Drosophila Amphiphysin is implicated in protein localization and membrane morphogenesis but not in synaptic vesicle endocytosis.

Amphiphysin family members are implicated in synaptic vesicle endocytosis, actin localization and one isoform is an autoantigen in neurological autoimmune disorder; however, there has been no genetic analysis of Amphiphysin function in higher eukaryotes. We show that Drosophila Amphiphysin is localized to actin-rich membrane domains in many cell types, including apical epithelial membranes, the intricately folded apical rhabdomere membranes of photoreceptor neurons and the postsynaptic density of glutamatergic neuromuscular junctions. Flies that lack all Amphiphysin function are viable, lack any observable endocytic defects, but have abnormal localization of the postsynaptic proteins Discs large, Lethal giant larvae and Scribble, altered synaptic physiology, and behavioral defects. Misexpression of Amphiphysin outside its normal membrane domain in photoreceptor neurons results in striking morphological defects. The strong misexpression phenotype coupled with the mild mutant and lack of phenotypes suggests that Amphiphysin acts redundantly with other proteins to organize specialized membrane domains within a diverse array of cell types.

Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila.

Amphiphysins 1 and 2 are enriched in the mammalian brain and are proposed to recruit dynamin to sites of endocytosis. Shorter amphiphysin 2 splice variants are also found ubiquitously, with an enrichment in skeletal muscle. At the Drosophila larval neuromuscular junction, amphiphysin is localized postsynaptically and amphiphysin mutants have no major defects in neurotransmission; they are also viable, but flightless. Like mammalian amphiphysin 2 in muscles, Drosophila amphiphysin does not bind clathrin, but can tubulate lipids and is localized on T-tubules. Amphiphysin mutants have a novel phenotype, a severely disorganized T-tubule/sarcoplasmic reticulum system. We therefore propose that muscle amphiphysin is not involved in clathrin-mediated endocytosis, but in the structural organization of the membrane-bound compartments of the excitation-contraction coupling machinery of muscles.

A role for ultraspiracle, the Drosophila RXR, in morphogenetic furrow movement and photoreceptor cluster formation.

Many of the same genes needed for proper eye and limb development in vertebrates, such as hairy, hedgehog, patched and cyclic AMP-dependent protein kinase A, are responsible for patterning Drosophila imaginal discs, the tissues that will give rise to the adult cuticle structures. This is well demonstrated in the control of morphogenetic furrow movement and differentiation in the eye imaginal disc. We report that ultraspiracle, the gene encoding the Drosophila cognate of the Retinoid X Receptor, is required for normal morphogenetic furrow movement and ommatidial cluster formation. Examination of the expression of genes involved in regulating the furrow suggests that ultraspiracle defines a novel regulatory pathway in eye differentiation.

Seven-up inhibits ultraspiracle-based signaling pathways in vitro and in vivo.

Seven-up (Svp), the Drosophila homolog of the chicken ovalbumin upstream transcription factor (COUP-TF); Ultraspiracle (Usp), the Drosophila homolog of the retinoid X receptor; and the ecdysone receptor are all members of the nuclear/steroid receptor superfamily. COUP-TF negatively regulates hormonal signaling involving retinoid X receptor in tissue culture systems. Here we demonstrate that Svp, like COUP-TF, can modulate Ultraspiracle-based hormonal signaling both in vitro and in vivo. Transfection assays in CV-1 cells demonstrate that Seven-up can inhibit ecdysone-dependent transactivation by the ecdysone receptor complex, a heterodimeric complex of Usp and ecdysone receptor. This repression depends on the dose of Svp and occurs with two different Drosophila ecdysone response elements. Ectopic expression of Svp in vivo induces lethality during early metamorphosis, the time of maximal ecdysone responsiveness. Concomitant overexpression of Usp rescues the larvae from the lethal effects of Svp. DNA binding studies show that Svp can bind to various direct repeats of the sequence AGGTCA but cannot bind to one of the ecdysone response elements used in the transient transfection assays. Our results suggest that Svp-mediated repression can occur by both DNA binding competition and protein-protein interactions.

Identification and characterization of a Drosophila nuclear receptor with the ability to inhibit the ecdysone response.

In a search for retinoid X receptor-like molecules in Drosophila, we have identified an additional member of the nuclear receptor superfamily, XR78E/F. In the DNA-binding domain, XR78E/F is closely related to the mammalian receptor TR2, as well as to the nuclear receptors Coup-TF and Seven-up. We demonstrate that XR78E/F binds as a homodimer to direct repeats of the sequence AGGTCA. In transient transfection assays, XR78E/F represses ecdysone signaling in a DNA-binding-dependent fashion. XR78E/F has its highest expression in third-instar larvae and prepupae. These experiments suggest that XR78E/F may play a regulatory role in the transcriptional cascade triggered by the hormone ecdysone in Drosophila.

Possible involvement of the long terminal repeat of transposable element 17.6 in regulating expression of an insecticide resistance-associated P450 gene in Drosophila.

P450-A and P450-B are electrophoretically defined subsets of cytochrome P450 enzymes in Drosophila melanogaster. P450-A is present among all strains tested, whereas expression of P450-B is associated with resistance to insecticides. Monoclonal antibodies were used to obtain cDNA clones for an enzyme from each P450 subset (i.e., P450-A1 and P450-B1). The P450-B1 cDNA was sequenced and shown to code for a P450 of 507 amino acids. Its gene has been named CYP6A2. Comparative molecular analyses of a pair of susceptible, 91-C, and resistant, 91-R, Drosophila strains were made. There was 20-30 times more P450-B1 mRNA in 91-R than in 91-C, and the small amount of P450-B1 mRNA in 91-C was significantly larger in size than that in 91-R. The P450-B1 gene in 91-R was structurally different from that in 91-C but was not amplified. The P450-B1 gene in 91-C contained a solitary long terminal repeat of transposable element 17.6 in its 3' untranslated region. It was absent in the P450-B1 gene of 91-R. On the basis of features of the long terminal repeat and its location in the gene of the susceptible fly, we propose that a posttranscriptional mechanism involving mRNA stability could be involved in regulating P450-B1 gene expression.