DOI: 10.5061/dryad.8w9ghx3n7
title: Single molecule tracking videos: SOX18 and its dominant-negative mutant SOX18RaOp

description: Few genetically dominant mutations involved in human disease have been
 fully explained at the molecular level. In cases where the mutant gene
 encodes a transcription factor, the dominant-negative mode of action of
 the mutant protein is particularly poorly understood. Here, we studied the
 genome-wide mechanism underlying a dominant-negative form of the SOX18
 transcription factor (SOX18RaOp) responsible for both the classical mouse
 mutant Ragged Opossum and the human genetic disorder
 Hypotrichosis-Lymphedema-Telangiectasia-Renal Syndrome. Combining three
 single-molecule imaging assays in living cells together with genomics and
 proteomics analysis, we found that SOX18RaOp disrupts the system through
 an accumulation of molecular interferences which impair several functional
 properties of the wild-type SOX18 protein, including its target gene
 selection process. The dominant-negative effect is further amplified by
 poisoning the interactome of its wild-type counterpart, which perturbs
 regulatory nodes such as SOX7 and MEF2C. Our findings explain in
 unprecedented detail the multi-layered process that underpins the
 molecular aetiology of dominant-negative transcription factor function.
description: <b>Single Molecule Tracking – Imaging </b> Immediately prior to imaging cells were washed twice and replaced with Fluorobrite DMEM (Glibco) imaging media. JF549 dye was a gift from Dr. Luke Lavis (Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States). 2 drops/mL of NucBlue Live ReadyProbes Reagent (Hoechst 33342) was added directly to the media and cells were incubated for 5 min at 37 <sup>o</sup>C with 5 % CO<sub>2</sub>, prior to adding 2 nM of JF549 Halo-tag dye directly to the media and cells and incubation for a further 15 min at 37 <sup>o</sup>C with 5 % of CO<sub>2</sub>. Following incubation, cells were washed twice and replaced with Fluorobrite DMEM (Glibco). Images were acquired on an Elyra single molecule imaging (PALM/STORM) total internal reflection fluorescence (TIRF) microscope, with an Andor 897 EMCCD camera, SR Cube 05 RL – BP 420-480 / BP 570-640 / LP 740 filter set and 100 X oil 1.46 NA TIRF objective using ZEISS ZEN blue software. Cells were imaged using a 561 nm excitation laser (power = 11.6 µW oblique illumination, 1.96 mW epi illumination; power density = 0.728 W/cm<sup>2</sup> oblique illumination, 123.24 W/cm<sup>2</sup> epi illumination) with a high-power TIRF filter (TIRF_HP). Using these parameters, we performed two different acquisition techniques; fast SMT which uses a 20 ms acquisition speed to acquire 6000 frames without intervals, and slow SMT which uses a 500 ms acquisition speed to acquire 500 frames without intervals. A low laser power was used to achieve a good signal-to-noise ratio with minimal photobleaching during imaging. Target and surrounding cells were prebleached for approximately 3 minutes (total across cells) prior to imaging to reduce the density of HALO-tagged molecules, background fluorescence, and the fluorescence interference from surrounding cells. <b>Single Molecule Tracking – Video generation</b> Fast (20 ms acquisition, 6000 frames) and slow (500 ms acquisition, 500 frames) SMT raw image stacks were cropped in ImageJ to reduce their file size and therefore minimize analysis time. Example image stacks for each condition were subsequently converted into AVI files using ImageJ in order to generate single molecule tracking videos.