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. 2010 Jun 6;7 Suppl 3(Suppl 3):S321-30.
doi: 10.1098/rsif.2010.0039.focus. Epub 2010 Mar 31.

Emergence of a Prestressed Eukaryotic Nucleus During Cellular Differentiation and Development

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Free PMC article

Emergence of a Prestressed Eukaryotic Nucleus During Cellular Differentiation and Development

Aprotim Mazumder et al. J R Soc Interface. .
Free PMC article

Abstract

Nuclear shape and size are emerging as mechanistic regulators of genome function. Yet, the coupling between chromatin assembly and various nuclear and cytoplasmic scaffolds is poorly understood. The present work explores the structural organization of a prestressed nucleus in a variety of cellular systems ranging from cells in culture to those in an organism. A combination of laser ablation and cellular perturbations was used to decipher the dynamic nature of the nucleo-cytoplasmic contacts. In primary mouse embryonic fibroblasts, ablation of heterochromatin nodes caused an anisotropic shrinkage of the nucleus. Depolymerization of actin and microtubules, and inhibition of myosin motors, resulted in the differential stresses that these cytoplasmic systems exert on the nucleus. The onset of nuclear prestress was then mapped in two contexts--first, in the differentiation of embryonic stem cells, where signatures of prestress appeared with differentiation; second, at an organism level, where nuclear or cytoplasmic laser ablations of cells in the early Drosophila embryo induced a collapse of the nucleus only after cellularization. We thus show that the interplay of physical connections bridging the nucleus with the cytoplasm governs the size and shape of a prestressed eukaryotic nucleus.

Figures

Figure 1.
Figure 1.
Chemical depolymerization of cytoplasmic filaments or inhibition of associated motor proteins cause a variation in nuclear size in PMEF cells. (a) Representative images of drug-treated and fixed nuclei with the DNA-stained with Hoechst dye. Scale bar, 20 µm. (b) Statistics for approximately 100 nuclei each. The error bars are standard errors. Cont, control; Noc, nocodazole; CytoD, cytochalasin D; Blebb, blebbistatin; IsoNuc, isolated nucleus. Also shown are the xy projected area of nuclei isolated from PMEF cells (n = 19) and the estimated hydrodynamic radius of the genome in these cells. The error bars shown are standard deviations. Student's t-test: *p < 0.05; **p < 0.001.
Figure 2.
Figure 2.
Effects of heterochromatin laser ablation on nuclear size. (a) The fluorescence image of the cell nucleus is super-imposed on differential interference contrast images of the cells. After ablation, cell shape does not change as much as the nucleus, although there are indications of a loss of membrane tension. Scale bar, 20 µm. (b) A further proof for reduction in nuclear volume comes from the rise in intensity of confocal slices with a reduction in nuclear area. We show it here for the shrinkage of a representative PMEF cell nucleus expressing histone H2B–EGFP as the chromatin marker. Images from every time point in the graph are presented in the panel above. Scale bar, 5 µm. (c) The fall in nuclear area is accompanied by a corresponding rise in mean pixel intensity in the area covered by the nucleus in (b). (d) Fractional change in area upon heterochromatin ablation for indicated drug treatments (n = 15, each). The error bars are standard deviations. Student's t-test, **p < 0.001.
Figure 3.
Figure 3.
Effects of heterochromatin laser ablation on nuclear shape. (a) Shape changes upon heterochromatin (het) and cytoplasmic (cyt) ablations—representative images (time in seconds is indicated on top of the panel; scale bar, 5 µm), (b) representative time traces for change in circularity and (c) statistics of change in circularity (n = 10). Inset: elongated nuclei align along actin stress fibres (DNA was stained with Hoechst dye (1 µg ml−1) (green) and actin with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin (1 µM) (red), cells fixed in 4% paraformaldehyde (PFA) and imaged). Scale bar, 20 µm. All error bars are standard deviations.
Figure 4.
Figure 4.
Effects of laser ablation on structural filaments. (a) PMEF cells were cotransfected with H1e–mRFP and either one of actin–EGFP or Tau–EGFP. Forty-eight hours post-transfection ablation experiments were performed (80 mW, 3.4 s) to sever actin stress fibres or microtubules locally. Images of representative cells reconstructed by z projection from confocal stacks are shown. Note the large shrinkage in nuclear size when actin is perturbed. (b) Ratio of the largest cross-sectional areas after and before ablation are plotted for perturbation of the actin and microtubule cytoskeletons (n = 8 and 6). The relative shrinkage is greater for perturbation of the actin stress fibres. (c) EGFP–Lamin B1 and H1e–mRFP cotransfected PMEF cells were ablated at the heterochromatin. Time course in a merged image is presented. Note the indentations in the lamin scaffold. The time points are indicated above the panel. Scale bar, 5 µm. (d) HeLa cells expressing H2B–EGFP were cotransfected with an siRNA against Lamin B1, and a plasmid-expressing mRFP. Forty-eight hours post-transfection, such cells showed a lesser response to heterochromatin ablation (120 mW, 3.4 s) than control cells transfected just with the mRFP plasmid (n = 8 each). mRFP-expressing cells were considered in both cases to identify transfected cells. The error bars shown are standard deviations. Student's t-test: *p < 0.05; **p < 0.001. The error bars are standard deviations.
Figure 5.
Figure 5.
Emergence of nuclear prestress in differentiation. (a) Isolated nuclei from both R1 ES cells and PMEF cells show reversible swelling under conditions of low salt. Nuclear volumes are plotted for nuclei inside ES or PMEF cells (hatched bars), or nuclei isolated from such cells in PBS (black bars) or the same nuclei in water (open bars) (n = 7 each). Note that the relative size of the nucleus is larger in a cellular context for PMEF cells, while the level of water-induced swelling is similar and probably represents an upper bound for nuclear swelling. (b) Representative images of R1 ES cells and differentiated R1 ES cells upon heterochromatin ablation. Time in seconds is indicated on top of the panel. Scale bar, 5 µm. (c) Fractional change in area upon heterochromatin ablation for indicated cell types (n = 17 each). Student's t-test: *p < 0.05; **p < 0.001. The error bars are standard deviations.
Figure 6.
Figure 6.
(a) Two distinct time points during Drosophila gastrulation—just after the 13th mitotic cycle (i) and after the onset of gastrulation (ii). The embryo is viewed from the dorsal side with the anterior to the left. The white rectangle indicates the nuclei of the germ band and amnioserosa cells. Scale bar, 20 µm. (b) Elongated cells of the germ band and early amnioserosa were ablated at the point indicated by the white arrow in the cytoplasm. Circularization and chromatin condensation were evident in the ablated cell, but not in control cells in the same neighbourhood. Scale bar, 2 µm. (c) The fall in nuclear volume is shown in the graph, before and after such ablations (n = 5). All error bars are standard deviations. Student's t-test, **p < 0.001.

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