Fig 2: Myosin VI is linked to oestrogen signalling. a Pull-down of 2 µM CBD by 10 µM GST-ER. P and S represent pellet and supernatant fractions. b Expression of ER gene targets following siRNA knockdown of MVI in MCF-7 cells. Expression is plotted as a percentage of expression in mock cells. ER (ESR1) and ß-actin were used to reflect global changes in transcription. c Luciferase reporter assay driven by the ERE promoter in MCF-7 cells. Estradiol led to a 5-fold increase in promoter activity. siRNA knockdown of MVI led to a 3-fold reduction in activity. Ethanol was used as a carrier control for the experiments (error bars represent SEM from three independent experiments **p < 0.001 by two-tailed t-test). d Working model of MVI recruitment by the ER. MVI binds to the ER and then gets activated by NDP52 (or CoCoA), which enables binding to RNAPII through actin. Association with NDP52, or CoCoA, ties MVI with the general transcription co-activators to initiate recruitment of RNAPII

Fig 3: NDP52-dependent dimerisation of myosin VI. a FRET titration of FITC-MVITAIL against 1 µM AF555-MVITAIL ± DNA (20 µM) and NDP52 (20 µM). Data fitting was performed as described in Methods giving a K d as plotted in b. See Supplementary Fig. 7a–c for raw intensity data. b Plot of K d from titrations in a and Supplementary Fig. 7d–g (error bars represent SEM from three independent experiments). c Velocity histogram from sliding filament assay with MVI immobilised alone (blue), through antibody (red) and NDP52 (green). Insert shows first frame (red) and after 60 s (green). d Two routes of NDP52-dependent dimerisation with different stoichiometry. (i) NDP52 unfolds MVI then directly recruits a second molecule. (ii) Each MVI is unfolded by an individual NDP52

Fig 4: NDP52 association kinetics with the myosin VI tail.(A) Schematic of the experiments for rapid mixing of the FITC labelled MVITAIL and Alexa-Fluor555 labelled NDP52. Experiments were performed as described in the methods. (B) Representative stopped-flow fluorescence trace depicting both changes in FRET signal, as described in the text for 10 µM NDP52 (dimer). Each process was then analysed separately over the corresponding time scales. (C) Representative stopped-flow fluorescence traces and exponential fitting for the initial increase in fluorescence at the stated concentrations of NDP52 dimers. (D) Representative stopped-flow fluorescence traces and exponential fitting for the decrease in fluorescence at the stated concentrations of NDP52 dimers. (E) The individual traces were fitted to single exponentials and the dependence of the rate constants on concentration was then fitted to a straight line, as shown. The points shown are averages of at least three measurements, where error bars represent SEM. The fit for the first phase gives a slope of 1.65 µM−1 s−1 and an intercept of 3.2 s−1. The second phase is independent of NDP52 concentration and gives an average rate constant of 2.2 s−1. (F) Cartoon depicting the two processes reported by the stopped-flow experiments. Step A represents NDP52 binding to the MVITAIL, which is dependent upon NDP52 concentration. Step B represents the subsequent unfolding of MVI with a rate constant of 2.2 s−1, which is independent of NDP52 concentration. The dotted line represents an alternative model where NDP52 would bind to spontaneously unfolding MVITAIL. (G) Schematic of the experiments for rapid mixing of 1 µM Cy3B-calmodulin-bound MVITAIL(NI) (pre-mix molar ratio 2 : 1) and unlabelled NDP52. Experiments were performed as described in the methods. (H) Representative stopped-flow fluorescence traces and exponential fitting to the fluorescence decrease when Cy3B-calmodulin MVITAIL(NI) is mixed with the stated concentrations of NDP52 dimers. (I) The individual traces were fitted to single exponentials and the dependence of the rate constants on NDP52 concentration was then fitted to a straight line, as shown. The points shown are averages of at least three measurements, where error bars represent SEM. The rate constants are independent of NDP52 concentration, with an average value of 1.95 s−1.

Fig 5: Backfolding of the myosin VI tail.(A) Cartoon depiction of the key regions of MVI, as discussed in the text. UI Unique Insert; 3HB Three Helix Bundle; SAH Stable Alpha Helix; CC Coiled-coil; CBD Cargo Binding Domain. This highlights position of the large insert (LI), small insert (SI), along with NDP52 and Dab2 binding sites. (B) FRET titration of Alexa555-CBD against 1 µM of FITC-MVITAIL(LI), large insert (LI) containing MVITAIL (residues 814–1091), in the presence of 10 µM NDP52 (dimer) or 10 µM tDab2. (C) FRET titration of Alexa555-CBD against 1 µM of FITC-MVITAIL(NI) non-insert (NI) MVITAIL (residues 814–1060), in the presence of 10 µM NDP52 (dimer) or 10 µM tDab2. All titration data fitting was performed as described in Methods (error bars represent SEM from three independent experiments). (D) Schematic representation of FRET assay to measure backfolding of the MVITAIL. (E) Representative fluorescence spectra of 1 µM GFP-MVITAIL(NI)-RFP ± 5 µM NDP52 (dimer), or 20 µM tDab2. (F) Representative fluorescence spectra of 1 µM GFP-MVITAIL(LI)-RFP (right) ± 5 µM NDP52 (dimer), or tDab2.