1926 Research Article 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 and Kathleen M. Beckingham* Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 South Main Street, Houston, TX 77005, USA *Author for correspondence (e-mail: [email protected])

Accepted 20 March 2008 Journal of Cell Science 121, 1926-1936 Published by The Company of Biologists 2008 doi:10.1242/jcs.026559

Summary Males of the genus Drosophila produce sperm of remarkable the yuri mutant, late clusters of syncytial nuclei are deformed length. Investigation of giant sperm production in Drosophila and disorganized. The basal bodies are also mispositioned on melanogaster has demonstrated that specialized actin and the nuclei, and the association of a specialized structure, the microtubule structures play key roles. The gene yuri gagarin centriolar adjunct (CA), with the basal body is lost. Some of (yuri) encodes a novel protein previously identified through its these nuclear defects might underlie a further unexpected role in gravitaxis. A male-sterile mutation of yuri has revealed abnormality: sperm nuclei occasionally locate to the wrong ends roles for Yuri in the functions of the actin and tubulin structures of the spermatid cysts. The structure of the axonemes that grow of spermatogenesis. Yuri is a component of the motile actin cones out from the basal bodies is affected in the yuri mutant, that individualize the spermatids and is essential for their suggesting a possible role for the CA in axoneme formation. formation. Furthermore, Yuri is required for actin accumulation in the dense complex, a microtubule-rich structure on the sperm Key words: Drosophila, Spermatogenesis, Actin, Tubulin, Basal nuclei thought to strengthen the nuclei during elongation. In body, Chordotonal organ, Centriole

Introduction sperm. The gene is only highly conserved in the genus Drosophila, A unique feature of the genus Drosophila is the formation of suggesting specialized roles in these organisms. Interestingly, yuri unusually long sperm tails. Sperm lengths of millimeters are was initially identified through its function in another specialized common within this group, with the 1.8 mm sperm of D. organ system of insects and arthropods: the chordotonal organs.

Journal of Cell Science melanogaster being fairly typical. This marked expansion in sperm These are complex mechanosensory structures with roles in length reflects an unusual aspect of spermatogenesis in these proprioception and graviperception. The first mutation at the locus, organisms: in contrast to other species in which an intraflagellar yuric263, was identified in a screen for mutants affecting gravitaxis. transport system is used for growth of the sperm flagellum (Scholey, Altered gravitaxis was shown to result from perturbed expression 2006), Drosophila sperm axonemes are assembled in syncytial cysts of yuri in subsets of chordotonal neurons (Armstrong et al., 2006). by a mechanism that does not require, and is not limited by, this The molecular functions of the locus identified here suggest that system (Han et al., 2003; Sarpal et al., 2003). This unusual sperm yuri mediates specialized actin- and microtubule-related activities axoneme development and the resulting expansion of sperm tail in Drosophila tissues. length have led to distinctive features of spermatogenesis not found in other species. In D. bifurca, a special ‘sperm roller’ has evolved Results to package its 6-centimeter-long gametes (Joly et al., 2003). In D. The yuri locus in D. melanogaster and other Drosophilids melanogaster, a highly evolved individualization process that In addition to the cDNA (GH14032) encoding a ~30 kDa protein generates 64 individual sperm from an elongate cyst containing 64 that we used previously (Armstrong et al., 2006), we identified 11 syncytial spermatids has been identified and studied (Noguchi and further yuri ESTs/cDNAs from adult testis, ovary, S2 cells and Miller, 2003; Tokuyasu et al., 1972a). The distinctive molecular embryos through FlyBase. Sequencing of these new cDNAs mechanisms needed for this process include a motile filamentous established that three major transcript classes are generated from actin system (the investment, or actin, cones) that traverses the entire yuri (Fig. 1). Two promoters are used, with the medium transcripts length of the sperm tails, removing excess cytoplasm and investing initiated at the proximal promoter and the short and long classes each sperm in its own plasma membrane. A specialized microtubule- from the distal promoter. However, all isoforms begin at one of rich structure (the dense complex) is also associated with the sperm two closely positioned ATGs. The short transcript class encodes nuclei and functions to position the basal body and also possibly the ~30 kDa protein identified previously. The medium class to strengthen the nuclei as they undergo extreme condensation encodes isoforms of 64-65 kDa that extend ~400 amino acids (A. D. Tates, Cytodifferentiation during spermatogenesis in further at the C-terminus. The long class, encoding proteins of 101- Drosophila melanogaster, PhD thesis, Rijksuniversiteit Leiden, The 107 kDa, extends an additional ~300 amino acids C-terminally. Netherlands, 1971) (Tokuyasu, 1974). The short yuri isoform is novel, with only a single recognizable We have identified a locus, yuri gagarin (yuri), that we show motif (a polyproline stretch). However, the two longer forms here has multiple roles in the generation of elongate individualized contain coiled-coil motifs with weak similarity (~20% identity) to yuri gagarin function in spermatogenesis 1927

Fig. 1. 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 1bЈ and 1bЉ. Exon 4, the 5Ј 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.

those in many fibrillar proteins that dimerize, such as myosin heavy Ubiquitous expression of the three major Yuri isoforms chain and CLIP-190. The strongest match is to the coiled-coil of To investigate Yuri expression, we generated antibodies against Sticky, the Drosophila citron kinase (Sweeney et al., 2008). the sequences common to all isoforms (see Materials and yuri is unique in the D. melanogaster genome, once the weak Methods). Immunoblots of yuri+ embryos and embryos lacking similarities to coiled-coil regions are disregarded. Thus, to avoid yuri established the specificity of our antisera and their ability to spurious similarities, the shortest yuri isoform was used to find yuri detect the three predicted Yuri isoform classes (Fig. 3A). These orthologs in other organisms. Significant matches were found in blots also demonstrated that only the short Yuri isoform is all 11 sequenced Drosophila genomes (Drosophila 12 Genomes maternally loaded into the embryo, with the longer isoforms Consortium, 2007), but none was identified in other evolutionary appearing later in embryogenesis (Fig. 3A,B). In later stages, all orders or other insects, including the closest Dipteran relatives, the three isoform classes are ubiquitously expressed (Fig. 3C). The Culicidae (mosquitoes) (Fig. 2). Sequence conservation within the ~65 kDa class is most abundant in most situations, although in Drosophila genus was high (91-37% sequence identity, 93-57% testis and thorax the other isoforms are also highly expressed (Fig. similarity) across the entire ~100 kDa isoform of D. melanogaster. 3C). The existence of at least two isoforms for both the ~100 kDa

Journal of Cell Science yuri therefore appears to be a Drosophila-specific gene. Most species and ~65 kDa classes was confirmed by these experiments. have one yuri gene, but two related genes are present in D. Additional bands were sometimes present that probably represent pseudoobscura and D. persimilis. specific degradation products, as they were largely missing in

Fig. 2. 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). 1928 Journal of Cell Science 121 (11)

Fig. 3. 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.

embryos lacking yuri (Fig. 3A) and in the yuriF64 mutant (Fig. mutation on the yuri locus. In order to determine whether yuriF64 3C) (see below). affects overall viability, the survival of yuriF64 homozygous progeny versus heterozygous progeny (yuriF64/CyO Roi) was quantitated for A yuri mutant that lacks Yuri ~65 kDa isoform(s) a cross of yuriF64 females with heterozygous (yuriF64/CyO Roi) The yuric263 mutation from our gravitaxic screen is an insertion of males. Of 649 progeny, 51% were yuriF64 homozygotes, indicating P{GawB} just upstream of the transcription start site for the that yuriF64 has no effects on survival to adulthood.

Journal of Cell Science medium length transcripts. We generated further mutations by The Drosophila testis contains a stem cell system at its apical tip imprecise excision of P{GawB} and of a second transposon, from which spermatogonial cells are budded off to proceed through KG03019 (Roseman et al., 1995), inserted three residues spermatogenesis. A somatic stem cell system is also present that downstream of the yuric263 P element. Three excisions that delete produces so-called cyst cells. A pair of cyst cells encases the division the relevant transposon and adjacent genomic DNA were identified. products of each spermatogonial cell throughout spermatogenesis One of these is lethal (yuriLE1), but the deletion extends upstream and post-meiotic spermiogenesis. Each spermatogonial cell generates into an adjacent gene (cullin3; guftagu) known to affect viability a cyst of 64 spermatids, linked by cytoplasmic bridges, which (Mistry et al., 2004). In yuriL5, a short region of yuri upstream undergoes dramatic elongation. At completion, each cyst has a highly sequence is deleted, causing reduced expression of all Yuri isoforms. elongate cytoplasm (~1.8 mm in length) with the 64 condensed nuclei Nevertheless, homozygous yuriL5 animals are viable with no positioned at the seminal vesicle end and 64 axonemes extending obvious phenotype. Only one deletion, yuriF64, removes transcribed from the nuclei along the length of the cyst towards the apical tip. sequences from the locus. Most of the 5Ј UTR of the ~65 kDa Two giant mitochondrial derivatives, generated by fusion of the isoforms is deleted, with only ten residues upstream of the first mitochondria within each post-meiotic spermatid, extend along the initiator ATG remaining (Fig. 1A). The yuriF64 deletion lead to length of each axoneme. The later stages of spermiogenesis involve complete loss of ~65 kDa isoforms in all tissues and stages a specialized process termed individualization (see below) in which examined (Fig. 3C). The ~100 kDa isoforms remained strongly the 64 syncytial spermatids are converted into 64 individual sperm. expressed, but expression of the ~30 kDa isoform was decreased Finally, a coiling process retracts the sperm down to the entrance to in several tissues and undetectable in the testis (Fig. 3C). the seminal vesicle. Highly elongate spermatid cysts were present in yuriF64 testes, Male sterility is associated with the yuriF64 mutation some of which were attempting to coil, but it was unclear whether Homozygous yuriF64 mutants (yuriF64) are viable with normal mature sperm were formed. To address this question, we introduced external morphology. However, yuriF64 males are completely sterile, a don juan-GFP fusion construct into the yuriF64 background. Don whereas females are fertile (data not shown). Flies heterozygous Juan protein is produced in the giant sperm tail mitochondria and for yuriF64 and deficiency Df(2L)do1, which deletes yuri, were also persists into mature sperm. Don Juan-GFP (Santel et al., 1997) male sterile and female fertile. The testis phenotype (see below) provides a marker for late spermiogenesis (Civetta, 1999; Gao et was identical in yuriF64 homozygotes and hemizygotes (data not al., 2003). We examined 8-day-old virgin males, which should have shown), demonstrating that it results from the effects of the yuriF64 large quantities of sperm in the seminal vesicles. In yuriF64/CyO yuri gagarin function in spermatogenesis 1929

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 F64 Fig. 4. Sperm elongate but show individualization and coiling defects in yuriF64. and waste bags were detected, the yuri testes contained (A) Sperm tails, marked with Don Juan-GFP (green), fill the seminal vesicle neither (Fig. 4). Instead, elongated ‘sleeves’ of actin were seen (arrow) in control testes. (B) In yuriF64 hemizygotes [yuriF64/Df(2L)do1], the around the periphery of some spermatid cysts. These appeared seminal vesicle (arrow) is empty, and sperm cysts show abortive coiling in the as solid tubes in normal fluorescence imaging (Fig. 4BЉ), but Ј Љ testis proper (arrowhead). (A -B ) Phalloidin staining (red) identifies actin cones as hollow structures in confocal sections (Fig. 5A). We and waste bags in control testis (AЈ, red arrow; as shown at higher magnification in AЉ). Mutant testis is devoid of these structures (BЈ), and F-actin sleeves are established that these sleeves are actually present in the somatic present instead (BЈ, red arrow; as shown at higher magnification in BЉ). Scale bars: cyst cells surrounding the cysts, rather than in the cysts 200 μm. 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 Roi heterozygotes carrying don juan-GFP, the seminal vesicles were and GFP in the cyst cells precisely overlapped (Fig. 5A). Having full of fluorescent sperm and the basal testis carried masses of identified these sleeves in yuriF64, we discovered similar structures fluorescent coiling sperm (Fig. 4A). In yuriF64, no fluorescence was present at a lower frequency in control testes (Fig. 5B). In controls, detectable in the seminal vesicles and the basal testis contained these sleeves are always in the basal regions of the testis where sperm curled structures, thicker than individual sperm with aberrant coiling takes place, whereas in yuriF64 they form throughout the testis. coiling (Fig. 4B). Squashes of seminal vesicles confirmed the We address the significance of these structures in the Discussion. presence of motile sperm in the controls and their complete absence The major conclusion here is that in yuriF64 no actin cone sets or F- in the mutant (data not shown). actin structures of any kind are present in the germline cysts proper.

Individualization fails in yuriF64 Actin cone initiation and nuclear behavior are aberrant in Phase-contrast examination of testis squashes revealed no defects yuriF64

Journal of Cell Science in spermatogenesis up to the post-meiotic stages; ‘onion stage’ The formation of the F-actin cones of individualization has been spermatids appeared normal. The structures undergoing abortive studied previously (Fabrizio et al., 1998; Lindsley and Tokuyasu,

Fig. 5. Spermatogenesis defects in yuriF64. (A-AЉ) 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-AЉ,C,D, 100 μm in B,E,F, 50 μm in G. 1930 Journal of Cell Science 121 (11)

1980; Noguchi et al., 2006). Initially, actin fibers accrete along tip. In the final stages of nuclear maturation, first the stripe the lengths of the condensed sperm nuclei in the basal testis. The disappeared and then the dot was also lost. actin then moves to form cones, flaring off the apical ends of the Tokuyasu has described the ultrastructural changes to the nuclei nuclei before release to move up the axonemes. Nuclei in all during elongation (Tokuyasu, 1974). Part of the nuclear membrane stages of this process are present in the basal region of wild-type is fenestrated with nuclear pores during this process. Initially, this testes. In yuriF64, although the nuclear sets were seen to descend region forms a cap over one hemisphere of the round post-meiotic to this level and undergo some condensation, they were clearly nucleus, with dense material aggregating over this region between more disorganized, with individual nuclei trailing behind, the nuclear membrane and adjacent endoplasmic reticulum. As the apparently detached from the main cluster. In some late-stage nuclei elongate, this cap and associated material transform to a stripe clusters, almost all the nuclei were distorted in shape, some in a along the long axis of the nucleus. More of the dense material helical or circular configuration (Fig. 5D). No nuclei ever accumulates along with microtubules, with the whole complex condensed to the tight bundles seen in controls (Fig. 5C). sinking inwards to form a groove filled with dense cytoplasm and Furthermore, no well-formed sets of cones were ever detected, a microtubule bundle (collectively the ‘dense complex’) that runs although a little F-actin accumulated around some nuclei (Fig. the length of the nucleus. The nuclei are actually horseshoe-shaped 5E,F). Interestingly, small under-developed cones were in cross-section at this stage. In the final stages of nuclear occasionally found singly or in clusters in this region. Some of maturation, the dense complex is dispersed and the nuclei regain a them were apparently mobile, as they appeared at some distance circular cross-section. The dense complex is thought to provide from any nuclei (Fig. 5G). structural rigidity to the nuclei during the elongation process (Tokuyasu, 1974). Early after meiosis, the single centriole of each Yuri protein localization in control testes spermatid embeds into the spherical nuclear membrane at the center Our antisera, which detect all Yuri isoforms, were used to examine of the dense complex and then converts into the basal body. During Yuri localization in control testes. Yuri was present at all stages of elongation, the basal body moves to the apical tip of the nucleus, germ cell development, peaking around meiosis, with most staining immediately adjacent to the stripe of dense complex (Fig. 6B). being cytoplasmic and diffuse (Fig. 6A). However, in addition, a The pattern of Yuri localization on the spermatid nuclei was striking and dynamic pattern of Yuri association with the post- strikingly similar to that of the dense complex and associated basal meiotic spermatid nuclei was seen as they condensed during body. To position Yuri relative to these structures, we co-stained elongation (Fig. 6B-F). While the nuclei were still round, Yuri was for γ-tubulin, Centrosomin (Cnn) and β-tubulin. γ-tubulin is a seen to accumulate as a cap over one hemisphere of each nucleus. component of the centriolar adjunct (CA) (Wilson et al., 1997), a As the nuclei became ellipsoid, the Yuri staining transformed into torus-shaped structure around the middle of the basal body during a stripe along the nuclear long axis and a dot at the apical nuclear elongation (Fig. 7C) (Tokuyasu, 1975). Centrosomin, a centriole Journal of Cell Science

Fig. 6. 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. yuri gagarin function in spermatogenesis 1931

Fig. 7. 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-EЉ) 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-FЉ) 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-EЉ, 20 μm in F-FЉ.

component, is present early in the transformation to the basal body sections Yuri appeared concentrated in the inner cone regions, but is subsequently lost (Li et al., 1998). β-tubulin is a general whereas actin was more peripheral. marker for microtubules. γ-tubulin/Yuri co-staining established that the basal body is at the center of the Yuri cap in round spermatids Roles of Yuri in dense complex and basal body assembly (Fig. 7A), providing evidence that the Yuri cap corresponds to the The yuriF64 mutation does not eliminate all isoforms of Yuri. accumulating dense complex. No round spermatid nuclei that co- Nevertheless, we determined that in yuriF64 the association of Yuri stained for the Yuri cap and Centrosomin were detected, suggesting with the dense complex is completely lost, and all elements of the that Centrosomin is lost before significant Yuri accumulation. We nuclear staining pattern – the cap, stripe and dot – are missing (Fig. F64

Journal of Cell Science were not able to detect a stripe of microtubules along the nuclei by 8A). Thus, the isoforms that are absent in yuri are essential for staining for β-tubulin. Very high general cytoplasmic staining and/or protein function at these sites. The absence of Yuri from the dense possibly the burying of the appropriate epitope could underlie this complex allowed us to determine whether Yuri is necessary for the failure. association of other components with this structure. In yuriF64, all Although γ-tubulin staining showed an apical dot on the elements of F-actin nuclear staining from the round spermatid stage elongating nuclei, interestingly, the Yuri dot and the γ-tubulin dot onwards were lost (Fig. 8C). Yuri is therefore required for the initial did not coincide. The Yuri dot, which at high magnification has a accumulation and subsequent maintenance of F-actin within the bell shape (Fig. 6F), was sandwiched between the dot of γ-tubulin dense complex. Similarly, γ-tubulin staining was never observed staining and the nuclear membrane. Thus, Yuri is probably not part on the early round nuclei or at the later elongate stages (Fig. 8B), of the basal body per se but lies between the basal body and the demonstrating that Yuri is required for attachment to, or possibly nuclear membrane. EM analysis has established that the basal body formation of, the CA of the basal body. is embedded into a ~0.5 μm indentation in the nuclear membrane This absence of the CA raised the issue of whether basal bodies (Tokuyasu, 1975) at this stage. It seems likely that the Yuri dot is are present at all on the spermatid nuclei in yuriF64. To address the residuum of the initial dense-complex cap that was always this question, a GFP-fusion construct for the PACT domain of the beneath the insertion point of the basal body, and that Yuri continues Drosophila Pericentrin-like protein (dPLP; Cp309) (Martinez- to fill the space between the membrane and the basal body during Campos et al., 2004) was introduced into the yuriF64 background. nuclear elongation. The PACT domain of both mammalian pericentrin and dPLP Given the complete failure of actin cone formation in yuriF64, provides targeting to the centrosomes/centrioles. In the Drosophila we also examined the relationship between Yuri and F-actin testis, GFP-PACT is an excellent fluorescent marker for the basal localization during spermiogenesis. We determined that the cap of body (Martinez-Campos et al., 2004). In control cysts (w1118 or dense complex at the round spermatid stage contains not only Yuri, w–; yuriF64/CyO Roi), small cylinders of GFP-PACT staining but also F-actin (Fig. 7D). Furthermore, the actin staining extended demonstrate the presence of the basal bodies tightly clustered at around the basal body. F-actin continued to colocalize with Yuri in the apical tips of condensing nuclei (Fig. 9A). GFP-PACT-marked the stripe and dot pattern as the nuclei elongated (Fig. 7E). We also basal bodies were also present on condensing nuclei in yuriF64. established that Yuri is a component of the F-actin cones used in However, they were not tightly localized at the apical tips but individualization (Fig. 7F). Yuri immunostaining was seen scattered along the nuclei. Indeed, in many clusters, a fraction of throughout the large cones moving up the testes, and in cross- the basal bodies were actually at the rostral rather than apical 1932 Journal of Cell Science 121 (11)

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 Fig. 8. yuriF64 effects on the dense complex and basal body. (A-C) In the the base of the basal body, even in round spermatids (Fig. 8D). F64 γ yuri mutant, the Yuri nuclear stripe and dot are lost (A), -tubulin is no Furthermore, in both round and elongating nuclei, a second dot of longer associated with the nuclei (B) and F-actin is no longer present on nuclei γ (C). (D,E) In Dynein light chain mutant Dlc90F05090, Yuri association with the Yuri was present (Fig. 8D,E). Co-staining with -tubulin nuclear cap (D) and stripe (E) is diminished (arrowheads), but the bell-shaped demonstrated that this dot is the region of the basal body distal to dot of Yuri (arrows) now appears precociously on round spermatid nuclei (D). the CA (Fig. 8F). 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) F64 is the region of the basal body distal to the CA. Scale bars: 20 μm in A-C, The axoneme-mitochondrial triads in yuri mutants and 10 μm in D-F. 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 nuclear tips (Fig. 9B). Quantitation of the GFP-PACT fluorescence protein Uncoordinated (Unc) now becomes associated with these associated with control or yuriF64 nuclear clusters (using structures (Baker et al., 2004). Mutations in cnn or unc affect basal Metamorph software) indicated that yuriF64 does not affect the body function and produce abnormalities in axoneme structure. level of GFP-PACT binding to the basal bodies. In the final stages Given the loss of the CA and the aberrant positioning of the core of nuclear condensation, the GFP-PACT fluorescence was lost basal bodies in yuriF64, we examined axoneme structure by TEM. from control nuclei. Similarly, although the nuclei never fully This analysis also confirmed the complete failure of condense in yuriF64, GFP-PACT was ultimately lost from these individualization in yuriF64 (Fig. 10C). In contrast to controls (Fig. nuclei too. 10A), the 64 axonemal ‘triads’ – the axonemes and their major and

Journal of Cell Science 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. F64 Fig. 9. Basal body positioning and aberrant nuclear migration in yuri . (A) In 10D,E). yuriF64 heterozygotes, GFP-PACT fluorescence reveals basal bodies clustered tightly at apical nuclear tips. GFP-PACT is lost in the final stages of nuclear Although the spermatids in elongating cysts are syncytial, the condensation (arrowhead). (B) In yuriF64 homozygotes, the basal bodies are links between them are narrow cytoplasmic bridges and the overall disarrayed with some positioned at the rostral nuclear tip (arrows). The most shape of each individual ‘cell’ is distinguishable in EM cross- condensed nuclei again show no GFP-PACT fluorescence (arrowheads). F64 sections. Each ‘cell’ typically contains a single axoneme-MD triad, (C,D) In both yuri heterozygotes (C) and homozygotes (D), subsets of although ‘fused’ cells with two-eight triads have been detected in 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, wild-type cysts (Stanley et al., 1972). The triad abnormalities in respectively. Scale bars: 20 μm. yuriF64 were largely within ‘cells’ that contained multiple triads (Fig. yuri gagarin function in spermatogenesis 1933

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 Fig. 10. TEM analysis of control and yuriF64 mutant sperm. (A) Individualized control elongating cyst so that their associated axonemes extend sperm (Sp/CyO Roi) each have one axoneme (Ax), one major mitochondrial derivative (M), along the cyst in the wrong direction. The latter and one minor mitochondrial derivative (m), contained within a single plasma membrane. explanation proved to be the case. Upon inspection, (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. Journal of Cell Science clusters of condensed sperm nuclei (ranging from one у Major mitochondrial derivatives look normal but minor derivatives are enlarged (arrows). or two to 20 nuclei) with attached basal bodies were (D,E) yuriF64 homozygotes and (F-I) heterozygotes showing that axonemes in elongating detected at the apical end of elongated cysts, close to cysts are sometimes associated with aberrant sets of mitochondrial derivatives, often sharing the stem cell tip (Fig. 9C,D). For the yuriF64 them or possessing multiple derivatives of the same type. P, paracrystalline body in major homozygote, four out of 40 testes examined showed this mitochondrial derivative. The outer ring of microtubule doublets is sometimes broken defect; for the heterozygotes, two out of nine testes had (arrows), and internal components (central-pair microtubules or linker arms) can be missing (arrowheads). Axonemes of apparently opposing chirality (curved arrows of differing color) these mispositioned nuclei. 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. Discussion Roles of the Yuri isoforms Our initial yuri mutant (yuric263) was identified by its altered ~100 kDa isoforms. The importance of the 30 kDa isoform is gravitaxic responses. Further studies indicated that these changed demonstrated by a consideration of the ovary (Fig. 3C). The ~100 responses arise from altered chordotonal neuron function, but kDa Yuri isoforms are not normally present in the ovary, so that in provided no information as to whether yuri is uniquely expressed yuriF64 the 30 kDa protein is the only isoform detectable in the in these neurons (Armstrong et al., 2006). Studies here reveal that tissue. Nevertheless, oogenesis and early embryogenesis proceed yuri is expressed ubiquitously, indicating that yuri is not dedicated normally. to gravitaxic responses but rather that the yuric263 mutation Although the major defects seen in yuriF64 homozygotes are disrupts yuri expression in a manner that specifically affects this largely absent from heterozygotes, some minor, incompletely function. penetrant defects (particularly in axoneme structure) are more All isoforms of Yuri are expressed ubiquitously and yuriF64 prevalent in the heterozygous than the homozygous condition. removes the major ~65 kDa isoform(s) from all tissues studied. Because yuriF64 causes loss of particular isoforms, the normal Surprisingly, the only obvious developmental defect is male sterility. stoichiometric balance between isoforms is disrupted in both In the yuriF64 testis, the 30 kDa isoform is also missing, whereas homozygotes and heterozygotes, but it is disrupted differently in in other tissues this isoform is less affected. Thus, the yuriF64 male the two situations. Thus, given that two classes of Yuri isoforms sterility reflects either unique roles for ~65 kDa isoforms, or the contain coiled-coil regions, altered dimerization or protein unique loss of the 30 kDa isoform. That loss of the ~65 kDa isoforms interactions that have more severe consequences for axoneme has no effects in other tissues might indicate redundancy with the assembly might be produced uniquely in the heterozygote. 1934 Journal of Cell Science 121 (11)

Yuri function and the defects in spermatogenesis Although Yuri appears to anchor tubulin structures, including the The various elements of the yuriF64 testis phenotype provide clues basal body, to the nuclear membrane, our findings for the dynein as to the molecular functions of the protein. One clear implication light chain mutant suggest that the initial positioning of Yuri on the is that Yuri regulates F-actin function. We show here for the first nuclear membrane is determined by dynein transport, presumably time that F-actin is associated with the dense complex on spermatid along microtubules. In the dynein light chain mutant, Yuri nuclei and that in yuriF64, F-actin never accumulates on the nuclei, localization is dramatically altered, with Yuri now primarily suggesting an initiating role for Yuri in dense-complex formation. associated with the basal body – a novel association not seen in the Yuri is also a component of the actin cones that mediate sperm wild type. The implication must be that an activity of dynein is individualization and is required for their formation. The actin cones required to prevent an interaction of Yuri with the basal body. are formed by a two-step process (Noguchi et al., 2006). Initially, The opposing orientations of some adjacent axonemes in yuriF64 parallel actin fibers are formed around the nuclei and then an actin reflects the unexpected positioning of sperm nuclei at the wrong meshwork is added at each apical nuclear tip. Given the absence ends of elongated cysts. Contacts that normally hold the nuclei of actin cone initiation in yuriF64, it seems likely that Yuri has an together in tight alignment appear to be missing in yuriF64, and this early role in F-actin deposition here too. could permit loose nuclei to migrate to the wrong location. The aberrant F-actin sleeves formed in the somatic cyst cells in Axonemes with opposite orientations in a single cyst have been yuriF64 led us to identify related actin sleeves around actively coiling reported for mutations in the Drosophila parkin homolog (Riparbelli sperm in control testes. Sperm coiling is executed within the and Callaini, 2007). Although these investigators did not report a confines of the head cyst cell, which completely engulfs the apical search for nuclei at the wrong ends of cysts, they did note occasional region of the cyst (Tokuyasu et al., 1972b). Elaborate microvilli, actin cones pointing in the wrong direction – a finding that suggests full of 50 Å filaments, project from the head cyst cell onto the cyst the same underlying cause for the two axoneme orientations in both walls and Tokuyasu and colleagues suggest that coiling largely their case and ours. represents the collapse of the intrinsically helical sperm tails into a flat pile of gyres as a result of contraction and shape change within Other genes that act in mechanosensory organs and the head cyst cell. We propose that the actin sleeves in control testes spermatogenesis are related to the 50 Å filaments seen by Tokuyasu et al. and that The finding that different mutations of yuri affect processes as in yuriF64, F-actin structures form at inappropriate positions in disparate as gravitaxis and spermatogenesis is initially surprising. association with abortive coiling. However, together with sperm, mechanoreceptor neurons, such In addition to regulating actin function, Yuri is implicated in as those affected by yuric263, are the only cell types in Drosophila microtubule/tubulin action. The stripe of dense complex along the that possess cilia, and genes that affect ciliary function have been elongating nuclei accretes a bundle of microtubules that are shown to affect both mechanosensory organs and spermatogenesis. thought to provide structural rigidity to the nuclei. Although we Mutations in touch insensitive larva B (tilB) are defective in were not able to image these microtubules, in yuriF64 many late- hearing and touch perception as a result of defects in the stage nuclei lose their rigidity and collapse into helical twirls, chordotonal organs (Eberl et al., 2000). Mutations in unc affect suggesting that the microtubules are no longer present. The both the chordotonal organs and the external sense organ (eso)

Journal of Cell Science presence of Yuri in the dense complex is also intimately associated class of mechanoreceptors (Eberl et al., 2000). Mutations at both with proper positioning, formation and functioning of the basal loci are also male sterile because they encode proteins with roles body. When Yuri is not present at this site, (1) the basal bodies are in cilia. TilB is a conserved ciliary protein with a leucine-rich scattered along the nuclei, or even mispositioned at the rostral region and a coiled-coil domain (Kavlie et al., 2007) and Unc is nuclear tips, (2) the CA element of the basal body is missing and associated with the basal bodies in sperm and mechanosensory (3) the axonemes show defects similar to those of other mutations neurons (Baker et al., 2004). Unc, like γ-tubulin, is a component (cnn and unc) that affect basal body function. Nevertheless, our of the CA and, like Yuri, is insect-specific and contains coiled- findings for the GFP-PACT marker indicate that dPLP is recruited coil regions (Baker et al., 2004). normally to the basal bodies in yuriF64. Interestingly, in mammalian These examples suggest that the yuri function affected in yuric263 systems, interaction between γ-tubulin and pericentrin is thought might be a role in positioning the ciliary basal bodies of the to underlie the targeting of γ-tubulin to centrosomes/centrioles chordotonal neurons, a role comparable to that identified here in (Young et al., 2000). dPLP is therefore implicated in promoting spermiogenesis. Furthermore, the intriguing possibility of molecular the presence of γ-tubulin and of the CA on the sperm basal body. interactions between Yuri and Unc is suggested. The proteins are Our evidence here that in yuriF64, dPLP is on the basal bodies but physically close at the basal body and their only distinguishing γ-tubulin is not, suggests a role for Yuri in the interaction of these features are coiled-coil domains that presumably facilitate protein- two proteins. protein interactions. It seems possible that these two proteins have At the end of elongation, prior to individualization, the nucleus- evolved to fulfil specialized roles associated with anchoring the basal basal body association is altered so that the axoneme and sperm bodies that could entail heterodimerization. head are locked in a permanent configuration relative to one another (Lindsley and Tokuyasu, 1980; Tokuyasu, 1975). This change Materials and Methods involves disappearance of the CA and movement of the basal body Yuri antibodies and immunoblots to lie in a shallow groove on one side of the nucleus. Predictably, 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 the CA components γ-tubulin and Unc are lost from the nuclei at recloned into the EcoRI and SalI sites of expression vector pET28a (Novagen). The this stage (Baker et al., 2004). We show here that both the Yuri dot recombinant His-tagged protein was purified by Ni2+ chromatography (Novagen) and the GFP-PACT marker also disappear at this point. The basal 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 body present on mature sperm is clearly stripped of many ancillary affinity purification. For immunoblots, samples were solubilized in SDS sample proteins. buffer, run on 12.5% polyacrylamide gels and blotted to Immobilon (Millipore) filters. yuri gagarin function in spermatogenesis 1935

Bands reacting with the affinity-purified antibody were detected with horseradish- We thank Dr R. P. Munjaal for contributions to the early phases of peroxidase-conjugated rabbit anti-chicken antibodies (Sigma) and the West Dura this work. We thank Dr Chris Bazinet for the dj-GFP line; Dr David reagent (Pierce). Caprette for EM help; Dr James Fabrizio for Flytrap lines he characterized as expressing GFP in the cyst cells; Dr Thomas Fertility testing, fly stocks and genetics у Kaufman for Centrosomin antibody; Dr Tatsuhiko Noguchi for For fertility testing, 20 individual males or virgin females were placed with three F64 w1118 partners in food vials for 7 days, after which adults were removed. The original critical insight into the actin sleeves in the yuri ; Dr Jordan Raff vials were checked for the presence of larvae, pupae and adults for a further 15 days. for the GFP-PACT line; Dr Kiyoteru Tokuyasu for helpful discussions Although eggs were laid, yuriF64 homozygous and hemizygous males never produced on the nuclear localization of Yuri. We are grateful to Kenneth Dunner, any viable progeny. A stock with deficiency Df(2L)do1, which removes yuri- Jr, Deborah Townley and Dr Wenhua Guo of the High Resolution containing region 35B1-35D2, balanced over a CyO-GFP balancer (Rudolph et al., EM Facility at MD Anderson, the Integrated Microscopy Core at 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- Baylor College of Medicine and the Smalley Institute of Nano Science GFP.Y}TR1] from the Bloomington Stock Center. To generate embryos homozygous and Technology at Rice. respectively, for their assistance with EM for Df(2L)do1 or homozygous for CyO-GFP, eggs were collected from the work. The help of Rice undergraduates, in particular Summer Bell, Df(2L)do1/CyO-GFP stock and left >24 hours to ensure that viable embryos hatched. Faraz Sultan and Anita Shankar, is gratefully acknowledged. These Fluorescent and non-fluorescent embryos were collected separately. Third studies were supported by NIH grant RO1 HD 39766, grant C-1119 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, from the Welch Foundation of Texas and NASA grant NCC2-1356. 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 References from the Bloomington Stock Center. Armstrong, J. D., Texada, M. J., Munjaal, R., Baker, D. A. and Beckingham, K. M. (2006). Gravitaxis in Drosophila melanogaster: a forward genetic screen. Genes Brain Sequence analysis and conservation of yuri Behav. 5, 222-239. The following yuri ESTs/cDNAs were sequenced: adult head GH14032; adult testis Baker, J. D., Adhikarakunnathu, S. and Kernan, M. J. (2004). Mechanosensory- AT03435, AT15480, AT15149, AT19027 and AT25733; adult ovary GM26781 and defective, male-sterile unc mutants identify a novel basal body protein required for GM25777; S2 cell line SD06513 and SD11641; embryo RE12523 and RE13793. ciliogenesis in Drosophila. Development 131, 3411-3422. Two clones from the testis, AT15149 and AT15480, end at the same 5Ј residue at a Caggese, C., Moschetti, R., Ragone, G., Barsanti, P. and Caizzi, R. (2001). dtctex-1, point between exons 3 and 5. The region immediately 5Ј to this point scores poorly the Drosophila melanogaster homolog of a putative murine t-complex distorter encoding in analyses designed to detect promoters. Given that these two cDNAs were prepared a dynein light chain, is required for production of functional sperm. Mol. Genet. Genomics 265, 436-444. from the same RNA, we assume they have an incomplete 5Ј terminus. However, Ј Civetta, A. (1999). Direct visualization of sperm competition and sperm storage in their 5 -most sequence, which is not present in other cDNAs, is part of an intron Drosophila. Curr. Biol. 9, 841-844. between exons 3 and 5 (Fig. 1). These clones thus either (1) provide evidence for Ј Crosby, M. A., Goodman, J. L., Strelets, V. B., Zhang, P. and Gelbart, W. M. (2007). the variable presence of an additional exon (labeled 4 in Fig. 1), the 5 boundary of Flybase: genomes by the dozen. Nucleic Acids Res. 35, D486-D491. which is not defined or (2) represent incompletely spliced transcripts. A paralog search Drosophila 12 Genomes Consortium (2007). Evolution of genes and genomes on the in D. melanogaster was performed using the Yuri ‘PD’ isoform (FlyBase) sequences Drosophila phylogeny. Nature 450, 203-218. and the BLASTP service at FlyBase. Because protein data sets are not available for Eberl, D. F., Hardy, R. W. and Kernan, M. J. (2000). Genetically similar transduction all sequenced insect species, and to avoid spurious matches to coiled-coil domains, mechanisms for touch and hearing in Drosophila. J. Neurosci. 20, 5981-5988. ortholog searches of translated DNA sequences were conducted with TBLASTN, Fabrizio, J. J., Himes, G., Lemmon, S. K. and Bazinet, C. (1998). Genetic dissection using the 239-residue protein encoded by the GH14032 cDNA as the query and both of sperm individualization in Drosophila melanogaster. Development 125, 1833-1843. the ‘nr/nt’ NCBI database and the 21 insect genome sequences searchable at FlyBase Gao, Z., Ruden, D. M. and Lu, X. (2003). PKD2 cation channel is required for directional as target data sets. The sequence identity computation for the ~100 kDa Yuri protein sperm movement and male fertility. Curr. Biol. 13, 2175-2178. in the 12 sequenced Drosophila sequences (Drosophila 12 Genomes Consortium, Han, Y.-G., Kwok, B. H. and Kernan, M. J. (2003). Intraflagellar transport is required 2007) was performed using BLASTP on the GLEANR consensus protein data in Drosophila to differentiate sensory cilia but not sperm. Curr. Biol. 13, 1679-1686. Joly, D., Bressac, C., Jaillard, D., Lachaise, D. and Lemullois, M. (2003). The sperm Journal of Cell Science 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 roller: a modified testicular duct linked to giant sperm transport within the male reproductive tract. J. Struct. Biol. 142, 348-355. searches. Kavlie, R. G., Kernan, M. J. and Eberl, D. F. (2007). touch insensitive larva B, a gene necessary for hearing and male fertility encodes a conserved ciliary protein. 48th Annual Imprecise excisions Drosophila Conference Abstract 642C, 305. c263 The P insertion yuri (Armstrong et al., 2006) and SUPor-P insertion KG03019 Kelso, R. J., Buszczak, M., Quinones, A. T., Castiblanco, C., Mazzalupo, S. and Cooley, (Roseman et al., 1995) were mobilized with the Δ2-3 transposase at 99B (Robertson L. (2004). Flytrap, a database documenting a GFP protein-trap insertion screen in et al., 1988). Standard genetic schemes generated stocks of viable excisions. For lethal Drosophila melanogaster. Nucleic Acids Res. 32, D418-D420. excisions, lines with GFP-marked balancers were prepared. Excisions were Li, K., Xu, E. Y., Cecil, J. K., Turner, F. R., Megraw, T. L. and Kaufman, T. C. (1998). characterized by PCR. Precise deletion endpoints were determined by sequencing. 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New York: incubated overnight at 4°C with rotation in BBX + 2% goat serum and one or more Academic Press. of the following antibodies: 1:100 affinity-purified Yuri antibody; 1:500 mouse Martinez-Campos, M., Basto, R., Baker, J., Kernan, M. and Raff, J. W. (2004). The monoclonal anti-γ-tubulin GTU-88 (Sigma); 1:200 rabbit polyclonal anti-Centrosomin Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to antibody R19 (gift of T. Kaufman, Indiana University, Bloomington, IN); 1:50 mouse be dispensable for mitosis. J. Cell Biol. 165, 673-683. monoclonal anti-β-tubulin E7 (Developmental Studies Hybridoma Bank). After two Mistry, H., Wilson, B. A., Roberts, I. A. H., O’Kane, C. J. and Skeath, J. B. (2004). washes each in BBX and BBX + 2% goat serum, appropriate Alexa Fluor-conjugated Cullin-3 regulates pattern formation, external sensor organ development and cell secondary antibodies (Invitrogen) were added at 1:500 in BBX + 2% goat serum and survival during Drosophila development. Mech. Dev. 121, 1495-1507. incubated for 2 hours. Rhodamine- or Alexa Fluor-conjugated phalloidin (Invitrogen) Noguchi, T. and Miller, K. G. (2003). A role for actin dynamics in individualization during at 1:50 dilution was included with the secondary antibody as appropriate. After four spermatogenesis in Drosophila melanogaster. Development 130, 1805-1816. washes with BBX, testes were mounted with 1:2000 Hoechst 33342 (Invitrogen) in Noguchi, T., Lenatowska, M. and Miller, K. G. (2006). Myosin VI stabilizes an actin 50% glycerol. Images were collected on a Zeiss Axioplan or on Zeiss LSM 410 and network during Drosophila spermatid individualization. Mol. Biol. 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