Fig. 5.
Injected EE-SNARE containing LUVs fuse with endogenous endosomes. (A) Effect of NEM (10 µM) and of a dominant negative αSNAP mutant (L294A, coinjected at 10 mg/mL) on colocalization between injected 4-EE-SNARE LUVs and endogenous endosomes, labeled with Alexa 488-Tfn. (B) Snapshots from a movie (Movie S7) showing approach between an Alexa 488-Tfn–labeled endosome (white arrow) and a 4-EE-SNARE LUV (white arrowhead). After contact (time: 0 s), the red and green spot merged and moved together. (Scale bar, 2.5 µm.) Movement trajectories of the endosome (green) and the LUV (red) are shown in the Upper Right plot. After contact the movements are identical. (Scale bar, 1 µm.) Lower Right shows the fluorescence intensities of the tracked endosome (green) and the LUV (red). Shortly after contact there is a sharp drop in red fluorescence, probably caused by the dilution of the labeled lipid after fusion and thus indicative of fusion. Only a slight change was observed in the intensity of the green dye, possibly due to the fact that the dilution factors between the membrane and the content markers are different. (C) Snapshots from a movie (Movie S8) showing increase of calcein fluorescence due to dilution-induced fluorescence dequenching upon fusion of a liposome with an endogenous vesicle. An endosome (blue) docked to a calcein-containing LUV reconstituted with 4-EE-SNAREs (red) exhibited a sudden increase in calcein fluorescence (green) signal (0 s time point) in the endosome. (Scale bar, 2 µm.) The graph shows the fluorescence intensities of the tracked endosome, labeled with Alexa 633-Tfn (blue) and calcein (green). There is a sharp increase in green fluorescence, caused by the dilution of calcein by fusion (0 s time point). (D) C12FDG, a fluorescein-based β-galactosidase substrate, was loaded into 4-EE-SNARE LUVs, followed by injection into HeLa cells that were preincubated with a β-galactosidase–Tfn fusion protein and with Alexa 633-Tfn. The cells were incubated for 15 min and fixed. A fluorescein (green) signal, generated by cleavage of C12FDG, was detectable in Alexa633-Tfn–positive endosomes (blue), whereas no such signal was observed when the injected and preloaded liposomes did not contain the four EE-SNARE proteins. (Scale bar, 5 µm.) (E) The fluorescent pH sensor pHrodo was incorporated into 4-EE-SNARE LUVs that were also labeled with 0.3% nitrobenzoxadiazole (NBD)-PE. These liposomes were injected into HeLa cells preincubated with Alexa 633-Tfn to label endogenous endosomes. Fifteen minutes after injection, an increase in pHrodo fluorescence was observable that overlapped with Alexa 633 and that was absent when the SNARE proteins were omitted. (Scale bar, 5 µm.) (F) The increase in pHrodo fluorescence is sensitive to the V-ATPase inhibitor bafilomycin. Cells injected with 4-EE-SNARE LUVs containing pHrodo were incubated for 15 min and then the capturing of time-lapse imaging was started. After addition of bafilomycin (200 nM) a decrease in fluorescence intensity was observable. The plot shows fluorescence intensity changes of pHrodo in individual cells before and 30 s after addition of bafilomycin. For all plots in this figure, error bars indicate SEM, *P < 0.05, **P < 0.01, ***P < 0.001, determined by unpaired t test.