Figure 4.

FRAP analysis reveals rapid recruitment of Nek2A to the centrosome. (A) EGFP-Nek2A U2OS cells induced with doxycycline for 24 h were mounted onto sterile glass coverslips, and FRAP was performed using an LSM 510 confocal microscope. One image was taken before bleaching (b and f, prebleach) and then the area within the indicated box was photobleached (c and g, bleach). An image was captured every second for 33 s (d and h, final image). FRAP was carried out in the presence (a–d, untreated) or absence (e–h, nocodazole-treated) of an intact microtubule cytoskeleton. U2OS cells treated in parallel were fixed and analyzed by indirect immunofluorescence microscopy (a and e) using anti-α tubulin antibodies (green) and anti-γ-tubulin (red) antibodies. Scale bar, 10 μm. (B–F) FRAP data were analyzed using LSM 510 software, and the relative GFP intensity within the bleached area was recorded as a measure of time; each data point represents the average intensity from 10 cells. A 50 × 50-pixel square was centered on paired centrosomes (B). A 250 × 250-pixel square was centered on paired centrosomes (C). A small spot was centered on one centriole of a split pair (D). A 50 × 50-pixel square was centered on one centriole of a split pair (E). A 250 × 250-pixel square was centered on both centrioles of a split pair (F). In each case, black diamonds represent untreated cells and open squares represent nocodazole treated cells. The centrosomal area bleached is shown in white in the top right corner. p values calculated using the Student's t test indicate the significance of the difference in half-life between untreated and nocodazole-treated samples in C and F.