Procentriole assembly without centriole disengagement: a paradox of male gametogenesis
Marco Gottardo, Giuliano Callaini* and Maria Giovanna Riparbelli
Department of Life Sciences, University of Siena, Via A. Moro 4, 53100 Siena, Italy
*Corresponding author: Giuliano Callaini; Fax: +39 577 234476; Email: [email protected]
© 2014. Published by The Company of Biologists Ltd.Jo
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JCS Advance Online Article. Posted on 17 June 2014
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Summary
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 override in insect gametogenesis where distinct procentrioles are generated during prophase of
the first meiosis when parent centrioles are still engaged. Moreover, the procentriole number
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, so
being unable to work as 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.
Running title: Procentriole assembly
Key Words: Centriole duplication, procentrioles, cartwheel, butterfly
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Introduction
The proper segregation of chromosomes during cell division into the two daughter cells
relies on the correct organization of the bipolar spindle by just duplicated centrosomes. This is a
crucial step, since 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). Since 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 make 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., 2009; Brito et al., 2012).
Recent studies have suggested that a core module of specific conserved proteins drives the
formation of daughter centrioles in the presence of pre-existing mothers or during de novo assembly
(Gönczy, 2012). However, ultrastructural data fail to unambiguously reveal the same intermediate
structures during the process of procentriole assembly in different organisms. It is still unclear if the
structural details observed at the onset of procentriole formation may differ in various animal
groups or if technical challenges may have prevented a careful analysis of the early steps of
centriole assembly. Therefore, an increased understanding of centriole biogenesis in different
animal systems is expected to shed light on the general mechanism of centriole duplication and
assembly. We report here that distinct procentrioles appear at the base of duplicated centrioles
during prophase of the first male meiosis in butterflies despite being engaged. Moreover, during the
following meiotic divisions 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
organize centrosomal material.
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Results and Discussion
Centriole engagement does not prevent procentriole assembly
As usual in insects, the butterfly primary spermatocytes had two centriole pairs disposed in V-
shaped configurations that represent the platform for the assembly of four long “cilium-like”
projections (Fig, 1a,b) (Henneguy, 1898; Meves, 1903; Friedlander and Wahrman, 1970; Wolf and
Traut, 1987, Yamashiki and Kawamura, 1998). Unlike most animal cells in which the cilia are
reabsorbed to release the centriole that will organize the spindle poles, the “cilium-like” projections
persist during insect spermatogenesis to later organize the sperm axoneme (Riparbelli et al., 2012;
Gottardo et al., 2013). Thus, the centriole performed the double role of spindle pole organizer and
axoneme constructor. While studying the dynamic of the “cilium-like” projections during
spermatogenesis and spermatid differentiation in the butterfly Pieris brassicae we found distinct
structures close to the basal region of each centriole (Fig. 1c,d). These structures appeared as short
cylinders of nearly constant size with an average diameter of 114.2 nm (4.1; n= 14) and an overall
length of 117.2 nm ( 3.7; n=9), longitudinally crossed by an inner tubule of about 21.3 nm (2.6;
n= 14) in diameter. The tubule is linked to the 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 animal cells. Procentrioles were lacking in young primary prophase
spermatocytes, but they were always found during late prophase. Thus, the primary spermatocytes
showed four fully elongated centrioles along with four procentrioles. This suggests that the
centrioles of primary prophase spermatocytes underwent an extra duplication cycle, in addition to
the usual duplication that occurs in early prophase soon after the last spermatogonial mitosis. This
represents an unconventional condition, since in animal cells the centrioles duplicate once and only
once in every cell cycle during transition from G1 to S of interphase (Wong and Stearns, 2003) to
avoid the formation of multipolar spindles. It is generally believed that breaking of the close
association, or disengagement, between mother and daughter centrioles during metaphase/anaphase
transition represents the licensing factor for the forthcoming duplication during interphase of the
next cell cycle (Tsou and Stearns, 2006; Tsou et al., 2009). Accordingly, ultrastructural analysis of
Drosophila primary spermatocytes (Tates, 1971; Fritzi-Nigli and Suda, 1972; Riparbelli et al.,
2012), in which centrioles are linked together by fibrous material (Stevens et al., 2010), failed to
reveal procentrioles. In butterfly primary spermatocytes, the centrioles within each pair were held
together by thin fibrous material, like in Drosophila centrioles, but they duplicate. This represents a
remarkable exception in the regulation of centriole duplication during the cell cycle and raises
questions on the universality of the licensing factor.
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The radial spokes connected the central hub to the wall of the procentriole (Fig. 1e) that was
formed by a thin peripheral ring surrounded by a cluster of dense material. The ring was more
evident at the distal end of the procentriole where the dense material was scarce (Fig. 1f). Short
microtubules contacted the outer side of the procentriole wall that was also crossed by curved
grooves those diameter was similar to that of single microtubules (Fig. 1e,g). Some of the grooves
underwent partial closure suggesting that they may represent intermediate stages in microtubule
assembly. The outer dense wall of the procentriole may be regarded, therefore, as the platform for
the assembly of microtubules and the curved grooves may denote nascent A-tubules. Strikingly,
electron-dense hook structures, of unknown composition, were observed on the tube of
Caenorhabditis prior to the presence of assembled microtubules (Dutcher, 2007). The A-tubule
formation in butterflies would require a process of lateral nucleation, such that described for B- and
C-tubules assembly during centriole formation in human cultured cells (Guichard et al., 2010). In
cultured cells, however, the B-tubule utilizes as template the A-tubule that is presumably nucleated
by a proximal cap of -tubulin.
A model of procentriole assembly
The parent centriole was surrounded by two concentric sheets of dense material: a complete inner
and an incomplete outer (Fig. 1h). The hub extended beyond the procentriole length and reached the
inner dense sheath that surrounded the base of the parent centriole (Fig. 1i,j). Radial projections
cannot be distinguished in the proximal region of the hub. Likewise, a distinct stalk connects the
central hub to the parent centriole in human cells (Guichard et al., 2010). These observations
suggest that the hub may emerge from the dense sheet at the base of the mother centriole to initiate
the organization of a central structural scaffold on which radial spokes assemble to originate the
primitive cartwheel. A thin cylinder may be then built around the radial spokes (Fig. 1k). Therefore,
the A-tubules do not attach to the distal end of the radial spokes by distinct pinheads, as reported in
other systems (Guichard et al., 2013), but contact a thin cylinder that is the scaffold on which the
material presumably involved in the nucleation of the A-tubule will be recruited.
Our observations are consistent with a model in which a small tube may represent a first step
in procentriole assembly. Consistently, the over-expression of Sas-6 in Drosophila led to the
appearance of tube-like structures (Rodrigues-Martins et al., 2007). Moreover, the concurrent over-
expression of Sas-6 with its binding partner Ana2 led to the formation of cartwheel-like structures
in Drosophila spermatocytes (Stevens et al., 2010). The assembly of a large tube that forms near the
parent centriole characterizes the first step of procentriole formation in Caenorhabditis (Pelletier et
al., 2006).
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Daughter centriole assembly does not require continuity with its mother
Each parent centriole has its procentriole during the first meiotic prophase (Fig. 1c,d). When the
cilium-like projections elongated further during prometaphase (Fig. 2a) two procentrioles were seen
in the proximity of each parent centriole (Fig. 2b,c; n=7). The spindle poles of secondary
spermatocytes during metaphase/anaphase contained only one centriole (Fig. 2d) at the base of the
cilium-like projections (Fig. 2e), but a cluster of two (Fig. 2f,g; n=9), three (Fig. 2h,i; n=5), and
sometimes four (n=2) procentrioles was found near the basal region of each parent centriole.
Therefore, multiple daughter procentrioles can be concurrently generated at the base of single
parent centrioles challenging the traditional view of a preferential site of assembly at the base of
each parent and its role in managing procentriole formation (discussed in Sluder and Khodjakov,
2010).
Besides the temporal control of the once and only once formation during the cell cycle,
animal cells also have a spatial control to yield the formation of only one procentriole at each
existing mother despite the large pool of cytoplasmic components available (Nigg and Stearns,
2011). Once the assembly of the new procentrioles 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 that the numerical control of procentriole formation is based on a
balanced control among protein-protein interactions and /or activations. Therefore, procentriole
formation would be restricted to the vicinity of the existing centrioles that maintain a compact
region of pericentriolar material, the only area in the cell where new centrioles can form (Loncarek
et al., 2008, 2010). Antibodies against DSas-6, Ana1 and Bld10/Cep135 that recognize a
procentriole-like structure (PCL) at the base of the mother centriole in Drosophila elongating
spermatids (Blachon et al., 2009, 2014; Mottier-Pavie and Megraw, 2009) did not cross-react with
butterfly centriolar proteins. By contrast, an antibody against Sak/Plk4 that plays in Drosophila
main roles in the assembly of the PCL (Blachon et al., 2009) recognised in Pieris additional spots
for each centriole (Fig. 2j).
The concurrent appearance of multiple centrioles has been described under experimental
conditions, in which the concentration of proteins involved in centriole biogenesis or cell-cycle
regulation has been modified (Brownlee and Rogers, 2012). Clusters of procentrioles in flower-like
or rosette disposition are, indeed, observed around each mother after the overexpression 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 also induced by down-regulation of the ubiquitin
ligase SCFSlimb complex that regulates Plk4 levels (Cunha-Ferreira et al., 2009; Rogers et al., 2009).
The rosette phenotype is also generated by the overexpression of Sas-6 (Strnad et al., 2007), a
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protein needed for cartwheel assembly, or by the overexpression of STIL, the putative partner of
Sas-6 (Tang et al, 2011; Arquint et al., 2012;). More than one daughter centriole at single mother
also forms following proteasome inhibition or HPV-16 E7 oncoprotein transfection (Duensing et
al., 2007). In addition, some cultured vertebrate cells assemble multiple daughter centrioles around
each mother, when blocked in S-phase (Balczon et al., 1995). Multiple basal bodies also arise from
a single mother in a flower-like disposition in mammalian differentiated epithelial cells such as the
ones of the respiratory and reproductive tracts (Anderson and Brenner, 1971; Dirksen, 1971).
However, under normal circumstances only one daughter forms in cycling cells. Therefore, the
finding of multiple procentrioles at each spindle pole in butterflies seems to create a paradox and
suggests that the centriole duplication control is down regulated. The rule of “only one centriole
forming per mother” may overcome, suggesting that the assembly of a daughter does not
necessarily prevent the formation of additional ones.
Procentrioles appear in butterfly during late primary prophase, when the parent centrioles
attained their full size and do not elongate further. Moreover, they did not undergo significant shape
changes and a complete set of A-tubule never forms. This suggests that centriole elongation is
correlated to the cell cycle with full elongation taking place at the end of the first meiotic prophase.
The failure of procentriole growth and maturation in butterfly spermatocytes may represent the
limiting factor to avoid the assembly of functional centrosomes and the formation of supernumerary
spindles. Therefore, the assembly of additional centrioles is blocked not at the beginning, but at the
tubule addition stage. Pieris brassicae spermatids lack a distinct centriole adjunct. Thus, it could be
easier to detect procentrioles close to basal bodies. One or two procentrioles 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 often found next to the nuclear membrane (Fig. 3d).
Procentrioles found in early spermatids had similar diameter than those found during preceding
meiotic divisions (112.4 nm (3.2;n=5) vs. 114.2 nm (4.1; n= 14), respectively) and an invariable
cartwheel structure, but were slightly shorter (81.7 nm (2,9; n=6) vs. 117.2 nm ( 3.7; n=9).
Procentrioles were not longer detected in elongating spermatids. These results are consistent with
the gradual reduction of the supernumerary procentrioles and point to a redundant role of these
structures during butterfly gametogenesis.
A procentriole at the core of the parent centriole
Parent centrioles at the end of the first prophase had a nearly constant size with an average diameter
of 195 nm and an overall length of 730 nm (Fig. 4a). The proximal end of the mature centriole
showed a dense ring connected to the central hub by nine radial spokes (Fig. 4b). This structure that
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we termed here the “inner ring complex”, i.e. IRC, can be recognized in longitudinal sections as a
short cylinder within the basal region of the centriole (Fig. 4a). Microtubule triplets appeared in
slightly high sections. Triplets were 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 the distal region of the centriole (Fig. 4e). The hub increased diameter
moving from the basal end of the centriole (Fig. 4b,c,d) and disappeared concurrently with the
peripheral ring. Sections in which we had the opportunity to simultaneously find the basal end of
the parent centrioles and associated procentrioles (Fig. 4f,g,h) showed that the overall fitting of
these structures is excellent. Thus, the procentrioles may correspond to distinct platforms, on which
the future centrioles will be assembled. A cartwheel is transiently present at the base of young
centrioles in vertebrate centrosomes, but disappears in mature centrioles (Alvey, 1986; Strnad and
Gönczy, 2008; Azimzadeh and Marshall, 2010).
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Materials and Methods
Insects
A culture of Pieris brassicae was obtained from a laboratory strain and reared on ventilated cages at
20°C. Larvae were fed with cabbage leaves until pupation.
Antibodies
We used the following antibodies: rabbit anti Sak-Plk4 (1:200; Bettencourt-Dias et al., 2005),
chicken anti-DSAS-6 (1:1000, Rodriguez-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 488 or Alexa 555.
Immunofluorescence preparations
For immunostaining testes from pupae of the lepidopteran Pieris brassicae were dissected and fixed
according to Riparbelli e al. (2012). For localization of axonemal microtubules and centriolar
proteins the samples were processed as described previously (Riparbelli et al. 2013). DNA was
visualized with Hoechst 33258. Images were taken with an EC Plan-Neofluar 100x/1.30 objective
by using an AxioImager Z1 (Carl Zeiss) microscope equipped with an AxioCam HR camera (Carl
Zeiss).
Transmission electron microscopy
Testes 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).
Acknowledgements
We are grateful to Monica Bettencourt-Dias and Timothy L. Megraw for generously providing
antibodies .This work was supported by PRIN2012 to GC.
Author contribution
M.G., M.G.R. and G.C. conceived the project. M.G. and M.G.R. performed experiments. G.C.
wrote the manuscript.
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Figure legends
Fig. 1. Procentriole in primary spermatocytes. (a) Primary butterfly spermatocytes have four
elongated cilium-like projections (green); DNA is red. (b) The axoneme of the cilium-like
projections is nucleated by elongated centrioles. (c,d) One procentriole (arrow) is found near the
proximal region of parent centrioles. (e,f) Consecutive cross sections through the proximal and
distal ends of the procentriole shown in c: a single microtubule is visible on the procentriole wall
(asterisk); a thin inner ring (double arrow) is more apparent where the dense material surrounding
the procentriole is scarce. (g) Curved grooves are visible on the wall of the procentriole (asterisks).
(h) The parent centriole is surrounded by inner (double arrowhead) and outer (arrow) sheets of
dense 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 bar =
2.5 μm in a, 500 nm in b, 200 nm in c-d, 50 nm in e-j.
Fig. 2. Multiple procentrioles form at the base of single mothers. Prometaphase of the first
meiosis: (a) Cilium-like projections are very elongated; microtubules are green, DNA is red. (b)
Details of centriole and (d) procentrioles at one spindle poles. Metaphase of the second meiosis:
The spindle poles contain only one centriole (d) that nucleates a cilium-like structure (e;
microtubules are green, DNA is red). Details of centrioles (f,h) and procentrioles (g,i) visible in
associated serial sections during metaphase/anaphase of the second meiosis. (j) Localization of Sak-
Plk4 cross-reacting antigens (red) at the base of the cilium-like projections (green) during
metaphase of the second meiosis: two small spots are visible (arrow). Scale bar = 2.5 μm in a and e;
200 nm in b, f and h; 500 nm in d; 50 nm in c, g and i; 1.5 μm in j.
Fig. 3. Procentrioles persist in young spermatids. (a) Early spermatids have a round nucleus (red,
DNA) and an elongate axoneme (green, microtubules). (b) The centriole that nucleates the
spermatid axoneme contacts the nucleus. (c,d) Procentrioles are often close to the nuclear envelope
(arrow). Scale bar = 2.5 μm in a, 200 nm in b, 100 nm in c and d.
Fig. 4. Procentrioles within the parent centrioles. (a) The basal region of the centriole contains a
short cylinder (arrowheads). (b-e) Consecutive cross sections through a parent centriole: the basal
end shows a thick ring (arrowheads) on which microtubule triplets assemble. The central hub
increases diameter moving far from the proximal end of the centriole (small arrowheads). The ring
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disappears in further sections. (f-h) Longitudinal and cross sections of two orthogonal centrioles:
note the overlapping of the basal end of the parent centriole (arrows) and the procentriole
(arrowhead). Scale bar = 100 nm.
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