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SHORT REPORT

Procentriole assembly without centriole disengagement 2

a paradox of male gametogenesis

Marco Gottardo, Giuliano Callaini* and Maria Giovanna Riparbelli

ABSTRACT

Disengagement of parent centrioles represents the licensing

process to restrict centriole duplication exactly once during the

cell cycle. However, we provide compelling evidence that this

general rule is overridden in insect gametogenesis, when distinct

procentrioles are generated during prophase of the first meiosis

while parent centrioles are still engaged. Moreover, the number of

procentrioles increases during the following meiotic divisions, and

up to four procentrioles were found at the base of each mother

centriole. However, procentrioles fail to organize a complete set of

A-tubules and are thus unable to function as a template for centriole

formation. Such a system, in which procentrioles form but

halt growth, represents a unique model to analyze the process of

cartwheel assembly and procentriole formation.

KEY WORDS: Centriole duplication, Procentrioles, Cartwheel,

Butterfly

INTRODUCTIONDuring cell division, the correct segregation of chromosomes into

the two daughter cells relies on the correct organization of the

bipolar spindle by centrosomes that had just duplicated. This is a

crucial step, because abnormal centrosome numbers may lead to

multipolar spindles with ensuing aneuploidy and chromosome

fragmentation, forms of genomic instability that represent an

established hallmark of cancer cells (Ganem et al., 2009; Crasta

et al., 2012; Vitre and Cleveland, 2012). Because centrioles

mark the sites where the centrosomal material is recruited their

number determines the number of centrosomes (Sluder and

Rieder, 1985) and, therefore, centriole duplication has to be

accurately regulated. Although, vertebrate cells have temporal

and spatial constraints to ensure that only one daughter is

assembled for each mother once during the cell cycle, the

mechanisms managing these controls are not fully understood.

Centriole disengagement during metaphase/anaphase has been

proposed to represent the licensing factor to allow centriole

duplication during the following interphase (Tsou et al., 2009;

Wang et al., 2011). Moreover, a newborn daughter centriole

seems to be sufficient to inhibit the formation of additional sisters

at the basal end of the mother by locally limiting the availability

of structural and/or regulatory proteins needed for their assembly

(Uetake et al., 2007; Brito et al., 2012).

Recent studies have suggested that a core module of specific

conserved proteins drives the formation of daughter centriolesin the presence of pre-existing mothers or during de novo

assembly (Gonczy, 2012). However, ultrastructural data fail to

unambiguously reveal the same intermediate structures during theprocess of procentriole assembly in different organisms. It is stillunclear whether the structural details observed at the onset of

procentriole formation can differ in various animal groups orwhether technical challenges have prevented a careful analysisof the early steps of centriole assembly. Therefore, increasedunderstanding of centriole biogenesis in different animal systems

is expected to shed light on the general mechanism of centrioleduplication and assembly. We report here that distinctprocentrioles appear at the base of duplicated centrioles during

prophase of the first male meiosis in butterflies, despite thecentrioles being engaged. Moreover, during the following meioticdivisions the procentrioles increase in number and form distinct

clusters at the proximal end of each mother centriole. However,procentrioles fail to grow and are, thus, unable to organizecentrosomal material.

RESULTS AND DISCUSSIONCentriole engagement does not prevent procentrioleassemblyAs usual in insects, the butterfly primary spermatocytes have twocentriole pairs disposed in V-shape configuration that represent theplatform for the assembly of four long ‘cilium-like’ projections

(Fig. 1a,b) (Henneguy, 1898; Meves, 1902; Friedlander andWahrman, 1970; Wolf and Traut, 1987, Yamashiki andKawamura, 1998). Unlike most animal cells, in which the ciliaare reabsorbed to release the centriole that then organizes the

spindle poles, the cilium-like projections persist during insectspermatogenesis in order to later organize the sperm axoneme(Riparbelli et al., 2012; Gottardo et al., 2013). Thus, the centriole

performs the double role of spindle pole organizer and axonemeconstructor. While studying the dynamics of the cilium-likeprojections during spermatogenesis and spermatid differentiation

in the butterfly Pieris brassicae, we found distinct structures closeto the basal region of each centriole (Fig. 1c,d). These structuresappeared as short cylinders of almost constant size with an average

diameter of 114.2 nm (64.1; n514) and an overall length of117.2 nm (63.7; n59), longitudinally crossed by an inner tubuleof about 21.3 nm (62.6; n514) in diameter. The tubule is linked tothe wall of the cylinder by thin radial projections (Fig. 1e). Such

architecture is reminiscent of the cartwheel found in procentrioles,the early intermediates in centriole assembly of most animalcells. Procentrioles were lacking in young primary prophase

spermatocytes but were always found during late prophase. Thus,the primary spermatocytes showed four fully elongated centriolestogether with four procentrioles. This suggests that the centrioles of

primary prophase spermatocytes undergo an extra duplication

Department of Life Sciences, University of Siena, Via A. Moro 4, 53100 Siena,Italy.

*Author for correspondence (callaini@unisi.it)

Received 10 March 2014; Accepted 2 June 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3434–3439 doi:10.1242/jcs.152843

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cycle, in addition to the usual duplication that occurs in earlyprophase soon after the last spermatogonial mitosis. This

represents an unconventional condition because, to avoid theformation of multipolar spindles, centrioles in animal cellsduplicate only once per cell cycle during transition from G1 to

S of interphase (Wong and Stearns, 2003). It is generallybelieved that the interruption of the close association, i.e. thedisengagement, between mother and daughter centrioles duringmetaphase/anaphase transition enables the forthcoming duplication

during interphase of the next cell cycle (Tsou and Stearns, 2006;Tsou et al., 2009). Accordingly, ultrastructural analysis ofDrosophila melanogaster primary spermatocytes (Tates, 1971;

Fritz-Nigli and Suda, 1972; Riparbelli et al., 2012), in whichcentrioles are linked together by fibrous material (Stevens et al.,

2010), failed to reveal procentrioles. In butterfly primaryspermatocytes, the centrioles within each pair are held together

by thin fibrous material – as in Drosophila centrioles – but theyduplicate. This represents a remarkable exception in the regulationof centriole duplication during the cell cycle and raises questions

on the universality of the licensing factor.The radial spokes connected the central hub to the wall of the

procentriole (Fig. 1e) that was formed by a thin peripheral ringsurrounded by a cluster of dense material. The ring was more

evident at the distal end of the procentriole where the densematerial was scarce (Fig. 1f). Short microtubules contacted theouter side of the procentriole wall, which was also crossed by

curved grooves whose diameter was similar to that of singlemicrotubules (Fig. 1e,g). Some of the grooves underwent partial

Fig. 1. Procentriole in primary spermatocytes. (a) Primary butterfly spermatocytes have four elongated cilium-like projections (green); DNA is shown in red.(b) The axoneme of the cilium-like projections is nucleated by elongated centrioles. (c,d) One procentriole (arrows) is found near the proximal region of parentcentrioles. (e,f) Consecutive cross sections through the proximal and distal ends of the procentriole shown in c. A single microtubule is visible on theprocentriole wall (asterisk) (e); a thin inner ring (double arrow) is more apparent where the dense material surrounding the procentriole is scarce (f). (g) Curvedgrooves are visible on the wall of the procentriole (asterisks). (h) The parent centriole is surrounded by inner (double arrowhead) and outer (arrow) sheets ofdense material; the proximal end of the procentriole leans against the outer sheet, whereas its distal end contacts a flat cistern. (i,j) The hub (arrowheads)reaches the inner sheet that surrounds the basal end of the mother centriole. (k) Possible steps in butterfly procentriole assembly. Scale bars: 2.5 mm(a), 500 nm (b), 200 nm (c–d), 50 nm (e–j).

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closure, suggesting that they represent intermediate stages ofmicrotubule assembly. The dense outer wall of the procentriole

might, therefore, be regarded as the platform for the assemblyof microtubules, and the curved grooves might denote nascentA-tubules, the first tubules of the pericentriolar tripletmicrotubules to form. Strikingly, electron-dense hook-like

structures of unknown composition, were observed on the tubeof Caenorhabditis elegans prior to the presence of assembledmicrotubules (Dutcher, 2007). The formation of A-tubules in

butterflies would require a process of lateral nucleation, such asthat described for the assembly of B- and C-tubules duringcentriole formation in human KE-37 cells (Guichard et al., 2010).

In these cells, however, the A-tubule, which is presumablynucleated by a proximal cap of c-tubulin, is used by the B-tubuleas a template.

A model of procentriole assemblyThe parent centriole was surrounded by two concentric sheetsof dense material: a complete inner and an incomplete outer

(Fig. 1h). The hub extended beyond the length of the procentrioleand reached the inner dense sheath that surrounded the base ofthe parent centriole (Fig. 1i,j). Radial projections could not be

distinguished in the proximal region of the hub. Likewise, adistinct stalk connects the central hub to the parent centriole inhuman cells (Guichard et al., 2010). These observations suggest

that the hub emerged from the dense sheet at the base of themother centriole to initiate the organization of a central structuralscaffold on which radial spokes assemble to originate the

primitive cartwheel. A thin cylinder might then be built aroundthe radial spokes (Fig. 1k). Therefore, the A-tubules do not attach

to the distal end of the radial spokes through distinct pinheads, asreported in other systems (Guichard et al., 2013), but contact a

thin cylinder that is the scaffold on which the material that ispresumably involved in the nucleation of the A-tubule will berecruited.

Our observations are consistent with a model in which a small

tube might represent a first step in procentriole assembly.Consistently, the overexpression of Sas-6 in Drosophila led tothe appearance of tube-like structures (Rodrigues-Martins et al.,

2007). Moreover, concurrent overexpression of Sas-6 and itsbinding partner Ana2 led to the formation of cartwheel-likestructures in Drosophila spermatocytes (Stevens et al., 2010). The

assembly of a large tube that forms near the parent centriolecharacterizes the first step of procentriole formation in C. elegans

(Pelletier et al., 2006).

Assembly of a daughter centriole does not require continuitywith the mother centrioleEach parent centriole has its procentriole during the first meiotic

prophase (Fig. 1c,d). When the cilium-like projections elongatedfurther during prometaphase (Fig. 2a) two procentrioles wereseen in the proximity of each parent centriole (Fig. 2b,c; n57).

The spindle poles of secondary spermatocytes during metaphaseand/or anaphase contained only one centriole (Fig. 2d) at the baseof the cilium-like projections (Fig. 2e), but a cluster of two

(Fig. 2f,g; n59), three (Fig. 2h,i; n55) and sometimes four(n52) procentrioles was found near the basal region of eachparent centriole. Therefore, multiple daughter procentrioles can

be concurrently generated at the base of single parent centrioles,which challenges the traditional view of a preferential site of

Fig. 2. Multiple procentrioles form at the base ofsingle mother centrioles. Prometaphase of the firstmeiosis: (a) cilium-like projections are very elongated;microtubules are shown in green, DNA is shown in red.Details of a centriole (b) and procentrioles (c) at onespindle pole are shown. Metaphase of the secondmeiosis: the spindle poles contain only one centriole(d) that nucleates a cilium-like structure (e); microtubulesare shown in green, DNA is shown in red. Details ofcentrioles (f,h) and procentrioles (g,i) visible in associatedserial sections during metaphase/anaphase of thesecond meiosis are shown. (j) Localization of Sak/Plk4cross-reacting antigens (red) at the base of the cilium-likeprojections (green) during metaphase of the secondmeiosis – two small spots are visible (arrow). Scale bars:2.5 mm (a,e), 200 nm (b,f,h), 500 nm (d), 50 nm(c,g,i), 1.5 mm (j).

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assembly at the base of each parent centriole and its role inmanaging the formation of procentrioles (discussed in Sluder and

Khodjakov, 2010).Besides the temporal control of the ‘once and only once’

formation during the cell cycle, vertebrate cells also have a spatialcontrol to yield formation of only one procentriole at each existing

mother centriole, despite the large pool of cytoplasmic componentsavailable (Nigg and Stearns, 2011). Once the assembly of the newprocentrioles has been initiated, further centriole duplication is

inhibited until the cells pass through mitosis (Wong and Stearns,2003; La Terra et al., 2005; Uetake et al., 2007). It is possible thatthe numerical control of procentriole formation is based on a

balanced control of protein–protein interactions and/or activations.Therefore, procentriole formation would be restricted to thevicinity of the existing centrioles that maintain a compact region

of pericentriolar material, the only area in the cell where newcentrioles can form (Loncarek et al., 2008; Loncarek et al., 2010).Antibodies against Drosophila Sas-6, Ana1 and Bld10 (a homologof Chlamydamonas reinhardtii Bld10p and human CEP135) that

recognize a procentriole-like structure (PCL) at the base of themother centriole in Drosophila elongating spermatids (Blachonet al., 2009; Blachon et al., 2014; Mottier-Pavie and Megraw,

2009) did not crossreact with butterfly centriolar proteins. Bycontrast, an antibody against Drosophila SAK, a homolog ofvertebrate PLK4, which – in Drosophila – has main roles in the

assembly of the PCL (Blachon et al., 2009), recognized in Pieris

brassicae additional spots for each centriole (Fig. 2j).The concurrent appearance of multiple centrioles has

been described under experimental conditions, in which theconcentration of proteins involved in centriole biogenesis or cell-cycle regulation has been modified (Brownlee and Rogers, 2013).Clusters of procentrioles in flower-like or rosette disposition

were, indeed, observed around each mother after theoverexpression of Plk4 kinase (Habedanck et al., 2005;Kleylein-Sohn et al., 2007), a process enhanced by cyclin-E–

CDK2 (Duensing et al., 2007). A similar phenotype is alsoinduced by downregulating the ubiquitin ligase SCFSlimb

complex, which regulates levels of Plk4 (Cunha-Ferreira et al.,

2009; Rogers et al., 2009). The rosette phenotype is alsogenerated by overexpression of Sas-6 (Strnad et al., 2007), aprotein needed for cartwheel assembly, or by the overexpressionof STIL, the putative partner of Sas-6 (Tang et al., 2011; Arquint

et al., 2012;). More than one daughter centriole also forms at asingle mother centriole following inhibition of proteasomes ortransfection with oncoprotein HPV-16 E7 (Duensing et al., 2007).

In addition, when blocked in S-phase, some cultured vertebratecells assemble multiple daughter centrioles around each mother(Balczon et al., 1995). Multiple basal bodies also arise from a

single mother centriole in a flower-like disposition in mammaliandifferentiated epithelial cells, such as those of the respiratory andreproductive tracts (Anderson and Brenner, 1971; Dirksen, 1971).

However, under normal circumstances only one daughter formsin cycling cells. Therefore, the finding of multiple procentrioles ateach spindle pole in butterflies seems to create a paradox andsuggests that the centriole duplication control is downregulated.

The rule of ‘only one centriole per mother’ may be overcome,suggesting that the assembly of a daughter centriole does notnecessarily prevent the formation of additional ones.

Procentrioles appear in butterfly during late primary prophase,when the parent centrioles attained their full size and do notelongate further. Moreover, they did not undergo significant

changes in shape and a complete set of A-tubules never forms.

This suggests that centriole elongation is correlated to the cellcycle, with full elongation taking place at the end of the

first meiotic prophase. The failure of procentriole growth andmaturation in butterfly spermatocytes may represent the limitingfactor to avoid the assembly of functional centrosomes and theformation of supernumerary spindles. Therefore, the assembly of

additional centrioles is blocked not at the beginning but at thestage when tubules are added. Pieris brassicae spermatids lack adistinct centriole adjunct. Thus, it might be easier to detect

procentrioles that are close to basal bodies. One or twoprocentrioles were observed in early spermatids (Fig. 3a,b).However, they lost the close association with parent centrioles

that nucleated the spermatid axoneme (Fig. 3c) and were oftenfound next to the nuclear membrane (Fig. 3d). Procentriolesfound in early spermatids had a diameter similar to those found

during preceding meiotic divisions [112.4 nm (63.2; n55)versus 114.2 nm (64.1; n514), respectively] as well as theinvariable cartwheel structure, but were slightly shorter [81.7 nm(62.9; n56) versus 117.2 nm (63.7; n59)]. Procentrioles were

no longer detected in elongating spermatids. These results areconsistent with the gradual reduction of the supernumeraryprocentrioles and point to a redundant role of these structures

during butterfly gametogenesis.

A procentriole at the core of the parent centrioleParent centrioles at the end of the first prophase had a nearlyconstant size, with an average diameter of 195 nm and an overalllength of 730 nm (Fig. 4a). The proximal end of the mature

centriole showed a dense ring connected to the central hub bynine radial spokes (Fig. 4b). This structure, which we call the

Fig. 3. Procentrioles persist in young spermatids. (a) Early spermatidshave a round nucleus (DNA in red) and an elongated axoneme (microtubulesin green). (b) The centriole that nucleates the spermatid axoneme contactsthe nucleus. (c,d) Procentrioles are often close to the nuclear envelope(arrows). Scale bars: 2.5 mm (a), 200 nm (b), 100 nm (c,d).

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‘inner ring complex’ (IRC), can be recognized in longitudinalsections as a short cylinder within the basal region of the

centriole (Fig. 4a). Microtubule triplets appeared in sectionstaken away from the basal region of the centriole. These tripletswere outline by a second outer ring of dense material (Fig. 4c).The IRC disappeared in further distal sections, whereas the outer

ring became undulated (Fig. 4d) and was barely detectable in thedistal region of the centriole (Fig. 4e). The hub increased indiameter, moving away from the basal end of the centriole

(Fig. 4b–d) and disappeared together with the peripheral ring.Sections in which we were able to detect both the basal end ofthe parent centrioles and associated procentrioles at the same

time (Fig. 4f–h) showed that the two were structurally verysimilar. Thus, the procentrioles can correspond to distinctplatforms on which the future centrioles will be assembled. A

cartwheel is transiently present at the base of young centrioles invertebrate centrosomes, but disappears in mature centrioles

(Alvey, 1986; Strnad and Gonczy, 2008; Azimzadeh andMarshall, 2010).

MATERIALS AND METHODSInsectsA culture of Pieris brassicae was obtained from a laboratory strain and

reared in ventilated cages at 20 C. Larvae were fed with cabbage leaves

until pupation.

AntibodiesWe used the following antibodies: rabbit anti-Sak/Plk4 (1:200;

Bettencourt-Dias et al., 2005), chicken anti-DSAS-6 (1:1000,

Rodrigues-Martins et al., 2007), rabbit anti-Bld10 (1:500, Mottier-Pavie

and Megraw, 2009), mouse anti-acetylated tubulin (1:100; Sigma-

Aldrich). The secondary antibodies used (1:800, Invitrogen) were

conjugated with Alexa Fluor 488 or Alexa Fluor 555.

Immunofluorescence preparationsFor immunostaining testes from pupae of the lepidopteran Pieris

brassicae were dissected and fixed according to Riparbelli et al.

(Riparbelli et al. 2012). For localization of axonemal microtubules and

centriolar proteins, samples were processed as described previously

(Riparbelli et al., 2013). DNA was visualized by using Hoechst 33258.

Images were taken with an EC Plan-Neofluar 1006/1.30 objective by

using an AxioImager Z1 (Carl Zeiss) microscope equipped with an

AxioCam HR camera (Carl Zeiss).

Transmission electron microscopyTestes isolated from pupae were fixed in 2.5% glutaraldehyde in PBS

overnight at 4 C and further processed as described previously (Gottardo

et al., 2013). Thin sections were stained routinely with uranyl acetate and

lead citrate, and then observed with a Tecnai Spirit EM (FEI) equipped

with a Morada CCD camera (Olympus).

AcknowledgementsWe are grateful to Monica Bettencourt-Dias (Instituto Gulbenkian de Ciencia,Oeiras, Portugal) and Timothy L. Megraw (Florida State University, Tallahassee,FL) for generously providing antibodies.

Competing interestsThe authors declare no competing interests.

Author contributionsM.G., M.G.R. and G.C. conceived the project. M.G. and M.G.R. performedexperiments. G.C. wrote the manuscript.

FundingThis work was supported by PRIN2012 to G.C.

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