Live-cell imaging of circular and long noncoding RNAs associated with FUS pathological aggregates by Pepper fluorescent RNA

Visualization of exogenous circ-HDGFRP3 colocalizing with FUS^P525L aggregates in living HEK 293T cells. (A) Representative HEK 293T cells transfected with p-circ-Ex4 expressing both circ-HDGFRP3 labeled with HBC620 (magenta) and GFP-FUS^P525L (green). Lower panel shows a magnification view of the green dotted box with some circ-HDGFRP3 and FUS^P525L particles interacting over time (duration 10 min; interval ∼11 sec). Spots and tracks detected with TrackMate Plug-in on Fiji-ImageJ are overlaid respectively as magenta (circ-HDGFRP3) and green (FUS^P525L) circles and lines. Scale bars, 5 μm. (B) Scatter interval plot representing circ-HDGFRP3 (magenta) and ROMO1 (gray) fraction of interacting particles with FUS^P525L over time. Dots represent individual measurements of number of interacting particles/total number of particles per cell in an interval of 1 min. Error bars represent ± SEM. Magenta and black lines represent mean values. Average and SEM values were calculated from 10 time-lapse measurements (duration 10 min; interval 11–13 sec) collected from four independent biological replicates for circ-HDGFRP3/FUS^P525L interactions and from six measurements from two independent biological replicates for ROMO1/FUS^P525L interactions. (C) Single-particle tracking of circ-HDGFRP3 and FUS^P525L particles shown in A over time. The plot indicates circ-HDGFRP3 (magenta) and FUS^P525L (green) xy-coordinates (micron) as a function of time (min). (D) Representative FUS^P525L aggregate containing circ-HDGFRP3. Starting from left, signals of circ-HDGFRP3 labeled with HBC620 imaged with Widefield or with 3D-SIM are compared. Intensity profiles (Arbitrary Unit) along the reference blue dotted line of circ-HDGFRP3 signal imaged with Widefield (cyan) or with 3D-SIM (magenta) microscopy are plotted in second panel. Third panel shows FUS^P525L signal (green) merged with circ-HDGFRP3 signal (magenta) imaged with 3D-SIM. Respective resolution values calculated on the image are indicated at the bottom of the image. In the last panel, intensity profiles (arbitrary unit) of circ-HDGFRP3 and FUS^P525L imaged with 3D-SIM along the blue dotted line are represented. Scale bars, 1 μm.