(A and B) Using a 3-step serial recombineering strategy, two genomic P[acman] clones were upgraded with a fluorescent tag, either on the N (A) or C terminus (B), and a selection marker expression cassette conferring resistance to G418 or blasticidin S. The bacterial ampicillin resistance marker (AmpR) was used to select successful recombineering reactions in the first step. This marker is removed via Cre-recombinase-mediated reduction in the third step. A short, flexible peptide sequence links the fluorescent tag in frame to the encoded gene (Figure S8).
(C and D) Resulting selected flies were verified via immunofluorescent staining for tagged P[acman] BAC products.
(C) Staining data for N-terminal EGFP tagged cystein string protein (Csp) shows strong neuropil expression throughout the adult fly brain (C′). In the larval brain, staining against tagged Csp shows clear neuropil expression in the visual system, including the optic lobes (OLs) as well as expression in the VNC (C″).
(D) Staining for C terminus mCherry-tagged Neurexin IV (NrxIV) shows brain surface glial expression in the adult fly brain (D′). Similarly, staining shows glial expression in the larval brain with clear labeling of septate junctions formed at the borders of the surface subperineural glial (arrows) in the cerebral hemispheres and VNC (D″). Scale bars represent 50 μm.
(E) Schematic of the workflow of the co-immunoprecipitation of GFP-tagged Csp from isolated fly heads followed by proteomic analysis.
(F) Volcano plot shows differentially enriched proteins (by NCBI gene symbol) between tagged Csp and GFP control pull-downs. Colored dots represent peptides (291/624) that were found to be significantly different between the two groups at a false discovery rate (FDR) < 0.05 and enrichment of ≥4 fold.
(G) Interactome analysis was performed with STRING. Shown is a selected interactome focusing on Csp and putative connections to vesicle recycling (purple) and other neuronal proteins (teal). Proteins in olive indicate known protein-protein interactions with Csp.
See also Figures S8 and S9.