ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3434–3439 doi:10.1242/jcs.152843
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
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 (Go¨nczy, 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 whether the structural details observed at the onset of procentriole formation can differ in various animal groups or whether technical challenges have prevented a careful analysis of the early steps of centriole assembly. Therefore, 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 the centrioles 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.
INTRODUCTION
Department of Life Sciences, University of Siena, Via A. Moro 4, 53100 Siena, Italy. *Author for correspondence ([email protected]) Received 10 March 2014; Accepted 2 June 2014
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RESULTS AND DISCUSSION Centriole engagement does not prevent procentriole assembly
As usual in insects, the butterfly primary spermatocytes have two centriole pairs disposed in V-shape configuration that represent the platform for the assembly of four long ‘cilium-like’ projections (Fig. 1a,b) (Henneguy, 1898; Meves, 1902; Friedla¨nder 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 then organizes the spindle poles, the cilium-like projections persist during insect spermatogenesis 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 axoneme constructor. While studying the dynamics 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 almost constant size with an average diameter of 114.2 nm (64.1; n514) and an overall length of 117.2 nm (63.7; n59), longitudinally crossed by an inner tubule of about 21.3 nm (62.6; n514) 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 were always found during late prophase. Thus, the primary spermatocytes showed four fully elongated centrioles together with four procentrioles. This suggests that the centrioles of primary prophase spermatocytes undergo an extra duplication
Journal of Cell Science
During 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).
SHORT REPORT
Journal of Cell Science (2014) 127, 3434–3439 doi:10.1242/jcs.152843
cycle, in addition to the usual duplication that occurs in early prophase soon after the last spermatogonial mitosis. This represents an unconventional condition because, to avoid the formation of multipolar spindles, centrioles in animal cells duplicate only once per cell cycle during transition from G1 to S of interphase (Wong and Stearns, 2003). It is generally believed that the interruption of the close association, i.e. the disengagement, between mother and daughter centrioles during metaphase/anaphase transition enables the forthcoming duplication during interphase of the next cell cycle (Tsou and Stearns, 2006; Tsou et al., 2009). Accordingly, ultrastructural analysis of Drosophila melanogaster primary spermatocytes (Tates, 1971; Fritz-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 are held together by thin fibrous material – as 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. 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, which was also crossed by curved grooves whose diameter was similar to that of single microtubules (Fig. 1e,g). Some of the grooves underwent partial 3435
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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 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) (e); a thin inner ring (double arrow) is more apparent where the dense material surrounding the procentriole is scarce (f). (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 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 of microtubule assembly. The dense outer wall of the procentriole might, therefore, be regarded as the platform for the assembly of microtubules, and the curved grooves might denote nascent A-tubules, the first tubules of the pericentriolar triplet microtubules to form. Strikingly, electron-dense hook-like structures of unknown composition, were observed on the tube of Caenorhabditis elegans prior to the presence of assembled microtubules (Dutcher, 2007). The formation of A-tubules in butterflies would require a process of lateral nucleation, such as that described for the assembly of B- and C-tubules during centriole formation in human KE-37 cells (Guichard et al., 2010). In these cells, however, the A-tubule, which is presumably nucleated by a proximal cap of c-tubulin, is used by the B-tubule as a template.
to the distal end of the radial spokes through distinct pinheads, as reported in other systems (Guichard et al., 2013), but contact a thin cylinder that is the scaffold on which the material that is presumably involved in the nucleation of the A-tubule will be recruited. 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 to the appearance of tube-like structures (Rodrigues-Martins et al., 2007). Moreover, concurrent overexpression of Sas-6 and 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 C. elegans (Pelletier et al., 2006).
A model of procentriole assembly
Assembly of a daughter centriole does not require continuity with the mother centriole
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 length of the procentriole and reached the inner dense sheath that surrounded the base of the parent centriole (Fig. 1i,j). Radial projections could not 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 emerged 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 might then be built around the radial spokes (Fig. 1k). Therefore, the A-tubules do not attach
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; n57). The spindle poles of secondary spermatocytes during metaphase and/or 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; n59), three (Fig. 2h,i; n55) and sometimes four (n52) 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, which challenges the traditional view of a preferential site of
Journal of Cell Science
Fig. 2. Multiple procentrioles form at the base of single mother centrioles. Prometaphase of the first meiosis: (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 one spindle pole are shown. Metaphase of the second meiosis: the spindle poles contain only one centriole (d) that nucleates a cilium-like structure (e); microtubules are shown in green, DNA is shown in red. Details of centrioles (f,h) and procentrioles (g,i) visible in associated serial sections during metaphase/anaphase of the second meiosis are shown. (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 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 in managing 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 spatial control to yield formation of only one procentriole at each existing mother centriole, 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 of 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 (Loncˇarek et al., 2008; Loncˇarek et al., 2010). Antibodies against Drosophila Sas-6, Ana1 and Bld10 (a homolog of Chlamydamonas reinhardtii Bld10p and human CEP135) that recognize a procentriole-like structure (PCL) at the base of the mother centriole in Drosophila elongating spermatids (Blachon et al., 2009; Blachon et al., 2014; Mottier-Pavie and Megraw, 2009) did not crossreact with butterfly centriolar proteins. By contrast, an antibody against Drosophila SAK, a homolog of vertebrate 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 the concentration of proteins involved in centriole biogenesis or cellcycle regulation has been modified (Brownlee and Rogers, 2013). Clusters of procentrioles in flower-like or rosette disposition were, 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 downregulating the ubiquitin ligase SCFSlimb complex, which regulates levels of Plk4 (Cunha-Ferreira et al., 2009; Rogers et al., 2009). The rosette phenotype is also generated by overexpression of Sas-6 (Strnad et al., 2007), a 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 also forms at a single mother centriole following inhibition of proteasomes or transfection with oncoprotein HPV-16 E7 (Duensing et al., 2007). In addition, when blocked in S-phase, some cultured vertebrate cells 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 mammalian differentiated epithelial cells, such as those 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 downregulated. The rule of ‘only one centriole per mother’ may be overcome, suggesting that the assembly of a daughter centriole 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 changes in shape and a complete set of A-tubules never forms.
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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 stage when tubules are added. Pieris brassicae spermatids lack a distinct centriole adjunct. Thus, it might be easier to detect procentrioles that are 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 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 the invariable 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 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, which we call the
Fig. 3. Procentrioles persist in young spermatids. (a) Early spermatids have a round nucleus (DNA in red) and an elongated axoneme (microtubules in green). (b) The centriole that nucleates the spermatid axoneme contacts the 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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SHORT REPORT
SHORT REPORT
Journal of Cell Science (2014) 127, 3434–3439 doi:10.1242/jcs.152843
(Alvey, 1986; Strnad and Go¨nczy, 2008; Azimzadeh and Marshall, 2010). MATERIALS AND METHODS Insects
A 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. Antibodies
We 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; SigmaAldrich). The secondary antibodies used (1:800, Invitrogen) were conjugated with Alexa Fluor 488 or Alexa Fluor 555. Immunofluorescence preparations
For 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 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 (Instituto Gulbenkian de Ciencia, Oeiras, Portugal) and Timothy L. Megraw (Florida State University, Tallahassee, FL) for generously providing antibodies.
Fig. 4. Procentrioles within the parent centrioles. (a) The basal region of the centriole contains a short cylinder-like structure (arrowheads). (b–e) Consecutive cross-sections through a parent centriole. The basal end shows a thick ring (arrowheads in b) on which microtubule triplets assemble. The central hub increases in diameter, moving away from the proximal end of the centriole (asterisks). The ring disappears in further sections. (f–h) Longitudinal and cross-sections of two orthogonal centrioles: note the overlap of the basal end of the parent centriole (arrows) and the procentriole (arrowhead). Scale bars: 100 nm.
Competing interests The authors declare no competing interests.
Author contributions M.G., M.G.R. and G.C. conceived the project. M.G. and M.G.R. performed experiments. G.C. wrote the manuscript.
Funding This work was supported by PRIN2012 to G.C.
‘inner ring complex’ (IRC), can be recognized in longitudinal sections as a short cylinder within the basal region of the centriole (Fig. 4a). Microtubule triplets appeared in sections taken away from the basal region of the centriole. These 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 in diameter, 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 of the parent centrioles and associated procentrioles at the same time (Fig. 4f–h) showed that the two were structurally very similar. Thus, the procentrioles can 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 3438
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Nigg, E. A. and Stearns, T. (2011). The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries. Nat. Cell Biol. 13, 1154-1160. Pelletier, L., O’Toole, E., Schwager, A., Hyman, A. A. and Mu¨ller-Reichert, T. (2006). Centriole assembly in Caenorhabditis elegans. Nature 444, 619-623. Riparbelli, M. G., Callaini, G. and Megraw, T. L. (2012). Assembly and persistence of primary cilia in dividing Drosophila spermatocytes. Dev. Cell 23, 425-432. Riparbelli, M. G., Cabrera, O. A., Callaini, G. and Megraw, T. L. (2013). Unique properties of Drosophila spermatocyte primary cilia. Biol. Open 2, 1137-1147. Rodrigues-Martins, A., Bettencourt-Dias, M., Riparbelli, M., Ferreira, C., Ferreira, I., Callaini, G. and Glover, D. M. (2007). DSAS-6 organizes a tubelike centriole precursor, and its absence suggests modularity in centriole assembly. Curr. Biol. 17, 1465-1472. Rogers, G. C., Rusan, N. M., Roberts, D. M., Peifer, M. and Rogers, S. L. (2009). The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication. J. Cell Biol. 184, 225-239. Sluder, G. and Khodjakov, A. (2010). Centriole duplication: analogue control in a digital age. Cell Biol. Int. 34, 1239-1245. Sluder, G. and Rieder, C. L. (1985). Centriole number and the reproductive capacity of spindle poles. J. Cell Biol. 100, 887-896. Stevens, N. R., Dobbelaere, J., Brunk, K., Franz, A. and Raff, J. W. (2010). Drosophila Ana2 is a conserved centriole duplication factor. J. Cell Biol. 188, 313-323. Strnad, P. and Go¨nczy, P. (2008). Mechanisms of procentriole formation. Trends Cell Biol. 18, 389-396. Strnad, P., Leidel, S., Vinogradova, T., Euteneuer, U., Khodjakov, A. and Go¨nczy, P. (2007). Regulated HsSAS-6 levels ensure formation of a single procentriole per centriole during the centrosome duplication cycle. Dev. Cell 13, 203-213. Tang, C. J., Lin, S. Y., Hsu, W. B., Lin, Y. N., Wu, C. T., Lin, Y. C., Chang, C. W., Wu, K. S. and Tang, T. K. (2011). The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation. EMBO J. 30, 4790-4804. Tates, A. D. (1971). Cytodifferentiation During Spermatogenesis in Drosophila Melanogaster: An Electron Microscopy Study. PhD thesis, Rijksuniversiteit de Leiden, Netherlands. Tsou, M. F. and Stearns, T. (2006). Controlling centrosome number: licenses and blocks. Curr. Opin. Cell Biol. 18, 74-78. Tsou, M. F., Wang, W. J., George, K. A., Uryu, K., Stearns, T. and Jallepalli, P. V. (2009). Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells. Dev. Cell 17, 344-354. Uetake, Y., Loncˇarek, J., Nordberg, J. J., English, C. N., La Terra, S., Khodjakov, A. and Sluder, G. (2007). Cell cycle progression and de novo centriole assembly after centrosomal removal in untransformed human cells. J. Cell Biol. 176, 173-182. Vitre, B. D. and Cleveland, D. W. (2012). Centrosomes, chromosome instability (CIN) and aneuploidy. Curr. Opin. Cell Biol. 24, 809-815. Wang, W. J., Soni, R. K., Uryu, K. and Tsou, M. F. (2011). The conversion of centrioles to centrosomes: essential coupling of duplication with segregation. J. Cell Biol. 193, 727-739. Wolf, K. W. and Traut, W. (1987). Ultrastructure of distal flagellar swellings in spermatocytes and spermatids of Ephestia kuehniella Z. (Pyralidae, Lepitoptera). Cell Tissue Res. 250, 421-424. Wong, C. and Stearns, T. (2003). Centrosome number is controlled by a centrosome-intrinsic block to reduplication. Nat. Cell Biol. 5, 539-544. Yamashiki, N. and Kawamura, N. (1998). Behavior of centrioles during meiosis in the male silkworm, Bombyx mori (Lepidoptera). Dev. Growth Differ. 40, 619630.
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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
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 (Go¨nczy, 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 whether the structural details observed at the onset of procentriole formation can differ in various animal groups or whether technical challenges have prevented a careful analysis of the early steps of centriole assembly. Therefore, 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 the centrioles 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.
INTRODUCTION
Department of Life Sciences, University of Siena, Via A. Moro 4, 53100 Siena, Italy. *Author for correspondence ([email protected]) Received 10 March 2014; Accepted 2 June 2014
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RESULTS AND DISCUSSION Centriole engagement does not prevent procentriole assembly
As usual in insects, the butterfly primary spermatocytes have two centriole pairs disposed in V-shape configuration that represent the platform for the assembly of four long ‘cilium-like’ projections (Fig. 1a,b) (Henneguy, 1898; Meves, 1902; Friedla¨nder 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 then organizes the spindle poles, the cilium-like projections persist during insect spermatogenesis 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 axoneme constructor. While studying the dynamics 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 almost constant size with an average diameter of 114.2 nm (64.1; n514) and an overall length of 117.2 nm (63.7; n59), longitudinally crossed by an inner tubule of about 21.3 nm (62.6; n514) 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 were always found during late prophase. Thus, the primary spermatocytes showed four fully elongated centrioles together with four procentrioles. This suggests that the centrioles of primary prophase spermatocytes undergo an extra duplication
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During 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).
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cycle, in addition to the usual duplication that occurs in early prophase soon after the last spermatogonial mitosis. This represents an unconventional condition because, to avoid the formation of multipolar spindles, centrioles in animal cells duplicate only once per cell cycle during transition from G1 to S of interphase (Wong and Stearns, 2003). It is generally believed that the interruption of the close association, i.e. the disengagement, between mother and daughter centrioles during metaphase/anaphase transition enables the forthcoming duplication during interphase of the next cell cycle (Tsou and Stearns, 2006; Tsou et al., 2009). Accordingly, ultrastructural analysis of Drosophila melanogaster primary spermatocytes (Tates, 1971; Fritz-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 are held together by thin fibrous material – as 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. 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, which was also crossed by curved grooves whose diameter was similar to that of single microtubules (Fig. 1e,g). Some of the grooves underwent partial 3435
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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 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) (e); a thin inner ring (double arrow) is more apparent where the dense material surrounding the procentriole is scarce (f). (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 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 of microtubule assembly. The dense outer wall of the procentriole might, therefore, be regarded as the platform for the assembly of microtubules, and the curved grooves might denote nascent A-tubules, the first tubules of the pericentriolar triplet microtubules to form. Strikingly, electron-dense hook-like structures of unknown composition, were observed on the tube of Caenorhabditis elegans prior to the presence of assembled microtubules (Dutcher, 2007). The formation of A-tubules in butterflies would require a process of lateral nucleation, such as that described for the assembly of B- and C-tubules during centriole formation in human KE-37 cells (Guichard et al., 2010). In these cells, however, the A-tubule, which is presumably nucleated by a proximal cap of c-tubulin, is used by the B-tubule as a template.
to the distal end of the radial spokes through distinct pinheads, as reported in other systems (Guichard et al., 2013), but contact a thin cylinder that is the scaffold on which the material that is presumably involved in the nucleation of the A-tubule will be recruited. 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 to the appearance of tube-like structures (Rodrigues-Martins et al., 2007). Moreover, concurrent overexpression of Sas-6 and 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 C. elegans (Pelletier et al., 2006).
A model of procentriole assembly
Assembly of a daughter centriole does not require continuity with the mother centriole
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 length of the procentriole and reached the inner dense sheath that surrounded the base of the parent centriole (Fig. 1i,j). Radial projections could not 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 emerged 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 might then be built around the radial spokes (Fig. 1k). Therefore, the A-tubules do not attach
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; n57). The spindle poles of secondary spermatocytes during metaphase and/or 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; n59), three (Fig. 2h,i; n55) and sometimes four (n52) 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, which challenges the traditional view of a preferential site of
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Fig. 2. Multiple procentrioles form at the base of single mother centrioles. Prometaphase of the first meiosis: (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 one spindle pole are shown. Metaphase of the second meiosis: the spindle poles contain only one centriole (d) that nucleates a cilium-like structure (e); microtubules are shown in green, DNA is shown in red. Details of centrioles (f,h) and procentrioles (g,i) visible in associated serial sections during metaphase/anaphase of the second meiosis are shown. (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 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 in managing 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 spatial control to yield formation of only one procentriole at each existing mother centriole, 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 of 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 (Loncˇarek et al., 2008; Loncˇarek et al., 2010). Antibodies against Drosophila Sas-6, Ana1 and Bld10 (a homolog of Chlamydamonas reinhardtii Bld10p and human CEP135) that recognize a procentriole-like structure (PCL) at the base of the mother centriole in Drosophila elongating spermatids (Blachon et al., 2009; Blachon et al., 2014; Mottier-Pavie and Megraw, 2009) did not crossreact with butterfly centriolar proteins. By contrast, an antibody against Drosophila SAK, a homolog of vertebrate 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 the concentration of proteins involved in centriole biogenesis or cellcycle regulation has been modified (Brownlee and Rogers, 2013). Clusters of procentrioles in flower-like or rosette disposition were, 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 downregulating the ubiquitin ligase SCFSlimb complex, which regulates levels of Plk4 (Cunha-Ferreira et al., 2009; Rogers et al., 2009). The rosette phenotype is also generated by overexpression of Sas-6 (Strnad et al., 2007), a 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 also forms at a single mother centriole following inhibition of proteasomes or transfection with oncoprotein HPV-16 E7 (Duensing et al., 2007). In addition, when blocked in S-phase, some cultured vertebrate cells 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 mammalian differentiated epithelial cells, such as those 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 downregulated. The rule of ‘only one centriole per mother’ may be overcome, suggesting that the assembly of a daughter centriole 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 changes in shape and a complete set of A-tubules never forms.
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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 stage when tubules are added. Pieris brassicae spermatids lack a distinct centriole adjunct. Thus, it might be easier to detect procentrioles that are 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 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 the invariable 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 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, which we call the
Fig. 3. Procentrioles persist in young spermatids. (a) Early spermatids have a round nucleus (DNA in red) and an elongated axoneme (microtubules in green). (b) The centriole that nucleates the spermatid axoneme contacts the 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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(Alvey, 1986; Strnad and Go¨nczy, 2008; Azimzadeh and Marshall, 2010). MATERIALS AND METHODS Insects
A 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. Antibodies
We 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; SigmaAldrich). The secondary antibodies used (1:800, Invitrogen) were conjugated with Alexa Fluor 488 or Alexa Fluor 555. Immunofluorescence preparations
For 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 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 (Instituto Gulbenkian de Ciencia, Oeiras, Portugal) and Timothy L. Megraw (Florida State University, Tallahassee, FL) for generously providing antibodies.
Fig. 4. Procentrioles within the parent centrioles. (a) The basal region of the centriole contains a short cylinder-like structure (arrowheads). (b–e) Consecutive cross-sections through a parent centriole. The basal end shows a thick ring (arrowheads in b) on which microtubule triplets assemble. The central hub increases in diameter, moving away from the proximal end of the centriole (asterisks). The ring disappears in further sections. (f–h) Longitudinal and cross-sections of two orthogonal centrioles: note the overlap of the basal end of the parent centriole (arrows) and the procentriole (arrowhead). Scale bars: 100 nm.
Competing interests The authors declare no competing interests.
Author contributions M.G., M.G.R. and G.C. conceived the project. M.G. and M.G.R. performed experiments. G.C. wrote the manuscript.
Funding This work was supported by PRIN2012 to G.C.
‘inner ring complex’ (IRC), can be recognized in longitudinal sections as a short cylinder within the basal region of the centriole (Fig. 4a). Microtubule triplets appeared in sections taken away from the basal region of the centriole. These 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 in diameter, 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 of the parent centrioles and associated procentrioles at the same time (Fig. 4f–h) showed that the two were structurally very similar. Thus, the procentrioles can 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 3438
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