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yuri gagarin is required for actin, tubulin and basal body functions in Drosophila spermatogenesis
Michael J. Texada, Rebecca A. Simonette, Cassidy B. Johnson, William J. Deery, Kathleen M. Beckingham
Journal of Cell Science 2008 121: 1926-1936; doi: 10.1242/jcs.026559
Michael J. Texada
Rebecca A. Simonette
Cassidy B. Johnson
William J. Deery
Kathleen M. Beckingham
Males of the genus Drosophila produce sperm of remarkable length. Investigation of giant sperm production in Drosophila melanogaster has demonstrated that specialized actin and microtubule structures play key roles. The gene yuri gagarin (yuri) encodes a novel protein previously identified through its role in gravitaxis. A male-sterile mutation of yuri has revealed roles for Yuri in the functions of the actin and tubulin structures of spermatogenesis. Yuri is a component of the motile actin cones that individualize the spermatids and is essential for their formation. Furthermore, Yuri is required for actin accumulation in the dense complex, a microtubule-rich structure on the sperm nuclei thought to strengthen the nuclei during elongation. In the yuri mutant, late clusters of syncytial nuclei are deformed and disorganized. The basal bodies are also mispositioned on the nuclei, and the association of a specialized structure, the centriolar adjunct (CA), with the basal body is lost. Some of these nuclear defects might underlie a further unexpected abnormality: sperm nuclei occasionally locate to the wrong ends of the spermatid cysts. The structure of the axonemes that grow out from the basal bodies is affected in the yuri mutant, suggesting a possible role for the CA in axoneme formation.
Basal body
Chordotonal organ
Centriole
A unique feature of the genus Drosophila is the formation of unusually long sperm tails. Sperm lengths of millimeters are common within this group, with the 1.8 mm sperm of D. melanogaster being fairly typical. This marked expansion in sperm length reflects an unusual aspect of spermatogenesis in these organisms: in contrast to other species in which an intraflagellar transport system is used for growth of the sperm flagellum (Scholey, 2006), Drosophila sperm axonemes are assembled in syncytial cysts by a mechanism that does not require, and is not limited by, this system (Han et al., 2003; Sarpal et al., 2003). This unusual sperm axoneme development and the resulting expansion of sperm tail length have led to distinctive features of spermatogenesis not found in other species. In D. bifurca, a special `sperm roller' has evolved to package its 6-centimeter-long gametes (Joly et al., 2003). In D. melanogaster, a highly evolved individualization process that generates 64 individual sperm from an elongate cyst containing 64 syncytial spermatids has been identified and studied (Noguchi and Miller, 2003; Tokuyasu et al., 1972a). The distinctive molecular mechanisms needed for this process include a motile filamentous actin system (the investment, or actin, cones) that traverses the entire length of the sperm tails, removing excess cytoplasm and investing each sperm in its own plasma membrane. A specialized microtubule-rich structure (the dense complex) is also associated with the sperm nuclei and functions to position the basal body and also possibly to strengthen the nuclei as they undergo extreme condensation (A. D. Tates, Cytodifferentiation during spermatogenesis in Drosophila melanogaster, PhD thesis, Rijksuniversiteit Leiden, The Netherlands, 1971) (Tokuyasu, 1974).
We have identified a locus, yuri gagarin (yuri), that we show here has multiple roles in the generation of elongate individualized sperm. The gene is only highly conserved in the genus Drosophila, suggesting specialized roles in these organisms. Interestingly, yuri was initially identified through its function in another specialized organ system of insects and arthropods: the chordotonal organs. These are complex mechanosensory structures with roles in proprioception and graviperception. The first mutation at the locus, yuric263, was identified in a screen for mutants affecting gravitaxis. Altered gravitaxis was shown to result from perturbed expression of yuri in subsets of chordotonal neurons (Armstrong et al., 2006). The molecular functions of the locus identified here suggest that yuri mediates specialized actin- and microtubule-related activities in Drosophila tissues.
The yuri locus in D. melanogaster and other Drosophilids
In addition to the cDNA (GH14032) encoding a ∼30 kDa protein that we used previously (Armstrong et al., 2006), we identified 11 further yuri ESTs/cDNAs from adult testis, ovary, S2 cells and embryos through FlyBase. Sequencing of these new cDNAs established that three major transcript classes are generated from yuri (Fig. 1). Two promoters are used, with the medium transcripts initiated at the proximal promoter and the short and long classes from the distal promoter. However, all isoforms begin at one of two closely positioned ATGs. The short transcript class encodes the ∼30 kDa protein identified previously. The medium class encodes isoforms of 64-65 kDa that extend ∼400 amino acids further at the C-terminus. The long class, encoding proteins of 101-107 kDa, extends an additional ∼300 amino acids C-terminally. The short yuri isoform is novel, with only a single recognizable motif (a polyproline stretch). However, the two longer forms contain coiled-coil motifs with weak similarity (∼20% identity) to those in many fibrillar proteins that dimerize, such as myosin heavy chain and CLIP-190. The strongest match is to the coiled-coil of Sticky, the Drosophila citron kinase (Sweeney et al., 2008).
Transcripts, proteins and mutations at the Drosophila yuri locus. (A) Two promoters (proximal and distal) generate three classes of yuri transcripts. The two medium transcripts differ by the presence of an intron between exons 1b′ and 1b″. Exon 4, the 5′ boundary of which is not defined (Materials and Methods), is included in some long transcripts. The original P{GawB} insertion (yuric263) and the DNA deleted in three imprecise excisions (LE1, L5 and F64) are shown. (B) Three Yuri isoform classes arise from the three transcript classes. Structural motifs are indicated.
yuri is unique in the D. melanogaster genome, once the weak similarities to coiled-coil regions are disregarded. Thus, to avoid spurious similarities, the shortest yuri isoform was used to find yuri orthologs in other organisms. Significant matches were found in all 11 sequenced Drosophila genomes (Drosophila 12 Genomes Consortium, 2007), but none was identified in other evolutionary orders or other insects, including the closest Dipteran relatives, the Culicidae (mosquitoes) (Fig. 2). Sequence conservation within the Drosophila genus was high (91-37% sequence identity, 93-57% similarity) across the entire ∼100 kDa isoform of D. melanogaster. yuri therefore appears to be a Drosophila-specific gene. Most species have one yuri gene, but two related genes are present in D. pseudoobscura and D. persimilis.
Evolutionary conservation of yuri in Drosophila species. Yuri orthologs are detectable in 12 Drosophila species, but not outside the genus. The ∼100 kDa isoform is more conserved than the ∼30 kDa isoform. Similarity is computed as the global fraction of residues of the D. melanogaster protein that are present as similar residues in orthologs; these are lower than the local similarity scores from BLAST programs. The GLEANR data set contains consensus sets of predicted proteins for the 12 Drosophila species and was searched using the protein-to-protein BLASTP program. Because protein predictions are not available (NA) for non-Drosophila species, the 30 kDa search was repeated for all sequenced insect species using the protein-to-DNA TBLASTN program. Tree image is from FlyBase (Crosby et al., 2007).
Ubiquitous expression of the three major Yuri isoforms
To investigate Yuri expression, we generated antibodies against the sequences common to all isoforms (see Materials and Methods). Immunoblots of yuri+ embryos and embryos lacking yuri established the specificity of our antisera and their ability to detect the three predicted Yuri isoform classes (Fig. 3A). These blots also demonstrated that only the short Yuri isoform is maternally loaded into the embryo, with the longer isoforms appearing later in embryogenesis (Fig. 3A,B). In later stages, all three isoform classes are ubiquitously expressed (Fig. 3C). The ∼65 kDa class is most abundant in most situations, although in testis and thorax the other isoforms are also highly expressed (Fig. 3C). The existence of at least two isoforms for both the ∼100 kDa and ∼65 kDa classes was confirmed by these experiments. Additional bands were sometimes present that probably represent specific degradation products, as they were largely missing in embryos lacking yuri (Fig. 3A) and in the yuriF64 mutant (Fig. 3C) (see below).
The distribution of Yuri isoforms throughout development. Immunoblots for Yuri isoforms are shown. (A) Specificity of Yuri antibodies. Lane 1, 30 unfertilized eggs from Df(2L)do1/CyO-GFP mothers [Df(2L)do1 removes yuri]. Lane 2, 30 terminal homozygous Df(2L)do1 embryos. Lane 3, 30 terminal homozygous CyO-GFP (homozygous yuri+) embryos. The two large isoforms are not present in unfertilized eggs or embryos lacking yuri, but are zygotically expressed in the yuri+ embryos. (B) Yuri isoforms during embryogenesis. The larger Yuri isoforms appear late in embryogenesis in embryos from control (w1118) and yuriF64 mothers mated to w1118 males. (C) Yuri isoforms present in various tissues and stages. Samples from w1118 control and yuriF64 animals. Sample sizes: ovaries, 8 pairs; testes, 7.5 pairs; heads, 3; thoraces, 0.5; third instar larvae, 0.5. Bands that might be degradation products are marked with an asterisk.
A yuri mutant that lacks Yuri ∼65 kDa isoform(s)
The yuric263 mutation from our gravitaxic screen is an insertion of P{GawB} just upstream of the transcription start site for the medium length transcripts. We generated further mutations by imprecise excision of P{GawB} and of a second transposon, KG03019 (Roseman et al., 1995), inserted three residues downstream of the yuric263 P element. Three excisions that delete the relevant transposon and adjacent genomic DNA were identified. One of these is lethal (yuriLE1), but the deletion extends upstream into an adjacent gene (cullin3; guftagu) known to affect viability (Mistry et al., 2004). In yuriL5, a short region of yuri upstream sequence is deleted, causing reduced expression of all Yuri isoforms. Nevertheless, homozygous yuriL5 animals are viable with no obvious phenotype. Only one deletion, yuriF64, removes transcribed sequences from the locus. Most of the 5′ UTR of the ∼65 kDa isoforms is deleted, with only ten residues upstream of the first initiator ATG remaining (Fig. 1A). The yuriF64 deletion lead to complete loss of ∼65 kDa isoforms in all tissues and stages examined (Fig. 3C). The ∼100 kDa isoforms remained strongly expressed, but expression of the ∼30 kDa isoform was decreased in several tissues and undetectable in the testis (Fig. 3C).
Male sterility is associated with the yuriF64 mutation
Homozygous yuriF64 mutants (yuriF64) are viable with normal external morphology. However, yuriF64 males are completely sterile, whereas females are fertile (data not shown). Flies heterozygous for yuriF64 and deficiency Df(2L)do1, which deletes yuri, were also male sterile and female fertile. The testis phenotype (see below) was identical in yuriF64 homozygotes and hemizygotes (data not shown), demonstrating that it results from the effects of the yuriF64 mutation on the yuri locus. In order to determine whether yuriF64 affects overall viability, the survival of yuriF64 homozygous progeny versus heterozygous progeny (yuriF64/CyO Roi) was quantitated for a cross of yuriF64 females with heterozygous (yuriF64/CyO Roi) males. Of 649 progeny, 51% were yuriF64 homozygotes, indicating that yuriF64 has no effects on survival to adulthood.
The Drosophila testis contains a stem cell system at its apical tip from which spermatogonial cells are budded off to proceed through spermatogenesis. A somatic stem cell system is also present that produces so-called cyst cells. A pair of cyst cells encases the division products of each spermatogonial cell throughout spermatogenesis and post-meiotic spermiogenesis. Each spermatogonial cell generates a cyst of 64 spermatids, linked by cytoplasmic bridges, which undergoes dramatic elongation. At completion, each cyst has a highly elongate cytoplasm (∼1.8 mm in length) with the 64 condensed nuclei positioned at the seminal vesicle end and 64 axonemes extending from the nuclei along the length of the cyst towards the apical tip. Two giant mitochondrial derivatives, generated by fusion of the mitochondria within each post-meiotic spermatid, extend along the length of each axoneme. The later stages of spermiogenesis involve a specialized process termed individualization (see below) in which the 64 syncytial spermatids are converted into 64 individual sperm. Finally, a coiling process retracts the sperm down to the entrance to the seminal vesicle.
Highly elongate spermatid cysts were present in yuriF64 testes, some of which were attempting to coil, but it was unclear whether mature sperm were formed. To address this question, we introduced a don juan-GFP fusion construct into the yuriF64 background. Don Juan protein is produced in the giant sperm tail mitochondria and persists into mature sperm. Don Juan-GFP (Santel et al., 1997) provides a marker for late spermiogenesis (Civetta, 1999; Gao et al., 2003). We examined 8-day-old virgin males, which should have large quantities of sperm in the seminal vesicles. In yuriF64/CyO Roi heterozygotes carrying don juan-GFP, the seminal vesicles were full of fluorescent sperm and the basal testis carried masses of fluorescent coiling sperm (Fig. 4A). In yuriF64, no fluorescence was detectable in the seminal vesicles and the basal testis contained curled structures, thicker than individual sperm with aberrant coiling (Fig. 4B). Squashes of seminal vesicles confirmed the presence of motile sperm in the controls and their complete absence in the mutant (data not shown).
Sperm elongate but show individualization and coiling defects in yuriF64. (A) Sperm tails, marked with Don Juan-GFP (green), fill the seminal vesicle (arrow) in control testes. (B) In yuriF64 hemizygotes [yuriF64/Df(2L)do1], the seminal vesicle (arrow) is empty, and sperm cysts show abortive coiling in the testis proper (arrowhead). (A′-B″) Phalloidin staining (red) identifies actin cones and waste bags in control testis (A′, red arrow; as shown at higher magnification in A″). Mutant testis is devoid of these structures (B′), and F-actin sleeves are present instead (B′, red arrow; as shown at higher magnification in B″). Scale bars: 200 μm.
Individualization fails in yuriF64
Phase-contrast examination of testis squashes revealed no defects in spermatogenesis up to the post-meiotic stages; `onion stage' spermatids appeared normal. The structures undergoing abortive coiling in yuriF64 testes were full-width spermatid cysts, indicating a failure of individualization. Individualization begins after formation of a cone of F-actin around the attachment site of each axoneme to the sperm nucleus, with the flat edge of the cone facing up the length of the sperm tail. All 64 cones within a cyst then travel in unison up the testis. In their wake they leave individual axonemes, each encased in a plasma membrane, and, ahead of the set, excess cytoplasm and organelles are pushed up the testis to be discarded as a `waste bag'. The actin cones are the only significant F-actin structures in the testis and are easily visualized with rhodamine-phalloidin (Fabrizio et al., 1998). Whereas in control (yuriF64/CyO Roi or w1118) testes, multiple sets of actin cones and waste bags were detected, the yuriF64 testes contained neither (Fig. 4). Instead, elongated `sleeves' of actin were seen around the periphery of some spermatid cysts. These appeared as solid tubes in normal fluorescence imaging (Fig. 4B″), but as hollow structures in confocal sections (Fig. 5A). We established that these sleeves are actually present in the somatic cyst cells surrounding the cysts, rather than in the cysts themselves, by use of GFP `exon trap' insertions (Kelso et al., 2004) that express GFP in the cyst cells (Materials and Methods). In the yuriF64 background, the actin sleeve staining and GFP in the cyst cells precisely overlapped (Fig. 5A). Having identified these sleeves in yuriF64, we discovered similar structures present at a lower frequency in control testes (Fig. 5B). In controls, these sleeves are always in the basal regions of the testis where sperm coiling takes place, whereas in yuriF64 they form throughout the testis. We address the significance of these structures in the Discussion. The major conclusion here is that in yuriF64 no actin cone sets or F-actin structures of any kind are present in the germline cysts proper.
Spermatogenesis defects in yuriF64. (A-A″) The actin sleeves in yuriF64 testes are within the cyst cells that encase the spermatid bundles. Phalloidin staining (red) coincides with GFP fluorescence (green) in a cyst cell expressing a GFP `exon trap' construct (cyst-GFP line G0147). (B) Longer actin sleeves are seen at the base of control testes in coiling sperm bundles. (C) Late-stage sperm nuclei in controls are straight and tightly bundled (arrow). (D) Nuclei in yuriF64 sperm are frequently bent or helically coiled (arrows) and never condense to tight bundles. (E) Nascent actin cones are visible on the tips of mature nuclei in controls (arrow). (F) Very little F-actin accumulates on yuriF64 mutant nuclei (arrow). (G) Small, individual actin cones are sometimes scattered along yuriF64 mutant cysts. Scale bars: 10 μm in A-A″,C,D, 100 μm in B,E,F, 50 μm in G.
Actin cone initiation and nuclear behavior are aberrant in yuriF64
The formation of the F-actin cones of individualization has been studied previously (Fabrizio et al., 1998; Lindsley and Tokuyasu, 1980; Noguchi et al., 2006). Initially, actin fibers accrete along the lengths of the condensed sperm nuclei in the basal testis. The actin then moves to form cones, flaring off the apical ends of the nuclei before release to move up the axonemes. Nuclei in all stages of this process are present in the basal region of wild-type testes. In yuriF64, although the nuclear sets were seen to descend to this level and undergo some condensation, they were clearly more disorganized, with individual nuclei trailing behind, apparently detached from the main cluster. In some late-stage clusters, almost all the nuclei were distorted in shape, some in a helical or circular configuration (Fig. 5D). No nuclei ever condensed to the tight bundles seen in controls (Fig. 5C). Furthermore, no well-formed sets of cones were ever detected, although a little F-actin accumulated around some nuclei (Fig. 5E,F). Interestingly, small under-developed cones were occasionally found singly or in clusters in this region. Some of them were apparently mobile, as they appeared at some distance from any nuclei (Fig. 5G).
Yuri protein localization in control testes
Our antisera, which detect all Yuri isoforms, were used to examine Yuri localization in control testes. Yuri was present at all stages of germ cell development, peaking around meiosis, with most staining being cytoplasmic and diffuse (Fig. 6A). However, in addition, a striking and dynamic pattern of Yuri association with the post-meiotic spermatid nuclei was seen as they condensed during elongation (Fig. 6B-F). While the nuclei were still round, Yuri was seen to accumulate as a cap over one hemisphere of each nucleus. As the nuclei became ellipsoid, the Yuri staining transformed into a stripe along the nuclear long axis and a dot at the apical nuclear tip. In the final stages of nuclear maturation, first the stripe disappeared and then the dot was also lost.
Yuri immunolocalization in control (yuriF64/CyO) testes. (A) General cytoplasmic staining is seen, peaking in primary spermatocytes and meiotic stages. (B) Positioning of the dense complex and basal body during spermatid nuclear condensation (for comparison with C-F). (Adapted from A. D. Tates, Cytodifferentiation during spermatogenesis in Drosophila melanogaster, PhD thesis, Rijksuniversiteit Leiden, The Netherlands, 1971.) (C) In post-meiotic spermatids with round nuclei, Yuri forms a cap over one nuclear hemisphere. (D) In elongating nuclei, Yuri forms a stripe along the nuclear long axis and a dot at the extreme apical tip where the axoneme connects to the nucleus. (E,F) The Yuri stripe narrows and disappears as the nuclei mature, leaving only the bell-shaped dot (inset in F) at the nuclear apex. By the onset of actin cone formation (right-hand nuclear set in F), all Yuri staining is lost from the nuclei. Scale bars: 10 μm.
Tokuyasu has described the ultrastructural changes to the nuclei during elongation (Tokuyasu, 1974). Part of the nuclear membrane is fenestrated with nuclear pores during this process. Initially, this region forms a cap over one hemisphere of the round post-meiotic nucleus, with dense material aggregating over this region between the nuclear membrane and adjacent endoplasmic reticulum. As the nuclei elongate, this cap and associated material transform to a stripe along the long axis of the nucleus. More of the dense material accumulates along with microtubules, with the whole complex sinking inwards to form a groove filled with dense cytoplasm and a microtubule bundle (collectively the `dense complex') that runs the length of the nucleus. The nuclei are actually horseshoe-shaped in cross-section at this stage. In the final stages of nuclear maturation, the dense complex is dispersed and the nuclei regain a circular cross-section. The dense complex is thought to provide structural rigidity to the nuclei during the elongation process (Tokuyasu, 1974). Early after meiosis, the single centriole of each spermatid embeds into the spherical nuclear membrane at the center of the dense complex and then converts into the basal body. During elongation, the basal body moves to the apical tip of the nucleus, immediately adjacent to the stripe of dense complex (Fig. 6B).
The pattern of Yuri localization on the spermatid nuclei was strikingly similar to that of the dense complex and associated basal body. To position Yuri relative to these structures, we co-stained for γ-tubulin, Centrosomin (Cnn) and β-tubulin. γ-tubulin is a component of the centriolar adjunct (CA) (Wilson et al., 1997), a torus-shaped structure around the middle of the basal body during elongation (Fig. 7C) (Tokuyasu, 1975). Centrosomin, a centriole component, is present early in the transformation to the basal body but is subsequently lost (Li et al., 1998). β-tubulin is a general marker for microtubules. γ-tubulin/Yuri co-staining established that the basal body is at the center of the Yuri cap in round spermatids (Fig. 7A), providing evidence that the Yuri cap corresponds to the accumulating dense complex. No round spermatid nuclei that co-stained for the Yuri cap and Centrosomin were detected, suggesting that Centrosomin is lost before significant Yuri accumulation. We were not able to detect a stripe of microtubules along the nuclei by staining for β-tubulin. Very high general cytoplasmic staining and/or possibly the burying of the appropriate epitope could underlie this failure.
Yuri localization relative to γ-tubulin and actin in controls. (A) γ-tubulin staining positions the centriole/basal body at the center of the Yuri nuclear cap in round spermatids. (B) On elongating nuclei, the Yuri dot lies between the body of the nucleus and the CA, as identified by γ-tubulin. (C) Diagram of the proposed location of Yuri on elongating nuclei. Adapted from Lindsley and Tokuyasu (Lindsley and Tokuyasu, 1980) with permission. (D) F-actin localization on round spermatid nuclei. (E-E″) Colocalization of Yuri and F-actin in the stripe and dot pattern seen on elongating nuclei. Arrow indicates actin/Yuri staining overlap on a single nucleus. (F-F″) Colocalization of actin and Yuri in moving actin cones. A cross-section of a set of large moving cones is shown. Yuri, green; nuclei, blue; γ-tubulin, red in A-C; actin, red in D-F. Scale bars: 10 μm in A-D,E-E″, 20 μm in F-F″.
Although γ-tubulin staining showed an apical dot on the elongating nuclei, interestingly, the Yuri dot and the γ-tubulin dot did not coincide. The Yuri dot, which at high magnification has a bell shape (Fig. 6F), was sandwiched between the dot of γ-tubulin staining and the nuclear membrane. Thus, Yuri is probably not part of the basal body per se but lies between the basal body and the nuclear membrane. EM analysis has established that the basal body is embedded into a ∼0.5 μm indentation in the nuclear membrane (Tokuyasu, 1975) at this stage. It seems likely that the Yuri dot is the residuum of the initial dense-complex cap that was always beneath the insertion point of the basal body, and that Yuri continues to fill the space between the membrane and the basal body during nuclear elongation.
Given the complete failure of actin cone formation in yuriF64, we also examined the relationship between Yuri and F-actin localization during spermiogenesis. We determined that the cap of dense complex at the round spermatid stage contains not only Yuri, but also F-actin (Fig. 7D). Furthermore, the actin staining extended around the basal body. F-actin continued to colocalize with Yuri in the stripe and dot pattern as the nuclei elongated (Fig. 7E). We also established that Yuri is a component of the F-actin cones used in individualization (Fig. 7F). Yuri immunostaining was seen throughout the large cones moving up the testes, and in cross-sections Yuri appeared concentrated in the inner cone regions, whereas actin was more peripheral.
Roles of Yuri in dense complex and basal body assembly
The yuriF64 mutation does not eliminate all isoforms of Yuri. Nevertheless, we determined that in yuriF64 the association of Yuri with the dense complex is completely lost, and all elements of the nuclear staining pattern – the cap, stripe and dot – are missing (Fig. 8A). Thus, the isoforms that are absent in yuriF64 are essential for protein function at these sites. The absence of Yuri from the dense complex allowed us to determine whether Yuri is necessary for the association of other components with this structure. In yuriF64, all elements of F-actin nuclear staining from the round spermatid stage onwards were lost (Fig. 8C). Yuri is therefore required for the initial accumulation and subsequent maintenance of F-actin within the dense complex. Similarly, γ-tubulin staining was never observed on the early round nuclei or at the later elongate stages (Fig. 8B), demonstrating that Yuri is required for attachment to, or possibly formation of, the CA of the basal body.
This absence of the CA raised the issue of whether basal bodies are present at all on the spermatid nuclei in yuriF64. To address this question, a GFP-fusion construct for the PACT domain of the Drosophila Pericentrin-like protein (dPLP; Cp309) (Martinez-Campos et al., 2004) was introduced into the yuriF64 background. The PACT domain of both mammalian pericentrin and dPLP provides targeting to the centrosomes/centrioles. In the Drosophila testis, GFP-PACT is an excellent fluorescent marker for the basal body (Martinez-Campos et al., 2004). In control cysts (w1118 or w–; yuriF64/CyO Roi), small cylinders of GFP-PACT staining demonstrate the presence of the basal bodies tightly clustered at the apical tips of condensing nuclei (Fig. 9A). GFP-PACT-marked basal bodies were also present on condensing nuclei in yuriF64. However, they were not tightly localized at the apical tips but scattered along the nuclei. Indeed, in many clusters, a fraction of the basal bodies were actually at the rostral rather than apical nuclear tips (Fig. 9B). Quantitation of the GFP-PACT fluorescence associated with control or yuriF64 nuclear clusters (using Metamorph software) indicated that yuriF64 does not affect the level of GFP-PACT binding to the basal bodies. In the final stages of nuclear condensation, the GFP-PACT fluorescence was lost from control nuclei. Similarly, although the nuclei never fully condense in yuriF64, GFP-PACT was ultimately lost from these nuclei too.
yuriF64 effects on the dense complex and basal body. (A-C) In the yuriF64 mutant, the Yuri nuclear stripe and dot are lost (A), γ-tubulin is no longer associated with the nuclei (B) and F-actin is no longer present on nuclei (C). (D,E) In Dynein light chain mutant Dlc90F05090, Yuri association with the nuclear cap (D) and stripe (E) is diminished (arrowheads), but the bell-shaped dot of Yuri (arrows) now appears precociously on round spermatid nuclei (D). In addition, a second dot (*) of Yuri is now found at the apex of both round (D) and elongate (E) nuclei. γ-tubulin staining (F) reveals that this dot (arrow) is the region of the basal body distal to the CA. Scale bars: 20 μm in A-C, 10 μm in D-F.
Basal body positioning and aberrant nuclear migration in yuriF64. (A) In yuriF64 heterozygotes, GFP-PACT fluorescence reveals basal bodies clustered tightly at apical nuclear tips. GFP-PACT is lost in the final stages of nuclear condensation (arrowhead). (B) In yuriF64 homozygotes, the basal bodies are disarrayed with some positioned at the rostral nuclear tip (arrows). The most condensed nuclei again show no GFP-PACT fluorescence (arrowheads). (C,D) In both yuriF64 heterozygotes (C) and homozygotes (D), subsets of nuclei sometimes migrate to the apical end of the cyst (arrows). Asterisks and white arrow indicate the position and direction of the stem cell tip, respectively. Scale bars: 20 μm.
Previous work has implicated cytoplasmic dynein and the related protein Dynactin in the formation of the dense complex (Li et al., 2004). Like Yuri, dynein heavy chain accumulates in the hemispherical cap on round spermatid nuclei but, in contrast to Yuri and the components examined here, its nuclear positioning is transient and it is not detectable in the dense-complex stripe during nuclear elongation. This brief association has a role in basal body functioning, however, because in a null mutant for the 14 kDa dynein light chain (Dlc90F), dynein heavy chain does not accumulate on the nuclei and, later, some nuclei lack a CA as judged by γ-tubulin staining.
In Dlc90F05090, an RNA-null in the testis (Caggese et al., 2001), the nuclear localization pattern of Yuri was found to be dramatically altered. The initial hemispherical cap of Yuri and the later stripe were highly attenuated and in some cases barely detectable (Fig. 8D,E). However, the bell-shaped dot of Yuri was now present at the base of the basal body, even in round spermatids (Fig. 8D). Furthermore, in both round and elongating nuclei, a second dot of Yuri was present (Fig. 8D,E). Co-staining with γ-tubulin demonstrated that this dot is the region of the basal body distal to the CA (Fig. 8F).
The axoneme-mitochondrial triads in yuriF64 mutants and aberrant nuclear migration
As the centrioles mature into basal bodies, a transition in protein composition occurs: Centrosomin is lost (Li et al., 1998) and the protein Uncoordinated (Unc) now becomes associated with these structures (Baker et al., 2004). Mutations in cnn or unc affect basal body function and produce abnormalities in axoneme structure. Given the loss of the CA and the aberrant positioning of the core basal bodies in yuriF64, we examined axoneme structure by TEM. This analysis also confirmed the complete failure of individualization in yuriF64 (Fig. 10C). In contrast to controls (Fig. 10A), the 64 axonemal `triads' – the axonemes and their major and minor mitochondrial derivatives (MDs) – all shared a single cytoplasm. Furthermore, terminal differentiation of the minor MDs was imperfect. In controls, this derivative undergoes dramatic expansion/disruption during individualization (Tokuyasu et al., 1972a) and collapses to a tiny structure in mature sperm (Fig. 10A). In the most developed cysts in yuriF64, the minor MD was less condensed than normal (Fig. 10C).
We examined axoneme structure in younger elongating cysts. Gross axonemal structure (the typical `9+2' arrangement) was normal in yuriF64. Of more than 750 studied, only two damaged axonemes were found, showing breaks in the outer circle of nine doublets (Fig. 10D). However, rarely, aberrant arrangements of axoneme-MD triads were found. These included: single axonemes with two major or two minor MDs, as judged by the presence/absence of a paracrystalline body, a marker for the major MD (Fig. 10D); sharing of a major or minor MD between two axonemes (Fig. 10E); major MDs with two or more paracrystalline bodies (Fig. 10E); and major MDs undergoing the expansion typically associated with the minor MD during individualization (Fig. 10D,E).
Although the spermatids in elongating cysts are syncytial, the links between them are narrow cytoplasmic bridges and the overall shape of each individual `cell' is distinguishable in EM cross-sections. Each `cell' typically contains a single axoneme-MD triad, although `fused' cells with two-eight triads have been detected in wild-type cysts (Stanley et al., 1972). The triad abnormalities in yuriF64 were largely within `cells' that contained multiple triads (Fig. 10E). However, our analysis of testis squashes provided no evidence that these arose as a result of cytokinesis defects in meiosis (see above).
We also examined sperm tails of yuriF64 heterozygotes in two genetic backgrounds (w–; yuriF64/CyO Roi and w–; yuriF64/+). Surprisingly, in both backgrounds, almost all mature cysts (∼90%) had a few imperfectly individualized triads (Fig. 10B), with a few cysts in which <50% of the sperm tails were still in syncytial cytoplasm. Thus, although fertile, yuriF64 heterozygotes clearly have individualization defects. Defects in triad development similar to those seen in yuriF64 homozygotes were also detected and, unexpectedly, were somewhat more prevalent in the heterozygotes, with ∼30% of the cysts in one testis showing these defects. More-pronounced axonemal abnormalities were also detected: in addition to broken sets of outer doublets, axonemes with no central doublet were present (Fig. 10G,I).
In both the yuriF64 homozygotes and heterozygotes, occasional examples were found of adjacent axonemes with their outer arms pointing in opposite orientations (Fig. 10D). Such cysts always had the correct number of axoneme profiles (64), and one cyst with 32 axonemes in one orientation and 32 in the other was found (data not shown). We could therefore exclude the possibility that these two orientations represented axonemes that were folded back on themselves. Two alternative explanations remained: either a fraction of the basal bodies have an altered chirality, or some of the 64 sperm nuclei migrate to the wrong end of the elongating cyst so that their associated axonemes extend along the cyst in the wrong direction. The latter explanation proved to be the case. Upon inspection, clusters of condensed sperm nuclei (ranging from one or two to ⩾20 nuclei) with attached basal bodies were detected at the apical end of elongated cysts, close to the stem cell tip (Fig. 9C,D). For the yuriF64 homozygote, four out of 40 testes examined showed this defect; for the heterozygotes, two out of nine testes had these mispositioned nuclei.
Roles of the Yuri isoforms
Our initial yuri mutant (yuric263) was identified by its altered gravitaxic responses. Further studies indicated that these changed responses arise from altered chordotonal neuron function, but provided no information as to whether yuri is uniquely expressed in these neurons (Armstrong et al., 2006). Studies here reveal that yuri is expressed ubiquitously, indicating that yuri is not dedicated to gravitaxic responses but rather that the yuric263 mutation disrupts yuri expression in a manner that specifically affects this function.
All isoforms of Yuri are expressed ubiquitously and yuriF64 removes the major ∼65 kDa isoform(s) from all tissues studied. Surprisingly, the only obvious developmental defect is male sterility. In the yuriF64 testis, the 30 kDa isoform is also missing, whereas in other tissues this isoform is less affected. Thus, the yuriF64 male sterility reflects either unique roles for ∼65 kDa isoforms, or the unique loss of the 30 kDa isoform. That loss of the ∼65 kDa isoforms has no effects in other tissues might indicate redundancy with the ∼100 kDa isoforms. The importance of the 30 kDa isoform is demonstrated by a consideration of the ovary (Fig. 3C). The ∼100 kDa Yuri isoforms are not normally present in the ovary, so that in yuriF64 the 30 kDa protein is the only isoform detectable in the tissue. Nevertheless, oogenesis and early embryogenesis proceed normally.
TEM analysis of control and yuriF64 mutant sperm. (A) Individualized control sperm (Sp/CyO Roi) each have one axoneme (Ax), one major mitochondrial derivative (M), and one minor mitochondrial derivative (m), contained within a single plasma membrane. (B) yuriF64/CyO Roi cysts contain mixtures of individualized (upper half of image) and non-individualized (lower half) sperm. (C) No individualization is seen in yuriF64 homozygotes. Major mitochondrial derivatives look normal but minor derivatives are enlarged (arrows). (D,E) yuriF64 homozygotes and (F-I) heterozygotes showing that axonemes in elongating cysts are sometimes associated with aberrant sets of mitochondrial derivatives, often sharing them or possessing multiple derivatives of the same type. P, paracrystalline body in major mitochondrial derivative. The outer ring of microtubule doublets is sometimes broken (arrows), and internal components (central-pair microtubules or linker arms) can be missing (arrowheads). Axonemes of apparently opposing chirality (curved arrows of differing color) are visible in D-F, and the central microtubule pair is seen to be `escaping' the opened axoneme in I (arrow). Scale bars: 500 nm in A-G, 250 nm in H,I.
Although the major defects seen in yuriF64 homozygotes are largely absent from heterozygotes, some minor, incompletely penetrant defects (particularly in axoneme structure) are more prevalent in the heterozygous than the homozygous condition. Because yuriF64 causes loss of particular isoforms, the normal stoichiometric balance between isoforms is disrupted in both homozygotes and heterozygotes, but it is disrupted differently in the two situations. Thus, given that two classes of Yuri isoforms contain coiled-coil regions, altered dimerization or protein interactions that have more severe consequences for axoneme assembly might be produced uniquely in the heterozygote.
Yuri function and the defects in spermatogenesis
The various elements of the yuriF64 testis phenotype provide clues as to the molecular functions of the protein. One clear implication is that Yuri regulates F-actin function. We show here for the first time that F-actin is associated with the dense complex on spermatid nuclei and that in yuriF64, F-actin never accumulates on the nuclei, suggesting an initiating role for Yuri in dense-complex formation. Yuri is also a component of the actin cones that mediate sperm individualization and is required for their formation. The actin cones are formed by a two-step process (Noguchi et al., 2006). Initially, parallel actin fibers are formed around the nuclei and then an actin meshwork is added at each apical nuclear tip. Given the absence of actin cone initiation in yuriF64, it seems likely that Yuri has an early role in F-actin deposition here too.
The aberrant F-actin sleeves formed in the somatic cyst cells in yuriF64 led us to identify related actin sleeves around actively coiling sperm in control testes. Sperm coiling is executed within the confines of the head cyst cell, which completely engulfs the apical region of the cyst (Tokuyasu et al., 1972b). Elaborate microvilli, full of 50 Å filaments, project from the head cyst cell onto the cyst walls and Tokuyasu and colleagues suggest that coiling largely represents the collapse of the intrinsically helical sperm tails into a flat pile of gyres as a result of contraction and shape change within the head cyst cell. We propose that the actin sleeves in control testes are related to the 50 Å filaments seen by Tokuyasu et al. and that in yuriF64, F-actin structures form at inappropriate positions in association with abortive coiling.
In addition to regulating actin function, Yuri is implicated in microtubule/tubulin action. The stripe of dense complex along the elongating nuclei accretes a bundle of microtubules that are thought to provide structural rigidity to the nuclei. Although we were not able to image these microtubules, in yuriF64 many late-stage nuclei lose their rigidity and collapse into helical twirls, suggesting that the microtubules are no longer present. The presence of Yuri in the dense complex is also intimately associated with proper positioning, formation and functioning of the basal body. When Yuri is not present at this site, (1) the basal bodies are scattered along the nuclei, or even mispositioned at the rostral nuclear tips, (2) the CA element of the basal body is missing and (3) the axonemes show defects similar to those of other mutations (cnn and unc) that affect basal body function. Nevertheless, our findings for the GFP-PACT marker indicate that dPLP is recruited normally to the basal bodies in yuriF64. Interestingly, in mammalian systems, interaction between γ-tubulin and pericentrin is thought to underlie the targeting of γ-tubulin to centrosomes/centrioles (Young et al., 2000). dPLP is therefore implicated in promoting the presence of γ-tubulin and of the CA on the sperm basal body. Our evidence here that in yuriF64, dPLP is on the basal bodies but γ-tubulin is not, suggests a role for Yuri in the interaction of these two proteins.
At the end of elongation, prior to individualization, the nucleus-basal body association is altered so that the axoneme and sperm head are locked in a permanent configuration relative to one another (Lindsley and Tokuyasu, 1980; Tokuyasu, 1975). This change involves disappearance of the CA and movement of the basal body to lie in a shallow groove on one side of the nucleus. Predictably, the CA components γ-tubulin and Unc are lost from the nuclei at this stage (Baker et al., 2004). We show here that both the Yuri dot and the GFP-PACT marker also disappear at this point. The basal body present on mature sperm is clearly stripped of many ancillary proteins.
Although Yuri appears to anchor tubulin structures, including the basal body, to the nuclear membrane, our findings for the dynein light chain mutant suggest that the initial positioning of Yuri on the nuclear membrane is determined by dynein transport, presumably along microtubules. In the dynein light chain mutant, Yuri localization is dramatically altered, with Yuri now primarily associated with the basal body – a novel association not seen in the wild type. The implication must be that an activity of dynein is required to prevent an interaction of Yuri with the basal body.
The opposing orientations of some adjacent axonemes in yuriF64 reflects the unexpected positioning of sperm nuclei at the wrong ends of elongated cysts. Contacts that normally hold the nuclei together in tight alignment appear to be missing in yuriF64, and this could permit loose nuclei to migrate to the wrong location. Axonemes with opposite orientations in a single cyst have been reported for mutations in the Drosophila parkin homolog (Riparbelli and Callaini, 2007). Although these investigators did not report a search for nuclei at the wrong ends of cysts, they did note occasional actin cones pointing in the wrong direction – a finding that suggests the same underlying cause for the two axoneme orientations in both their case and ours.
Other genes that act in mechanosensory organs and spermatogenesis
The finding that different mutations of yuri affect processes as disparate as gravitaxis and spermatogenesis is initially surprising. However, together with sperm, mechanoreceptor neurons, such as those affected by yuric263, are the only cell types in Drosophila that possess cilia, and genes that affect ciliary function have been shown to affect both mechanosensory organs and spermatogenesis. Mutations in touch insensitive larva B (tilB) are defective in hearing and touch perception as a result of defects in the chordotonal organs (Eberl et al., 2000). Mutations in unc affect both the chordotonal organs and the external sense organ (eso) class of mechanoreceptors (Eberl et al., 2000). Mutations at both loci are also male sterile because they encode proteins with roles in cilia. TilB is a conserved ciliary protein with a leucine-rich region and a coiled-coil domain (Kavlie et al., 2007) and Unc is associated with the basal bodies in sperm and mechanosensory neurons (Baker et al., 2004). Unc, like γ-tubulin, is a component of the CA and, like Yuri, is insect-specific and contains coiled-coil regions (Baker et al., 2004).
These examples suggest that the yuri function affected in yuric263 might be a role in positioning the ciliary basal bodies of the chordotonal neurons, a role comparable to that identified here in spermiogenesis. Furthermore, the intriguing possibility of molecular interactions between Yuri and Unc is suggested. The proteins are physically close at the basal body and their only distinguishing features are coiled-coil domains that presumably facilitate protein-protein interactions. It seems possible that these two proteins have evolved to fulfil specialized roles associated with anchoring the basal bodies that could entail heterodimerization.
Yuri antibodies and immunoblots
The entire coding region of the 30 kDa Yuri isoform from clone GH14032 was amplified by PCR, cloned in Topo vector pCR2.1 (Invitrogen) and sequenced, then recloned into the EcoRI and SalI sites of expression vector pET28a (Novagen). The recombinant His-tagged protein was purified by Ni2+ chromatography (Novagen) and used to raise antibodies in chickens (Aves Labs). Recombinant Yuri protein cross-linked to NHS-activated Sepharose 4 Fast Flow (Amersham) was used for affinity purification. For immunoblots, samples were solubilized in SDS sample buffer, run on 12.5% polyacrylamide gels and blotted to Immobilon (Millipore) filters. Bands reacting with the affinity-purified antibody were detected with horseradish-peroxidase-conjugated rabbit anti-chicken antibodies (Sigma) and the West Dura reagent (Pierce).
Fertility testing, fly stocks and genetics
For fertility testing, ⩾20 individual males or virgin females were placed with three w1118 partners in food vials for 7 days, after which adults were removed. The original vials were checked for the presence of larvae, pupae and adults for a further 15 days. Although eggs were laid, yuriF64 homozygous and hemizygous males never produced any viable progeny. A stock with deficiency Df(2L)do1, which removes yuri-containing region 35B1-35D2, balanced over a CyO-GFP balancer (Rudolph et al., 1999), was generated from crosses of stocks 3212 [Df(2L)do1, pr1 cn1/In(2LR)Gla, wgGla-1 DNApol-γ352] and 5702 [w1; nocSco/CyO, P{GAL4-Hsp70.PB}TR1, P{UAS-GFP.Y}TR1] from the Bloomington Stock Center. To generate embryos homozygous for Df(2L)do1 or homozygous for CyO-GFP, eggs were collected from the Df(2L)do1/CyO-GFP stock and left >24 hours to ensure that viable embryos hatched. Fluorescent and non-fluorescent embryos were collected separately. Third chromosomes carrying (1) a don juan-GFP construct (Santel et al., 1997) or (2) a GFP-PACT construct (Martinez-Campos et al., 2004) or (3) Flytrap lines ZCL0931, ZCL2183, ZCL2155 and G0147 (Kelso et al., 2004) were introduced into a w–; yuriF64 background. Mutation ms(3)05090 at the Dlc90F gene (Caggese et al., 2001) was from the Bloomington Stock Center.
Sequence analysis and conservation of yuri
The following yuri ESTs/cDNAs were sequenced: adult head GH14032; adult testis AT03435, AT15480, AT15149, AT19027 and AT25733; adult ovary GM26781 and GM25777; S2 cell line SD06513 and SD11641; embryo RE12523 and RE13793. Two clones from the testis, AT15149 and AT15480, end at the same 5′ residue at a point between exons 3 and 5. The region immediately 5′ to this point scores poorly in analyses designed to detect promoters. Given that these two cDNAs were prepared from the same RNA, we assume they have an incomplete 5′ terminus. However, their 5′-most sequence, which is not present in other cDNAs, is part of an intron between exons 3 and 5 (Fig. 1). These clones thus either (1) provide evidence for the variable presence of an additional exon (labeled 4 in Fig. 1), the 5′ boundary of which is not defined or (2) represent incompletely spliced transcripts. A paralog search in D. melanogaster was performed using the Yuri `PD' isoform (FlyBase) sequences and the BLASTP service at FlyBase. Because protein data sets are not available for all sequenced insect species, and to avoid spurious matches to coiled-coil domains, ortholog searches of translated DNA sequences were conducted with TBLASTN, using the 239-residue protein encoded by the GH14032 cDNA as the query and both the `nr/nt' NCBI database and the 21 insect genome sequences searchable at FlyBase as target data sets. The sequence identity computation for the ∼100 kDa Yuri protein in the 12 sequenced Drosophila sequences (Drosophila 12 Genomes Consortium, 2007) was performed using BLASTP on the GLEANR consensus protein data sets. Coiled-coil predictions were made using the COILS program at http://www.ch.embnet.org/software/COILS_form.html. Default settings were used in searches.
Imprecise excisions
The P insertion yuric263 (Armstrong et al., 2006) and SUPor-P insertion KG03019 (Roseman et al., 1995) were mobilized with the Δ2-3 transposase at 99B (Robertson et al., 1988). Standard genetic schemes generated stocks of viable excisions. For lethal excisions, lines with GFP-marked balancers were prepared. Excisions were characterized by PCR. Precise deletion endpoints were determined by sequencing.
Immunocytochemistry
Testes were dissected in ice-cold phosphate-buffered saline pH 7.2 (PBS), fixed with 3% paraformaldehyde in PBS for 10 minutes and permeabilized by four washes in BBX (PBS + 0.3% Triton X-100 + 0.1% BSA) for 10 minutes. They were then incubated overnight at 4°C with rotation in BBX + 2% goat serum and one or more of the following antibodies: 1:100 affinity-purified Yuri antibody; 1:500 mouse monoclonal anti-γ-tubulin GTU-88 (Sigma); 1:200 rabbit polyclonal anti-Centrosomin antibody R19 (gift of T. Kaufman, Indiana University, Bloomington, IN); 1:50 mouse monoclonal anti-β-tubulin E7 (Developmental Studies Hybridoma Bank). After two washes each in BBX and BBX + 2% goat serum, appropriate Alexa Fluor-conjugated secondary antibodies (Invitrogen) were added at 1:500 in BBX + 2% goat serum and incubated for 2 hours. Rhodamine- or Alexa Fluor-conjugated phalloidin (Invitrogen) at 1:50 dilution was included with the secondary antibody as appropriate. After four washes with BBX, testes were mounted with 1:2000 Hoechst 33342 (Invitrogen) in 50% glycerol. Images were collected on a Zeiss Axioplan or on Zeiss LSM 410 and 510 confocal microscopes and processed with Metamorph (Molecular Devices) or Zeiss software.
Transmission electron microscopy (TEM)
TEM analysis was as described previously (Tokuyasu et al., 1972a), with minor modifications. Sections were cut at 700Å and stained with uranyl acetate and lead citrate. JEOL 1010, JEOL 1230 and Hitachi H-7500 electron microscopes were used.
We thank Dr R. P. Munjaal for contributions to the early phases of this work. We thank Dr Chris Bazinet for the dj-GFP line; Dr David Caprette for EM help; Dr James Fabrizio for Flytrap lines he characterized as expressing GFP in the cyst cells; Dr Thomas Kaufman for Centrosomin antibody; Dr Tatsuhiko Noguchi for critical insight into the actin sleeves in the yuriF64; Dr Jordan Raff for the GFP-PACT line; Dr Kiyoteru Tokuyasu for helpful discussions on the nuclear localization of Yuri. We are grateful to Kenneth Dunner, Jr, Deborah Townley and Dr Wenhua Guo of the High Resolution EM Facility at MD Anderson, the Integrated Microscopy Core at Baylor College of Medicine and the Smalley Institute of Nano Science and Technology at Rice. respectively, for their assistance with EM work. The help of Rice undergraduates, in particular Summer Bell, Faraz Sultan and Anita Shankar, is gratefully acknowledged. These studies were supported by NIH grant RO1 HD 39766, grant C-1119 from the Welch Foundation of Texas and NASA grant NCC2-1356.
Accepted March 20, 2008.
© The Company of Biologists Limited 2008
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