The tumor suppressor archipelago E3 ligase is required for spermatid differentiation in Drosophila testis

The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelago (ago) gene. Ago is an F-box protein that gives substrate specificity to its SCF ubiquitin ligase complex. It has a central role in multiple biological processes in a tissue-specific manner such as cell proliferation, cellular differentiation, hypoxia-induced gene expression. Here we present a previously unknown tissue-specific role of Ago in spermatid differentiation. We identified a classical mutant of ago which is semi-lethal and male-sterile. During the characterization of ago function in testis, we found that ago plays role in spermatid development, following meiosis. We confirmed spermatogenesis defects by silencing ago by RNAi in testes. The ago mutants show multiple abnormalities in elongating and elongated spermatids, including aberration of the cyst morphology, malformed mitochondrial structures, and individualization defects. Additionally, we determined the subcellular localization of Ago protein with mCherry-Ago transgene in spermatids. Our findings highlight the potential roles of Ago in different cellular processes of spermatogenesis, like spermatid individualization, and regulation of mitochondrial morphology.

Here we present the role of the pleiotropic Ago protein, during the post-meiotic stages of Drosophila spermatogenesis. We show that lack of Ago resulted in male sterility and abnormal individualization of spermatids, including abnormal nuclear and mitochondrial structure.

Results
Lack of ago cause male sterility in Drosophila. We identified a male-sterile allele of the archipelago gene, ago 5-HA-2760 (ago ms ). In ago ms there is a P{RS5}-element insertion in the intronic region of the 5′ UTR of the ago gene (Fig. 1a). We tested genetically ago ms mutant line and found that ago ms is semi-lethal and male-sterile in homozygous and in hemizygous combination with the overlapping Df(3L)BSC370 deficiency and lethal in transheterozygous combination with the previously described ago 1 and ago 3 alleles 24 . These results suggest ago ms is a strong hypomorph allele. Precise excision of the P-element in ago ms restored wild type fertility. Next, we investigated how ago transcripts could be affected by the transposon insertion. We performed quantitative RT-PCR using mRNA samples of wild type and ago ms testes to measure the levels of ago transcripts. Three different ago transcripts are annotated, however, a single polypeptide is encoded by ago (Fig. 1a). We found a strong decrease of ago mRNA level in ago ms mutant homozygotes and hemizygotes measuring the three ago transcripts together ( Fig. 1a,b). When we tested the ago transcripts individually and we found a dramatic reduction of ago-RB and ago-RC transcript levels and also a moderate reduction of ago-RA transcript level (Fig. 1a,c). High througput experiments reported the ago transcripts mainly enriched in the apical region but present in later stages as well 9,28 . To investigate the expression profile of the ago isoforms we isolated RNA from the apical and basal parts of the testis. We used CG3927 as an apically and CG10252 as a post-meiotically enriched controls, and normalized the gene expression to rp49 (Fig. S1 a) 3,29 . The RT-qPCR results shows the presence of ago transcripts both in the apical and basal parts, and suggests a more stable transcript level than CG3927 and rp49.
To study the role of Ago exclusively during spermatogenesis, without the potential somatic influence 30 and to overcome the semi-lethality of the ago ms mutant, we utilized the P{TRiP.HMS00111}attP2ago (ago TRiP ) transgenic RNAi line. ago TRiP was driven by the germline-specific Bam-Gal4 driver in the presence of Df(3L)BSC370 deficiency (ago TRiPDf ) to increase the silencing effect (Fig. 1a). We used the driverless + / + ; Df(3L)BSC370/ P{TRiP. HMS00111}attP2ago flies as control unless we state otherwise. RNAi knockdown of ago recapitulates the classical mutant phenotype, resulting in ~ 70% of sterility in the male offspring (Fig. 1d). We confirmed the downregulation of ago transcripts with quantitative RT-PCR in ago TRiPDf testis (Fig. 1e).

Disrupted spermatid individualization in ago mutant testes.
To understand the function of Ago during spermatogenesis we analyzed the morphology of both ago ms and ago TRiPDf testes ( Supplementary Fig. S1 b-g' , Fig. 2a,b,f,g,h,i). The seminal vesicles are empty in the ago ms mutants, but cyst elongation occurs after meiosis, which suggests that the elongated spermatids are failed to individualize ( Supplementary Fig. S1 b,c). Visualizing DNA with DAPI and investment cones with Texas Red-X phalloidin staining revealed that individualization is disturbed in both ago ms  We visualized the activation of the Caspase cascade with α-cleaved Caspase-3 antibody staining. In wild type testis, active-Caspase-3 signal mostly restricted to cystic bulge and the preindividualised parts of the spermatids and finally accumulate in the waste bags following individualization (Fig. 2f,g). The asynchronous migration of actin cones results in abnormal cystic bulges and waste bags and dispersed active-Caspase-3 signal through the elongated individualizing cysts in the ago TRiPDf testes (Fig. 2f,g).
DAPI staining showed disturbed nuclear bundles in elongated cysts of ago ms mutant. ( Supplementary Fig. S1 d,d' ,e,e'). It was shown previously, that Archipelago has a nuclear localization during larva development 16 . To test the possibility that nuclear phenotype is the consequence of the abnormal chromatin reorganization in the post-meiotic stages, we investigated the displacement of histones by protamines. We found normal Protamine-GFP accumulation, in the nuclei onward of late canoe stage both in control and ago TRiPDf flies (Fig. 2h,i). Consistently with DAPI staining, many Protamine-GFP positive nuclei are scattered in the elongated cysts ( Fig. 2h,i,j,k,l,l'), the nuclei distal to the nuclear bundle, that are scattered along the cysts tail cannot elongate and become hypercondensed.
Mitochondrial abnormalities in ago mutants. Several lines of evidence suggest, that mitochondria are the key organelles connected to the spermatid elongation, individualization, and protein degradation in Drosophila [31][32][33] . Therefore, we tested the mitochondrial structure in ago TRiPDf flies. First, we visualized the mitochondrial derivatives by anti-ATP5 staining and found that the ago TRiPDf spermatids showed severe alteration in mitochondrial morphology (Fig. 3a,b). Mitochondrial bulges are present next to the elongation zone at the basal end in ago TRiPDf elongating cysts. Similar mitochondrial bulges are observable in parkin, clueless and fascetto mutants, which function is described earlier in mitochondria microtubule interactions 31,33 . Furthermore, in elongated ago TRiPDf cysts we observed large vacuolar structures at the basal end, which we identified as swollen megamitochondria ( Fig. 3c-f). This phenotype is mild compared to bb8 ms mutants where the mitochondrial  29 . To clarify if the observed alterations affect spermatid elongation itself in ago TRiPDf testis, we measured the length of the late elongated cysts by utilizing AXO49 antibody staining, which identifies the polyglycilated tubulin, representing the fully matured axoneme in the individualizing cysts ( Fig. 3g-i) 5 . The mitochondrial alterations do not affect the elongation process of ago TRiPDf spermatids. To test the ultrastructure of mitochondrial derivatives we utilized transmission electron microscopy on ago TRiPDf testes. We found normal axoneme formation, however, we observed different mitochondrial abnormalities, which manifest in morphology, size, and paracrystalline material accumulation phenotype ( Fig. 3j-m).  www.nature.com/scientificreports/ Ago localization during spermatogenesis. To investigate the potential spatial and temporal presence of Ago function in testis, we established a transgenic fly line with P{β2-tub-mCherry-Ago} (mCherry-Ago), where the testis-specific expression of the transgene was driven by the β2-tub promoter 5,34,35 . The functionality of the mCherry-Ago was tested in ago ms background and we found that the transgene was able to partially restore the fertility of ago ms mutant (Fig. 1d). Next, we investigated the subcellular localization of the transgene and found that mCherry-Ago protein is localized to the nuclei of primary spermatocytes (Fig. 4a,a' ,a''), which is consistent with the localization pattern of Ago in body wall muscle 16 . However, in the elongated spermatids, mCherry-Ago became cytoplasmic with a continuous enrichment towards the basal end of the cysts (Fig. 4b).
During individualization, the mCherry-Ago accumulates in the cystic bulges and shows a specific enrichment around the investment cones ( Fig. 4b-d,d' ,d''), and after the signal persists in the waste bags (Fig. 4b,c). The stage-specific different localization pattern of Ago suggests a pleiotropic role of it even in spermatogenesis.

Discussion
Directed protein degradation is a crucial requirement of the spermatid individualization in Drosophila. Multiple data sources suggest, that at least two E3 ligase complexes are involved in caspase activation: the Klhl10/Cul3/ Roc1b, and the Ntc/Cul1/SkpA E3 complex 8,13 . Key elements of these complexes are the testis-specific isoform of Cul3 and the testis-specific Ntc proteins. Moreover, it seems the Ntc protein might have a conserved function during spermatogenesis; Fbxo7 the mouse orthologue of Ntc is also required for mouse spermatid development 36 . (e,f) Anti-ATP5alpha staining on wild type (e) and ago TRiPDf (f) of elongated cysts: staining reveals mitochondrial swelling in ago TRiPDf spermatids. (g,h) Anti-AXO49 (green) staining on control (g) and on ago TRiPDf (h). Cysts from the ago TRiPDf testis elongate normally compared to control (g,h,i). Statistical significance was determined by Welch two-sample t-test (i).( j-m) Transmission electron micrographs of wild type (j), ago TRiPDf (k-m). We can observe multiple mitochondrial abnormalities in ago TRiPDf , like paracrystalline accumulation in both mitochondrial derivatives (k,l,m yellow arrows), mitochondrial swelling (l, red arrows) and misshaped mitochondria (m red arrow) The charts was created in Microsoft Excel 2016 MSO ver. 16 www.nature.com/scientificreports/ A previous analysis of the Drosophila testis transcriptome highlighted the existence of several other testis-specific members of the ubiquitin-proteasome system with higher transcript accumulation in later developmental stages, including multiple F-box proteins, and modulator subunits, like the SKP family 9 . All these data suggest the importance of targeted protein degradation in the late stages of spermatogenesis.
Here, we present a previously unknown function of the F-Box protein Ago in the post-meiotic stages of Drosophila melanogaster spermiogenesis. Multiple aberrations are present during spermatid differentiation both in classical ago ms and in the ago TRiPDf testis. In ago ms mutant testes, all three ago transcript levels are decreased, however, ago-RA shows only a moderate decrease, suggesting that ago-RC and ago-RB could have critical role during spermatogenesis, similarly to what was shown in the case of ago-RC function in tracheal differentiation 16 .
We did not observe abnormalities in the early stages of spermiogenesis, however, mCherry-Ago first localizes to the nuclei in primary spermatocytes. The nuclear localization of Ago was demonstrated in ventrolateral body wall muscle, therefore the observed localization in testis suggests a probable redundant function of Ago in the early stages of spermiogenesis 16 . During elongation, the mCherry-Ago protein becomes cytosolic, accumulates at the basal end of the cysts during individualization, and is followed by enrichment in the cystic bulge. Similar to the other known E3 ligase complexes, such as Culin3 and ubiquitin itself, mCherry-Ago also has been transferred to the waste bags 13 . mCherry-Ago localization pattern is also similar to another testis-specific SCF complex member, the Ntc/Cul1/SkpA (SCF Ntc ) 8 , however in contrast to the SCF Ntc , mCherry-Ago is not present in the proximal region of the nuclei, it is localized to the more matured cystic bulges. The co-localization of cleaved caspase signal and mCherry-Ago signal suggests that mCherry-Ago protein might not be a target of caspase activity. Ago mutant phenotypes also indicate that Ago is not required for caspase activation. However, it is also tempting to hypothesize that the Ago protein plays role in the restriction of activated caspases to the cystic bulge. This is potentially reinforced by Ago function by restricting the apoptotic activity of the rbf1/de2f1 pathway 15 . www.nature.com/scientificreports/ Our observation on mitochondrial abnormalities suggests that Ago protein could play a direct or indirect role in the unfurling and elongation of mitochondrial derivatives. Drosophila Parkin is a member of a ubiquitin ligase complex that was already shown to have a very similar phenotype to ago TRiPDf testis, with enrichment of mitochondrial bulges in the elongating cysts 7,33 . Direct approaches show the hSel-10 (hAgo, FBW7) protein interacts with Parkin in human neurons and targets CycE 37 . A more recent publication showed that Parkin targets the SCF substrate adapter Fbw7β (hAgo beta isoform) for proteasomal degradation in dopaminerg neurons, where lack of Parkin causes mitochondrial oxidative stress due to the unregulated SCF Fbw7β -mediated ubiquitylationdependent proteolysis of Mcl-1 38 . While Ago was shown to have a role in the oxidative stress response in Drosophila tracheal development 16 , there is no proven interaction between Ago and Parkin in Drosophila. However, the involvement of both Ago and hAgo in oxidative responses raises the possibility of a conserved mechanism, moreover, the similar phenotype of mitochondrial derivatives in parkin mutant and ago TRiPDf testis suggest a potential connection between them in Drosophila as well.
Ago protein role was proposed in growth regulation as an indirect effect on S6kinase (S6K) levels, which place Ago between metabolic signaling and protein synthesis regulation 39,40 . Ago's role as a metabolic regulator is reinforced by the mitochondrial abnormalities we observed. Furthermore, it was reported the CycE levels depend on mitochondrial morphology 41 , which also makes the Ago a potential link to metabolism since its role in CycE level regulation 24 . The mitochondrial connection is also explicable due to Ago function in hypoxicinduced gene expression 16 . All of these data suggest Ago protein's role in the differentiation of mitochondrial derivatives during spermatogenesis.
Despite of the abnormal mitochondrial development in ago TRiPDf testes, there was no measurable elongation defect of the cysts. On the other hand, the integrity of the elongated nuclear bundles was compromised and malformed nuclei were present in the mutants, without effecting the histone-protamine transition. The hypercondensed nuclei is not exclusive for ago mutants, they are characteristic to several male-sterile mutants, such as the dBruce, Mst77F and Rae1 mutants 42,43 .
The mitochondrial abnormalities could be sufficient to cause the observed defective individualization complex, however, the mCherry-Ago signal localization in the cystic bulges and in the basal end of elongating cysts suggests a more direct role of Ago in cytoskeletal reorganization, therefore Ago's function as a mediator between the mitochondria and cytoskeletal elements. In this report, we proposed an additional role of the F-box Ago protein in spermatid differentiation and maturation.
Staining and microscopy. Testis preparations and staining were performed as earlier described by White-Cooper, 2004 45 .
Electron microscopic analysis of testes was done as described in Laurinyecz  Molecular biology. β2-tub-mCherry-Ago transgenic construct was established by amplifying ago cDNA (BDGP DGC clone LD21322) and mCherry cDNA (mCherry LIC cloning vector, Addgene) with Phusion High-