the ciliogenesis defectsobserved in HsFOR20p depleted human cells. Finally, we

observed that PtFOR20p still marks the site of basal bodyattachment at sites of programmed disassembly-reassembly, andthus might provide a cortical pattern landmark.

ResultsTwo PtFOR20p-encoding genes are present in Paramecium

First described as small FOP-like proteins (Azimzadeh et al.,2008), FOR20 (FOPNL) was subsequently identified as a

centrosomal protein involved in primary cilium assembly(Sedjaı̈ et al., 2010). This protein, which is detected only inciliated organisms, has similarities to the N-terminal part of the

FOP proteins and presents the three specific sequence features,the TOF and LisH domains and the PLL motif. Using Blasthomology search, two FOR20 orthologs, derived from the lastwhole genome duplication (Aury et al., 2006) were identified in

the Paramecium genome. Multiple alignment of the twoPtFOR20 proteins, which differ by a single amino acid, withnumerous other FOR20 proteins show that this protein is strongly

conserved through evolution (supplementary material Fig. S1).The analysis of transcriptome microarray data which areavailable in the Paramecium database (http://paramecium.cgm.

cnrs-gif.fr/cgi/browse/microarray) indicates that these two geneshave an identical expression pattern.

Both GFP-PtFOR20 and GFP-HsFOR20 localise at basalbodies in Paramecium

In order to localise PtFOR20p in living cells, we examined

transformants expressing the GFP-tagged protein. We alsoexamined the localisation of the human protein. In bothPtFOR20p and HsFOR20p expressing cells, which exhibited a

slightly reduced growth rate (2–3 instead of 3–4 divisions per dayat 22 C̊) but no abnormal morphology, discrete spots offluorescence were aligned at the cell surface, following a

pattern similar to that of the basal bodies (Fig. 1A). Double-labelling with the ID5 antibody, which decorates the basal bodies(Ruiz et al., 1999), showed that this surface pattern of the GFP

signal precisely overlapped that of the basal bodies (Fig. 1B).When probed with the monoclonal antibody 17E2 directedagainst HsFOR20p (Sedjaı̈ et al., 2010), only the HsFOR20p

expressing cells reacted, yielding the same dotted cortical pattern

as revealed by the ID5 labelling (not shown). In contrast, control

cells were negative, indicating that the endogenous Paramecium

protein did not cross-react with the antibody.

The GFP-PtFOR20 pattern does not correlate with ciliation

Because FOR20 had been shown to control ciliation in human

RPE1 cell (Sedjaı̈ et al., 2010), we examined the spatial

correlation between the presence of PtFOR20p and cilia. In

their vegetative stage, the cells are densely ciliated but not all

basal bodies bear a cilium (Fig. 1C). Double labelling with two

antibodies specific for basal bodies and cilia respectively is

necessary to ascertain the ciliated or non-ciliated status of basal

bodies. On the ventral surface, single and paired basal bodies

alternate along the antero-posterior rows. A careful examination

Fig. 1. GFP-PtFOR20 localises at basal bodies, whether they are ciliated

or not. (A). GFP labelling observed by epifluorescence on living cells

expressing either GFP-PtFOR20 (top) or GFP-HsFOR20 (bottom). In both

cases, the GFP signal is detected as dots at the cell surface. Scale bar: 2 mm.(B) Double labelling of a GFP-PtFOR20-expressing cell with GFP (green)

and ID5 (red). Confocal merge image of the ventral side of the cell showing

the antero-posterior rows of basal bodies, which encircle the oral groove

(arrow). The GFP-PtFOR20 protein overlaps the ID5 labelling of the basal

bodies. Inserts correspond to the cell area delimited by a grey square on the

merge image. Scale bar: 20 mm (inserts:64.3). (C) Double labelling of awild-type cell with TAP952 (grey) and1D5 (red) and showing cilia and basal

bodies, respectively. Confocal images of the ventral side of the cell. On this

side, the labelling of cilia shows that the anterior invariant field (dashed blue

line) is more densely ciliated than other cell areas. In this area, basal bodies

are paired (dashed line in red image and corresponding insert), and both are

ciliated (merged insert). This area, together with the posterior one where all

basal bodies are single (dotted line), correspond respectively to the anterior

and the posterior ‘invariant’ fields in which no basal body duplication occurs

during cell division (see supplementary material Fig. S2 for further details). In

the rest of the cell, singled and paired basal bodies alternate along the rows

(red insert) and only the posterior basal body is ciliated (merge insert). Scale

bar: 20 mm (inserts:65).

Journal of Cell Science 125 (18)4396

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shows that, although all the basal bodies are labelled by GFP-

PtFOR20, the anterior basal bodies of some pairs are not ciliated

(Fig. 1C): PtFOR20p is thus associated with both ciliated and

non-ciliated basal bodies. This observation means that, if FOR20

is necessary for cilium assembly, as shown for RPE1 cells, its

presence at the tip of a basal body is not sufficient, at least for

motile cilia in Paramecium.

GFP-PtFOR20 localises to the transition zone

To ascertain the precise localisation of PtFOR20p in the basal

bodies, we compared two sets of cells: (1) control cells double-

labelled with ID5 and an antibody directed against the epiplasm,

a sub-membrane cytoskeletal layer segmented into units in the

centre of which the basal bodies are inserted (Nahon et al., 1993),

and (2) GFP-PtFOR20 expressing cells double-labelled with an

anti-GFP and the anti-epiplasm antibody (Fig. 2A,B). Transverse

optical sections show that the ID5 and anti-GFP labelling do not

colocalise: while the ID5 labelling extends beneath the epiplasm

(Fig. 2A), the GFP labelling is confined at the level of the

epiplasm (Fig. 2B), indicating that PtFOR20p localises at the

distal part of the basal bodies.

The localisation of PtFOR20p was then investigated at the

ultrastructural level by immunolabelling of GFP-PtFOR20

expressing cells. The ultrastructure of the transition zone, located

between the basal body and the 9+2 region of the cilium of

Paramecium has been well described (Dute and Kung, 1978). This

transition zone comprises three plates: a proximal thick terminal

plate, a thin intermediate plate and a distal axosomal plate on which

lies the axosome. The structure of the distal part of anchored but

non-ciliated basal bodies is less well described. Fig. 3A shows that

it displays almost the same morphology as ciliated ones, but that the

region between the intermediate plate and the axosomal plate is

thinner suggesting that ciliogenesis involves a reorganisation of this

region. In GFP-PtFOR20 expressing cells, we did not detect any

defect in the morphology of either ciliated or non-ciliated basal

bodies, except for a slight increase of the terminal plate thickness

(Fig. 3B) indicating that the expression of the GFP-tagged protein

has little or no effect on basal body ultrastructure. Byimmunolabelling of whole permeabilised cells (pre-embedding)and of sections (post-embedding), we detected the GFP-labelling at

the level of the transition zone, close to the microtubular scaffold(Fig. 3C,D). In post-embedding processing, immunogold particleswere detected on about 24% of basal bodies, in most cases justabove the terminal plate (Fig. 3C, supplementary material Table

S1). After pre-embedding processing, 38% of basal bodies weredecorated, with a labelling restricted to the external and apical partof the terminal plate (Fig. 3D, supplementary material Table S1).

On transverse sections, in both cases, the gold particles weredetected at the level of microtubule doublets, close to fibres linkingthese doublets to the membrane (Fig. 3C,D). The presence of this

membrane in the pre-embedding protocol (Fig. 3D), whichincludes a treatment with Triton X100, shows that it is detergentresistant at that level. This particularity has already been observed

in Chlamydomonas where this membrane portion endowed withspecific biochemical properties has been called ‘transitionmembrane’ (Kamiya and Witman, 1984). Altogether, theseresults show that PtFOR20p is located at the level of the

transition zone. A similar localisation at the tip of the basalbodies was described both in Tetrahymena by electron microscopyfor the homologous protein called Bbc20 (Kilburn et al., 2007) and

in mouse trachea epithelia by immunofluorescence (Sedjaı̈ et al.,2010). In Giardia, a protein homologous to Bbc20/FOR20 isdetected along the intracytoplasmic part of axonemes, suggesting

an interaction with microtubule doublets (Lauwaet et al., 2011).The fact that, at the ultrastructural level, the labelling was observedonly on a fraction of basal bodies by pre-embedding labelling on

whole permeabilised cells as well as by post-embedding labellingon sections of non permeabilised cells, suggests that the protein isnot abundant and/or located on discrete structures, thus not foundon all longitudinal sections of basal bodies. A similar discrete

labelling has previously been reported in Chlamydomonas forCep290, another protein of the transition zone (Craige et al., 2010).This situation might explain why no labelling was detected at the

base of the primary cilium in RPE1 cells (Sedjaı̈ et al., 2010).

PtFOR20p is detected early in basal body assembly

During cell division in Paramecium, basal bodies duplicate

according to a precise spatio-temporal pattern (Iftode et al.,1989), allowing unambiguous identification of pre-existing andnewly assembled basal bodies. A new basal body assembles next

to and anterior to its mother, and elongates as it tilts up to dock atthe cell surface (Dippell, 1968). The time of appearance ofPtFOR20p with respect to these steps was followed in cellsexpressing GFP-PtFOR20 by double labelling with ID5 and an

anti-GFP antibody. In dividing cells, spots of GFP-PtFOR20 weredetected close to the proximal part of parental basal bodies, at thesite of new basal body assembly, before the developing basal

bodies could be detected by ID5 (Fig. 4A). As ID5 is specific forpolyglutamylated tubulin, it can be concluded that PtFOR20pis associated with the nascent basal bodies before their

polyglutamylation.

PtFOR20p stably associates with the cell surface

During division, the duplication of basal bodies and epiplasm

units starts at the cell equator and spreads as a wave towards thetwo cell poles (Iftode et al., 1989). However, in the ‘invariantanterior field’ (Fig. 1C, supplementary material Fig. S2), where

Fig. 2. GFP-PtFOR20 localises to the distal part of basal bodies.

(A) Ventral side of a wild-type cell double-labelled with anti-epiplasm (grey)

and 1D5 (red). (B) GFP-PtFOR20-expressing cell double-labelled with an

anti-GFP (green) and the anti-epiplasm (grey). Central inserts correspond to

the area marked on the corresponding whole cell. Inserts at the bottom

correspond to one optical transverse section (top, outside, bottom, inside the

cell), performed across the cell margin. In control cells, the ID5 labelling

(red), which marks basal bodies, is located at the centre of the epiplasmic

units (grey) and beneath the cell surface (bottom insert). By contrast, in cells

expressing GFP-PtFOR20, the epiplasmic units are less regularly organised

around the basal bodies (in green) and GFP labelling is located at the level of

the epiplasm (bottom insert). Scale bar: 20 mm (inserts:66).

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all epiplasm units harbour two basal bodies, these units do not

duplicate but, as the wave of basal body proliferation reaches

them, the anterior basal body of each pair disappears and a new

one is reassembled and anchored at the same place (Romero and

Torres, 1993) (supplementary material Fig. S2). Surprisingly,

when the anterior basal body of each pair disappears, the GFP-

PtFOR20 labelling still marks its site (Fig. 4B). This observation

suggests that, once PtFOR20p is anchored at the membrane after

basal body docking, it may become dynamically independent of

the microtubule shaft and may guide the anchoring of a newly

assembled basal body.

Depletion of PtFOR20p prevents basal body docking at thecell surface

In order to analyse its role in the process of basal body and cilium

assembly, PtFOR20p was depleted by RNAi in wild-type and in

GFP-PtFOR20-expressing cells (see Materials and Methods). In

both types of cells, a strong effect was observed within two

divisions, yielding poorly ciliated, abnormal small cells with non

functional oral groove and an altered basal body pattern. In

addition, many basal bodies were detected inside the cells

(Fig. 5A).

In cells expressing GFP-PtFOR20 examined during the first

division under RNAi conditions, the GFP signal was still present

on the parental basal bodies, but was absent from newly

assembled ones, detected by ID5 antibody (supplementary

material Fig. S3), indicating the effective silencing of

PtFOR20. After two divisions, in both wild-type and GFP-

PtFOR20-expressing cells, the same phenotype was observed:

many basal bodies, all devoid of GFP were present within the

cytoplasm (not shown). The only basal bodies retaining the GFP

signal, presumably the parental ones, were anchored at the

surface (Fig. 5B). These observations demonstrate that: (1)

PtFOR20p depletion does not prevent the assembly of new

basal bodies and (2) the GFP-tagged protein is stably

incorporated into the basal bodies, since it remains associated

with parental basal bodies over at least two cell generations.

To understand how intracytoplasmic basal bodies and altered

basal body pattern were generated, the steps leading to this

phenotype were further analysed by double-labelling of wild-type

cells with antibodies specific for basal bodies and for the

epiplasm. The first phenotypic effects were detected at the first

division under inactivation conditions. The new basal bodies

were slightly mis-positioned in the epiplasm, more anterior than

normal and occasionally between two units (Fig. 6B), while in

the controls (Fig. 6A) they were clustered in the centre of the

unit.

At the ultra-structural level, many basal bodies, whether

undocked or partially docked at the membrane, were found next

to the parental ciliated basal bodies (Fig. 7A). Particularly

significant are the upper images of Fig. 7A which account for

the slight mis-positioning observed at the photonic microscope

level (Fig. 6B). Free basal bodies were also present deep in the

cytoplasm. These internal basal bodies all exhibit a distal end

Fig. 3. PtFOR20p localises to the transition zone. (A,B) Ultrastructure of ciliated and non-ciliated basal bodies in wild-type (A) and GFP-PtFOR20-expressing

cells (B). (C,D) Localisation of the GFP tag after post-embedding (C) and pre-embedding (D) immunolabelling. (A) Longitudinal sections of ciliated

(left) and unciliated (right) basal bodies in a wild-type cell. In both basal bodies, the three plates of the transition zone are detected (white arrowheads): the

terminal plate (large arrowhead), the intermediate plate (intermediate arrowhead) and the axosomal plates (small arrowhead). eu, epiplasm unit; er, ring of

epiplasm encircling the basal body; cn, ciliary necklace. Transition fibres and y-links are not visible on this section orientation. Scale bar: 200 nm.

(B) Longitudinal section of two paired basal bodies, one ciliated, the other one unciliated in a cell expressing GFP-PtFOR20. Expression of GFP-PtFOR20 does

not induce abnormalities in the basal body ultrastructure. The only observed difference is a faint thickening of the terminal plate as compared to the control

(4064 nm instead of 3565 nm, n530 basal bodies for each cell type). (C,D). Anti-GFP immunogold labelling on longitudinal (left) and transverse (right) sections

(C) and on whole, permeabilised cells (D). On longitudinal sections, the labelling is located just above the terminal plate (arrows in C and D). On transverse

sections, the labelling is detected at the level of thin fibres linking the microtubule doublets to the membrane (arrows in C and D). Note that in the pre-embedding

protocol, which includes a Triton X-100 treatment, the membrane is detected around the transversal sections of the axonemes. Scale bars: 200 nm.


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closed with structures resembling an incomplete transition zone.

In some internal basal bodies, microtubules extending beyond the

distal end could also be observed. As intracytoplasmic basal

bodies are never found in control cells, these observations

demonstrate that PtFOR20p is essential for basal body docking at

the cell surface and/or for completion of its maturation before

docking. Such observations indicate that, as already described by

Dippell (Dippell, 1968), some structures of the transition zone are

assembled before basal body anchoring to the cell surface. In

addition, in such undocked basal bodies, depletion of PtFOR20p

allows microtubules to extend beyond the incomplete transition

zone.

PtFOR20p and PtCen2p, but not PtCen3p, are necessary to

build the transition zone

Undocked intracytoplasmic basal bodies have previously been

observed under depletion of PtCen2p and Ptcen3p (Ruiz et al.,

2005), the two Paramecium basal body specific centrin isoforms,

orthologs to the mammalian centriolar centrins HsCen2 and

HsCen3 (Middendorp et al., 1997; Bornens and Azimzadeh,

2007). In order to compare the roles of these two proteins with

that of PtFOR20p, we re-investigated their localisation and the

effects of their depletion in Paramecium. GFP-PtCen2p which

localises within the basal body cylinder (Ruiz et al., 2005) was

shown here to be more abundant at its distal part, at the level of

the terminal plate (supplementary material Table S1). The same

localisation has also been reported in Tetrahymena (Stemm-Wolf

et al., 2005). A new observation was also that, under depletion of

PtCen2p, many basal bodies not properly docked or undocked

were longer than normal (Fig. 7B).

While the majority of GFP-PtCen3 is localised outside the

centriolar cylinder, anteriorly and apposed to its proximal end

(Ruiz et al. 2005), a faint labelling is also detected at the terminal

plate (supplementary material Table S1). A novel interesting

observation is that, under PtCen3p depletion, all internal basal

bodies develop a distal structure similar to the transition zone of

non-ciliated anchored basal bodies, with terminal, intermediate

and axosomal plates (Fig. 7C).

These observations, taken together with our results on

PtFOR20p localisation and depletion presented above, allow us

to propose the following sequence in basal body biogenesis:

PtCen2p acts first in basal body elongation and is essential for the

initiation of the transition zone assembly (Fig. 7B, supplementary

material Table S2). This step is upstream of the action of

PtFOR20p, which is involved in the completion of a structural cap

evoking intermediate or axosomal plates (Fig. 7A, supplementary

material Table S2). Last in the sequence is PtCen3p, whose major

role is not in the building of the transition zone, but in the docking

of the mature basal body to the cortex.

In order to investigate more precisely the functional

interactions between PtFOR20p and the centrins during basal

body assembly, GFP-PtFOR20 expressing cells were depleted for

either PtCen2p or PtCen3p. In both cases, the GFP-FOR20p

signal remained associated with the parental basal bodies over

generations. But while depletion of PtCen2p induced a loss of

GFP-PtFOR20 in all new basal bodies, the GFP fluorescence was

Fig. 4. GFP-PtFOR20 is both an early marker of new basal body assembly and a cortical remnant of disassembled basal bodies. (A) Early stage and

(B) middle stage of division of a GFP-PtFOR20-expressing cell decorated with 1D5 (red) and anti-GFP (green) antibodies. Inserts show higher magnifications of

the grey squares on the corresponding cells. (A) On the ventral side of the cell at an early stage of division, a new oral apparatus assembles (arrow). On both sides

of the future fission line (dashed line), basal body duplication takes place. In inserts below main panel (63), new GFP dots (arrows) are detected anterior toparental basal bodies. In inserts to the right of main panel (63.5), optical sections, transverse to the cell surface at the cell margin, show that the GFP labelling(green) localises at the proximal part of the parental basal bodies (red) at sites where new basal bodies are expected to be assembled. (B) At this later division

stage, the two oral apparatuses have pulled apart and the fission line is readily detected. The inserts (63) show that only one basal body (red) is detected in theanterior invariant field (arrows) when the anterior basal body of each pair is disassembled, whereas two GFP-labelled dots are present (green). Scale bars: 20 mm.


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retained on all basal bodies under depletion of PtCen3p (Fig. 8).

These results show that PtCen2p, but not PtCen3p, is necessary

for the recruitment and/or stabilisation of PtFOR20p at the basal

body. Acccordingly, in GFP-PtCen2-expressing cells, the GFP

signal associated with the basal bodies did not disappeared after

PtFOR20 depletion (data not shown).

DiscussionCiliogenesis requires the assembly and maturation of the basal

body, its docking at the cell surface and the growth of the ciliary

axoneme. In this paper, we demonstrate that, in the multiciliated

model Paramecium, the PtFOR20 protein is recruited very early

in the course of basal body biogenesis, cooperates with PtCen2p

to build the transition zone, and is required, in conjunction with

PtCen3p for basal body docking at the cell surface. These results

identify successive steps of basal body biogenesis, as well as

multiple roles of the transition zone.

PtFOR20p is an essential constituent of the transition zone

required for basal body anchoring to the membrane

Immunofluorescence and immunogold labelling on GFP-

PtFOR20 expressing cell lines show that PtFOR20p is stably

incorporated at the transition zone of basal bodies. By definition,

this region is localised between the basal body and the cilium, its

distal and proximal ends being clearly defined only in motile cilia

by the transition between triplets and doublets of microtubules

and the beginning of the two central axonemal microtubules,

respectively. In contrast, in non-motile cilia with the 9+0

architecture, this area remains weakly delimited and recent

observations suggest that the whole cilium could correspond to a

transition zone (Gluenz et al., 2010). In motile cilia, the transition

zone displays a variable architecture depending upon the species;

nevertheless some ultrastructural elements conserved during

evolution can be recognised, in particular the transition fibres

and the Y links which connect the microtubule doublets to the

membrane at the level of the ciliary necklace (reviewed by Fisch

and Dupuis-Williams, 2011). In Paramecium cells expressing

GFP-PtFOR20, the immunogold labelling is found on

microtubule/membrane links, at the level of microtubule

doublets, that is on the transitional fibres and/or the Y links

described by Dute and Kung (Dute and Kung, 1978). This

observation fits well the proposed microtubule-associated

functions of the LisH domain (Ames and Ponting, 2001).

The localisation of PtFOR20p at the transition zone of

assembled cilia is in agreement with the observed impairment

of basal body docking at the cell surface after PtFOR20p

depletion and can account for the phenotype observed after

HsFOR20p depletion in RPE1 cells (Sedjaı̈ et al., 2010). The

biogenesis of the primary cilium is a two step process, leading

first to the assembly of a short axoneme as the mother centriole

binds the ciliary vesicle, and then to axoneme elongation after

docking of the centriole at the cell surface (Sorokin, 1962;

Rohatgi and Snell, 2010). HsFOR20p depletion could thus

interfere with primary cilium assembly by preventing the second

Fig. 5. Depletion of PtFOR20p prevents basal body docking at the cell surface. (A) Confocal section at the intracytoplasmic level of a control cell (left) and a

cell after two divisions under PtFOR20p depletion (right), double labelled with ID5 (red) and anti-epiplasm antibody (grey). Many internal basal bodies are

detected after depletion, whereas none are detected in the control cell. Arrow indicates oral apparatus. (B) The surface of a GFP-PtFOR20-expressing cell after

three divisions under PtFOR20p depletion, labelled with ID5 (red) and the anti-GFP antibody (green). The basal body pattern is completely disorganised. Arrow

indicates abnormal oral apparatus. The GFP signal is detected on very few basal bodies (right inserts), which correspond to the labelled basal bodies of the parental

cell, assembled before depletion. Scale bar: 20 mm (inserts62).

Fig. 6. Depletion of PtFOR20p alters basal body

positioning at the cortex. Projections of optical sections

acquired at the level of the dorsal side of the cells double

labelled with ID5 (green) and the anti-epiplasm antibody

(red). (A) Control dividing cell. (B) Cell in the first division

during depletion. No abnormality is detected in the global

morphology of the depleted cell (B) compared with the

control (A). High magnification of the future fission line

shows that the new basal bodies, which appear in the centre

of the unit, close to parental basal bodies in the control cell

(insert A), are less precisely localised within the epiplasmic

units and often far from parental basal bodies after

PtFOR20p depletion (arrows). Scale bar: 20 mm(inserts:66).


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step of the process. In addition, the reduction of ciliary length

observed in ciliated cells could also be explained by a defect in

the assembly of the microtubule/membrane links, known to

control the intraflagellar transport (Craige et al., 2010; Williams

et al., 2011).

The respective roles of PtFOR20p, PtCen2p and PtCen3p

identify four steps in basal body assembly

Beyond the nine triplet nucleation by the ancestral centriole/basal

body set of core proteins (Carvalho-Santos et al., 2011; Cottee

et al., 2011), the above functional and cytological dissection

circumscribes four steps in basal body biogenesis: elongation of

the triplet microtubules, capping as an initiation of transition

zone assembly, maturation of the transition zone, then basal body

docking. Each of these steps can be identified by the sequential

requirement of PtCen2p, PtFOR20p and PtCen3p.

PtCen2p is required to initiate the transition zone assembly and

is involved in the control of basal body elongation

Long basal bodies were often observed after PtCen2p depletion,

showing that PtCen2p is required for control of basal body length.

In addition, after PtCen2p depletion, the cap which closes the distal

part of the basal body is missing. This strongly suggests that

PtCen2p is necessary to recruit cap proteins, which in turn would

be required to control pro-basal body elongation and assembly of

the transition zone. A similar role of this centrin in transition zone

assembly has already been noted in Chlamydomonas in the case of

the centrin mutant vfl2 (Jarvik and Suhan, 1991). However, a role

of this protein in the control of basal body elongation has not yet

been documented. In this context, it will be interesting to test the

interaction of PtCen2p with other proteins shown to control

centriole length such as POC1, POC5 or OFD1 (Keller et al., 2009;

Azimzadeh et al., 2009; Singla et al., 2010).

Fig. 7. Differential ultrastructural effects of PtFOR20p, PtCen2p and PtCen3p depletion on basal body maturation and docking. (A) Upon PtFOR20p

depletion, undocked or partially docked basal bodies are detected close to the cell surface (top panel) or deep in the cytoplasm (bottom panel). A partial terminal

plate can be detected on partially docked basal bodies on the side where they attach to the cell surface, and on intracytoplasmic basal bodies (arrows); in the latter

case, microtubule extensions are often detected on the free, uncapped side of the basal body. Vestigial structures resembling intermediate and axosomal plates are

detected on all basal bodies (arrowheads). (B) Upon PtCen2p depletion, basal bodies longer than normal were often observed, as in the top panel where the new

basal bodies (arrows) are longer than their parents (to the right). In addition, their transition zones are abnormal (arrows). Many intracytoplasmic long basal bodies

without or with an incomplete (arrow) transition zone are also detected (lower panel). (C) Upon PtCen3p depletion, the partially undocked and the

intracytoplasmic basal bodies have an associated transition zone in which the three plates (terminal, intermediate and axosomal) can be identified (arrows). Scale

bars: 200 nm.

Fig. 8. Recruitment of PtFOR20p requires PtCen2p. After PtCen2p

depletion, some newly assembled basal bodies (arrows) lack the associated

GFP-PtFOR20 signal, whereas after PtCen3p depletion, all basal bodies (in

red) have their associated GFP-PtFOR20 cap (green). Scale bar: 2 mm.


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PtFOR20p is required for transition zone maturation

PtCen2p depletion prevents the appearance of PtFOR20p in basalbodies, indicating that the former protein is required for therecruitment/stabilisation of the latter. Accordingly, under

PtFOR20p depletion, basal body biogenesis is stopped at a laterstep, when basal bodies exhibit a normal length and are closedwith a structure resembling an incomplete transition zone.

Ultrastructural data suggest that it is the terminal plate which isthe most affected, but molecular data for the incompletetransition zone would be necessary to confirm this hypothesis.The fact that upon PtFOR20p depletion microtubules of

intracytoplasmic basal bodies are able to grow beyond the capsuggests that recruitment of PtFOR20p is necessary to controlmicrotubule elongation. The presence of the LisH domain

suggests that, as proposed for OFD1 (Singla et al., 2010),PtFOR20p could regulate the dynamics of basal bodymicrotubules.

PtCen3p is required for basal body docking of fully maturebasal bodies

Undocked basal bodies resulting from PtCen3p depletion have anormal length and a fully assembled transition zone, at least

according to ultrastructural criteria. These results show that thetransition zone can be assembled independently of basal bodydocking at the cell surface. These undocked basal bodies resultfrom a defect in the tilting up movement of the new basal body

toward its docking site at the cell surface, in agreement with thefact that PtCen3p has been mainly localised outside the basalbody at its proximal part (Ruiz et al., 2005) (M.J.-D. et al.,

unpublished results). The same localisation has been observed inanother ciliate, Paraurostyla (Lemullois et al., 2004). We showhere that, in addition to this proximal localisation, some PtCen3p

is also detected distally, within the basal body at the transitionzone. In ciliated epithelia, HsCen3p has been exclusivelydetected at the distal part of basal bodies (Laoukili et al.,2000). This distal localisation suggests that this protein could also

be required for basal body docking at the cell surface. PtCen3pcould thus be involved in different functions in association withdiverse centrin-binding proteins, such as Sfi1 (Kilmartin, 2003)

or hPOC5 (Azimzadeh et al., 2009) whose homologs exist inParamecium (Gogendeau et al., 2007).

The basal body as a cell compartment

It is striking that proteins localised at the transition zone of the

basal body are recruited very early during its biogenesis, asdemonstrated here for PtFOR20p, and in other studies for Vlf1 inChlamydomonas (Silflow et al., 2001), and a 210kD protein inSpermatopsis similis (Lechtreck et al., 1999). This observation is

paralleled by ultrastructural data showing that nascent basalbodies display a distal cap at the very beginning of their assembly(Dippell, 1968; Allen, 1969). These molecular and ultrastructural

observations strongly suggest that the distal cap corresponds tothe first step of transition zone assembly. The nascent basal bodywould thus be equipped with a nascent transition zone.

A growing set of data shows that the mature transition zone

acts as a barrier or a filter for the cilium compartment (Craigeet al., 2010; Williams et al., 2011; Garcia-Gonzalo et al., 2011)(for a review, see Ishikawa and Marshall, 2011). Moreover, it

maintains basal body integrity by preventing entry of ciliaryspecific components: in the centrin vfl2 mutant ofChlamydomonas which has a defective transition region, the

central pair of the cilium runs into the lumen of the basal body(Jarvik and Suhan, 1991). In the same way, as soon as assembled,

the nascent transition zone could act as a diffusion barrier tocreate a nascent basal body compartment, thus maintaining itsmolecular individuality until its docking at the surface. The

CP110 cap complex assembled at the distal part of the elongatingcentriole in mammalian cells (Kleylein-Sohn et al., 2007) mightfulfil a similar function. Accordingly, the internal texture of

centrioles and basal bodies differs from that of the surroundingcytoplasm (for a review, see Azimzadeh and Marshall, 2010)suggesting that these two cellular spaces, inside and outside thecentriole/basal body compartment, would not have the same

composition.

FOP-like proteins as components of an ancestral moduleinvolved in microtubule membrane interaction

Our results show that, in Paramecium, once assembled, theFOR20 protein remains at the cell surface after basal bodydisappearance, and thus could act as a structural landmark for

subsequent morphogenetic events as for example duringreciliation after injury of epithelia (Carson et al., 1981). Such astructural landmark has already been observed for FASS, acentrosomal protein conserved in land plants and involved in the

assembly of the pre-prophase band (Wright et al., 2009), atransient cortical microtubular array required for determination ofthe subsequent cell division plane in plants.

Several other proteins involved in centriolar and ciliaryfunctions are conserved in acentriolar plants (Laligné et al.,

2010; Hodges et al., 2011). Many of them have, or are supposedto have, a role in cytoskeletal/microtubule organisation (Gardinerand Marc, 2011; Hodges et al., 2011) suggesting that they are

maintained for microtubular organisation in non-ciliated plants(Hodges et al., 2011). The member of the centrosomal FOPfamily conserved in land plants, TONNEAU1, is known tointeract with centrin and to be required for correct assembly of

the preprophase band (Azimzadeh et al., 2008). The commonproperties of FOR20 and TONNEAU1 suggest an ancestralevolutionarily conserved function of the FOP proteins in the

control of the interactions between microtubular complexes andthe cell surface.

Materials and MethodsStrains and culture conditions

Stock d4-2 of Paramecium tetraurelia, the wild-type reference strain, was used inRNAi experiments. The mutant nd7-1 (Skouri and Cohen, 1997) which carries arecessive monogenic mutation preventing trichocyst discharge, a dispensablefunction under laboratory conditions, was used for transformations. Cells weregrown at 27 C̊ in a wheat grass infusion, BHB (L’arbre de vie, Luçay Le Male,France) or WGP (Pines International, Lawrence, KS), bacterised with Klebsiellapneumoniae and supplemented with 0.8 mg/ml b-sitosterol according to standardprocedures (Sonneborn, 1970).

Gene identification

By BLAST search, we identified two Paramecium genes encoding proteinshomologous to FOR20: PtFOR20a (PTETG1700017001) and PtFOR20b(PTETG4900011001) (Arnaiz and Sperling, 2011). These two genes result fromthe last whole genome duplication which occurred in Paramecium, and are 92.4%identical: they code for 165 amino acid long proteins which differ by a singleamino acid, at position 22.

Gene cloning

For expression of the GFP-tagged PtFOR20 protein, the entire 595 base pair longPtFOR20a gene was amplified from genomic DNA by PCR, using primers intowhich linkers containing the specific sequence of the KpnI enzyme were added.Two GGT glycine codons were also added in the sequence of the 59 primerbetween the KpnI restriction sequence and the ATG sequence of the PtFOR20


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gene. After KpnI restriction digest, the fragment was cloned into the KpnIrestriction site located at the 39 end of the GFP synthetic gene that was designed byEric Meyer and Jean Cohen (personal communication) and which had beenintroduced into the pPXV vector (Haynes et al., 1995), the recombinant gene beingunder the control of the Paramecium calmodulin regulators. After cloning, thegene was entirely sequenced to ensure that no error was introduced during theamplification. For gene silencing, only a part of the PtFOR20-b gene (from base 3to 453) was amplified by PCR and cloned into the XhoI and HindIII site of theL4440 vector. This vector allows the synthesis of double-stranded RNAcorresponding to the cloned gene from two T7 promoters. The possible off-target effect of this sequence was analysed using the RNAi off-target tool in the P.tetraurelia genome database (http://paramecium.cgm.cnrs-gif.fr/cgi/tool/alignment/off_target.cgi) that searches the genome for stretches of 23 identicalnucleotides (the size of Paramecium siRNA). The only genomic sequencematching with PtFOR20a was PtFOR20b, thus ruling out possible off-targetingeffect, but indicating that both genes will be co-silenced by the use of a singleRNAi vector. For PtCen2 and PtCen3 silencing, we used the same vectors as thosedescribed previously (Ruiz et al., 2005).

Paramecium transformation

nd7-1 mutant cells were transformed by micro-injection into their macronucleus offiltered and concentrated plasmid DNA containing a mixture of the plasmidsof interest (5 mg/ml) digested by Sfi1 and of plasmid DNA directing the expressionof the ND7 wild-type gene (Skouri and Cohen, 1997). Transformants were firstscreened for their ability to discharge their trichocysts and if so, further analysed.Microinjection was made under an inverted Nikon phase-contrast microscope,using a Narishige micromanipulation device and an Eppendorf air pressuremicroinjector.

Gene silencing

Gene silencing was performed by the feeding method as previously described(Galvani and Sperling, 2002). Stationary wild-type cells or log-phase transformedcells were fed with the appropriate double-stranded RNA-expressing HT115bacteria and transferred daily into fresh feeding medium as needed. Control cellswere fed with bacteria transformed by the L4440 plasmid or the L4440 plasmidcarrying the complete coding region of the ND7 gene.

Fluorescence microscopy

Immunostaining of cells was carried out as previously described (Iftode et al.,1989), except for cilia labelling, where saponin 0.5% was used instead of Triton X-100 to permeabilise the cells, with saponin 0.25% maintained in all subsequentbuffers. Cells were observed under a Zeiss Axioskop 2-plus fluorescencemicroscope equipped with a Roper Coolsnap-CF intensifying camera with GFPfilters. Images were processed with Metamorph software (Universal Imaging).Alternatively, they were observed with a Confocal Nikon eclipse TE 2000-Umicroscope equipped with argon and Helium-Neon lasers using the EZ-C1 3.30software for acquisitions. Unless specified, images are projections of opticalsections acquired at the level of one cell side. Projections were calculated usingImageJ (NIH) and images were treated with Photoshop CS2 (Adobe) software.

Electron microscopy

For ultrastructural observations, the depleted and control cells were fixed in 1%(v/v) glutaraldehyde and 1% OsO4 (v/v) in 0.05 M cacodylate buffer, pH 7.4 for30 min. After rinsing and dehydration in ethanol and propylene oxide series, theywere embedded in Epon resin. For pre-embedding immunolocalisation, theimmunostaining process was carried out as described for immunofluorescenceusing a gold-coupled instead of fluorochrome-coupled secondary antibody. Thencells were rinsed, fixed and embedded as previously described. For post-embedding immunolocalisation, cells were fixed in 3% paraformaldehyde, 0.15%glutaraldehyde, in 0.05 M cacodylate buffer, pH 7.4 at 4 C̊ for 1 h. After washesand dehydration in ethanol, they were embedded in LR White (London Resin).Thin sections were collected on nickel grids and treated with 0.1 M NH4Cl in0.1 M PBS and then saturated with 3% BSA and 0.1 M glycine in PBS. Sectionswere incubated with anti-GFP polyclonal antibody at room temperature for 45 min.After several washes in PBS a gold-labelled anti-rabbit IgG (GAR G10, Aurion)diluted 1:50 was applied for 30 min. The grids were rinsed in PBS and distilledwater, and finally stained with uranyl acetate. All ultrathin sections werecontrasted with uranyl acetate and lead citrate. The sections were examined withJeol 1400 (120 kV) or Philips CM10 transmission electron microscopes.

Antibodies

The following antibodies were used: the monoclonal 17E2 directed againstHsFOR20p (Sedjaı̈ et al., 2010) diluted 1:1000; the monoclonal anti-glutamylatedtubulin ID5 (Wehland and Weber, 1987) diluted 1:500 to label basal bodies; themonoclonal TAP952 directed against monoglycylated tubulin (Callen et al., 1994;Janke and Bulinski, 2011) diluted 1:10 to decorate the cilia; the monoclonalCTS32 directed against epiplasmins (Nahon et al., 1993) diluted 1:20, to decorate

the epiplasm, and the polyclonal anti-GFP antibody (Interchim, Montluçon,France) diluted 1:500 to amplify the GFP signal. Appropriate secondary (mouse orrabbit) antibodies from Jackson ImmunoResearch labs (West Grove, PA) Alexa488 or Alexa 568 were used at a dilution of 1:200.

AcknowledgementsWe thank A. M. Tassin, D. Gogendeau, M. Bornens for criticalreading of the manuscript and the ‘Signalisation cellulaire etmorphogenèse’ team (CGM) for confocal facilities. Electronmicroscopy was performed at the Imagif plateforme, CNRS, Gifsur Yvette (France).

FundingThis work was supported by the CNRS, INSERM and Institut Paoli-Calmettes and by a grant from the Agence Nationale de la Recherche[grant number 2010 BLAN 1122 03]. M.J.-D. was supported bystatute grants from The Ministry of Science and Higher Education(Poland) to the M. Nencki Institute of Experimental Biology, PolishAcademy of Sciences (Warsaw, Poland). C.L. was supported by theFondation pour la Recherche Médicale.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.108639/-/DC1

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