The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus

Abstract

Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface.

Fig. 1.

spag4 expression, spag4 knockout and fertility assays. (A) spag4 mRNA (~800bp) is limited to males, but low levels of the primary transcript (~1000 bp) are present in male and female pupae and adults. (B) spag4 mRNA localization in wild-type testes by ISH. AS, antisense; S, sense probe control. Scale bar: 200 μm. White bracket: region of spag4 mRNA expression. Arrowheads indicate apical tips of testes. (C) Schematics (relatively proportional but not to scale) of spag4 transcription unit determined by 5′RLM-RACE and 3′RACE, and Spag4 protein and its domains. Newly identified regions of transcription unit are shown. Yellow and blue indicate 5′ and 3′ UTR, respectively. Amino acid boundaries of the TM and SUN domains are shown below. (D) Ends-out homologous recombination scheme for deletion of spag4. Gray bars: PstI fragments of wild-type (bottom strand) and mutant (top strand) DNA detected by Southern blot probe (green bar). The mini-white (w⁺) gene replaces spag4 upon recombination. (E) Genomic Southern blots of PstI-digested DNA from wild-type flies and the five spag4-null mutants isolated. PstI fragments and probe as in D. (F) Fertility assays for spag4-related lines. Error bars indicate s.d.; n=20 for each genotype. Blue bars, males; pink bars, females.

Fig. 2.

Dynamic localization of Spag4 during spermatogenesis. (A) Whole-mount testis (excluding seminal vesicle) expressing Spag4:6×MYC. Lettered arrowheads indicate approximate regions where data in the other panels were obtained. NE localization in polar and mature primary spermatocytes (B) and in round spermatids (E). Spindle-associated localization during meiosis I and II (C,D); arrowheads indicate spindle fibers. Caps of Spag4:6×MYC on late round spermatid nuclei with the centrioles (γ-tubulin stained) at their apices (F). Focus of Spag4:6×MYC at centriolar attachment site and along dense complex microtubules (e.g. arrowhead) in early elongating spermatids (G-G″). A concentration of Spag4:6×MYC at the apical tip of elongating spermatid nuclei (H,I′) is shown by the basal body marker YFP:Asl (I) to lie between the BB and the nuclei proper (I″). This Spag4:6×MYC focus persists on mature sperm in the seminal vesicle (J), similarly to the BB marker YFP:Asl (K). Red, Spag4:6×MYC; blue, DAPI; green, γ-tubulin in F, α-Tub:GFP in G″, YFP:Asl in I and K, Spag4:GFP in J. Scale bars: 150 μm (A), 20 μm (B-G,J,K) and 10 μm (H,I).

Fig. 3.

Nuclear-centriolar dissociation during elongation stages is the earliest visible defect in spag4 mutant testes. Centriolar association with primary spermatocyte (A), round spermatid (B) and elongating (C) nuclei in wild-type testes. In spag4 mutant testes, centriolar association is normal in primary spermatocytes (D) and mostly normal in round spermatids (E), and early elongating spermatids (F). (G) Fully penetrant nuclear-centriolar dissociation becomes apparent in later elongation stages. (H) Nuclei contort in advanced elongation stages. (I) The microtubules of the dense complex stripe are retained on spag4 spermatid nuclei. Scale bars: 20 μm (A,D), 10 μm (B,C,E,I) and 15 μm (F,G,H).

Fig. 4.

Individualization fails in the spag4 mutant testes. (A) Normal actin cones of the individualization complex (IC) form on wild-type elongated nuclei. (B) Abnormal actin deposition forms aberrant ICs in the spag4 mutant. (C) Lack of actin deposition on curled nuclei in the spag4 mutant. (D) TEM of wild-type elongated cyst cross-section. Black lines denote axis through the central doublet of the axoneme; within a wild-type cyst, spermatid cross-sections (red lines) lie at a constant angle (clockwise 45° to the central doublet. (E) In spag4 mutant cysts, aberrant angles demonstrate spermatid-axoneme misalignment. Red arrowhead indicates missing central doublet in spag4 axoneme. Images in D and E were taken in comparable regions of wild-type and mutant testes (towards terminal epithelium, immediately apical to cyst coiling region). Black arrows indicate major mitochondrial derivatives (MDs) and white arrows, minor MDs. Black arrowhead in D indicates plasma membrane. Both major and minor MDs are less condensed in spag4 mutant than in the wild type, and large regions of ribosome-rich cytoplasm persist in the cysts (blue dotted line). Scale bars: ~10 μm (A-C), 500 nm (D,E).

Fig. 5.

Linking the spermatid nucleus to the centriole or BB does not require a KASH domain protein. (A) Fertility assays for KASH-domain protein alleles. n=20 for each genotype. Error bars indicate s.d. All genotypes are at least partially fertile. The large error bars in msp300^ΔKASH; klar^CD4 double mutants are due to high (40%) male death during the assay. Compare with Fig. 1F for wild-type male fertility levels. Centriolar-nuclear attachment is normal in elongating nuclei bundles from klar^CD4 (B), msp300^ΔKASH (C) and msp300^ΔKASH; klar^CD4 (D) mutant males as shown by tight juxtaposition of γ-tubulin staining (red) of the centriolar adjunct to the dent component of Yuri staining (green, see text, Fig. 6J and supplementary material Fig. S7). Scale bar: 10 μm.

Fig. 6.

Spag4 and Yuri localize to nuclear envelopes, cap, BB dent and stripe during spermatogenesis. Spag4 and Yuri localization at the NE of primary spermatocytes (A,B), at the centriolar cap on round spermatids (C,D) and in the BB dent and stripe on elongating nuclei (E,F) are compellingly similar. F′ shows Yuri localization on a single elongate nucleus. In the spag4 mutant, Yuri fails to localize to the NE of primary spermatocytes (G) and to the centriolar cap (H) and the BB dent and stripe (I) at later stages. However, Yuri accumulates on the BB and overlays the CA (I). I′ also shows absence of Yuri stripe and Yuri accumulation on BB for a single nucleus. Upper arrows in F′, I′ show position of BB, lower arrows show position of stripe. Yuri accumulation on the centrioles of primary spermatocytes is also intermittently seen in the spag4 mutant (arrow, G). In yuri^F64, most elements of the Spag4 localization pattern remain (J,K) but accumulation at the BB dent is lost (L). Control genotypes: in A, w;; spag4:gfp; in C,E, w;; spag4:6xmyc; in B,D,F,F′, w¹¹¹⁸. Scale bars 25 μm (A,B,G,J), 15 μm (C,D,H,K), 10 μm (E), 6 μm (F,F′,I,I′,L).

Fig. 7.

Dynein-dynactin components require Spag4, but not Yuri, for localization during spermatogenesis. Spag4:6×MYC localization is essentially normal in (A) primary spermatocytes, (B) round spermatids and (C) elongating spermatids in the Dlc90F^e155 mutant background, although elongating nuclei fail to remain in register. Conversely, the p50:GFP dynactin subunit, which localizes to the centriolar cap on wild-type round spermatid nuclei (D-D″), fails to localize normally in the spag4-null mutant, aggregating instead on centrioles on round spermatid nuclei (E) and in puncta on the CAs of elongating nuclei (F). (G) Localization of p50:GFP to the centriolar cap is unaffected in the yuri^F64 mutant. Scale bars: 25 μm (A), 10 μm (B-D′,F,G), 8 μm (E).