Figure 1.
ReAsH labeling of Tc-tagged RyR1.
(A) ReAsH binding to an optimized Tc tag attached to a YFP-RyR1 fusion protein (FLN(YFP)RyR1) was assessed via determination of YFP/ReAsH colocalization in intact cells. Black bar indicates RyR1 primary sequence. (B) ReAsH labeling timecourse of HEK-293T cells expressing FLN(YFP)RyR1. YFP (upper left panel) and ReAsH fluorescence (subsequent panels) was recorded at the indicated timepoints after addition of ReAsH. Arrowheads indicate same cells in all panels. ReAsH labeling timecourse is also presented as aggregate ReAsH fluorescence at each timepoint normalized to initial fluorescence 5 min after ReAsH addition (F/F0) (lower right).
Figure 2.
Removal of nonspecific ReAsH labeling using BAL.
(A) HEK-293T cells expressing FLN(YFP)RyR1 labeled with ReAsH were incubated with 0.25 mM BAL. YFP (top left panel) or ReAsH fluorescence (subsequent panels) was recorded at the timepoints indicated after addition of BAL. Colocalization of YFP and ReAsH in these cells was plainly evident after 10′ incubation in BAL. (B) YFP (top panel) and ReAsH fluorescence (bottom) for ReAsH-treated HEK-293T cells expressing (YFP)RyR1 (which lacks a Tc tag) were recorded 15′ after treatment with 0.25 mM BAL. No specific YFP/ReAsH colocalization was observed. Arrows indicate same cells in both images in (B).
Figure 3.
FRET measurements of FLN(YFP)RyR1 labeled with ReAsH.
(A) BAL-washed HEK-293T cells expressing ReAsH-labeled FLN(YFP)RyR1 were examined for YFP (top panels) and ReAsH fluorescence (bottom panels) both before (left panels) and after (right panels) photobleaching of ReAsH. Donor fluorescence increased after acceptor photobleaching due to FRET only for those cells initially labeled with ReAsH (arrows) whereas cells that were not initially ReAsH-labeled did not display FRET (asterisks). (B) Mean FRET efficiencies are shown for FLN(YFP)RyR1 (black bar) or (YFP)RyR1 that had either been postwashed with 0.25 mM BAL for 15′ (green bar) or that was not treated with BAL (blue bar). Values represent mean +/− S.E.M. for the number of cells indicated in each bar.
Figure 4.
ReAsH binding stability to two different Tc tags on RyR1.
HEK-293T cells expressing FLN(YFP)RyR1 or CC(YFP)RyR1 labeled with ReAsH were incubated in increasing [BAL] for 15′ followed by quantification of YFP and ReAsH fluorescence. Values represent mean ReAsH/YFP fluorescence ratio normalized to this ratio acquired at 0 BAL for 9–12 cells per datum point.
Figure 5.
FlAsH labeling of Tc-tagged RyR1.
(A) Scheme for FlAsH labeling of RyR1 containing an optimized tetracysteine tag at the N-terminus (FLNRyR1). (B) Cells expressing FLNRyR1 were labeled with FlAsH and then washed with either 1 mM (left panels) or 10 mM BAL (right panels). Panels depict either the FlAsH label (top panels) or RyR1 detected via immunocytochemical analysis using the 34 C anti-RyR antibody (bottom panels). Arrows depict same cells in both the top and bottom panels of each set.
Figure 6.
FRET analysis of FlAsH-labeled RyR1 constructs.
HEK-293T cells expressing either FLN(+His)RyR1 (A) or FLNRyR1 (B) were labeled with FlAsH (acting as FRET donor) and Cy3NTA (FRET acceptor). FlAsH fluorescence was recorded before (top panels) and after (bottom panels) photobleaching of the Cy3NTA acceptor. FRET was observed as an increase in FlAsH fluorescence after acceptor photobleaching. (C) Mean FRET efficiencies are shown for FlAsH-labeled FLN(+His)RyR1 and FLNRyR1 using Cy3NTA as FRET acceptor. Values represent mean +/− S.E.M for the number of cells indicated in each bar.