Fig. 4. Vps35 is required for efficient endocytosis in non-neuronal cells but not for SV endocytosis. (A) Map of Drosophila vps35, showing exons with coding region (black boxes) and UTRs (open boxes), and insertion P{EPgy2}EY14200 in the 5' UTR. The sequence of cDNA clone RE65032, and alignment with the genomic sequence suggests that Drosophila vps35 has nine exons, with the coding sequence stretching from nucleotide 121 of RE65032, at the start of exon 2, to a stop codon at nucleotide 2524 in exon 8. (B) Levels of vps35 mRNA determined using real-time PCR, normalised to the wild type (WT). n=2. (C) Representative images of WT and vps35/Df mutant haemocytes (inside dotted circles) after mBSA-Texas-Red uptake (maximum-intensity projections of confocal stacks). (D) mBSA-Texas-Red uptake in haemocytes from animals with genotypes as shown. All comparisons are with WT; n=12-24 cells from 6-9 larvae; mean ± s.e.m. (E) Labelling of clathrin heavy chain (Chc) and the scavenger receptor Crq in WT and vps35 primary haemocytes, visualised using single confocal sections taken at equal distances (1 µm) from the base of each cell. Arrows indicate plasma membrane surrounding the body of the cell and arrowheads the leading edge of lamellopodia. (F) Representative images of NMJ boutons labelled with FM1-43FX in a 10-minute loading protocol. (G) FM1-43FX dye internalisation in WT and vps35 NMJs after 10 minutes or 5 seconds of loading. Non-specific staining of WT boutons in Ca²⁺-free medium was used as a background control. n=5-7 NMJs; mean ± s.e.m.