Figure 6.

HMGB2 and HMGB4 show relocalization from the nucleus to the cytoplasm, while HMGB1 remains nuclear. Up to five selected fluorescence images of BY-2 protoplasts are shown that expressed paGFP-HMGB2 (A–E), paGFP-HMGB4 (F–J), or paGFP-HMGB1 (K–M). N shows a bright-field image of the protoplast shown in K to M. Images were taken before (0 s), during (50–250 s), and after (300 s) photoactivation of paGFP in the nuclei at the time points indicated. The regions of interest, where photoactivation was performed in A to M, were located within the nuclei that are indicated by dashed lines. Before photoactivation, the average fluorescence intensity was barely detectable. Shortly after the onset of photoactivation (performed with a two-photon laser scanning microscope during 250 s using femtosecond laser bursts covering an area of 7 × 8 μm with 64 parallel laser foci, at 4 mW at 800 nm per focus), strong fluorescent signals were detected, and the dynamics of photoactivated paGFP was monitored continuously in each case. Fluorescence intensity panels and scale bars are shown to the left of each row. In O to X, photoactivation was performed in the cytoplasm. Five selected fluorescence images of BY-2 protoplasts are shown that expressed paGFP-HMGB2 (O–S) or paGFP-HMGB4 (T–X). The region of interest is indicated by a dashed line, and the nucleus is indicated by a dotted line.