Fig. 6.

TAT-5 localization to late endosomes increases in EV-releasing mutants. (A) GFP::TAT-5 (green) staining localizes primarily to the plasma membrane in control four-cell embryos. TAT-5 is also found in cytoplasmic vesicles, but they do not often colocalize with LMP-1 (red) staining in late endosomes or lysosomes, as shown in Insets. (B) Mislocalization of GFP::TAT-5 in rme-8 RNAi causes increased colocalization with LMP-1. (C) Large GFP::TAT-5-positive cytoplasmic vesicles colocalize with LMP-1 in a mon-2(xh22) mutant embryo treated with snx-6 RNAi. (A′–C′) Line scans of GFP::TAT-5 and LMP-1 intensity next to the dotted lines or circle in Insets. (D) The percentage of TAT-5 colocalized with LMP-1 is significantly increased in 2- to 12-cell embryos after rme-8 RNAi and in mon-2(xh22) mutants treated with snx-6 RNAi compared with control embryos. Number of embryos scored is indicated for each genotype. (E) The Pearson’s coefficient of GFP::TAT-5 colocalization with LMP-1 increases significantly from a negative correlation in control embryos to a positive correlation in rme-8 RNAi as well as in mon-2(xh22) mutants treated with snx-6 RNAi. (F) Cytoplasmic GFP::TAT-5 vesicles (green) rarely colocalize with mCherry::UNC-108 staining in Golgi and endosomes (red) in two-cell embryos, as shown in Insets. (G) TAT-5 colocalization with the Rab2 homolog UNC-108 increases after rme-8 RNAi. (H) TAT-5 colocalization with UNC-108 increased slightly in mon-2 mutants treated with snx-6 RNAi. (F′–H′) Line scans of GFP::TAT-5 and UNC-108 intensity next to the dotted lines or circle in Insets. (I) The percentage of TAT-5 colocalized with UNC-108 is significantly increased in 2- to 12-cell embryos after rme-8 RNAi and in mon-2(xh22) mutants treated with snx-6 RNAi compared with control embryos. Number of embryos scored is indicated for each genotype. (J) The Pearson’s coefficient of GFP::TAT-5 colocalization with LMP-1 increases significantly in rme-8 RNAi, but not in mon-2(xh22) mutants treated with snx-6 RNAi. (K) Enlarged GFP::TAT-5 vesicles colocalize significantly more often with LMP-1 (n = 54 vesicles) than the PI3P reporter 2xFYVE (n = 85) or UNC-108 (n = 89). Percentages are expressed in relation to the total number of large vesicles. Number of embryos scored is indicated for each genotype. Student’s t test with Bonferroni correction was used for statistical analysis. *P < 0.05; **P < 0.001. [Scale bars: A and F, 10 µm (also apply to B and C and G and H, respectively); Insets in A and F, 5 µm (also apply to Insets in B and C and G and H, respectively).]