Fig. 8

Overexpression of TSPAN6 compromises autophagy. a HEK293 cells overexpressing TSPAN6 analyzed by TEM show phagosomes (aa) multilamellar bodies (ad) and large vacuoles containing undegraded heterogeneous material in the lumen (ab-ac, arrowheads). In comparison, HEK293 cells expressing an empty vector present a reduced number of enlarged vesicles with homogeneous luminal material (ae-af, empty arrowheads). Nucleus is indicated with an N. b Representative experiment showing autophagy induction in control (empty vector) or TSPAN6-overexpressing HEK293 cells by 2 h and 4 h starvation. Lysates were analyzed by western blot with antibodies against TSPAN6, APP, P62, LC3β, PS1-CTF and actin. TSPAN6-overexpressing cells show no degradation of APP-CTF after 4 h starvation compared to control cells. Activation of autophagy was assessed by conversion of LC3-I into LC3-II and the degradation of P62. The levels of PS1-CTF and actin were used as negative controls. c Quantification of APP-CTF levels from western blots like in panel b (n = 3 experiments). d Quantification of P62 levels from western blots like in panel b (n = 3 experiments). e HEK293 cells co-transfected with LC3-GFP-RFP and an empty vector or myc-TSPAN6 were starved for 3 h to induce autophagy. The fusion of autophagosomes (red and green) with lysosomes (red) is reflected in the loss of the fluorescence of GFP protein due to the low pH found in lysosomes. TSPAN6 overexpression is detected with an anti-myc antibody (blue) (scale bar = 10 μm). f Ratio between the number of autophagosomes fused to lysosomes versus the total number of autophagosomes (2 independent experiment; n = 43 autophagosomes for control; n = 43 autophagosomes for TSPAN6). g The number of phagosomes not fused to lysosomes (red and green) per cell is increased in cells overexpressing TSPAN6 (2 independent experiments, n = 59 cells for control; n = 80 cells for TSPAN6). h Scheme showing the cellular pathways affected by TSPAN6. 1-Increased levels of TSPAN6 increment the secretion of exosomes by a syntenin-dependent mechanism. 2-TSPAN6 impairs the fusion between autophagosomes and lysosomes affecting the cellular proteostasis. Statistical significance was determined by t- test (*p <0.05, **p <0.01, ***p <0.001), Ɨ p <0.05)