Chen P et al. (2024), Nuclear growth and import can be uncoupled.

ECB-ART-52607

Mol Biol Cell 2024 Jan 01;351:ar1. doi: 10.1091/mbc.E23-04-0138.

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Nuclear growth and import can be uncoupled.

Chen P , Mishra S , Prabha H , Sengupta S , Levy DL .

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What drives nuclear growth? Studying nuclei assembled in Xenopus egg extract and focusing on importin α/β-mediated nuclear import, we show that, while import is required for nuclear growth, nuclear growth and import can be uncoupled when chromatin structure is manipulated. Nuclei treated with micrococcal nuclease to fragment DNA grew slowly despite exhibiting little to no change in import rates. Nuclei assembled around axolotl chromatin with 20-fold more DNA than Xenopus grew larger but imported more slowly. Treating nuclei with reagents known to alter histone methylation or acetylation caused nuclei to grow less while still importing to a similar extent or to grow larger without significantly increasing import. Nuclear growth but not import was increased in live sea urchin embryos treated with the DNA methylator N-nitrosodimethylamine. These data suggest that nuclear import is not the primary driving force for nuclear growth. Instead, we observed that nuclear blebs expanded preferentially at sites of high chromatin density and lamin addition, whereas small Benzonase-treated nuclei lacking DNA exhibited reduced lamin incorporation into the nuclear envelope. In summary, we report experimental conditions where nuclear import is not sufficient to drive nuclear growth, hypothesizing that this uncoupling is a result of altered chromatin structure.

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	FIGURE 1:. DNA amount influences nuclear growth in X. laevis egg extract. (A–F) Nuclei were assembled in X. laevis egg extract using X. laevis sperm chromatin. Assembled nuclei were treated with 2.5 U/µL Benzonase or an equivalent volume of buffer (Control). (A) Nuclei were incubated for the indicated lengths of time, fixed, and visualized by immunofluorescence using mAb414 to label the NPC (red) and Hoechst to label DNA (blue). NPC staining was used to quantify nuclear CS area for on average 72 nuclei at each time point. Representative 60-min images are shown. Scale bar, 50 µm. Also see Supplemental Figure S1D. (B) Nuclear assembly and growth were allowed to proceed for different lengths of time (30, 30, 90, 80, and 120 min for Experiments 1–5, respectively) to allow nuclei to grow to different initial sizes (Initial) before treatment with Benzonase or buffer (Control) for 1 h. Nuclear size was quantified for fixed nuclei as in (A) for on average 82 nuclei per condition. (C) Preassembled nuclei were treated with Benzonase or buffer (Control) for 10 min, and then GFP-NLS (2.9 µM) was added. After an additional 5-min incubation, live imaging was performed at 2-min intervals with the same exposure time. Nuclear size was quantified based on the GFP-NLS signal. Data are shown for nine control nuclei and five Benzonase-treated nuclei. (D) Preassembled nuclei were treated with Benzonase for 10 min, and then FITC-dextran (150 kDa, 4 mg/ml) was added. Live imaging was performed with the same exposure time. Representative images are shown. The white signal corresponds to FITC-dextran and dark regions correspond to intact nuclei that exclude FITC-dextran. Nuclei were identified by the import of mCherry-NLS, and Hoechst was used to verify the absence of DNA (unpublished data). Scale bar, 50 µm. (E) Preassembled nuclei were treated with Benzonase or buffer (Control) for 10 min, and then GFP-Lamin B3 (80 nM) was added. After a 90-min incubation, live imaging was performed with the same exposure time. Representative images are shown. Scale bar, 50 µm. Two Lamin B3 intensity line scans were acquired through the middle of each nucleus in ImageJ Fiji by randomly generating lines and using the “Intensity Profile” tool. To calculate the lamina-to-nucleoplasm Lamin B3 localization ratio, the mean edge intensity (average of points A and B) was divided by the middle intensity (point C). Ratio values greater than one correspond to increased incorporation of Lamin B3 into the nuclear lamina. Representative data from one of two independent experiments are presented. (F) After a 10-min control or Benzonase treatment, GFP-NLS (2.9 µM) was added and incubated for 5 min. Live images were then acquired at 2-min intervals with the same exposure time. The initial nuclear import rate and nuclear growth rate were quantified over 10 min as described in the Methods, in each case normalizing to controls. On average 12 nuclei were quantified per condition. (G) Nuclei were assembled by adding X. laevis sperm chromatin to X. laevis egg extract lacking Cytochalasin B (i.e., F-actin-intact egg extract). Nuclei were then treated with 2.5 U/µL Benzonase or an equivalent volume of buffer (Control) for the indicated lengths of time, fixed, and visualized by immunofluorescence as in (A). NPC staining was used to quantify nuclear size for on average 134 nuclei at each time point. (H–K) Nuclei were assembled in X. laevis egg extract using X. laevis or axolotl sperm chromatin. After incubation for 2 h, nuclei were fixed and visualized by immunofluorescence as described in (A). (H) Nuclear CS area was quantified for on average 113 fixed nuclei per condition. (I) Total DNA intensity was calculated based on Hoechst staining for on average 31 fixed nuclei per condition. (J) Based on data presented in (H) and (I), nuclear CS area is plotted as a function of total DNA intensity for nuclei assembled with axolotl sperm. Representative images are shown above the graph. Scale bar, 50 µm. (K) GFP-NLS (2.9 µM) was added to preassembled nuclei and incubated for 5 min. Live images were then acquired at 3-min intervals with the same exposure time. The initial nuclear import rate was quantified over 15 min as described in the Materials and Methods and normalized to controls. On average 13 nuclei were quantified per condition. Two-tailed Student’s t tests assuming equal variances: ***p < 0.001. Error bars represent SD.
	FIGURE 2:. DNA fragmentation by MNase reduces nuclear growth but has little effect on import in X. laevis egg extract. Nuclei were assembled in X. laevis egg extracts with X. laevis sperm. Assembled nuclei were treated with 40 U/µL MNase or an equivalent volume of buffer (Control). Similar results were obtained with MNase and MNase-NLS, so those data were combined. (A–C) One-hour MNase treatment. (D–F) 15-min MNase treatment. (A) After a 1-h incubation with MNase, nuclei were fixed and visualized by immunofluorescence using mAb414 (red) and Hoechst staining (blue). Left: Representative images are shown. Scale bar, 50 µm. Right: DNA was extracted from isolated nuclei, separated on a 2% agarose gel, and stained with ethidium bromide. The left lane shows a DNA ladder with sizes noted. DNA corresponding to sperm chromosomes runs at the very top of the gel. (B) After a 1-h MNase treatment, 2.9 µM GFP-NLS was added and incubated for 5 min. Live images were then acquired at 15-s intervals with the same exposure time. The initial nuclear import rates and nuclear growth rates were quantified over 1.5 min as described in the Materials and Methods and normalized to controls. On average 83 nuclei were quantified per condition. (C) Nuclear assembly and growth were allowed to proceed for different lengths of time (30, 30, 45, 60, and 90 min for Experiments 1–5, respectively) to allow nuclei to grow to different initial sizes (Initial) before treatment with MNase or buffer (Control) for 1 h. Nuclei were then fixed and visualized by immunofluorescence using mAb414 to label the NPC. Nuclear CS area was quantified for on average 100 nuclei per condition. (D) After a 10-min MNase or control treatment, GFP-NLS (2.9 µM) was added and incubated for 5 min. Live images were then acquired at 2-min intervals with the same exposure time. Nuclear CS area was quantified at each time point for seven control nuclei and eight MNase-treated nuclei. Representative images are shown at 134 min. Scale bars, 50 µm. (E) For the experiment presented in (D), the initial nuclear import rates and nuclear growth rates were quantified as described in the Methods and normalized to controls. (F) After a 10-min MNase treatment, 2.9 µM GFP-NLS was added and incubated for 5 min. Live images were then acquired at 15-s intervals with the same exposure time. The initial nuclear import rates and nuclear growth rates were quantified as described in the Materials and Methods and normalized to controls. On average 46 nuclei were quantified per condition. Two-tailed Student’s t tests assuming equal variances: ns, not significant; p < 0.01; *p < 0.001. Error bars represent SD.
	FIGURE 3:. Modifying chromatin structure can alter nuclear size while having little to no effect on nuclear import in X. laevis egg extract. Nuclei were assembled in X. laevis egg extract with X. laevis sperm. Assembled nuclei were treated as indicated: (A and B) VPA (0.5 mM), (C and D) DZNep (2 µM), (E and F) Methylstat (4.5 µM), (G and H) NDMA (0.135 M), (I and J) spermidine (10 µM), or the appropriately matched buffer control (Control). We tested a range of concentrations for each small molecule and here present data for the concentrations that maximally affected nuclear size. (A, C, E, G, and I) Nuclei were incubated with the indicated small molecules for 90 min and were then fixed and visualized by immunofluorescence using mAb414. Nuclear CS area was measured for on average 88 nuclei per condition and normalized to controls. One set of representative data is shown from three independent experiments. (B, D, F, H, and J) Nuclear import and growth rates for GFP-NLS were quantified as described in Figure 2F for on average 19 nuclei per condition. Two-tailed Student’s t tests assuming equal variances: ns, not significant; p < 0.05; p < 0.01; **p < 0.001. Error bars represent SD.
	FIGURE 4:. Nuclear growth and import can be uncoupled in vivo in sea urchin embryos. One-cell sea urchin embryos were microinjected with GFP-NLS protein and mRNAs encoding membrane-mCherry (mem-mCh) to label the plasma membrane and H2B-RFP to label chromatin. Embryos were treated with 64 mM NDMA or an equal volume of buffer. Live confocal z-stacks were acquired with a 2-µm step size at 2-min intervals starting at the four-cell stage and extending into the 32-cell stage. Quantification focused on the eight- and 16-cell stages, for which the entire cell cycle was imaged. (A) Representative maximum-intensity z-projections are shown for 16-cell embryos immediately before entry into mitosis. GFP-NLS is in green. H2B-RFP and membrane-mCherry are in red. Scale bar, 20 µm. (B) Nuclear CS areas were measured in the GFP-NLS channel for the entire time-lapses. Because nuclei grow continuously throughout the cell cycle in early embryos, we plotted maximum nuclear CS area within a given embryonic stage to simplify comparisons. (C) Initial nuclear CS area growth rates were calculated based on the rate of nuclear growth over 6–10 min after the first appearance of nuclear GFP-NLS accumulation. (D) Nuclear import rates were calculated based on GFP-NLS import as described in the Materials and Methods. (E) Cell cycle length, which refers to the time elapsed between consecutive cell divisions, was measured. (F) Representative images of anaphase chromosomes from control and NDMA-treated embryos at the 16-cell stage. Scale bar, 5 µm. (G) Anaphase chromosome length was measured in embryos at the 16-cell stage, focusing on distinct chromosomes that could be easily visualized. Number of nuclei for (B–E): 51 eight-cell control nuclei, 42 eight-cell NDMA nuclei, 83 16-cell control nuclei, 53 16-cell NDMA nuclei. Numbers of chromosomes measured for (G): 136 control and 173 NDMA. Cumulative data are shown from eight control and seven NDMA-treated embryos. Two-tailed Student’s t tests assuming equal variances: p <0.001; *p < 0.0001. Error bars represent SD.
	FIGURE 5:. Chromatin colocalizes with sites of nuclear blebs and lamin incorporation in X. laevis egg extracts. Nuclei were assembled in X. laevis egg extract with X. laevis sperm. (A and B) Preassembled nuclei were labeled with 1 µg/ml Hoechst (DNA). Live time-lapse images were acquired with the same exposure time. (A) Time points from two representative time-lapse series are shown. Scale bar, 50 µm. (B) Relative chromatin area was calculated as described in Supplemental Figure S2A (blue line). To quantify chromatin distribution, we measured the chromatin heterogeneity index as described in (Chen et al., 2019). Briefly, for each nucleus two Hoechst intensity line scans were acquired through the middle of the nucleus. The SD of all intensity values along each line was calculated and normalized to the average intensity to obtain the chromatin heterogeneity index (orange line). Larger values correspond to a more heterogeneous chromatin distribution. (C) Preassembled nuclei were labeled with 1 µg/ml Hoechst (DNA) and 81.6 nM GFP-Lamin B3. Live images were acquired with the same exposure time. Arrowheads indicate the region shown at 10 × magnification in the lower set of images. Intensity line scans were acquired where indicated (blue for chromatin and green for Lamin B3). (D) Preassembled nuclei were labeled with 1 µg/ml Hoechst (DNA) and 81.6 nM GFP-Lamin B3. Live images were acquired 10 min after Hoechst and GFP-Lamin B3 addition. Representative images are shown. As indicated, multiple rectangular regions of interest were selected randomly for NE regions that were clearly chromatin-rich (red) or chromatin-poor (orange). GFP-Lamin B3 intensity within these rectangles was measured. For each nucleus, GFP-Lamin B3 intensities were normalized to the average intensity within the chromatin-poor regions. Each line represents an individual nucleus. Data are shown for 34 nuclei based on three experiments. (E) Preassembled nuclei were labeled with 1 µg/ml Hoechst (DNA) and 44.5 nM GFP-Lamin B3. Live time-lapse images were acquired with the same exposure time. Time points from a representative time-lapse series are shown. Scale bar, 50 µm. Nonparametric Wilcoxon signed rank test: ***p < 0.001. Error bars represent SD.
	FIGURE 6:. Compiled data showing uncoupling of nuclear import and growth when chromatin is altered. Nuclear import rate versus nuclear growth rate was plotted for individual nuclei from multiple experiments. (A) Compiled data for control nuclei are from Figures 2, B and F, 3, B, D, F, and H, and Supplemental Figure S1B (n = 203). (B) Compiled data for chromatin-altered nuclei with both increased and decreased nuclear size are from Figures 2, B and F, 3, B, D, F, and H (n = 194). Comparing the slopes of the two linear regression lines in (A) and (B) in GraphPad Prism indicated that the differences between the slopes are extremely significant (p < 0.0001, two-tailed analysis of covariance).