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During spermatogenesis, the Golgi apparatus serves important roles including the formation of the acrosome, which is a sperm-specific organelle essential for fertilization. We have previously demonstrated that D. melanogaster ATP-dependent Citrate Lyase (ATPCL) is required for spindle organization, cytokinesis, and fusome assembly during male meiosis, mainly due to is activity on fatty acid biosynthesis. Here, we show that depletion of DmATPCL also affects the organization of acrosome and suggest a role for this enzyme in the assembly of Golgi-derived structures during Drosophila spermatogenesis.

Protein recycling is important for maintaining homeostasis of the Golgi and its cisternae. The Vps54 (Scat) protein, a subunit of the GARP tethering complex, is a central factor in retrograde transport to the trans-Golgi. We found the scat1 mutant to be male sterile in Drosophila with individualization problems occurring during spermatogenesis. Another typically observed phenotype was the abnormal nuclear structure in elongated mutant cysts. When examining the structure and function of the Golgi, a failure in acrosome formation and endosome-Golgi vesicular transport were found in the scat1 mutant. This acrosome formation defect was due to a fault in the trans-Golgi side of the acroblast ribbon. When testing a mutation in a second retrograde transport protein, Fws, a subunit of the conserved oligomeric Golgi (COG) tethering complex, the acroblast structure, was again disrupted. fwsP caused a similar, albeit milder, acrosome and sperm individualization phenotype as the scat1 mutant. In the case of fwsP the cis side of the acroblast ribbon was dispersed, in-line with the intra-Golgi retrograde function of COG. Our results highlight the importance of an intact acroblast for acrosome formation, nuclear elongation and therefore sperm maturation. Moreover, these results suggest the importance of retrograde tethering complexes in the formation of a functional Golgi ribbon.

Background: GGAs (Golgi-localised, g-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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

During spermatogenesis, the Golgi apparatus serves important roles including the formation of the acrosome, which is a sperm-specific organelle essential for fertilization. We have previously demonstrated that D. melanogaster ATP-dependent Citrate Lyase (ATPCL) is required for spindle organization, cytokinesis, and fusome assembly during male meiosis, mainly due to is activity on fatty acid biosynthesis. Here, we show that depletion of DmATPCL also affects the organization of acrosome and suggest a role for this enzyme in the assembly of Golgi-derived structures during Drosophila spermatogenesis. [ABSTRACT FROM AUTHOR]

Abstract Background 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. Results 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 Conclusion 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

Protein recycling is important for maintaining homeostasis of the Golgi and its cisternae. The Vps54 (Scat) protein, a subunit of the GARP tethering complex, is a central factor in retrograde transport to the trans-Golgi. We found the scat(1) mutant to be male sterile in Drosophila with individualization problems occurring during spermatogenesis. Another typically observed phenotype was the abnormal nuclear structure in elongated mutant cysts. When examining the structure and function of the Golgi, a failure in acrosome formation and endosome-Golgi vesicular transport were found in the scat(1) mutant. This acrosome formation defect was due to a fault in the trans-Golgi side of the acroblast ribbon. When testing a mutation in a second retrograde transport protein, Fws, a subunit of the conserved oligomeric Golgi (COG) tethering complex, the acroblast structure, was again disrupted. fws(P) caused a similar, albeit milder, acrosome and sperm individualization phenotype as the scat(1) mutant. In the case of fws(P) the cis side of the acroblast ribbon was dispersed, in-line with the intra-Golgi retrograde function of COG. Our results highlight the importance of an intact acroblast for acrosome formation, nuclear elongation and therefore sperm maturation. Moreover, these results suggest the importance of retrograde tethering complexes in the formation of a functional Golgi ribbon., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)