Fig. 3: Live-cell single-molecule tracking of HALO-tagged MITF.
From: Acetylation reprograms MITF target selectivity and residence time
a HALO-tagged MITF expression vectors. NLS; nuclear localization sequence. b Western blot of HALO-tagged MITF WT and mutants in 501mel cells with or without treatment with Doxycycline used to ensure similar expression levels performed in parallel with the SMT analysis shown in this figure. Vinculin was a loading control. Induction of HALO-MITF by Dox in 501mel cells has been performed independently at least 3 times with similar results. For Source Data see Supplementary Fig. S6. c Single molecule tracking movies collected at 100 fps tracks were extracted using the FiJi/ImageJ plugin TrackMate and analysed in terms of the distribution of single-molecule displacements between consecutive frames that was then fit with a three component model (one immobile component and two diffusing components), to generate quantitative estimates for HaloTag, WT MITF and mutants. d Quantitative estimates derived from SMT using WT and K206 HALO-tagged MITF for the fraction of molecules in the bound and slow states and the respective diffusion coefficients. Each point represents a single cell, the blue line the median, the box limits represent upper and lower quartiles, and whiskers extend between Q1 − 1.5 IQR and Q3 + 1.5 IQR, where IQR is the interquartile range. For HaloTag, WT MITF, K206R and K206Q mutants respectively Nreplicates = 2; Ncells = 19, 30; 30; 30; Njumps: 130387; 245200; 210929; 172286. Statistical test: non-parametric Kruskal-Wallis (two-sided). e Slower movies (frame rate 2 fps, laser exposure 200 ms) were acquired to calculate the distribution of residence times for immobile MITF molecules, following photobleaching correction using data collected on H2B-HaloTag (see Methods). f The fraction of bound molecules displaying a residence time longer than 100 s (left) and the (restricted) mean survival time (right). Nreplicates = 2; Ncells = 30, 30, 30, 30, 35, and 36, and Nmolecules = 3836; 3429; 3219; 1185; 3809; 1225 for WT MITF, K206R, K206Q, Δbasic, USF1 and p53 respectively; error bars (SEM) were calculated by the jacknife approach (see Methods); each SMT experiment was performed at least twice after adjustment of doxycycline concentration to ensure similar MITF expression levels.
