A potential role for the clathrin adaptor GGA in Drosophila spermatogenesis.
A potential role for the clathrin adaptor GGA in Drosophila spermatogenesis.
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DOI：
10.1186/1471-2121-12-22
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发表时间：
2011-05-20
期刊：
影响因子：
--
通讯作者：
Carmichael J
中科院分区：
文献类型：
--
作者：
Hirst J;Carmichael J
GGA（高尔基体定位的、含有γ-耳的、ADP核糖基化因子结合的）是网格蛋白衔接子家族，其将许多生物学上重要的跨膜蛋白分选到网格蛋白包被的囊泡中。确定GGA功能的敲除和敲低研究被哺乳动物细胞中存在3个GGA基因的事实混淆。因此，黑腹果蝇是一个有用的模型系统，研究组织表达谱和敲低表型，因为有一个单一的GGA直系同源物。在这里，我们定量了果蝇中的蛋白质表达，并表明GGA在雄性果蝇中的表达比雌性果蝇高>3倍。在雌性果蝇中，大多数GGA表达在头部。在雄性果蝇中，GGA不仅在头部以高水平表达，而且由于GGA在睾丸中的大量表达，存在性别特异性表达增加。使用高度特异性的抗体，我们本地化的内源性GGA蛋白在睾丸南瓜，并可视化它在体细胞和生殖细胞。我们发现，GGA在精子发育的多个阶段表达，并与trans-Golgi网络的标记共染色。这在早期精子细胞的顶母细胞中最为显著。尽管GGA在睾丸中高表达，但使用转基因RNAi果蝇品系将其表达敲低>95%并不影响雄性生育力。因此，雄性果蝇的精子发生似乎在GGA <5%的情况下正常进行，这很可能是因为替代衔接子可能能够部分或完全取代GGA的功能。我们还确定“cueball”是GGA的一种新型货物，并且cueball的突变体已被证明具有雄性不育表型。在果蝇中，我们发现了GGA在雄性果蝇睾丸中的潜在作用。GGA的性别特异性高表达，其在睾丸中的特异性富集及其在发育中的精母细胞和精子细胞的顶母细胞中的定位支持GGA在果蝇精子发生中的功能，即使当GGA表达被耗尽至<5%的对照时精子发生仍然发生。
GGAs (Golgi-localised, γ-ear containing, ADP ribosylation factor-binding) are a family of clathrin adaptors that sort a number of biologically important transmembrane proteins into clathrin-coated vesicles. Knockout and knockdown studies to determine GGA function are confounded by the fact that there are 3 GGA genes in mammalian cells. Thus Drosophila melanogaster is a useful model system to study tissue expression profiles and knockdown phenotypes as there is a single GGA ortholog. Here we have quantified protein expression in Drosophila and show that there is >3-fold higher expression of GGA in male flies relative to female flies. In female flies the majority of GGA expression is in the head. In male flies GGA is not only expressed at high levels in the head but there is a gender specific increased expression which is due to the abundant expression of GGA in the testes. Using a highly specific antibody we have localised endogenous GGA protein in testes squashes, and visualised it in somatic and germ line cells. We show that GGA is expressed during multiple stages of sperm development, and co-stains with a marker of the trans-Golgi Network. This is most striking at the acroblast of early spermatids. In spite of the high expression of GGA in testes, knocking down its expression by >95% using transgenic RNAi fly lines did not affect male fertility. Therefore spermatogenesis in the male flies appears to progress normally with <5% GGA, most likely because alternative adaptors may be able to substitute partially or completely for the function of GGA. We also identify 'cueball' as a novel cargo for GGA, and mutants of cueball have been shown to have a male sterility phenotype. In Drosophila we have uncovered a potential role for GGA in the testes of male flies. The gender specific higher expression of GGA, its specific enrichment in testes and its localisation to developing spermatocytes and at the acroblast of spermatids supports a role for GGA function in Drosophila spermatogenesis, even though spermatogenesis still occurs when GGA expression is depleted to <5% of control.
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