Synchronization of norrin secretion using the RUSH system. (
A) Schematic of the RUSH plasmid construct and system. Under physiological conditions, Ii-streptavidin-HA binds to NDP-SBP-EYFP to retain the protein in the ER. After addition of 40 µM biotin, NDP-SBP-EYFP is released and transported to the Golgi and subsequently to the plasma membrane. (
B) Fluorescence images of HeLa cells expressing RUSH-NDP-EYFP at various time points after biotin addition (from
Supplementary Video S1). At time 00:00 (minutes:seconds) of biotin addition, norrin remained in the ER. At time 15:00, Norrin starts to leave the ER and can be seen in the juxtanuclear region. At around time 25:00, Norrin is seen entirely in the juxtanuclear region. Starting around 35:00 Norrin can be seen in vesicle exiting the juxtanuclear region and moving toward the periphery. By time 65:00, the juxtanuclear region appears to be depleted of Norrin. (
C) HeLa cells were transfected with RUSH plasmids encoding WT Norrin or mutants. The localization of the different versions of RUSH constructs was analyzed after incubation with biotin for the indicated time. The highlighted and magnified area in the
red box indicates the presence of Norrin in the juxtanuclear region (Scale bar = 10 µm). (
D) Quantifications of the percentage of cells showing juxta-nuclear–accumulated EYFP signal after incubation with biotin for the indicated time (mean ± SD;
n = 3; >50 cells counted for each time point). (
E) Quantification of the time from the appearance to the disappearance of the juxta-nuclear region EYFP signal, mean ± SD;
n = 50 cells counted for each condition. (
F) Representative immunofluorescence images of Ii-streptavidin-HA (anti-HA,
magenta), NDP-SBP-EYFP (anti-GFP,
green) in HeLa cells transfected with RUSH constructs (WT or mutants) before incubation with biotin (Scale bar = 10 µm). (
G) Representative immunofluorescence images of Ii-streptavidin-HA (anti-HA,
magenta), NDP-SBP-EYFP (anti-GFP,
green) and GM130 (anti-GM130,
red) in HeLa cells transfected with RUSH constructs (WT or mutants) after incubation with biotin for 25 minutes (Scale bar = 10 µm). Two-way ANOVA with Dunnett's multiple comparisons test was performed to compare each mutant and WT at the indicated time for (
D). One-way ANOVA with Tukey's multiple comparisons test was performed to compare each construct at the indicated time for (
E). ****
P < 0.0001