A forward genetic screen in Drosophila melanogaster to identify mutations affecting INAD localization in photoreceptor cells.

In Drosophila photoreceptors, the multivalent PDZ protein INAD interacts with multiple signaling components and localizes complexes to the rhabdomere, a subcellular compartment specialized for phototransduction. Since this localization is critical for signaling, we conducted a genetic screen of the third chromosome for mutations that result in mislocalization of an INAD-GFP fusion protein. We identified seven mutant lines that fall into two complementation groups, idl (INAD localization)-A and idl-B. We show that idl-A mutants fail to complement with chaoptic (chp) mutants. Since chaoptin is a structural component of the rhabdomere, mislocalization of INAD may be a secondary effect of the retinal degeneration in chp and idl-A mutants. Genetic complementation and DNA sequencing reveal that the two idl-B mutants represent new alleles of trp, a gene encoding the major light-activated channel. The molecular change in each allele affects a highly conserved residue in either an ankyrin domain on the N-terminus or in the S6 transmembrane domain of TRP. These changes lead to the loss of TRP protein. TRP has previously been shown to anchor INAD in the rhabdomeres, therefore the independent identification of two trp alleles validates our screen for INAD-GFP localization. One possibility is that a limited number of proteins are required for localizing INAD-signaling complexes. A similar screen of the X and second chromosomes may be required to find the remaining players involved.

DLGS97/SAP97 is developmentally upregulated and is required for complex adult behaviors and synapse morphology and function.

The synaptic membrane-associated guanylate kinase (MAGUK) scaffolding protein family is thought to play key roles in synapse assembly and synaptic plasticity. Evidence supporting these roles in vivo is scarce, as a consequence of gene redundancy in mammals. The genome of Drosophila contains only one MAGUK gene, discs large (dlg), from which two major proteins originate: DLGA [PSD95 (postsynaptic density 95)-like] and DLGS97 [SAP97 (synapse-associated protein)-like]. These differ only by the inclusion in DLGS97 of an L27 domain, important for the formation of supramolecular assemblies. Known dlg mutations affect both forms and are lethal at larval stages attributable to tumoral overgrowth of epithelia. We generated independent null mutations for each, dlgA and dlgS97. These allowed unveiling of a shift in expression during the development of the nervous system: predominant expression of DLGA in the embryo, balanced expression of both during larval stages, and almost exclusive DLGS97 expression in the adult brain. Loss of embryonic DLGS97 does not alter the development of the nervous system. At larval stages, DLGA and DLGS97 fulfill both unique and partially redundant functions in the neuromuscular junction. Contrary to dlg and dlgA mutants, dlgS97 mutants are viable to adulthood, but they exhibit marked alterations in complex behaviors such as phototaxis, circadian activity, and courtship, whereas simpler behaviors like locomotion and odor and light perception are spared. We propose that the increased repertoire of associations of a synaptic scaffold protein given by an additional domain of protein-protein interaction underlies its ability to integrate molecular networks required for complex functions in adult synapses.

Cell-nonautonomous function of ceramidase in photoreceptor homeostasis.

Neutral ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila ceramidase. We show that secreted ceramidase functions in a cell-nonautonomous manner to maintain photoreceptor homeostasis. In the absence of ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of rhodopsin, and do not respond to light stimulus. Consistent with a cell-nonautonomous function, overexpression of ceramidase in tissues distant from photoreceptors suppresses photoreceptor degeneration in an arrestin mutant and facilitates membrane turnover in a rhodopsin null mutant. Furthermore, our results show that secreted ceramidase is internalized and localizes to endosomes. Our findings establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function.

Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors.

Here, we reveal a novel feature of the dynamic organization of signaling components in Drosophila photoreceptors. We show that the multi-PDZ protein INAD and its target proteins undergo light-induced recruitment to detergent-resistant membrane (DRM) rafts. Reduction of ergosterol, considered to be a key component of lipid rafts in Drosophila, resulted in a loss of INAD-signaling complexes associated with DRM fractions. Genetic analysis demonstrated that translocation of INAD-signaling complexes to DRM rafts requires activation of the entire phototransduction cascade, while constitutive activation of the light-activated channels resulted in recruitment of complexes to DRM rafts in the dark. Mutations affecting INAD and TRP showed that PDZ4 and PDZ5 domains of INAD, as well as the INAD-TRP interaction, are required for translocation of components to DRM rafts. Finally, selective recruitment of phosphorylated, and therefore activatable, eye-PKC to DRM rafts suggests that DRM domains are likely to function in signaling, rather than trafficking.