Panel A: Confocal fluorescence images showing photoconversion dynamics of a nuclear protein, with pre-converted signal in green and post-converted signal in red, and merged views highlighting spatial redistribution over time, with the targeted irradiation region indicated. Panel B: A line graph showing changes in normalized nuclear intensity over time for one histone variant in control and deficient conditions, with a marked transition point corresponding to a key cell cycle event. Panel C: A line graph showing similar nuclear intensity dynamics for a second histone variant under the same conditions, allowing comparison between control and deficient samples. Panel D: Heat map images displaying maximum intensity projections of chromatin-associated signal across multiple nuclear cycles in control and deficient embryos, with color scale indicating relative intensity. Panel E: A scatter plot summarizing total chromatin-associated signal intensity for the first histone variant across nuclear cycles, comparing control and deficient conditions with statistical significance indicated. Panel F: Heat map images showing chromatin-associated signal for the second histone variant across nuclear cycles in control and deficient embryos. Panel G: A scatter plot summarizing total chromatin-associated signal intensity for the second histone variant across nuclear cycles, comparing conditions, and indicating statistical differences.
NASP-deficient embryos have less nucleoplasmic and chromatin-associated H3. (A) Representative images showing photoconversion of H3.2-Dendra2. Non–photo-switched Dendra2 is shown in green (λex = 488 nm), and the photo-converted Dendra2 is shown in red (λex = 568 nm). The yellow circle represents the nucleus irradiated by the 405 nm laser. Scale bar represents 20 µm. (B) Nuclear export measurement for H3.2-Dendra2 in WT and NASP-deficient embryos in NC12. Time point 0 represents Nuclear envelop breakdown (NEB). N = 3 biological replicates. Data represent the mean ± standard deviation (SD). (C) Nuclear export measurement for H3.3-Dendra2 in WT and NASP-deficient embryos in NC12. N = 3 biological replicates. Data represent the mean ± standard deviation (SD). (D) Maximum intensity projections of metaphase chromatin from WT (top) and NASP-deficient (bottom) embryos expressing H3.2-Dendra over NC10-13. Heat map created by pseudo-coloring images using nonlinear Thermal LUT on FIJI. Scale bar represents 10 µm. (E) Total intensities of H3.2-Dendra2 on mitotic chromatin of WT and NASP-deficient embryos for metaphase 10–13. N > 4 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between groups were analyzed by employing a two-way ANOVA Sídák’s multiple comparison test. (F) Maximum intensity projections of metaphase chromatin from WT (top) and NASP-deficient (bottom) embryos expressing H3.3-Dendra over NC10-13. Heat map created by pseudo-coloring images using nonlinear thermal LUT on FIJI. Scale bar represents 10 µm. (G) Total intensities of H3.3-Dendra2 on mitotic chromatin of WT and NASP-deficient embryos for metaphase 10–13. N > 4 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between groups was analyzed by employing a two-way ANOVA Sídák’s multiple comparison test. In the figure panels, ****P < 0.0001.