Figure 6.

Expression of GFP-DGKα affects the formation of mature MVBs and the distribution of LBPA⁺ vesicles, but does not inhibit the accumulation of CD63 at the plasma membrane. Jurkat cells expressing GFP-VPSwt, GFP-VPS4EQ mutant or GFP-DGKα, together with CFP-CD63, were stimulated with CCh to induce the formation and traffic of MVBs, and LBPA was stained as indicated in Materials and Methods. The endosomal sorting complex required for transport (ESCRT) machinery is involved in the formation of the intraluminal vesicles of MVBs, and the AAA-ATPase vacuolar protein-sorting VPS4 regulates the recycling of the ESCRT complexes.³⁴ The expression of a VPS4 ATPase-defective mutant, VPS4EQ, inhibits the inward budding at the limiting membrane of MVBs and the subsequent formation of ILVs.^{22, 35} These effects result in the formation of immature, large endosomes,^{22, 23} and thus the expression of VPS4EQ mutant constitutes an appropriate control for aberrant MVBs maturation. (a) Epifluorescence microscopy images (GFP-X, CFP-CD63 and LBPA, respectively) of control and CCh-stimulated (8 h) cells. In GFP-VPS4EQ-expressing cells, the dispersion of LBPA⁺ granules that was induced by CCh in GFP-VPS4EQ cells or in cells expressing GFP-VPS4wt was inhibited, and the large LBPA⁺ structures accumulated in the perinuclear area. (b) Cells expressing GFP-DGKα (upper rows) or GFP-VPS4EQ (lower rows), together with CFP-CD63 were stimulated with CCh for the indicated times to visualise the accumulation of CFP-CD63 at the plasma membrane by fluorescence microscopy. CD63 cell surface labelling is a consequence of the transport, docking and fusion of MVBs with the plasma membrane (see also Figure 5b, Supplementary Videos 3 and 4). (c) The average number per cell and the mean diameter (±S.D.) of LBPA⁺ vesicles were measured in cells from three independent experiments similar to that described in panel a (in a total of 11 control, untransfected cells; 8 GFP-DGKα⁺ cells; and 19 GFP-VPS4EQ⁺ cells) after 6 h of CCh stimulation. In a significant fraction (40–50%, n=20 cells) of GFP-DGKα-expressing cells, the number of LBPA⁺ vesicles was lower to that found in GFP-DGKα⁻ cells stimulated with CCh (Supplementary Figure S5, panel A, third row). In approximately 20% of GFP-DGKα⁺ cells, the LBPA⁺ structures were condensed in some areas but not dispersed throughout the cytosol (Supplementary Figure S5, panel A, lower row), as observed in GFP-VPS4EQ⁺ cells (Supplementary Figure S5, panel A, second row). See also Supplementary Figure S5, panel C