One of the most significant morphogenic events in the development of Drosophila is the elongation of imaginal discs during puparium formation. This macroscopic event is accompanied by the formation of Golgi stacks from small Golgi larval clusters of vesicles and tubules that are present prior to the onset of disc elongation. The fly steroid hormone 20-hydroxyecdysone triggers both the elongation itself and the formation of Golgi stacks. Using mRNA in situ hybridisation, it has been shown that ecdysone triggers the upregulation of a subset of genes encoding Golgi-related proteins (such as dnsf1, dsec23, Syx5, and drab1) and downregulates the expression of others (such as dergic53, dbeta'COP, and drab6). The transcription factor Broad-complex, itself an "early" ecdysone target, mediates this regulation. The ecdysone-independent upregulation of dnsf1 and dsnap prior to the ecdysone peak leads to a precocious formation of large Golgi stacks. The ecdysone-triggered biogenesis of Golgi stacks at the onset of imaginal disc elongation offers the exciting possibility of advancing understanding of the relationship between gene expression and organelle biogenesis (Dunne, 2002).
To confirm the significance of the two-hybrid analysis that detected an interaction between alpha-SNAP and Syt5, a GST fusion protein was generated with a Syx5 cDNA lacking the transmembrane domain (Syx5delta) and the interaction of Syx5delta with increasing amounts of recombinant His6-alpha-SNAP was examined. Western blots of eluted protein were probed with anti-GST to confirm the presence of GST-Syx5delta on the glutathione-agarose beads, and with anti-alpha-SNAP to measure alpha-SNAP binding. Under nonsaturating binding conditions, alpha-SNAP binds in a dose-dependent manner. alpha-SNAP from adult fly homogenates is also able to bind to immobilized recombinant Syx5delta. These results establish that Syx5 acts as an alpha-SNAP receptor and define the H3 domain as the likely region for this interaction (H. Xu, 2002).
Another predicted function of a SNARE protein is its ability to interact with other members of the SNARE pathway. The phenotype that results from the overexpression of a dominant-negative form of Drosophila NSF2 at the developing wing margin has been described (Stewart, 2001). To determine whether Syx5 interacts genetically with NSF2, a single copy of a null mutation in Syx5 was introduced into the flies expressing dominant-negative NSF2 along the wing margin; a significant enhancement of wing notching was seen. This enhancement was as strong as any of the other known interactors identified previously (Stewart, 2001) and provides further evidence that Syx5 functions as a SNARE in vivo (H. Xu, 2002).
In situ hybridization was used to analyze the expression pattern of Syx5; the gene is broadly expressed throughout the developing embryo in virtually all tissues and is present at high levels in 1-h-old embryos, indicative of a significant maternal contribution (H. Xu, 2002).
In mammals, syntaxin 5 has been localized to a perinuclear compartment likely to be the cis portion of the Golgi complex (Bennett, 1993). To determine whether Syx5 in Drosophila behaves the same as its mammalian ortholog, it was determined whether antibodies against rat syntaxin 5 would recognize Drosophila Syx5. This antibody indeed recognizes an IPTG-inducible band that represents the GST-Syx5 fusion. This antibody was then used for immunocytochemistry on Schneider S2 cells. Anti-syntaxin 5 antibodies revealed a punctate, perinuclear pattern of staining that overlapped extensively with the Golgi marker p120. To further confirm this colocalization, the full-length cDNA was subcloned into the pRmHa-3 expression vector in frame with an N-terminal Myc epitope. This was transfected into S2 cells, and the cells were costained with antibodies against Myc and p120. Again, significant overlap of the Myc and p120 signals was seen. Hence, the Drosophila Syx5 protein, like its mammalian ortholog, predominantly resides in the Golgi complex (H. Xu, 2002).
The Syx5 gene maps near cornichon and extensive EMS mutagenic analysis of the locus has identified several lethal complementation groups that appear to be in the vicinity of the Syx5 gene. It was determined that complementation group l(2)35Ff was likely to correspond to Syt5. To determine whether any of the members of this complementation group contain mutations in Syx5, RT-PCR was used to amplify the Syx5 cDNAs from this group with four alleles (dSyx5AR113, dSyx5AE48, dSyx5AA73, and dSyx5AE73). Sequence analysis reveals that the line dSyx5AR113 contains an amber mutation at glutamine residue 153. This mutation would create a truncated N-terminal peptide that should not interact with the SNARE proteins or alpha-SNAP and would therefore be expected to be functionally null. Analysis of the homozygous or transheterozygous combinations of the complementation group revealed lethality during the first larval instar. This indicates that, although the molecular basis for the other mutations is not known, all are likely to be null or severe hypomorphic alleles (H. Xu, 2002).
Since Syx5 is implicated in the traffic of membranes and membrane proteins through the Golgi complex, exocytosis of an exogenous fluorescent transgene was monitored in Oregon R (Ore-R) and Syx5 mutant flies. For this purpose, UAS-mCD8-GFP flies were used. mCD8-GFP is a protein fusion between the murine lymphocyte receptor CD8 and EGFP. It accumulates at the plasma membrane of most cells, and in epithelial cells is found to accumulate at the apical membranes. To express the mCD8-GFP construct broadly, the UAS-mCD8-GFP and a GAL4 gene driven by the daughterless promoter (da-GAL4) were introduced into the dSyx5AR113 or wild type backgrounds. In the embryonic salivary gland of heterozygous dSyx5AR113/CyO flies, mCD8-GFP can be seen to accumulate on the apical surface during embryogenesis. In contrast, flies homozygous for the Syx5 mutation have little if any mCD8-GFP on the salivary gland apical surface and instead appear to accumulate fluorescence throughout the cytoplasm. Hence, Syx5 function appears necessary for proper transport through the secretory pathway (H. Xu, 2002).
In addition to the EMS alleles of Syx5, an EP line [EP(2)2313] was obtained that resulted from an insertion into the Syx5 promoter region. EP elements are modified P transposable elements that contain a GAL4 binding site and a weak promoter, allowing the directional expression of adjacent genes. EP(2)2313 is 803 bp upstream of the Syx5 ATG and only 46 bp upstream of the longest cDNA in the dbEST database. In addition, EP(2)2313 is in the correct orientation to permit overexpression by GAL4. The location of EP(2)2313 also suggested that it might be hypomorphic for Syx5. EP(2)2313 is homozygous lethal, but this lethality is likely due to second-site mutations or the effect of the EP on adjacent genes, since it is only semilethal in combination with the Syx5 mutant allele dSyx5AR113. When da-GAL4 is introduced into the EP(2)2313/Syx5AR113 background, expression of Syx5 is increased and the semilethality is fully rescued. Similar results were obtained by using da-GAL4 to rescue another allele of Syx5, Syx5AE48 as a transheterozygote with EP(2)2313. In contrast, da-GAL4 cannot rescue the lethality of the EP(2)2313 homozygotes, indicating that their lethality is not due to lack of Syx5. Hence, it appears that EP(2)2313 is hypomorphic for Syx5 (H. Xu, 2002).
The incomplete penetrance of the lethal phenotype exhibited by EP(2)2313/dSyx5AR113 permitted analysis of the adult escapers. Whereas the females appeared normal, the males were sterile when crossed to female escapers or Ore-R females, suggesting that Syx5 mutant flies have a defect in some aspect of spermatogenesis (H. Xu, 2002).
Initial examination of the testes of the Syx5 mutant flies revealed that they have no motile sperm and appear to be defective in spermatid elongation. In contrast to wild type testes, Syx5 mutant testes contained predominantly large, oval cysts and few elongated bundles. In situ hybridization was used to examine when during spermatogenesis Syx5 is expressed. Syx5 is expressed in the primary spermatocytes, but expression does not persist past the meiosis stage and is essentially absent from the elongating bundles (H. Xu, 2002).
Examination of the Golgi marker Lava lamp (Lva) reveals that, in those cysts that do undergo elongation, Lva staining appeared diffuse with a mixture of large and small punctate structures. In contrast, wild type flies display exclusively large punctate structures. These results indicate that spermatogenesis may be a developmental process that is particularly sensitive to the function of Syt5 (H. Xu, 2002).
Closer analysis of the spermatids reveals that more than 80% of those from the Syx5 mutant flies have abnormally large mitochondrial derivatives that are associated with multiple nuclei. Since mitochondria fuse together following meiosis, the presence of a large mitochondrial derivative surrounded by multiple nuclei is consistent with cells having failed to complete meiotic cytokinesis (H. Xu, 2002).
It is important to note that membrane fusion often occurs during the preparation of these cells for microscopy, causing nuclei to become colocalized. However, the presence of a single large mitochondrial derivative surrounded by nuclei is diagnostic of failed cytokinesis events, whereas multiple mitochondria and nuclei of the same size within a cell have arisen as an artifact in the unfixed squashed preparations. Typical mutant spermatids have an enlarged mitochondrial derivative associated with two or more nuclei (H. Xu, 2002).
Quantification of the cytokinesis failure obtained from examining a large number of cysts reveals that, of a total of 1361 mutant nuclei counted, the vast majority are present in multinucleated cells. In contrast, most cysts from control flies have mitochondrial derivatives and nuclei of approximately the same size and fewer than 0.5% of cysts contain multiple nuclei. Spermatids with 2 nuclei likely failed a single meiotic division while those with four probably failed to divide during both meioses. Occasionally, some spermatids are observed that have 8 nuclei, suggestive of an additional failure at an earlier mitotic division step. In addition, some spermatids have numbers of nuclei that could have arisen if a multinucleated parental cell divided unequally (H. Xu, 2002).
These phenotypes are similar to those observed in spermatids from several male meiosis mutants including four wheel drive (fwd) (Brill, 2000). fwd encodes a phosphatidylinositol 4-kinase that is required for completion of cytokinesis, particularly during the meiotic divisions, and the similarity of the two phenotypes suggests that Syx5 is also required for this process (H. Xu, 2002).
If the defects in cytokinesis are due to the lack of Syx5, then it should be possible to rescue this by increasing Syx5 expression. da-GAL4 was introduced into the EP(2)2313/dSyx5AR113 background to drive Syx5 expression from EP(2)2313 chromosome. It was first determined whether the EP line could lead to elevated Syx5 expression in the presence of the GAL4 driver. To measure this, a pair of PCR primers was generated, one from the EP element and one from the 3' end of the Syx5 gene, to measure transcripts generated from the EP. The EP primer would be included in transcripts that arose from the EP element when activated by GAL4 and would measure only those transcripts. As a control, primers from Syx5 from the 5' and 3' ends of the Syx5 coding sequence were generated. These would detect all Syx5 transcripts, since the primer sites are within both the wild type and EP-derived mRNAs. Indeed, using RT-PCR, fusion transcripts could be detected from the EP only when the da-GAL4 driver was present, but not in the control flies. Cysts from the da-GAL4;EP(2)2313/dSyx5AR113 flies that overexpress Syx5 reveal significant rescue of the multinucleated phenotype. Occasional cells with two nuclei are observed, but quantification of these data clearly reveals a nearly complete rescue of cytokinesis by Syx5 expression. Only 3.9% of the spermatids counted contained multiple nuclei (compared with 80% without rescue), and viability was completely restored (H. Xu, 2002).
In addition to a failure in cytokinesis, EP(2)2313/ dSyx5AR113 testes also exhibit an accumulation of unusual ovoid spermatid cysts. Expression of Syx5 from the EP chromosome with da-GAL4 rescues both the cytokinesis and these bundle elongation defects. Interestingly, few motile sperm were observed and the flies remained infertile. This suggests that either insufficient levels of Syx5 were reached to achieve complete rescue, or other unrelated causes were responsible for the late stage maturation defects. In support of the former idea, introducing the stronger actin-GAL4 driver into the EP(3)2313/dSyx5AR113 background achieves complete rescue of the spermatid maturation defect, resulting in fertile flies with motile sperm. Thus, strong expression of Syx5 from the EP is sufficient to rescue all spermatogenesis defects associated with mutations in dSyx5 (H. Xu, 2002).
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date revised: 20 May 2003
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