Mukherjee RN et al. (2020), The Perinuclear ER Scales Nuclear Size Independ...

XB-ART-57034

Dev Cell 2020 Aug 10;543:395-409.e7. doi: 10.1016/j.devcel.2020.05.003.

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The Perinuclear ER Scales Nuclear Size Independently of Cell Size in Early Embryos.

Mukherjee RN , Sallé J , Dmitrieff S , Nelson KM , Oakey J , Minc N , Levy DL .

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Nuclear size plays pivotal roles in gene expression, embryo development, and disease. A central hypothesis in organisms ranging from yeast to vertebrates is that nuclear size scales to cell size. This implies that nuclei may reach steady-state sizes set by limiting cytoplasmic pools of size-regulating components. By monitoring nuclear dynamics in early sea urchin embryos, we found that nuclei undergo substantial growth in each interphase, reaching a maximal size prior to mitosis that declined steadily over the course of development. Manipulations of cytoplasmic volume through multiple chemical and physical means ruled out cell size as a major determinant of nuclear size and growth. Rather, our data suggest that the perinuclear endoplasmic reticulum, accumulated through dynein activity, serves as a limiting membrane pool that sets nuclear surface growth rate. Partitioning of this local pool at each cell division modulates nuclear growth kinetics and dictates size scaling throughout early development.

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Species referenced: Xenopus laevis
GO keywords: nucleus [+]
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Figure 7. Perinuclear ER Volume Scales Nuclear Size In Vitro (A and B) Blastomeres from different stage Xenopus laevis embryos were dissociated, fixed, and stained with antibodies against perinuclear ER sheet marker KTN1 and the NPC (mAb414). (A) Representative confocal images are shown. (B) Perinuclear ER volume was quantified from confocal z stacks (see Figure S4C; STAR Methods). Nuclear surface area was extrapolated from CS area. Cumulative data are shown from three different batches of embryos. n = 11 (stage 8), n = 46 (stage 9), and n = 14 (stage 10). (Câ€“G) Nucleus and ER formation were induced in fractionated interphase X. laevis egg extract supplemented with membrane dye DiI and GST-GFP-NLS protein. Extract and nuclei were encapsulated in droplets of differing volumes using microfluidic devices. Where indicated, extracts were supplemented with 67 nM recombinant Rtn4b protein, 3.2 Î¼M recombinant p150CC1 dynein inhibitor, or 10% X. laevis light membranes. Confocal z stacks were acquired after a 3-h incubation at 16Â°C. n = 55 (control), n = 52 (Rtn4b), n = 27 (p150CC1), and n = 26 (more light membrane). (C) The experimental approach. The image of the microfluidic device was adapted with permission from Hazel et al. (2013). (D) Representative images of different sized droplets. Imaging of the droplet periphery verified that Rtn4b addition induced a more tubulated cortical ER (data not shown). The droplet boundary is outlined in white in the merged images. (E) Perinuclear ER volume was quantified from DiI images for different size droplets (see Figure S4C; STAR Methods). Previous studies have established that DiI stains ER membranes in Xenopus extract (Dreier and Rapoport, 2000). Correlation coefficients: 0.60 for control (p < 0.0001), 0.87 for Rtn4b (p < 0.0001), and 0.90 for p150CC1 (p < 0.0001). (F) Nuclear CS area was quantified from GFP-NLS images for different size droplets and extrapolated to surface area (see STAR Methods). For individual droplets, nuclear surface area was plotted as a function of the pER volume measured in (E). Correlation coefficient 0.88 for all data (p < 0.0001). (G) Perinuclear ER volume and nuclear surface area were quantified as in (E) and (F). Focusing on smaller droplets, a subset of the control data (90â€“710-pL droplets) is shown in comparison to the â€œmore light membranesâ€ data (110â€“720-pL droplets). Error bars represent SD. âˆ—âˆ—âˆ—p < 0.005; âˆ—p < 0.05. Scale bars, 20 Î¼m. See also Figure S6.

Figure S6. Additional data related to Xenopus in vitro experiments and comparisons of nuclear size scaling in sea urchins and Xenopus, Related to Figure 7. (A) Nucleus formation was induced in fractionated interphase X. laevis egg extract supplemented with GST-GFP-NLS protein. Extract and nuclei were encapsulated in 80-90 Î¼m diameter droplets using microfluidics. Once intranuclear GFP-NLS was apparent indicating formation of an intact NE, confocal images were acquired at 20-minute intervals. At each time point nuclear CS area was measured for 8-12 nuclei (10 nuclei on average), and nuclear surface area was extrapolated for the nearly spherical nuclei. Nuclear growth is plotted as a function of time. (B) Nuclear surface area as a function of perinuclear ER volume is plotted for the droplet data shown in Fig. 7F with 95% confidence ellipses. (C) To validate that recombinant Rtn4b added to Xenopus extract properly incorporates into reconstituted ER, crude interphase egg extract was supplemented with 1 Î¼M DiI and 67 nM recombinant Rtn4b. To label the His-tagged Rtn4b, an Alexa Fluor 488 labeled anti-His antibody was added (see Methods for details). Representative images are shown. The reticular localization of Rtn4b co-localizing with DiI demonstrates its proper integration into the ER. (D) To determine the concentration of exogenously added Rtn4b that most limits nuclear growth, we assembled nuclei de novo in crude X. laevis egg extract and titrated in recombinant Rtn4b at the indicated concentrations. After a 30-minute incubation, nuclear CS areas were quantified based on NPC staining and extrapolated to surface areas (see Methods for details). 37-66 nuclei were quantified per condition (53 nuclei on average). Because nuclei were smallest with 67 nM added Rtn4b, we used this concentration of Rtn4b throughout the study. (E) Nuclear volume is plotted as a function of cell volume for different urchin and Xenopus developmental stages. Urchin data are from Fig. 1D. Frog data were published previously (Jevtic and Levy, 2015). N/C volume ratios were calculated for individual urchin blastomeres and averaged for a given stage based on the data in Fig. 1D. X. laevis N/C volume ratios were published previously (Jevtic and Levy, 2015). The timing of zygotic genome activation (ZGA) in each species is noted based on published reports (Poccia, et al., 1985; Newport and Kirschner, 1982; Nemer, 1963). Error bars represent SD. ***, p<0.005; *, p<0.05; ns, not significant. Scale bars: 20 Î¼m.

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Abrams, Dynamic assembly of brambleberry mediates nuclear envelope fusion during early development. 2012, Pubmed