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First published online December 5, 2007
doi: 10.1242/10.1242/jcs.012336

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Drosophila Vps35 function is necessary for normal endocytic trafficking and actin cytoskeleton organisation

Viktor I. Korolchuk^*,,, Martin M. Schütz^*,¶, Carolina Gómez-Llorente, João Rocha, Nico R. Lansu^**, Stephanie M. Collins, Yogesh P. Wairkar, Iain M. Robinson^¶¶ and Cahir J. O'Kane

Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK

View larger version (39K): [in this window] [in a new window]   Fig. 1. Uptake of mBSA-Texas-Red by clathrin-mediated endocytosis in Drosophila S2 cells. (A) Schematic diagram of the assay. (B) Representative images of cells treated with RNAi and assayed for uptake of mBSA-Texas-Red. Maximum projection of several Z-sections collected using a wide-field fluorescence microscope is shown. Dynamin, -adaptin and Chc images have approximately the same number of cells per field as control ones (not shown). (C) Fluorescence levels in cells treated with RNAi against known or proposed endocytic proteins. Quantification was performed using data from 3-4 experiments, each averaged from 10-12 images (average integrated fluorescence per cell), normalised using untreated cells (`Water') in the same experiment, and compared statistically with untreated cells. In this and all subsequent figures, *P<0.05, **P<0.01, ***P<0.005, Student's t-test; all other comparisons are not significant (NS); error bars indicate s.e.m.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Efficiency of protein knockdown. (A) Representative images of cells immunostained with antibodies against endocytic proteins 3-4 days after RNAi (RNAi, bottom row) or cells left untreated (Control, top row). Maximum-intensity projections of several Z-sections, collected using fluorescent microscopy, are shown. (B) Protein levels in S2 cells after RNAi knockdown. Images similar to those in A were used to quantify average fluorescence per cell, data represent mean ± s.e.m. of three independent experiments. (C) Primary haemocytes (inside dotted circles) from eps15 and endoA mutant animals internalise mBSA normally. Haemocytes harvested from wild-type (WT), eps15 (EP(2)2513), endoA (endoA1) and dynamin (shits1) mutant third instar larvae (Guha et al., 2003) were incubated with mBSA-Texas-Red for 2 minutes, followed by a 4-minute chase. Assays were performed at 35°C with WT and shits1 cells, and at room temperature with eps15 and endoA mutant cells.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Vps35 knockdown affects endocytosis and tagged Vps35 localises to endosomes in S2 cells. (A) Uptake of fluorescent mBSA by cells after RNAi knockdown of novel candidate endocytic proteins. Only knockdowns that significantly inhibited endocytosis and relevant control data are shown; the complete data set is presented in supplementary material Fig. S1. Comparisons are to water control. (B) Single confocal sections showing localisation of Vps35-mRFP (magenta) in S2 cells. Colocalisation with markers of early endosome (anti-Rab5), late endosome (Rab7-EGFP) late endosome and lysosome (spinster-EGFP) and recycling endosome (Rab11 antibody) compartments (green) is shown. Cells were co-transfected with each combination of plasmids for 24 hours and expression was induced with 1 mM CuSO4 for 4 hours before imaging.

View larger version (30K): [in this window] [in a new window]   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.

View larger version (75K): [in this window] [in a new window]   Fig. 5. Proliferation and signalling defects in vps35 mutant haemocytes. (A) Mutant vps35 third instar larvae develop melanotic masses. One WT and two vps35 animals are shown. (B) Representative DIC images of haemocytes isolated from WT or vps35 third instar larvae. Note increased numbers of small round cells (plasmatocytes) in vps35 larvae, and flat lamellocytes (arrows) that are absent in WT haemolymph. (C) Increased numbers of haemocytes extracted from vps35/Df larvae, relative to WT, and rescue using Hml-Gal4. Numbers are normalised relative to WT; n=12-15 images from six larvae; mean ± s.e.m. (D) Toll signalling is upregulated in vps35 mutant haemocytes, judged by increased levels of Toll (arrowhead indicates leading edge of lamellopodia that is labelled with Toll in vps35 but not in WT cell) and nuclear localisation of Dorsal. Dashed circles demarcate nuclei (as defined by bright field image). (E) Toll levels per cell; n=14 cells from five larvae. (F) Percentage of cells in which Dorsal is enriched in nuclei. n300 cells isolated from six larvae; mean ± s.e.m.

View larger version (42K): [in this window] [in a new window]   Fig. 6. NMJ bouton phenotype of vps35. (A) Projections of confocal sections of wild-type (WT) and vps35 NMJs stained for the presynaptic marker Eps15 (green) and the largely postsynaptic marker Dlg (magenta). Insets show higher magnification of boxed areas. Arrowheads indicate examples of satellite boutons in vps35 NMJs, that are rare in WT; note that Eps15 labelling in these is completely surrounded by Dlg. (B) vps35 mutant larvae have approximately twice as many boutons as WT. The defect is partially rescued when Vps35 expression is driven by elav-Gal4 or BG57-Gal4 and completely rescued using a combination of these drivers. n=5-12 NMJs from 5-10 larvae; mean ± s.e.m. (C) Single confocal sections of NMJ boutons showing upregulation of pMad in vps35 mutant NMJs. Note the pMad puncta (green) predominantly at the inner surface of the mainly postsynaptic Dlg staining (magenta). (D) Quantification of pMad levels at the NMJ (n=10 boutons from five animals; mean ± s.e.m.). (E) The supernumerary bouton phenotype of vps35 larvae is suppressed by removal of a single copy of the BMP signalling components wit or Mad (wit/+, Mad/+ respectively) and more strongly by complete loss of Mad (Mad). n=5-11 NMJs from 5-9 larvae; statistical comparisons are with vps35. NMJs from muscles 6 and 7 in abdominal segment A3 were used for all analyses; mean ± s.e.m.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Involvement of actin cytoskeleton in vps35 mutant phenotypes. (A) Labelling of F-actin in WT and vps35 haemocytes, visualised using single confocal sections as in Fig. 4E. Arrow indicates plasma membrane surrounding the body of the cell. Arrowhead indicates leading edge of lamellopodia. (B) F-actin levels in WT, vps35, vps35/Df, vps35/Df;Hml-Gal4 and vps35/Df; Rac1/+ haemocytes and in vps35 haemocytes treated with 0.1 mM cytochalasin D (cytoD) (average fluorescence per cell). n=25-30 cells from four larvae; statistical comparisons are with vps35/Df. (C) Removal of a single copy of Rac1, or treatment with 0.1 mM cytochalasin D, partially rescues the loss of mBSA-Texas-Red uptake in haemocytes that lack Vps35. n=25-30 cells from four larvae. (D) The vps/Df supernumerary bouton phenotype is suppressed by removal of a single copy of Rac1, or by presynaptic or postsynaptic expression of dominant-negative Rac1 (UAS-Rac1.N17). Note that elav-GAL4 and BG57-GAL4 also drive Vps35 expression and partially rescue the vps35 mutant phenotype (see Fig. 6B). NMJs from muscles 6 and 7 in abdominal segment 3 were used for all analyses; n=3-8 larvae. All statistical comparisons are with WT except where indicated. All data are mean ± s.e.m.