Neurotransmitter Transporter-Like: a male germline-specific SLC6 transporter required for Drosophila spermiogenesis

Abstract

The SLC6 class of membrane transporters, known primarily as neurotransmitter transporters, is increasingly appreciated for its roles in nutritional uptake of amino acids and other developmentally specific functions. A Drosophila SLC6 gene, Neurotransmitter transporter-like (Ntl), is expressed only in the male germline. Mobilization of a transposon inserted near the 3' end of the Ntl coding region yields male-sterile mutants defining a single complementation group. Germline transformation with Ntl cDNAs under control of male germline-specific control elements restores Ntl/Ntl homozygotes to normal fertility, indicating that Ntl is required only in the germ cells. In mutant males, sperm morphogenesis appears normal, with elongated, individualized and coiled spermiogenic cysts accumulating at the base of the testes. However, no sperm are transferred to the seminal vesicle. The level of polyglycylation of Ntl mutant sperm tubulin appears to be significantly lower than that of wild type controls. Glycine transporters are the most closely related SLC6 transporters to Ntl, suggesting that Ntl functions as a glycine transporter in developing sperm, where augmentation of the cytosolic pool of glycine may be required for the polyglycylation of the massive amounts of tubulin in the fly's giant sperm. The male-sterile phenotype of Ntl mutants may provide a powerful genetic system for studying the function of an SLC6 transporter family in a model organism.

Figure 1. Amino acid alignment of Ntl with human and A.aeolicus SLC6 transporters.

Amino acid sequence alignment of Drosophila melanogaster Ntl (Ntl; NP_609135.1) with A.aeolicus Leu T_Aa (Leu; NP_214423), human homologues for Serotonin (5HT;P31645), Glycine (Gly, I57956), GABA (GABA; P30531), Dopamine (DOPA; Q01959) using Clustal W alignment [http://www.ebi.ac.uk/Tools/clustalw2/index.html]. Strictly conserved residues are highlighted in red; α coils and β sheets are depicted as blocks and arrows respectively. Open and filled green circles represent putative cationic gates at extra and intra cellular surfaces (EL and IL) respectively. Open and filled blue triangles indicate sites that interact with sodium ions in the LeuA structure. Tyrosine in TM3 is a critical residue present in Ntl, which is indispensable for substrate binding and transport. Adapted from Yamashita et al, 2005.

Figure 2. Generation of Ntl mutants.

A) Ntl transcript/CDR from 28C on the left arm of chromosome 2. P element is inserted at the very end of the protein coding region. B) Scheme for generation of Ntl deletion mutations by imprecise excision of the P element.

Figure 3. Ntl expression is testis specific and limited to the germline.

A) From the left: Ntl RT-PCR products from wt whole males, wt male heads, wt male testis, 129A whole male; Ntl RT-PCR products from wt whole females, wt female heads and wt female ovaries. The last lane is a negative control without RNA. B) Ntl RT-PCR products from Ntl/Ntl mutant males carrying a pTMR-Ntl cDNA construct (Lane 1; yw; Ntl^129A/Ntl^129A; pTMR-Ntl1/TM3,Sb) and a genomic pCaSpeR4 construct (Lane 2; yw; Ntl^129A/Ntl^129A; pCaSpeR 4- Ntl1/TM3,Sb). Lane 3: Ntl RT-PCR product from testes of male offspring of tud/tud females, which lack germ cells.

Figure 4. Ntl mutant sperm are immotile and are not transferred into seminal vesicles.

Panels A, B, E, and F: Phase contrast images of testes from Ntl⁺ (A, E) and Ntl^- (B, F) males. The major phenotypic feature of the mutants is the accumulation of coiled cysts at the base of the testis (asterisks), and the empty/shrunken state of the seminal vesicle (arrows). Panels, C, D, G, and H: don juan-GFP fluorescence images corresponding to phase images immediately above them, showing the disposition of elongated cysts and mature sperm in the testis and seminal vesicle. Note the complete absence of fluorescence from the seminal vesicle of Ntl/Ntl mutants (arrows) compared to the accumulated fluorescence in wild type seminal vesicles (arrowheads), and accumulation of coiled cysts in the base of the mutant testes (asterisks). In panels A and C, the letter M demotes dense masses of mature motile sperm which is not seen in the mutants. Left hand panels: wild type (Ntl/+); right hand panels: Ntl ^129A/Ntl ^129A mutants. Bars, 20 µm.

Figure 5. Individualization in Ntl mutants.

A, B: wt testis expressing don juan-GFP counterstained with TRITC phalloidin to visualize the actin cone-based individualization complexes. Arrowhead marks the actin cones of the complex. C, D: Ntl mutant testis preparations expressing dj-GFP, counterstained with TRITC-phalloidin. Formation and movement of actin cones/individualization complex along the mutant cysts appears normal. E, F: TRITC-phalloidin staining of W.T. (Ntl/+) (E) and Ntl mutant (F) cysts from males expressing βTub-GFP. Bars, 20 um.

Figure 6. Ultrastructure of Ntl mutant spermiogenesis.

A, B: TEM of cross-sections through wild-type pre-individualized and post-individualized cyst respectively. C and D: cross-sections through pre-individualized and individualized cyst of Ntl/Ntl mutant cyst. No obvious differences between the mutant and the wild type phenotype were observed at this level of resolution. ax, axoneme; M, major mitochondrial derivative; m, minor mitochondrial derivative. Bars, 200 um (A, C); 100 um (B, D).

Figure 7. Polyglycylation of tubulin is partially decreased in mutant testes.

A)Western Blot of Ntl/Ntl male abdomens compared to +/+ (yw) probed with anti Poly-G antiserum. B) Quantitation of 6 independent replicates of the Western analysis of panel A. Bar, standard error of the mean. N = 6.