Regulation of Rab5-positive vesicle formation in the endocytic compartment of fat cells.

(A) The Rab5:GFP tracer was expressed in control, w- or vap1 fat bodies using cg-Gal4. Mid-3rd instar larvae were starved for 4h before staining of the fat tissue with LysoTracker and live cells analysis. Compared to control, w- cells, vap1 cells shows enhanced vesicular Rab5 trafficking near the cell membrane (green arrows). Scale bars = 20 μm. (B, B’) Cortical and endocytic compartment organization of fat body cells. Control strain, w- was used to express a GFP:Spri transgene using a cg-Gal4 driver. Fed fat cells or 1h30’-starved cells of early-3rd instar larvae were analyzed after immunostaining for Rab5, and F-actin (Pha) plus nuclei (Hoe) labeling. Each panel shows: (Top) Z-sections images of dome-shaped fat cells reconstituted from serial XY optical sections exemplified below. (Below left images) Colored surface plan views shows the distribution of GFP:Spri present as spots close to the plasma membrane. (Below right images) Underneath plan section view of Rab5 (grey scale or red) carried along the dotted lines shown in the Z section views. Scale bars in Z and plan sections = 10 μm. The GFP:Spri labeling is imbricate with the cortical F-actin stain (blue) above the vesicular Rab5 staining (Rab5 signal overlapped 2,6% of GFP:Spri signal). Insets: high magnification images. Scale bars = 2.5 μm. In B’, Rab5-positive vesicles are clearly increased after short starvation period (Rab5 signal overlapped 11.7% of GFP:Spri signal). Scale bars as in B. (D-D”) GFP:Spri and myc-tagged Vap were coexpressed from an Act-Gal4 driver in fat cell clones of fed control, w- animals. A plan surface view reveals the GFP:Spri spots together with immunostained Vap:myc proteins in red (also shown in the respective gray scale images). Vap-specific staining overlapped 8% of GFP:Spri signal (yellow arrows) as determined in wide field images. Spri proteins were found engaged with several partners of the cell cortex [79] and only phospho-Tyrosylated Spri associated to Vap [48]. This may account for the relatively low coincidence of the two proteins. Scale bar = 2 μm. (C, C’) The density of endocytic Rab5-positive vesicles was quantified in control and mutant fat bodies. Experimental setting was exactly as in B, but used the genotype given below. Single plan images along the dotted line in the Z views were used to measure Rab5-positive vesicle densities (numbers of vesicles per μm2 of cellular area) as taken from wide field images. Values were normalized to the fed control, w-; cg-Gal4/+. Data using three different GFP:Spri transgenes were pooled. spri6G1-null cells were analyzed as above but the GFP:Spri transgene was omitted. In fed cells, the absence of vap causes in an elevation of ca. 2 fold of the Rab5-positive vesicle density, whereas the loss of spri results in a 6 fold reduction of vesicle density. Starvation is associated with a rise of 2.2 fold of vesicular Rab5 in control cells and this is not significantly different in the vap or spri mutant cells.(w- fed, n = 13; vap1 fed, n = 5; spri6G1 fed, n = 3; w- sta, n = 13; vap1 sta, n = 5; spri6G1 sta, n = 3). Error bars are standard errors; significances are from ANOVA. Genotypes. (A) Control: w1118/Y; cg-GAL4/ UAS-Rab5:GFP/+. Assay: vap1/Y; cg-GAL4/ UAS-Rab5:GFP/+. (B, B’) Assay: w1118/Y; cg-GAL4/ UASp-GFP:Spri9M. (C, C’) Control: w1118/Y; cg-GAL4/+; UASp-GFP7M or 8M or 9M/+. Assay: vap1/Y; cg-GAL4/+; UASp-GFP:Spri7M or 9M/+. spri6G1/Y; cg-GAL4/+. (D) Assay: w1118/ hsFLP12; UAS- Vap:myc16.4/ UASp-GFP:Spri9M; Act>CD2>GAL4/+.