Figure 1.

Method to trace telomere movement after oxidative damage. (a) Scheme of the method. Cells with transient transfection of KR-TRF1 for telomere labeling were tracked over 1 min with 0.429 s time intervals. KillerRed in half of the cell nucleus was activated using 559 nm laser scanning. Telomere movements were recorded in a video and were analyzed by modified single molecular movement analysis in a 2D program in MATLAB. (b) System drift was tested in fixed cells with transient transfection of KR-TRF1 for telomere labeling. The representative image at an enlarged pixel-level of KR-TRF1 and tracking map of a single telomere at pixel level is shown (Left). MSD over time of all of the telomeres in the fixed sample is shown in the quantified graph (Right). (c) Representative tracking maps of individual telomeres immediately after damage induction with a time interval 0.429 s for 120 frames: Some telomeres show potential directional movement (a), while the majority exhibit a random walk or random walk within a confined region. (b) MSD over time is fit to a power function model $r^2=A{t}^{\alpha }+b$. The α value is relevant to the pattern of movement: α > 1 is directional movement (Red); α < 1 is random walk in a confined region (blue); and α = 1 is random walk (grey). (d) Movement of telomeres with KR-TRF1 with/without damage was analyzed as described in Fig. 1c. Red curve represents directional movement; blue curve represents random walk. MSD of undamaged telomeres is lower than 5 × 10⁴ nm², which is indicated as a gray dotted line in the graph. (e) The MSD-time fit; MSD of damaged telomeres (red) versus the undamaged ones (black) was plotted.