Figure 7.
Single-molecule analysis of protein mobility relative to RNAP2 Ser2ph-enriched regions. The mobility of proteins that are highly and lowly associated with RNAP2 Ser2ph-mintbody–enriched regions was quantified using single-molecule trajectories of HaloTag-tagged proteins (RPB3, BRD4, CDK9, p300, H2B, and PCNA) stained with HaloTag TMR ligand recorded at 33.33 ms/frame, which were superimposed upon high-pass-filtered RNAP2 Ser2ph-mintbody images in living HeLa cells. (A) Representative single-molecule trajectories of RPB3 superimposed upon RNAP2 Ser2ph-mintbody–enriched regions. RNAP2 Ser2ph-mintbody images were time averaged, high-pass filtered, and normalized to define locally enriched areas. The corresponding histograms of pixel intensities are shown. Right: Trajectories of tracked single molecules are overlaid on the normalized grayscale image. Green and magenta lines indicate trajectories of mobile and bound molecules, respectively. Scale bar, 5 µm. (B) Magnified views of RNAP2 Ser2ph-mintbody images for 0.5 s averaging (left), 16.67 s averaging (middle), and processed with trajectories (right). Scale bars, 1 µm. (C) Representative trajectories and MSD curves of bound RPB3 molecules that were highly associated (i) and lowly associated (ii) with RNAP2 Ser2ph-mintbody–enriched regions. Yellow points represent the tracking points with the top two RNAP2 Ser2ph-mintbody intensities, which were averaged and used as the relative RNAP2 Ser2ph-mintbody intensity (Irel_Ser2ph) of the trajectory. The D value (μm2/s) was obtained by linear fitting of the first six steps of MSD (≤0.2 s). Scale bars, 200 nm. (D) The distributions of D of the HaloTag-tagged proteins (top) and schematic representation (bottom) showing how to classify the mobile (right) and bound (left) molecules using two-component Gaussian fitting of the distribution of log10(D). Fitted lines for two-component Gaussian (solid line) and each component (green broken and magenta dotted lines) are indicated. A blue vertical line indicates the threshold Dthr (0.065 µm2/s) by which 97.5% of mobile molecules fall into the mobile fraction. Numbers of analyzed trajectories were H2B, 4,290; p300, 5,655; BRD4, 8,298; CDK9, 4,343; RPB3, 4,097; and PCNA, 6,144. (E and F) Relative intensities (int.) of RNAP2 Ser2ph-mintbody in a locally selected area at the position of tracked single HaloTag-tagged protein are plotted during the tracked period. Frequencies of trajectories that showed the local relative intensity >0.5 (cyan lines) are indicated. (E) Graphs corresponding to the molecules, (i) and (ii), shown in C. (F) Trajectories moving into and out of RNAP2 Ser2ph-minbody–enriched regions are shown with the relative intensity by time. Scale bars, 200 nm. (G)D and Irel_Ser2ph of RPB3 are plotted. Each dot represents a single trajectory (molecule). The whole plot (top; 4,097 trajectories) and only the bound fraction (bottom, left; 619 trajectories) are shown with the histogram of Irel_Ser2ph (bottom, right). Dots (i) and (ii) correspond to molecules shown in C. The top and bottom 25% Irel_Ser2ph are shown in magenta and green, respectively. (H) Frequencies of trajectories that showed local relative RNAP2 Ser2ph-minbody intensity >0.5 in the first to fourth quarters from the highest Irel_Ser2ph are box plotted (n = 619). Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; means are indicated by ×; gray dots indicate individual data points.

Single-molecule analysis of protein mobility relative to RNAP2 Ser2ph-enriched regions. The mobility of proteins that are highly and lowly associated with RNAP2 Ser2ph-mintbody–enriched regions was quantified using single-molecule trajectories of HaloTag-tagged proteins (RPB3, BRD4, CDK9, p300, H2B, and PCNA) stained with HaloTag TMR ligand recorded at 33.33 ms/frame, which were superimposed upon high-pass-filtered RNAP2 Ser2ph-mintbody images in living HeLa cells. (A) Representative single-molecule trajectories of RPB3 superimposed upon RNAP2 Ser2ph-mintbody–enriched regions. RNAP2 Ser2ph-mintbody images were time averaged, high-pass filtered, and normalized to define locally enriched areas. The corresponding histograms of pixel intensities are shown. Right: Trajectories of tracked single molecules are overlaid on the normalized grayscale image. Green and magenta lines indicate trajectories of mobile and bound molecules, respectively. Scale bar, 5 µm. (B) Magnified views of RNAP2 Ser2ph-mintbody images for 0.5 s averaging (left), 16.67 s averaging (middle), and processed with trajectories (right). Scale bars, 1 µm. (C) Representative trajectories and MSD curves of bound RPB3 molecules that were highly associated (i) and lowly associated (ii) with RNAP2 Ser2ph-mintbody–enriched regions. Yellow points represent the tracking points with the top two RNAP2 Ser2ph-mintbody intensities, which were averaged and used as the relative RNAP2 Ser2ph-mintbody intensity (Irel_Ser2ph) of the trajectory. The D value (μm2/s) was obtained by linear fitting of the first six steps of MSD (≤0.2 s). Scale bars, 200 nm. (D) The distributions of D of the HaloTag-tagged proteins (top) and schematic representation (bottom) showing how to classify the mobile (right) and bound (left) molecules using two-component Gaussian fitting of the distribution of log10(D). Fitted lines for two-component Gaussian (solid line) and each component (green broken and magenta dotted lines) are indicated. A blue vertical line indicates the threshold Dthr (0.065 µm2/s) by which 97.5% of mobile molecules fall into the mobile fraction. Numbers of analyzed trajectories were H2B, 4,290; p300, 5,655; BRD4, 8,298; CDK9, 4,343; RPB3, 4,097; and PCNA, 6,144. (E and F) Relative intensities (int.) of RNAP2 Ser2ph-mintbody in a locally selected area at the position of tracked single HaloTag-tagged protein are plotted during the tracked period. Frequencies of trajectories that showed the local relative intensity >0.5 (cyan lines) are indicated. (E) Graphs corresponding to the molecules, (i) and (ii), shown in C. (F) Trajectories moving into and out of RNAP2 Ser2ph-minbody–enriched regions are shown with the relative intensity by time. Scale bars, 200 nm. (G)D and Irel_Ser2ph of RPB3 are plotted. Each dot represents a single trajectory (molecule). The whole plot (top; 4,097 trajectories) and only the bound fraction (bottom, left; 619 trajectories) are shown with the histogram of Irel_Ser2ph (bottom, right). Dots (i) and (ii) correspond to molecules shown in C. The top and bottom 25% Irel_Ser2ph are shown in magenta and green, respectively. (H) Frequencies of trajectories that showed local relative RNAP2 Ser2ph-minbody intensity >0.5 in the first to fourth quarters from the highest Irel_Ser2ph are box plotted (n = 619). Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; means are indicated by ×; gray dots indicate individual data points.

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