1926 Research Article

IntroductionA unique feature of the genus Drosophila is the formation ofunusually long sperm tails. Sperm lengths of millimeters arecommon within this group, with the 1.8 mm sperm of D.melanogaster being fairly typical. This marked expansion in spermlength reflects an unusual aspect of spermatogenesis in theseorganisms: in contrast to other species in which an intraflagellartransport system is used for growth of the sperm flagellum (Scholey,2006), Drosophila sperm axonemes are assembled in syncytial cystsby a mechanism that does not require, and is not limited by, thissystem (Han et al., 2003; Sarpal et al., 2003). This unusual spermaxoneme development and the resulting expansion of sperm taillength have led to distinctive features of spermatogenesis not foundin other species. In D. bifurca, a special ‘sperm roller’ has evolvedto package its 6-centimeter-long gametes (Joly et al., 2003). In D.melanogaster, a highly evolved individualization process thatgenerates 64 individual sperm from an elongate cyst containing 64syncytial spermatids has been identified and studied (Noguchi andMiller, 2003; Tokuyasu et al., 1972a). The distinctive molecularmechanisms needed for this process include a motile filamentousactin system (the investment, or actin, cones) that traverses the entirelength of the sperm tails, removing excess cytoplasm and investingeach sperm in its own plasma membrane. A specialized microtubule-rich structure (the dense complex) is also associated with the spermnuclei and functions to position the basal body and also possiblyto strengthen the nuclei as they undergo extreme condensation(A. D. Tates, Cytodifferentiation during spermatogenesis inDrosophila melanogaster, PhD thesis, Rijksuniversiteit Leiden, TheNetherlands, 1971) (Tokuyasu, 1974).

We have identified a locus, yuri gagarin (yuri), that we showhere 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, yuriwas initially identified through its function in another specializedorgan system of insects and arthropods: the chordotonal organs.These are complex mechanosensory structures with roles inproprioception 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 expressionof yuri in subsets of chordotonal neurons (Armstrong et al., 2006).The molecular functions of the locus identified here suggest thatyuri mediates specialized actin- and microtubule-related activitiesin Drosophila tissues.

ResultsThe yuri locus in D. melanogaster and other DrosophilidsIn addition to the cDNA (GH14032) encoding a ~30 kDa proteinthat we used previously (Armstrong et al., 2006), we identified 11further yuri ESTs/cDNAs from adult testis, ovary, S2 cells andembryos through FlyBase. Sequencing of these new cDNAsestablished that three major transcript classes are generated fromyuri (Fig. 1). Two promoters are used, with the medium transcriptsinitiated at the proximal promoter and the short and long classesfrom the distal promoter. However, all isoforms begin at one oftwo closely positioned ATGs. The short transcript class encodesthe ~30 kDa protein identified previously. The medium classencodes isoforms of 64-65 kDa that extend ~400 amino acidsfurther 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 recognizablemotif (a polyproline stretch). However, the two longer formscontain coiled-coil motifs with weak similarity (~20% identity) to

Males of the genus Drosophila produce sperm of remarkablelength. Investigation of giant sperm production in Drosophilamelanogaster has demonstrated that specialized actin andmicrotubule structures play key roles. The gene yuri gagarin(yuri) encodes a novel protein previously identified through itsrole in gravitaxis. A male-sterile mutation of yuri has revealedroles for Yuri in the functions of the actin and tubulin structuresof spermatogenesis. Yuri is a component of the motile actin conesthat individualize the spermatids and is essential for theirformation. Furthermore, Yuri is required for actin accumulationin the dense complex, a microtubule-rich structure on the spermnuclei thought to strengthen the nuclei during elongation. In

the yuri mutant, late clusters of syncytial nuclei are deformedand disorganized. The basal bodies are also mispositioned onthe nuclei, and the association of a specialized structure, thecentriolar adjunct (CA), with the basal body is lost. Some ofthese nuclear defects might underlie a further unexpectedabnormality: sperm nuclei occasionally locate to the wrong endsof the spermatid cysts. The structure of the axonemes that growout from the basal bodies is affected in the yuri mutant,suggesting a possible role for the CA in axoneme formation.

Key words: Drosophila, Spermatogenesis, Actin, Tubulin, Basalbody, Chordotonal organ, Centriole

Summary

yuri gagarin is required for actin, tubulin and basalbody functions in Drosophila spermatogenesisMichael J. Texada, Rebecca A. Simonette, Cassidy B. Johnson, William J. Deery andKathleen 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: kate@rice.edu)

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

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those in many fibrillar proteins that dimerize, such as myosin heavychain and CLIP-190. The strongest match is to the coiled-coil ofSticky, the Drosophila citron kinase (Sweeney et al., 2008).

yuri is unique in the D. melanogaster genome, once the weaksimilarities to coiled-coil regions are disregarded. Thus, to avoidspurious similarities, the shortest yuri isoform was used to find yuriorthologs in other organisms. Significant matches were found inall 11 sequenced Drosophila genomes (Drosophila 12 GenomesConsortium, 2007), but none was identified in other evolutionaryorders or other insects, including the closest Dipteran relatives, theCulicidae (mosquitoes) (Fig. 2). Sequence conservation within theDrosophila 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 specieshave one yuri gene, but two related genes are present in D.pseudoobscura and D. persimilis.

Ubiquitous expression of the three major Yuri isoformsTo investigate Yuri expression, we generated antibodies againstthe sequences common to all isoforms (see Materials andMethods). Immunoblots of yuri+ embryos and embryos lackingyuri established the specificity of our antisera and their ability todetect the three predicted Yuri isoform classes (Fig. 3A). Theseblots also demonstrated that only the short Yuri isoform ismaternally loaded into the embryo, with the longer isoformsappearing later in embryogenesis (Fig. 3A,B). In later stages, allthree isoform classes are ubiquitously expressed (Fig. 3C). The~65 kDa class is most abundant in most situations, although intestis and thorax the other isoforms are also highly expressed (Fig.3C). The existence of at least two isoforms for both the ~100 kDaand ~65 kDa classes was confirmed by these experiments.Additional bands were sometimes present that probably representspecific degradation products, as they were largely missing in

Fig. 1. Transcripts, proteins andmutations at the Drosophila yurilocus. (A) Two promoters(proximal and distal) generatethree classes of yuri transcripts.The two medium transcripts differby the presence of an intronbetween exons 1b� and 1b�. Exon4, the 5� boundary of which is notdefined (Materials and Methods),is included in some longtranscripts. The original P{GawB}insertion (yuric263) and the DNAdeleted in three impreciseexcisions (LE1, L5 and F64) areshown. (B) Three Yuri isoformclasses arise from the threetranscript classes. Structural motifsare indicated.

Fig. 2. Evolutionary conservation of yuri inDrosophila species. Yuri orthologs aredetectable in 12 Drosophila species, but notoutside the genus. The ~100 kDa isoform ismore conserved than the ~30 kDa isoform.Similarity is computed as the global fractionof residues of the D. melanogaster protein thatare present as similar residues in orthologs;these are lower than the local similarity scoresfrom BLAST programs. The GLEANR dataset contains consensus sets of predictedproteins for the 12 Drosophila species and wassearched using the protein-to-protein BLASTPprogram. Because protein predictions are notavailable (NA) for non-Drosophila species,the 30 kDa search was repeated for allsequenced insect species using the protein-to-DNA TBLASTN program. Tree image is fromFlyBase (Crosby et al., 2007).

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embryos lacking yuri (Fig. 3A) and in the yuriF64 mutant (Fig.3C) (see below).

A yuri mutant that lacks Yuri ~65 kDa isoform(s)The yuric263 mutation from our gravitaxic screen is an insertion ofP{GawB} just upstream of the transcription start site for themedium length transcripts. We generated further mutations byimprecise excision of P{GawB} and of a second transposon,KG03019 (Roseman et al., 1995), inserted three residuesdownstream of the yuric263 P element. Three excisions that deletethe relevant transposon and adjacent genomic DNA were identified.One of these is lethal (yuriLE1), but the deletion extends upstreaminto an adjacent gene (cullin3; guftagu) known to affect viability(Mistry et al., 2004). In yuriL5, a short region of yuri upstreamsequence is deleted, causing reduced expression of all Yuri isoforms.Nevertheless, homozygous yuriL5 animals are viable with noobvious phenotype. Only one deletion, yuriF64, removes transcribedsequences from the locus. Most of the 5� UTR of the ~65 kDaisoforms is deleted, with only ten residues upstream of the firstinitiator ATG remaining (Fig. 1A). The yuriF64 deletion lead tocomplete loss of ~65 kDa isoforms in all tissues and stagesexamined (Fig. 3C). The ~100 kDa isoforms remained stronglyexpressed, but expression of the ~30 kDa isoform was decreasedin several tissues and undetectable in the testis (Fig. 3C).

Male sterility is associated with the yuriF64 mutationHomozygous yuriF64 mutants (yuriF64) are viable with normalexternal morphology. However, yuriF64 males are completely sterile,whereas females are fertile (data not shown). Flies heterozygousfor yuriF64 and deficiency Df(2L)do1, which deletes yuri, were alsomale sterile and female fertile. The testis phenotype (see below)was identical in yuriF64 homozygotes and hemizygotes (data notshown), demonstrating that it results from the effects of the yuriF64

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mutation on the yuri locus. In order to determine whether yuriF64

affects overall viability, the survival of yuriF64 homozygous progenyversus heterozygous progeny (yuriF64/CyO Roi) was quantitated fora cross of yuriF64 females with heterozygous (yuriF64/CyO Roi)males. Of 649 progeny, 51% were yuriF64 homozygotes, indicatingthat yuriF64 has no effects on survival to adulthood.

The Drosophila testis contains a stem cell system at its apical tipfrom which spermatogonial cells are budded off to proceed throughspermatogenesis. A somatic stem cell system is also present thatproduces so-called cyst cells. A pair of cyst cells encases the divisionproducts of each spermatogonial cell throughout spermatogenesisand post-meiotic spermiogenesis. Each spermatogonial cell generatesa cyst of 64 spermatids, linked by cytoplasmic bridges, whichundergoes dramatic elongation. At completion, each cyst has a highlyelongate cytoplasm (~1.8 mm in length) with the 64 condensed nucleipositioned at the seminal vesicle end and 64 axonemes extendingfrom the nuclei along the length of the cyst towards the apical tip.Two giant mitochondrial derivatives, generated by fusion of themitochondria within each post-meiotic spermatid, extend along thelength of each axoneme. The later stages of spermiogenesis involvea specialized process termed individualization (see below) in whichthe 64 syncytial spermatids are converted into 64 individual sperm.Finally, a coiling process retracts the sperm down to the entrance tothe seminal vesicle.

Highly elongate spermatid cysts were present in yuriF64 testes,some of which were attempting to coil, but it was unclear whethermature sperm were formed. To address this question, we introduceda don juan-GFP fusion construct into the yuriF64 background. DonJuan protein is produced in the giant sperm tail mitochondria andpersists into mature sperm. Don Juan-GFP (Santel et al., 1997)provides a marker for late spermiogenesis (Civetta, 1999; Gao etal., 2003). We examined 8-day-old virgin males, which should havelarge quantities of sperm in the seminal vesicles. In yuriF64/CyO

Fig. 3. The distribution of Yuri isoforms throughout development. Immunoblots for Yuri isoforms are shown. (A) Specificity of Yuri antibodies. Lane 1, 30unfertilized 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 terminalhomozygous CyO-GFP (homozygous yuri+) embryos. The two large isoforms are not present in unfertilized eggs or embryos lacking yuri, but are zygoticallyexpressed 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.

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Roi heterozygotes carrying don juan-GFP, the seminal vesicles werefull of fluorescent sperm and the basal testis carried masses offluorescent coiling sperm (Fig. 4A). In yuriF64, no fluorescence wasdetectable in the seminal vesicles and the basal testis containedcurled structures, thicker than individual sperm with aberrantcoiling (Fig. 4B). Squashes of seminal vesicles confirmed thepresence of motile sperm in the controls and their complete absencein the mutant (data not shown).

Individualization fails in yuriF64

Phase-contrast examination of testis squashes revealed no defectsin 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. Individualizationbegins after formation of a cone of F-actin around theattachment site of each axoneme to the sperm nucleus, withthe 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 ina plasma membrane, and, ahead of the set, excess cytoplasmand organelles are pushed up the testis to be discarded as a‘waste bag’. The actin cones are the only significant F-actinstructures 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 conesand waste bags were detected, the yuriF64 testes containedneither (Fig. 4). Instead, elongated ‘sleeves’ of actin were seenaround the periphery of some spermatid cysts. These appearedas solid tubes in normal fluorescence imaging (Fig. 4B�), butas hollow structures in confocal sections (Fig. 5A). Weestablished that these sleeves are actually present in the somaticcyst cells surrounding the cysts, rather than in the cyststhemselves, by use of GFP ‘exon trap’ insertions (Kelso et al.,2004) that express GFP in the cyst cells (Materials andMethods). In the yuriF64 background, the actin sleeve staining

and GFP in the cyst cells precisely overlapped (Fig. 5A). Havingidentified these sleeves in yuriF64, we discovered similar structurespresent at a lower frequency in control testes (Fig. 5B). In controls,these sleeves are always in the basal regions of the testis where spermcoiling 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.

Actin cone initiation and nuclear behavior are aberrant inyuriF64

The formation of the F-actin cones of individualization has beenstudied previously (Fabrizio et al., 1998; Lindsley and Tokuyasu,

Fig. 4. 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], theseminal vesicle (arrow) is empty, and sperm cysts show abortive coiling in thetestis proper (arrowhead). (A�-B�) Phalloidin staining (red) identifies actin conesand waste bags in control testis (A�, red arrow; as shown at higher magnificationin A�). Mutant testis is devoid of these structures (B�), and F-actin sleeves arepresent instead (B�, red arrow; as shown at higher magnification in B�). Scale bars:200 μm.

Fig. 5. 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 thebase 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 arefrequently 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. Scalebars: 10 μm in A-A�,C,D, 100 μm in B,E,F, 50 μm in G.

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1980; Noguchi et al., 2006). Initially, actin fibers accrete alongthe lengths of the condensed sperm nuclei in the basal testis. Theactin then moves to form cones, flaring off the apical ends of thenuclei before release to move up the axonemes. Nuclei in allstages of this process are present in the basal region of wild-typetestes. In yuriF64, although the nuclear sets were seen to descendto this level and undergo some condensation, they were clearlymore disorganized, with individual nuclei trailing behind,apparently detached from the main cluster. In some late-stageclusters, almost all the nuclei were distorted in shape, some in ahelical or circular configuration (Fig. 5D). No nuclei evercondensed 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 wereoccasionally found singly or in clusters in this region. Some ofthem were apparently mobile, as they appeared at some distancefrom any nuclei (Fig. 5G).

Yuri protein localization in control testesOur antisera, which detect all Yuri isoforms, were used to examineYuri localization in control testes. Yuri was present at all stages ofgerm cell development, peaking around meiosis, with most stainingbeing cytoplasmic and diffuse (Fig. 6A). However, in addition, astriking and dynamic pattern of Yuri association with the post-meiotic spermatid nuclei was seen as they condensed duringelongation (Fig. 6B-F). While the nuclei were still round, Yuri wasseen to accumulate as a cap over one hemisphere of each nucleus.As the nuclei became ellipsoid, the Yuri staining transformed intoa stripe along the nuclear long axis and a dot at the apical nuclear

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tip. In the final stages of nuclear maturation, first the stripedisappeared and then the dot was also lost.

Tokuyasu has described the ultrastructural changes to the nucleiduring elongation (Tokuyasu, 1974). Part of the nuclear membraneis fenestrated with nuclear pores during this process. Initially, thisregion forms a cap over one hemisphere of the round post-meioticnucleus, with dense material aggregating over this region betweenthe nuclear membrane and adjacent endoplasmic reticulum. As thenuclei elongate, this cap and associated material transform to a stripealong the long axis of the nucleus. More of the dense materialaccumulates along with microtubules, with the whole complexsinking inwards to form a groove filled with dense cytoplasm anda microtubule bundle (collectively the ‘dense complex’) that runsthe length of the nucleus. The nuclei are actually horseshoe-shapedin cross-section at this stage. In the final stages of nuclearmaturation, the dense complex is dispersed and the nuclei regain acircular cross-section. The dense complex is thought to providestructural rigidity to the nuclei during the elongation process(Tokuyasu, 1974). Early after meiosis, the single centriole of eachspermatid embeds into the spherical nuclear membrane at the centerof the dense complex and then converts into the basal body. Duringelongation, 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 wasstrikingly similar to that of the dense complex and associated basalbody. To position Yuri relative to these structures, we co-stainedfor γ-tubulin, Centrosomin (Cnn) and β-tubulin. γ-tubulin is acomponent of the centriolar adjunct (CA) (Wilson et al., 1997), atorus-shaped structure around the middle of the basal body duringelongation (Fig. 7C) (Tokuyasu, 1975). Centrosomin, a centriole

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-meioticspermatids 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 atthe 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-shapeddot (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.

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component, is present early in the transformation to the basal bodybut is subsequently lost (Li et al., 1998). β-tubulin is a generalmarker for microtubules. γ-tubulin/Yuri co-staining established thatthe basal body is at the center of the Yuri cap in round spermatids(Fig. 7A), providing evidence that the Yuri cap corresponds to theaccumulating dense complex. No round spermatid nuclei that co-stained for the Yuri cap and Centrosomin were detected, suggestingthat Centrosomin is lost before significant Yuri accumulation. Wewere not able to detect a stripe of microtubules along the nuclei bystaining for β-tubulin. Very high general cytoplasmic staining and/orpossibly the burying of the appropriate epitope could underlie thisfailure.

Although γ-tubulin staining showed an apical dot on theelongating nuclei, interestingly, the Yuri dot and the γ-tubulin dotdid not coincide. The Yuri dot, which at high magnification has abell shape (Fig. 6F), was sandwiched between the dot of γ-tubulinstaining and the nuclear membrane. Thus, Yuri is probably not partof the basal body per se but lies between the basal body and thenuclear membrane. EM analysis has established that the basal bodyis embedded into a ~0.5 μm indentation in the nuclear membrane(Tokuyasu, 1975) at this stage. It seems likely that the Yuri dot isthe residuum of the initial dense-complex cap that was alwaysbeneath the insertion point of the basal body, and that Yuri continuesto fill the space between the membrane and the basal body duringnuclear elongation.

Given the complete failure of actin cone formation in yuriF64,we also examined the relationship between Yuri and F-actinlocalization during spermiogenesis. We determined that the cap ofdense complex at the round spermatid stage contains not only Yuri,but also F-actin (Fig. 7D). Furthermore, the actin staining extendedaround the basal body. F-actin continued to colocalize with Yuri inthe stripe and dot pattern as the nuclei elongated (Fig. 7E). We alsoestablished that Yuri is a component of the F-actin cones used inindividualization (Fig. 7F). Yuri immunostaining was seenthroughout 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 assemblyThe yuriF64 mutation does not eliminate all isoforms of Yuri.Nevertheless, we determined that in yuriF64 the association of Yuriwith the dense complex is completely lost, and all elements of thenuclear staining pattern – the cap, stripe and dot – are missing (Fig.8A). Thus, the isoforms that are absent in yuriF64 are essential forprotein function at these sites. The absence of Yuri from the densecomplex allowed us to determine whether Yuri is necessary for theassociation of other components with this structure. In yuriF64, allelements of F-actin nuclear staining from the round spermatid stageonwards were lost (Fig. 8C). Yuri is therefore required for the initialaccumulation and subsequent maintenance of F-actin within thedense complex. Similarly, γ-tubulin staining was never observedon the early round nuclei or at the later elongate stages (Fig. 8B),demonstrating that Yuri is required for attachment to, or possiblyformation of, the CA of the basal body.

This absence of the CA raised the issue of whether basal bodiesare present at all on the spermatid nuclei in yuriF64. To addressthis question, a GFP-fusion construct for the PACT domain of theDrosophila Pericentrin-like protein (dPLP; Cp309) (Martinez-Campos et al., 2004) was introduced into the yuriF64 background.The PACT domain of both mammalian pericentrin and dPLPprovides targeting to the centrosomes/centrioles. In the Drosophilatestis, GFP-PACT is an excellent fluorescent marker for the basalbody (Martinez-Campos et al., 2004). In control cysts (w1118 orw–; yuriF64/CyO Roi), small cylinders of GFP-PACT stainingdemonstrate the presence of the basal bodies tightly clustered atthe apical tips of condensing nuclei (Fig. 9A). GFP-PACT-markedbasal bodies were also present on condensing nuclei in yuriF64.However, they were not tightly localized at the apical tips butscattered along the nuclei. Indeed, in many clusters, a fraction ofthe basal bodies were actually at the rostral rather than apical

Fig. 7. Yuri localization relative to γ-tubulin and actin in controls. (A) γ-tubulin staining positions thecentriole/basal body at the center ofthe Yuri nuclear cap in roundspermatids. (B) On elongating nuclei,the Yuri dot lies between the body ofthe nucleus and the CA, as identifiedby γ-tubulin. (C) Diagram of theproposed location of Yuri onelongating nuclei. Adapted fromLindsley and Tokuyasu (Lindsley andTokuyasu, 1980) with permission.(D) F-actin localization on roundspermatid nuclei.(E-E�) Colocalization of Yuri andF-actin in the stripe and dot patternseen on elongating nuclei. Arrowindicates actin/Yuri staining overlapon a single nucleus.(F-F�) Colocalization of actin andYuri in moving actin cones. A cross-section of a set of large moving conesis shown. Yuri, green; nuclei, blue;γ-tubulin, red in A-C; actin, red inD-F. Scale bars: 10 μm in A-D,E-E�,20 μm in F-F�.

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nuclear tips (Fig. 9B). Quantitation of the GFP-PACT fluorescenceassociated with control or yuriF64 nuclear clusters (usingMetamorph software) indicated that yuriF64 does not affect thelevel of GFP-PACT binding to the basal bodies. In the final stagesof nuclear condensation, the GFP-PACT fluorescence was lostfrom control nuclei. Similarly, although the nuclei never fullycondense in yuriF64, GFP-PACT was ultimately lost from thesenuclei too.

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Previous work has implicated cytoplasmic dynein and the relatedprotein Dynactin in the formation of the dense complex (Li et al.,2004). Like Yuri, dynein heavy chain accumulates in thehemispherical cap on round spermatid nuclei but, in contrast to Yuriand the components examined here, its nuclear positioning istransient and it is not detectable in the dense-complex stripe duringnuclear elongation. This brief association has a role in basal bodyfunctioning, however, because in a null mutant for the 14 kDa dyneinlight chain (Dlc90F), dynein heavy chain does not accumulate onthe nuclei and, later, some nuclei lack a CA as judged by γ-tubulinstaining.

In Dlc90F05090, an RNA-null in the testis (Caggese et al., 2001),the nuclear localization pattern of Yuri was found to be dramaticallyaltered. The initial hemispherical cap of Yuri and the later stripewere highly attenuated and in some cases barely detectable (Fig.8D,E). However, the bell-shaped dot of Yuri was now present atthe base of the basal body, even in round spermatids (Fig. 8D).Furthermore, in both round and elongating nuclei, a second dot ofYuri was present (Fig. 8D,E). Co-staining with γ-tubulindemonstrated that this dot is the region of the basal body distal tothe CA (Fig. 8F).

The axoneme-mitochondrial triads in yuriF64 mutants andaberrant nuclear migrationAs the centrioles mature into basal bodies, a transition in proteincomposition occurs: Centrosomin is lost (Li et al., 1998) and theprotein Uncoordinated (Unc) now becomes associated with thesestructures (Baker et al., 2004). Mutations in cnn or unc affect basalbody function and produce abnormalities in axoneme structure.Given the loss of the CA and the aberrant positioning of the corebasal bodies in yuriF64, we examined axoneme structure by TEM.This analysis also confirmed the complete failure ofindividualization in yuriF64 (Fig. 10C). In contrast to controls (Fig.10A), the 64 axonemal ‘triads’ – the axonemes and their major andminor mitochondrial derivatives (MDs) – all shared a singlecytoplasm. Furthermore, terminal differentiation of the minor MDswas imperfect. In controls, this derivative undergoes dramaticexpansion/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 lesscondensed than normal (Fig. 10C).

We examined axoneme structure in younger elongating cysts.Gross axonemal structure (the typical ‘9+2’ arrangement) wasnormal in yuriF64. Of more than 750 studied, only two damagedaxonemes were found, showing breaks in the outer circle of ninedoublets (Fig. 10D). However, rarely, aberrant arrangements ofaxoneme-MD triads were found. These included: single axonemeswith 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 typicallyassociated with the minor MD during individualization (Fig.10D,E).

Although the spermatids in elongating cysts are syncytial, thelinks between them are narrow cytoplasmic bridges and the overallshape 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 inwild-type cysts (Stanley et al., 1972). The triad abnormalities inyuriF64 were largely within ‘cells’ that contained multiple triads (Fig.

Fig. 8. yuriF64 effects on the dense complex and basal body. (A-C) In theyuriF64 mutant, the Yuri nuclear stripe and dot are lost (A), γ-tubulin is nolonger 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 thenuclear cap (D) and stripe (E) is diminished (arrowheads), but the bell-shapeddot 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.

Fig. 9. Basal body positioning and aberrant nuclear migration in yuriF64. (A) InyuriF64 heterozygotes, GFP-PACT fluorescence reveals basal bodies clusteredtightly at apical nuclear tips. GFP-PACT is lost in the final stages of nuclearcondensation (arrowhead). (B) In yuriF64 homozygotes, the basal bodies aredisarrayed with some positioned at the rostral nuclear tip (arrows). The mostcondensed nuclei again show no GFP-PACT fluorescence (arrowheads).(C,D) In both yuriF64 heterozygotes (C) and homozygotes (D), subsets ofnuclei sometimes migrate to the apical end of the cyst (arrows). Asterisks andwhite arrow indicate the position and direction of the stem cell tip,respectively. Scale bars: 20 μm.

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10E). However, our analysis of testis squashes providedno evidence that these arose as a result of cytokinesisdefects 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 bothbackgrounds, almost all mature cysts (~90%) had a fewimperfectly individualized triads (Fig. 10B), with a fewcysts in which <50% of the sperm tails were still insyncytial cytoplasm. Thus, although fertile, yuriF64

heterozygotes clearly have individualization defects.Defects in triad development similar to those seen inyuriF64 homozygotes were also detected and,unexpectedly, were somewhat more prevalent in theheterozygotes, with ~30% of the cysts in one testisshowing these defects. More-pronounced axonemalabnormalities were also detected: in addition to brokensets of outer doublets, axonemes with no central doubletwere present (Fig. 10G,I).

In both the yuriF64 homozygotes and heterozygotes,occasional examples were found of adjacent axonemeswith their outer arms pointing in opposite orientations(Fig. 10D). Such cysts always had the correct numberof axoneme profiles (64), and one cyst with 32axonemes in one orientation and 32 in the other wasfound (data not shown). We could therefore exclude thepossibility that these two orientations representedaxonemes that were folded back on themselves. Twoalternative explanations remained: either a fraction ofthe basal bodies have an altered chirality, or some ofthe 64 sperm nuclei migrate to the wrong end of theelongating cyst so that their associated axonemes extendalong the cyst in the wrong direction. The latterexplanation proved to be the case. Upon inspection,clusters of condensed sperm nuclei (ranging from oneor two to �20 nuclei) with attached basal bodies weredetected at the apical end of elongated cysts, close tothe stem cell tip (Fig. 9C,D). For the yuriF64

homozygote, four out of 40 testes examined showed thisdefect; for the heterozygotes, two out of nine testes hadthese mispositioned nuclei.

DiscussionRoles of the Yuri isoformsOur initial yuri mutant (yuric263) was identified by its alteredgravitaxic responses. Further studies indicated that these changedresponses arise from altered chordotonal neuron function, butprovided no information as to whether yuri is uniquely expressedin these neurons (Armstrong et al., 2006). Studies here reveal thatyuri is expressed ubiquitously, indicating that yuri is not dedicatedto gravitaxic responses but rather that the yuric263 mutationdisrupts yuri expression in a manner that specifically affects thisfunction.

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, whereasin other tissues this isoform is less affected. Thus, the yuriF64 malesterility reflects either unique roles for ~65 kDa isoforms, or theunique loss of the 30 kDa isoform. That loss of the ~65 kDa isoformshas no effects in other tissues might indicate redundancy with the

~100 kDa isoforms. The importance of the 30 kDa isoform isdemonstrated by a consideration of the ovary (Fig. 3C). The ~100kDa Yuri isoforms are not normally present in the ovary, so that inyuriF64 the 30 kDa protein is the only isoform detectable in thetissue. Nevertheless, oogenesis and early embryogenesis proceednormally.

Although the major defects seen in yuriF64 homozygotes arelargely absent from heterozygotes, some minor, incompletelypenetrant defects (particularly in axoneme structure) are moreprevalent in the heterozygous than the homozygous condition.Because yuriF64 causes loss of particular isoforms, the normalstoichiometric balance between isoforms is disrupted in bothhomozygotes and heterozygotes, but it is disrupted differently inthe two situations. Thus, given that two classes of Yuri isoformscontain coiled-coil regions, altered dimerization or proteininteractions that have more severe consequences for axonemeassembly might be produced uniquely in the heterozygote.

Fig. 10. TEM analysis of control and yuriF64 mutant sperm. (A) Individualized controlsperm (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 elongatingcysts are sometimes associated with aberrant sets of mitochondrial derivatives, often sharingthem or possessing multiple derivatives of the same type. P, paracrystalline body in majormitochondrial 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 openedaxoneme in I (arrow). Scale bars: 500 nm in A-G, 250 nm in H,I.

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Yuri function and the defects in spermatogenesisThe various elements of the yuriF64 testis phenotype provide cluesas to the molecular functions of the protein. One clear implicationis that Yuri regulates F-actin function. We show here for the firsttime that F-actin is associated with the dense complex on spermatidnuclei 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 spermindividualization and is required for their formation. The actin conesare formed by a two-step process (Noguchi et al., 2006). Initially,parallel actin fibers are formed around the nuclei and then an actinmeshwork is added at each apical nuclear tip. Given the absenceof actin cone initiation in yuriF64, it seems likely that Yuri has anearly role in F-actin deposition here too.

The aberrant F-actin sleeves formed in the somatic cyst cells inyuriF64 led us to identify related actin sleeves around actively coilingsperm in control testes. Sperm coiling is executed within theconfines of the head cyst cell, which completely engulfs the apicalregion of the cyst (Tokuyasu et al., 1972b). Elaborate microvilli,full of 50 Å filaments, project from the head cyst cell onto the cystwalls and Tokuyasu and colleagues suggest that coiling largelyrepresents the collapse of the intrinsically helical sperm tails intoa flat pile of gyres as a result of contraction and shape change withinthe head cyst cell. We propose that the actin sleeves in control testesare related to the 50 Å filaments seen by Tokuyasu et al. and thatin yuriF64, F-actin structures form at inappropriate positions inassociation with abortive coiling.

In addition to regulating actin function, Yuri is implicated inmicrotubule/tubulin action. The stripe of dense complex along theelongating nuclei accretes a bundle of microtubules that arethought to provide structural rigidity to the nuclei. Although wewere 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. Thepresence of Yuri in the dense complex is also intimately associatedwith proper positioning, formation and functioning of the basalbody. When Yuri is not present at this site, (1) the basal bodies arescattered along the nuclei, or even mispositioned at the rostralnuclear 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, ourfindings for the GFP-PACT marker indicate that dPLP is recruitednormally to the basal bodies in yuriF64. Interestingly, in mammaliansystems, interaction between γ-tubulin and pericentrin is thoughtto underlie the targeting of γ-tubulin to centrosomes/centrioles(Young et al., 2000). dPLP is therefore implicated in promotingthe 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 butγ-tubulin is not, suggests a role for Yuri in the interaction of thesetwo proteins.

At the end of elongation, prior to individualization, the nucleus-basal body association is altered so that the axoneme and spermhead are locked in a permanent configuration relative to one another(Lindsley and Tokuyasu, 1980; Tokuyasu, 1975). This changeinvolves disappearance of the CA and movement of the basal bodyto lie in a shallow groove on one side of the nucleus. Predictably,the CA components γ-tubulin and Unc are lost from the nuclei atthis stage (Baker et al., 2004). We show here that both the Yuri dotand the GFP-PACT marker also disappear at this point. The basalbody present on mature sperm is clearly stripped of many ancillaryproteins.

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Although Yuri appears to anchor tubulin structures, including thebasal body, to the nuclear membrane, our findings for the dyneinlight chain mutant suggest that the initial positioning of Yuri on thenuclear membrane is determined by dynein transport, presumablyalong microtubules. In the dynein light chain mutant, Yurilocalization is dramatically altered, with Yuri now primarilyassociated with the basal body – a novel association not seen in thewild type. The implication must be that an activity of dynein isrequired 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 wrongends of elongated cysts. Contacts that normally hold the nucleitogether in tight alignment appear to be missing in yuriF64, and thiscould permit loose nuclei to migrate to the wrong location.Axonemes with opposite orientations in a single cyst have beenreported for mutations in the Drosophila parkin homolog (Riparbelliand Callaini, 2007). Although these investigators did not report asearch for nuclei at the wrong ends of cysts, they did note occasionalactin cones pointing in the wrong direction – a finding that suggeststhe same underlying cause for the two axoneme orientations in boththeir case and ours.

Other genes that act in mechanosensory organs andspermatogenesisThe finding that different mutations of yuri affect processes asdisparate as gravitaxis and spermatogenesis is initially surprising.However, together with sperm, mechanoreceptor neurons, suchas those affected by yuric263, are the only cell types in Drosophilathat possess cilia, and genes that affect ciliary function have beenshown to affect both mechanosensory organs and spermatogenesis.Mutations in touch insensitive larva B (tilB) are defective inhearing and touch perception as a result of defects in thechordotonal organs (Eberl et al., 2000). Mutations in unc affectboth the chordotonal organs and the external sense organ (eso)class of mechanoreceptors (Eberl et al., 2000). Mutations at bothloci are also male sterile because they encode proteins with rolesin cilia. TilB is a conserved ciliary protein with a leucine-richregion and a coiled-coil domain (Kavlie et al., 2007) and Unc isassociated with the basal bodies in sperm and mechanosensoryneurons (Baker et al., 2004). Unc, like γ-tubulin, is a componentof 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 thechordotonal neurons, a role comparable to that identified here inspermiogenesis. Furthermore, the intriguing possibility of molecularinteractions between Yuri and Unc is suggested. The proteins arephysically close at the basal body and their only distinguishingfeatures are coiled-coil domains that presumably facilitate protein-protein interactions. It seems possible that these two proteins haveevolved to fulfil specialized roles associated with anchoring the basalbodies that could entail heterodimerization.

Materials and MethodsYuri antibodies and immunoblotsThe entire coding region of the 30 kDa Yuri isoform from clone GH14032 wasamplified by PCR, cloned in Topo vector pCR2.1 (Invitrogen) and sequenced, thenrecloned into the EcoRI and SalI sites of expression vector pET28a (Novagen). Therecombinant His-tagged protein was purified by Ni2+ chromatography (Novagen)and used to raise antibodies in chickens (Aves Labs). Recombinant Yuri proteincross-linked to NHS-activated Sepharose 4 Fast Flow (Amersham) was used foraffinity purification. For immunoblots, samples were solubilized in SDS samplebuffer, run on 12.5% polyacrylamide gels and blotted to Immobilon (Millipore) filters.

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Bands reacting with the affinity-purified antibody were detected with horseradish-peroxidase-conjugated rabbit anti-chicken antibodies (Sigma) and the West Durareagent (Pierce).

Fertility testing, fly stocks and geneticsFor fertility testing, �20 individual males or virgin females were placed with threew1118 partners in food vials for 7 days, after which adults were removed. The originalvials 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 producedany 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 homozygousfor Df(2L)do1 or homozygous for CyO-GFP, eggs were collected from theDf(2L)do1/CyO-GFP stock and left >24 hours to ensure that viable embryos hatched.Fluorescent and non-fluorescent embryos were collected separately. Thirdchromosomes carrying (1) a don juan-GFP construct (Santel et al., 1997) or (2) aGFP-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) wasfrom the Bloomington Stock Center.

Sequence analysis and conservation of yuriThe following yuri ESTs/cDNAs were sequenced: adult head GH14032; adult testisAT03435, AT15480, AT15149, AT19027 and AT25733; adult ovary GM26781 andGM25777; 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 apoint between exons 3 and 5. The region immediately 5� to this point scores poorlyin analyses designed to detect promoters. Given that these two cDNAs were preparedfrom 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 intronbetween exons 3 and 5 (Fig. 1). These clones thus either (1) provide evidence forthe variable presence of an additional exon (labeled 4 in Fig. 1), the 5� boundary ofwhich is not defined or (2) represent incompletely spliced transcripts. A paralog searchin D. melanogaster was performed using the Yuri ‘PD’ isoform (FlyBase) sequencesand the BLASTP service at FlyBase. Because protein data sets are not available forall 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 boththe ‘nr/nt’ NCBI database and the 21 insect genome sequences searchable at FlyBaseas target data sets. The sequence identity computation for the ~100 kDa Yuri proteinin the 12 sequenced Drosophila sequences (Drosophila 12 Genomes Consortium,2007) was performed using BLASTP on the GLEANR consensus protein datasets. Coiled-coil predictions were made using the COILS program athttp://www.ch.embnet.org/software/COILS_form.html. Default settings were used insearches.

Imprecise excisionsThe 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 (Robertsonet al., 1988). Standard genetic schemes generated stocks of viable excisions. For lethalexcisions, lines with GFP-marked balancers were prepared. Excisions werecharacterized by PCR. Precise deletion endpoints were determined by sequencing.

ImmunocytochemistryTestes were dissected in ice-cold phosphate-buffered saline pH 7.2 (PBS), fixed with3% paraformaldehyde in PBS for 10 minutes and permeabilized by four washes inBBX (PBS + 0.3% Triton X-100 + 0.1% BSA) for 10 minutes. They were thenincubated overnight at 4°C with rotation in BBX + 2% goat serum and one or moreof the following antibodies: 1:100 affinity-purified Yuri antibody; 1:500 mousemonoclonal anti-γ-tubulin GTU-88 (Sigma); 1:200 rabbit polyclonal anti-Centrosominantibody R19 (gift of T. Kaufman, Indiana University, Bloomington, IN); 1:50 mousemonoclonal anti-β-tubulin E7 (Developmental Studies Hybridoma Bank). After twowashes each in BBX and BBX + 2% goat serum, appropriate Alexa Fluor-conjugatedsecondary antibodies (Invitrogen) were added at 1:500 in BBX + 2% goat serum andincubated for 2 hours. Rhodamine- or Alexa Fluor-conjugated phalloidin (Invitrogen)at 1:50 dilution was included with the secondary antibody as appropriate. After fourwashes with BBX, testes were mounted with 1:2000 Hoechst 33342 (Invitrogen) in50% glycerol. Images were collected on a Zeiss Axioplan or on Zeiss LSM 410 and510 confocal microscopes and processed with Metamorph (Molecular Devices) orZeiss software.

Transmission electron microscopy (TEM) TEM analysis was as described previously (Tokuyasu et al., 1972a), with minormodifications. Sections were cut at 700Å and stained with uranyl acetate and leadcitrate. JEOL 1010, JEOL 1230 and Hitachi H-7500 electron microscopes wereused.

We thank Dr R. P. Munjaal for contributions to the early phases ofthis work. We thank Dr Chris Bazinet for the dj-GFP line; Dr DavidCaprette for EM help; Dr James Fabrizio for Flytrap lines hecharacterized as expressing GFP in the cyst cells; Dr ThomasKaufman for Centrosomin antibody; Dr Tatsuhiko Noguchi forcritical insight into the actin sleeves in the yuriF64; Dr Jordan Rafffor the GFP-PACT line; Dr Kiyoteru Tokuyasu for helpful discussionson the nuclear localization of Yuri. We are grateful to Kenneth Dunner,Jr, Deborah Townley and Dr Wenhua Guo of the High ResolutionEM Facility at MD Anderson, the Integrated Microscopy Core atBaylor College of Medicine and the Smalley Institute of Nano Scienceand Technology at Rice. respectively, for their assistance with EMwork. The help of Rice undergraduates, in particular Summer Bell,Faraz Sultan and Anita Shankar, is gratefully acknowledged. Thesestudies were supported by NIH grant RO1 HD 39766, grant C-1119from the Welch Foundation of Texas and NASA grant NCC2-1356.

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