Raw single-molecule imaging data for "Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription"

Raw single-molecule imaging data for "Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription"

Shasha Chong1, Thomas G.W. Graham2, Claire Dugast-Darzacq2,5, Gina M. Dailey2, Xavier Darzacq2,5, Robert Tjian2,3,4,5*

1 Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA

2 Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.

3 Howard Hughes Medical Institute, University of California, Berkeley, CA, USA.

4 Li Ka Shing Center for Biomedical & Health Sciences, University of California, Berkeley, CA, USA.

5 CIRM Center of Excellence, University of California, Berkeley, CA. 

* Lead contact

Overview

This repository contains 1) movies of endogenously expressed EWS::FLI1-Halo in genome-edited A673 cells acquired using stroboscopic photo-activatable single particle tracking (spaSPT) and 2) images of exogenously expressed mNeonGreen-EWS-NPM1 fusion protein in the above cells before and after spaSPT movies were acquired. The uploaded files include data acquired from 80 live cells on 4 different days. The imaging data, after being processed, were used to generate Figure 4C-E of the manuscript in the title. 

Method details

The genome-edited A673 cells (described in https://www.science.org/doi/10.1126/science.aar2555) with inducible expression of mNeonGreen-EWS-NPM1 were grown on 25 mm circular No. 1.5 cover glasses (Azer Scientific, 200251) that were plasma-cleaned prior to use. We induced the cells with 200 ng/ml of doxycycline for 96 hours, stained the cells with 20 nM PA-JF646 and 200 nM JFX549 HaloTag ligands, and performed single-molecule imaging of EWS::FLI1-Halo on a custom-built Nikon (Nikon Instruments Inc.) TI microscope described in (https://elifesciences.org/articles/25776). We took images with a 100x/NA 1.49 oil-immersion TIRF objective (Nikon apochromat CFI Apo TIRF 100x Oil) under highly inclined and laminated optical sheet (HILO) illumination (https://www.nature.com/articles/nmeth1171) using following laser lines: 488 nm for mNG; 561 nm for JFX549; 405 nm and 633 nm for photo-activation and excitation of PA-JF646, respectively. The incubation chamber maintained a humidified 37°C atmosphere with 5% CO2 and the objective was similarly heated to 37°C for live-cell experiments. 

High-concentration JFX549 staining allows visualization of the intracellular distribution of EWS::FLI1-Halo. We chose cells with EWS::FLI1-Halo enriched in the nucleolus to perform spaSPT. The procedure of spaSPT largely follows what is described in (https://elifesciences.org/articles/25776). Both the excitation laser (633 nm) and the photo-activation laser (405 nm) for PA-JF646 were pulsed. Each frame consisted of a 7-ms camera exposure time followed by a ~500 μs camera ‘dead’ time. The excitation laser (633 nm) was pulsed for 1 ms starting at the beginning for the 7 ms camera exposure time. The photo-activation laser (405 nm) was pulsed during the ~500 μs camera ‘dead’ time, minimizing fluorescence background. Each cell was imaged for 20,000 frames corresponding to ~1.5 min. Images of mNeonGreen-EWS-NPM1 were collected with a camera exposure time of 500 ms before and after the acquisition of each spaSPT movie.