Immunocytochemical Localization of Synaptic Proteins to Photoreceptor Synapses of Drosophila Melanogaster

The location of proteins that contribute to synaptic function has been widely studied in vertebrate synapses, far more than at model synapses of the genetically manipulable fruit fly, Drosophila melanogaster. Drosophila photoreceptor terminals have been extensively exploited to characterize the actions of synaptic genes, and their distinct and repetitive synaptic ultrastructure is anatomically well suited for such studies. Synaptic release sites include a bipartite T-bar ribbon, comprising a platform surmounting a pedestal. So far, little is known about the composition and precise location of proteins at either the T-bar ribbon or its associated synaptic organelles, knowledge of which is required to understand many details of synaptic function. We studied the localization of candidate proteins to pre- or postsynaptic organelles, by using immuno-electron microscopy with the pre-embedding method, after first validating immunolabeling by confocal microscopy. We used monoclonal antibodies against Bruchpilot, epidermal growth factor receptor pathway substrate clone 15 (EPS-15), and cysteine string protein (CSP), all raised against a fly head homogenate, as well as sea urchin kinesin (antibody SUK4) and Discs large (DLG). All these antibodies labeled distinct synaptic structures in photoreceptor terminals in the first optic neuropil, the lamina, as did rabbit anti-DPAK (Drosophila p21 activated kinase) and anti-Dynamin. Validating reports from light microscopy, immunoreactivity to Bruchpilot localized to the edge of the platform, and immunoreactivity to SUK4 localized to the pedestal of the T-bar ribbon. Anti-DLG recognized the photoreceptor head of capitate projections, invaginating organelles from surrounding glia. For synaptic vesicles, immunoreactivity to EPS-15 localized to sites of endocytosis, and anti-CSP labeled vesicles lying close to the T-bar ribbon. These results provide markers for synaptic sites, and a basis for further functional studies.