Mobility of RNAP2 Ser2ph foci compared with PCNA foci and replication domains. (A–C) RNAP2 Ser2ph-mintbody and Halo-PCNA were expressed in HeLa cells and fluorescence images were acquired at 551 ms/frame using a high-resolution confocal system. RNAP2 Ser2ph-mintbody and Halo-PCNA foci in cells that exhibited euchromatic (A) and heterochromatic (B) PCNA patterns at the early and late S stages, respectively, were tracked to measure their mobilities. PCNA is concentrated in euchromatic foci in the nuclear interior region in the early S phase (A) and then in heterochromatic foci at the nuclear periphery and around nucleoli in the late S phase (B); see also Fig. 5 A. The whole nucleus, magnified views with indications of tracked foci (orange and cyan circles for mintbody and PCNA, respectively), and traces of individual foci are shown. (C) MSDs (means of all foci) are shown in linear scale (top) with SEM, D values (μm2/s), and anomalous exponents (α), and in double logarithmic scale (bottom) with means in single cells (thin lines). Numbers of analyzed foci are 3,993 (RNAP2 Ser2ph), 1,226 (euchromatic PCNA), and 925 (heterochromatic PCNA), from 12 cells for RNAP2 Ser2ph (143–468 foci/cell) and 6 cells each for euchromatic and heterochromatic PCNA foci (31–291 and 72–249 foci/cell, respectively). (D–F) HeLa cells stably expressing RNAP2 Ser2ph-mintbody were loaded with Cy3-dUTP to pulse-label chromatin domains that replicated just after loading, and cells were further grown for 2 d. Fluorescence images were acquired, foci were tracked, and MSDs were plotted, as in A–C. Cells were classified into euchromatic (D) and heterochromatic (E) Cy3-DNA patterns, just as were PCNA-expressing cells. (D) Numbers of analyzed foci are 1,329 (RNAP2 Ser2ph), 951 (euchromatic Cy3), and 623 (heterochromatic Cy3) from 15 cells for RNAP2 Ser2ph (41–194 foci/cell) and 9 cells each for euchromatic and heterochromatic Cy3-DNA foci (49–149 and 37–123 foci/cell, respectively). Scale bars, 5 µm; 1 µm for magnified views.
