ATL2-2 supports content mixing. (A) Schematic representation of the content mixing assay using ATL2-containing liposomes. (B) ATL2-bearing liposomes containing Biotin-PhycoE and ATL2-bearing liposomes containing Cy5-SA were mixed and incubated at 30°C for 10 min. Reactions were further incubated in the absence or presence of GTP or a GTP analog at 30°C for 30 min, and FRET signals between phycoerythrin and Cy5 were measured every minute for 30 min. To determine total fluorescence, 1% Thesit was added to the mixture of ATL2-bearing liposomes containing Biotin-PhycoE and ATL2-bearing liposomes containing Cy5-SA in the absence of streptavidin. Experiments were performed multiple times, and representative data are shown. (C) Experiments were performed as described in B. Fusion after incubation for 20 min was determined. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (D) Content mixing assays were performed in the presence of GTP and increasing concentrations of an anti-ATL2 antibody. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (E) The short isoform [ATL3 (19–541)] of ATL3 failed to support liposome fusion. Donor and acceptor liposomes bearing the long isoform [ATL3 (1–541)] or the short isoform [ATL3 (19–541)] of ATL3 at a protein:lipid ratio of 1:1,000 were mixed and incubated in the absence or presence of GTP. Fusion is represented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ****P < 0.0001, two-tailed unpaired Student’s t test. (F) Donor and acceptor liposomes bearing wild-type ATL3 (the long isoform) or mutant ATL3 (Y192C), which causes sensory neurodegeneration, at a protein/lipid ratio of 1:1,000 were mixed and incubated in the absence or presence of GTP. Fusion is represented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ***P < 0.001, two-way ANOVA with Tukey’s multiple comparisons test.
Figure 10.

ATL2-2 supports content mixing. (A) Schematic representation of the content mixing assay using ATL2-containing liposomes. (B) ATL2-bearing liposomes containing Biotin-PhycoE and ATL2-bearing liposomes containing Cy5-SA were mixed and incubated at 30°C for 10 min. Reactions were further incubated in the absence or presence of GTP or a GTP analog at 30°C for 30 min, and FRET signals between phycoerythrin and Cy5 were measured every minute for 30 min. To determine total fluorescence, 1% Thesit was added to the mixture of ATL2-bearing liposomes containing Biotin-PhycoE and ATL2-bearing liposomes containing Cy5-SA in the absence of streptavidin. Experiments were performed multiple times, and representative data are shown. (C) Experiments were performed as described in B. Fusion after incubation for 20 min was determined. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (D) Content mixing assays were performed in the presence of GTP and increasing concentrations of an anti-ATL2 antibody. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (E) The short isoform [ATL3 (19–541)] of ATL3 failed to support liposome fusion. Donor and acceptor liposomes bearing the long isoform [ATL3 (1–541)] or the short isoform [ATL3 (19–541)] of ATL3 at a protein:lipid ratio of 1:1,000 were mixed and incubated in the absence or presence of GTP. Fusion is represented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ****P < 0.0001, two-tailed unpaired Student’s t test. (F) Donor and acceptor liposomes bearing wild-type ATL3 (the long isoform) or mutant ATL3 (Y192C), which causes sensory neurodegeneration, at a protein/lipid ratio of 1:1,000 were mixed and incubated in the absence or presence of GTP. Fusion is represented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ***P < 0.001, two-way ANOVA with Tukey’s multiple comparisons test.

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