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Dev Growth Differ
2022 Dec 01;649:501-507. doi: 10.1111/dgd.12819.
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Differential contribution of nuclear size scaling mechanisms between Xenopus species.
Heijo H
,
Merten CA
,
Hara Y
.
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Size of the nucleus, a membrane-bound organelle for DNA replication and transcription in eukaryotic cells, varies to adapt nuclear functions to the surrounding environment. Nuclear size strongly correlates with cytoplasmic size and genomic content. Previous studies using Xenopus laevis have unraveled two modes, cytoplasmic and chromatin-based mechanisms, for controlling nuclear size. However, owing to limited comparative analyses of the mechanisms among eukaryotic species, the contribution of each mechanism in controlling nuclear size has not been comprehensively elucidated. Here, we compared the relative contribution utilizing a cell-free reconstruction system from the cytoplasmic extract of unfertilized eggs of Xenopus tropicalis to that of the sister species X. laevis. In this system, interphase nuclei were reconstructed in vitro from sperm chromatin and increased in size throughout the incubation period. Using extracts from X. tropicalis, growth rate of the reconstructed nuclei was decreased by obstructing the effective cytoplasmic space, decreasing DNA quantity, or inhibiting molecules involved in various cytoplasmic mechanisms. Although these features are qualitatively identical to that shown by the extract of X. laevis, the sensitivities of experimental manipulation for each cellular parameter were different between the extracts from two Xenopus species. These quantitative differences implied that the contribution of each mode to expansion of the nuclear envelope is coordinated in a species-specific manner, which sets the species-specific nuclear size for in vivo physiological function.
Figure 1. Nuclear growth dynamics depends on the MT-based mechanism. (a) DNA (magenta; stained with Hoechst 33342) and membrane (green; stained with DiOC6(3)) were visualized in the nuclei reconstructed from sperm chromatin of X. laevis obtained from cytoplasmic extracts. Scale bars, 50 μm. (b) Dynamics of the mean cross-sectional areas of nuclei reconstructed in the extract of X. laevis (XL; light blue) or X. tropicalis (XT; blue) extract from sperm chromatin of X. laevis. Averages of mean cross-sectional area (±standard deviation [SD]) from each extract preparation are connected utilizing a line in each dataset. The inset depicts mean calculated growth rate of nuclear cross-sectional area (±SD). (c) Mean values (large filled symbols; ±SD) of normalized nuclear diameters are plotted against the square root of cross-sectional area of the microchamber using a log–log plot. Data fitted to a linear regression with two segments. Data in the first segment (pink) only show a significant correlation between nuclear diameter and channel dimension (p < 0.05), whereas the remaining data (blue) do not (p = 0.41). The mean diameters (±SD) in individual experiment are represented as small open symbols. The black dashed line indicates the border (1.91 on the X-axis, corresponding to 82.15 μm) between the two segments. (d) Nuclei (blue; Hoechst 33342), membranes (green; DiOC6(3)), and MTs (magenta; immunostained using anti-α-tubulin antibody) visualized after incubating sperm chromatin of X. laevis in the cytoplasmic extract of X. laevis or X. tropicalis for 120 min. Scale bar, 100 μm. (e) Average length (along the long axis; ±SD) and width (along the short axis) of the MT-occupied space reconstructed from sperm chromatin of X. laevis in the extract of X. laevis or X. tropicalis. * represents a statistically significant difference with the extract of X. laevis; p < 0.05 by Wilcoxon signed-rank test
Figure 2. Inhibition of the cytoplasm-based mechanisms. (a, b, d, e) Dynamics of mean cross-sectional areas of the nuclei reconstructed in the extracts of X. tropicalis (XT; a, d) or X. laevis (XL; b, e) in the presence of dimethyl sulfoxide (DMSO) (control; blue), 500 nM nocodazole (light blue), 5 μM nocodazole (light green), 50 μM nocodazole (green), 50 μM ciliobrevin D (orange), 100 μM importazole (purple), or 200 μg/ml of WGA (pink) from sperm chromatin of X. laevis. The averages of mean cross-sectional area (±SD) from each extract are connected utilizing a line in each dataset. Each inset depicts the mean calculated growth rate of nuclear cross-sectional area (±SD) for each inhibitor treatment. (c, f) Mean values of fold-change in nuclear growth rate with inhibitor treatment to the control. The calculated growth rate was divided by that of the control condition for each preparation. * represents a statistically significant difference with the control condition; p < 0.05 by Wilcoxon signed-rank test
Figure 3. Nuclear growth dynamics in different DNA quantities. (a) Dynamics of the mean cross-sectional areas in reconstructed nuclei from sperm chromatin of X. laevis or X. tropicalis (XLSP or XTSP, respectively) in the presence of DMSO (control) or aphidicolin (APH) in the extract of X. tropicalis. XLSP + DMSO: blue; XLSP + APH: pink; XTSP + DMSO: green; and XTSP+APH: light blue. The growth dynamics of nuclei from XLSP + DMSO in the extract of X. laevis is shown in gray. Mean nuclear cross-sectional areas (±SD) from each extract are connected utilizing a line in each dataset. (b) Mean growth rates and (c) the maximum values of nuclear cross-sectional area during incubation are plotted against the DNA quantity. The data for X. laevis are identical to the data previously reported (Heijo et al., 2020). (d) Mean nuclear growth rates in the presence of each inhibitor in the extract of X. laevis are plotted against the DNA quantity. Average values (±SD) are plotted. Data from each Xenopus species or each inhibitor condition are fitted to the linear regression (the extract of X. laevis: open symbols, dotted line; the extract of X. tropicalis: filled symbols, solid line; supplemented with 5 μM nocodazole, light green; 100 μM importazole, purple; or 200 μg/ml WGA, pink)
