Fig. 1: Nuclear organisation of myosin VI.

A Cartoon depiction of the MVI key features including the cargo-binding domain (CBD). B Widefield Immunofluorescence staining against MVI (magenta) and DNA (cyan) in HeLa cells. Images were acquired at the mid-point of the nucleus (Scale bar 10 μm). (See Supplementary Figs. 1, 2 for confocal z stack and antibody staining control). C Example STORM render images of MVI under normal, serum+ and TIP-treated conditions (scale bar 2 μm). Dotted lines represent a region of interest (ROI) containing the nucleus which are taken forward for cluster analysis. The nucleus was identified using either Hoechst or RNAPII staining. D Depiction of molecular clustering and random distribution. We performed cluster analysis using the linearised form of Ripley’s K function²¹ L(r)-r, where r is the radius. A plot of L(r)-r versus r gives a value of zero for a random distribution (blue line), but deviates from zero, towards positive values, due to molecular clustering (red). The organisation of MVI is seen with peaks at 125 ± 45 nm (normal), 120 ± 25 nm (TIP) and 200 ± 30 nm (Serum). E Cluster maps based upon the selected ROI in (C). Clusters are shown in green (Normal) or red (Serum and TIP treatment). Selected regions are shown at higher magnification. F Cluster analysis of MVI nuclear organisation under normal, serum+ and TIP-treated conditions. Individual data points correspond to the average value for a cell ROI (n = 14 for normal, 11 for serum+ and TIP-treated). The values represent the mean from the ROIs for each condition (Only statistically significant changes are highlighted *p < 0.05; **p < 0.01 by two-tailed t test compared to normal conditions). G Number of localisations of MVI under the different conditions. Individual data points correspond to the value for a cell ROI (n = 14). The values represent the mean from the ROIs for each condition (****p < 0.0001 by two-tailed t test compared to normal conditions). H Western-blot against MVI under Normal, Serum and TIP-treated conditions. The expression levels relative to normal are 0.95 and 1.05 for serum and TIP-treated conditions, respectively.