Decision letter: Mitotic chromosomes scale to nuclear-cytoplasmic ratio and cell size in Xenopus

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to scale coordinately with mitotic spindles, but the underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle, and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be reset by cytoplasmic factors from earlier developmental stages. In vitro, increasing nuclear-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin α scales mitotic chromosomes to cell surface area/volume ratio (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and Hi-C data suggest that mitotic chromosomes shrink during embryogenesis through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter chromosome axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo. Editor's evaluation This study combines experiments in developing embryos and embryo extracts to investigate a fundamental relationship in biology – how the size of mitotic chromosomes scales with changes in cell size during development. Using the unique tools available in the Xenopus genus developmental biology system as well as modern genomic approaches, the authors convincingly demonstrate that mitotic chromosome scaling is mediated by differential loading of maternal chromatin remodeling factors during interphase. Although it remains unclear exactly how these factors impact chromosome size, the findings reported here will be of broad interest to the cell biology community and are likely to spawn new avenues of experimental inquiry aimed at understanding intracellular scaling relationships. https://doi.org/10.7554/eLife.84360.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Upon fertilization, embryos undergo a series of rapid cell divisions in the absence of cell growth, resulting in decreasing cell size. Subcellular structures including the nucleus and mitotic spindle scale to cell size through a defined set of mechanisms (Heald and Gibeaux, 2018; Levy and Heald, 2015). Mitotic chromosomes also shrink in size during development and scale with cell size across metazoans (Conklin, 1912; Kramer et al., 2021; Micheli et al., 1993), but the underlying mechanisms are poorly understood. In plants and in fly embryos, fused chromosomes with extended lengths were reported to mis-segregate during mitosis (Schubert and Oud, 1997; Sullivan et al., 1993). Similar experiments in budding yeast showed that an artificially lengthened chromosome was hyper-compacted during anaphase due to Aurora B kinase phosphorylation of substrates including condensin, a key regulator of mitotic chromosome condensation and resolution (Neurohr et al., 2011). In Caenorhabditis elegans, a screen for genes required for segregation of an extra-long, fused chromosome identified the centromeric histone CENP-A and topoisomerase II (topo II) as regulators of holocentric chromosome size (Ladouceur et al., 2017). However, it is unclear whether pathways that tune the length of an artificially long chromosome also operate during the physiological process of mitotic chromosome scaling during embryogenesis. Mechanisms that scale the spindle and nucleus during development have been well-characterized. As cell volume decreases, structural components become limiting (Good et al., 2013; Hazel et al., 2013; Levy and Heald, 2010). Additionally, some scaling factors are regulated by the nuclear transport factor importin α, which partitions between the cytoplasm and the cell membrane and serves as a sensor for the cell’s surface area to volume ratio (Brownlee and Heald, 2019). Previous studies of mitotic chromosome scaling, performed mainly in C. elegans, revealed that mitotic chromosome size correlates positively with cell size and nuclear size and negatively with intranuclear DNA density, as haploid embryos contain longer mitotic chromosomes than diploids, and knockdown of importin α or the chromatin-bound Ran guanine exchange factor RCC1 decrease both nuclear and mitotic chromosome size (Hara et al., 2013; Ladouceur et al., 2015). However, conserved relationships among genome size, nuclear size, and cell size complicate efforts to distinguish correlation from causation during mitotic chromosome scaling. Furthermore, it is unclear whether similar underlying mechanisms operate during embryogenesis of vertebrates that possess larger, monocentric chromosomes and more complex karyotypes. The African clawed frog Xenopus laevis provides a powerful system for studying the mechanisms of mitotic chromosome scaling. Female frogs produce thousands of eggs that enable isolation of undiluted and cell cycle-synchronized cytoplasm in the form of egg extracts that reproduce many cellular processes in vitro including mitotic chromosome condensation and individualization (Maresca and Heald, 2006). In addition, fertilized eggs divide synchronously, allowing extracts to be prepared from embryos at different stages of development. Our previous work suggested that embryo nuclei added to egg extracts can recapitulate a decrease in mitotic chromosome size during development (Kieserman and Heald, 2011), but did not uncover underlying scaling mechanisms. It was also unclear how this observation related to scaling of mitotic chromosomes and other subcellular structures observed in vivo. Here, we fully leverage the Xenopus system by systematically comparing changes in mitotic chromosome size observed in vivo with perturbations in vitro to distinguish the factors that regulate mitotic chromosome scaling including nuclear size, spindle size, cell size, cell-cycle stage, and nuclear-cytoplasmic (N/C) ratio. We find that mitotic chromosomes scale continuously with spindle size, even in the largest cells of early embryos. We show that scaling occurs primarily through differential recruitment of the DNA loop extruding motor condensin I, which alters DNA loop size and thus length-wise compaction of chromosomes. Finally, we describe how reductive divisions that progressively decrease cell size and increase N/C ratio operate during different phases of the cell cycle to reduce chromosome length over the course of development. Together, these results suggest a multiscale model for how mitotic chromosome size is set in an embryo and open new avenues for deeper exploration of how changes in chromosome compaction and organization contribute to genome functions during early vertebrate embryogenesis. Results Mitotic chromosomes scale continuously with cell, nuclear and spindle size We reasoned that mitotic chromosome size may relate to nuclear size and/or content due to chromatin factors associated with the DNA during interphase. Alternatively, mitotic chromosomes could scale with spindle size through mechanisms operating in mitosis. To distinguish between these possibilities, we performed a time course of whole-embryo immunofluorescence through the late blastula stages of X. laevis development and measured the dimensions of cells, spindles, and metaphase plates (Figure 1A and B, Figure 1—figure supplement 1). Although previous work showed that spindle lengths reach a plateau in cells larger than ~200 μm in diameter (Figure 1—figure supplement 2A; Wühr et al., 2008), measurement of spindle volumes by confocal microscopy revealed that spindles continue to scale at larger cell sizes (Figure 1C), consistent with observations that spindle width correlates more robustly with cell volume than spindle length in cultured cells (Figure 1—figure supplement 2B; Kletter et al., 2021). The combined volume of all mitotic chromosomes (the metaphase plate) also scaled continuously with cell size (Figure 1D), similar to published work describing nuclear scaling (Figure 1—figure supplement 3; Jevtić and Levy, 2015). To assess whether the metaphase plate scaled more with nuclear size or with mitotic spindle size, we binned the data by cell size and plotted average volumes of the different subcellular structures. We found that total mitotic chromosome volume scaled in size remarkably similarly with both spindles and nuclei (Figure 1—figure supplement 4). However, the decrease in nuclear volume during early development was far greater than for mitotic structures: nuclei decreased in size approximately 10-fold over early cleavage divisions, while metaphase plate and spindle volumes decreased by approximately 3-fold and 2-fold, respectively (Figure 1E and F). This comparison suggests that the fold-increase in chromosome compaction as a cell transitions from interphase to metaphase diminishes from 8-fold in early blastula embryos to 1.5-fold in late blastula stages (Figure 1—figure supplement 5). Overall, these results demonstrate that the metaphase plate scales continuously with cell size in the early embryo, suggesting that mitotic chromosomes share scaling features with both nuclei and mitotic spindles. Figure 1 with 5 supplements see all Download asset Open asset Metaphase plates scale continuously with cell size. (A) Experimental scheme for whole-embryo immunofluorescence. Blastula-stage embryos undergoing synchronous divisions were fixed during mitosis and stained with anti-histone H3 and anti-tubulin antibodies to visualize mitotic chromosomes and spindles, respectively. Representative image of two cells from a stage 6 embryo with white rectangles outlining mitotic spindles, scale bar = 100 μm. Inset: magnified view of one of the mitotic spindles, scale bar = 20 μm. (B) Dimensions of cells and spindles were either directly measured or calculated (for details, see ‘Materials and methods’). (C) Measurements of spindle volume or (D) total mitotic chromosome (metaphase plate) volume plotted against cell diameter, colored by developmental stage. (E) Volumes of spindles, nuclei, and metaphase plates all plotted against cell diameter, fit with linear models. 95% confidence intervals shown in gray. (F) Zoom-in of yellow panel shown in (E). n = 3 biological replicates. Fold-change in size of nuclei (approximately 10-fold), spindles (approximately 2-fold), and metaphase plates (approximately 3-fold) were estimated by calculating the difference in median size in stage 3 vs. stage 8 embryos. Source data are available in Figure 1—source data 1. Figure 1—source data 1 This file contains all of the source data for Figure 1 and related supplemental figures. https://cdn.elifesciences.org/articles/84360/elife-84360-fig1-data1-v2.zip Download elife-84360-fig1-data1-v2.zip Mitotic chromosomes scale predominantly through length-wise compaction To examine how morphologies of individual mitotic chromosomes change during development and how their size relates to metaphase plate volume, we prepared mitotic cell extracts from stage 3 (4-cell) or stage 8 (~4000-cell) embryos (Wilbur and Heald, 2013) and centrifuged single endogenous mitotic chromosomes onto coverslips for size measurements. We found that median chromosome lengths decreased approximately 2-fold between stage 3 and stage 8 (Figure 2A and B), while chromosome widths increased only by ~1.2-fold (Figure 2—figure supplement 1). These changes were consistent with the magnitude of metaphase plate scaling during this period estimated by whole-embryo immunofluorescence (approximately 2-3-fold, Figure 1D) and demonstrates that shortening of the long axis is the predominant metric underlying the change in mitotic chromosome size during early embryogenesis. We also observed that the median length of endogenous stage 3 mitotic chromosomes was not statistically different from that of replicated sperm chromosomes formed in egg extracts (Figure 2B), suggesting that replicated sperm chromosomes formed in egg extracts may serve as a proxy for mitotic chromosome size during the earliest cell divisions. Figure 2 with 1 supplement see all Download asset Open asset Mitotic chromosomes scale length-wise. (A) Mitotic extracts were prepared from stage 3 or stage 8 embryos, and single endogenous mitotic chromosomes were centrifuged onto coverslips and stained with Hoechst DNA dye. Representative images of stage 3 and stage 8 chromosomes are shown. (B) Length distributions of sperm mitotic chromosomes replicated in egg extract, mitotic chromosomes isolated from embryo extracts and stage 8 embryo extract chromosomes after mixing 1:1 with egg extract. (C) Stage 8 extracts were filtered to remove endogenous chromosomes, then unreplicated or replicated sperm nuclei were added to form mitotic chromosomes. Representative images of an endogenous stage 8 chromosome or replicated sperm chromosome formed in stage 8 extracts. (D) Quantification of chromosome lengths for the experiment shown in (C), for both replicated and unreplicated sperm conditions. n = 3 biological replicates, >50 chromosomes per replicate. Scale bar = 10 μm. ***p<0.001 by the Mann–Whitney U test. Source data are available as Figure 2—source data 1. Figure 2—source data 1 This file contains all of the source data for Figure 2 and the related supplemental figure. https://cdn.elifesciences.org/articles/84360/elife-84360-fig2-data1-v2.zip Download elife-84360-fig2-data1-v2.zip Previously, it was shown that mixing mitotic extracts prepared from early and late blastula stage embryos resulted in spindles of intermediate size due to equilibration of cytoplasmic spindle scaling factors (Wilbur and Heald, 2013). Likewise, combining interphase extracts at different ratios from two Xenopus species with different sized nuclei produced a graded effect on nuclear size (Levy and Heald, 2010). To test whether cytoplasmic factors similarly modulate mitotic chromosome size, we combined metaphase-arrested egg extracts in a 1:1 ratio with stage 8 mitotic embryo extracts containing endogenous mitotic chromosomes (Figure 2B). However, we observed no increase in chromosome length, indicating that mitotic chromosome scaling factors are not exchangeable in the cytoplasm during metaphase. To test whether cytoplasmic extract could alter mitotic chromosome size if added before the onset of chromosome condensation, we filtered stage 8 extracts to remove endogenous chromosomes, then added sperm nuclei either directly or following replication in interphase egg extracts (Figure 2C). In both cases, sperm chromosomes were at least 2-fold longer than the endogenous stage 8 chromosomes (Figure 2D), suggesting that stage 8 metaphase cytoplasm is sufficient to remodel sperm nuclei into mitotic chromosomes, but unable to recapitulate embryo chromosome size. Thus, mitotic chromosome size is predominantly set by factors loaded during interphase that are not readily exchangeable during metaphase, making mitotic chromosome scaling fundamentally distinct from nuclear or spindle size scaling. Mitotic chromosome size is determined by nuclear factors during interphase The results above indicated that mitotic chromosome size is largely determined by nuclear rather than cytoplasmic factors. We next confirmed previous results that G2-arrested nuclei isolated from blastula-stage embryos and added to metaphase egg extracts produced mitotic chromosomes approximately 2-fold shorter than replicated sperm chromosomes formed in the same extract (Figure 3A and B; Kieserman and Heald, 2011). This finding further supports the idea that mitotic chromosome size is determined prior to entry into metaphase, likely by chromatin factors loaded during interphase. A difference in chromosome size was also recapitulated in extracts depleted of membranes through ultracentrifugation, which are incapable of forming spindles but competent for mitotic chromosome assembly, indicating that spindle formation is not required for mitotic chromosome scaling (Figure 3—figure supplement 1). However, the magnitude decrease in chromosome size was diminished (2-fold in crude egg extracts compared to 1.4-fold in clarified extracts), suggesting that the spindle and/or membrane-associated factors may contribute to the decrease in mitotic chromosome size observed in blastula-stage embryos. Figure 3 with 6 supplements see all Download asset Open asset Egg extracts recapitulate mitotic chromosome scaling through differential recruitment of condensin I, topo II, and histone H1.8. (A) Experimental scheme (also see ‘Materials and methods’). Stage 8 embryos were arrested in G2 using cycloheximide, then fractionated to isolate cytoplasm containing nuclei. Embryo nuclei were pelleted and added to metaphase-arrested egg extracts to form mitotic spindles and chromosomes. (B) Lengths of replicated sperm chromosomes or stage 8 embryo chromosomes formed in metaphase egg extracts. (C) Representative images of mitotic chromosomes prepared by adding replicated sperm nuclei (top) or stage 8 embryo nuclei (bottom) to metaphase egg extracts, and stained with antibodies for condensin I (xCAP-G), topo II, or histone H1.8. Scale bar = 10 μm. (D) Abundances of topo II, condensin I, and histone H1.8 (calculated by normalizing immunofluorescence signal to Hoechst signal, see ‘Materials and methods’ for details) on short embryo chromosomes normalized to long sperm chromosomes (denoted by dotted line), from three different extracts. n = 3 biological replicates, >50 chromosomes per replicate. ***p<0.001 by the Mann–Whitney U test. Source data are available in Figure 3—source data 1. Figure 3—source data 1 This file contains all of the source data for Figure 3 and related supplemental figures. https://cdn.elifesciences.org/articles/84360/elife-84360-fig3-data1-v2.zip Download elife-84360-fig3-data1-v2.zip Previous work in C. elegans suggested that mitotic chromosome size correlates with intranuclear density and nuclear size (Hara et al., 2013). We observed that embryo nuclei were roughly 2-fold larger in diameter than interphase sperm nuclei (Figure 3—figure supplement 2A), consistent with the doubling of genome size due to the presence of both paternal and maternal genomes in embryo nuclei, suggesting that intranuclear DNA density is comparable between the two sources of nuclei. Yet, mitotic chromosomes formed by adding stage 8 embryo nuclei into egg extracts were 2-fold shorter than those formed from replicated sperm nuclei (Figure 3B), while mitotic spindles formed with either source of nuclei were barely distinguishable in size (Figure 3—figure supplement 2B). These data suggest that neither nuclear size, intranuclear DNA density during interphase, nor spindle size during metaphase determines mitotic chromosome size and further supports the idea that scaling of mitotic spindles, nuclei, and mitotic chromosomes are not necessarily coordinated. Interestingly, although mitotic chromosome scaling could be recapitulated by adding embryo nuclei to metaphase-arrested egg extracts, chromosome morphologies were distinct. The separation of sister chromatid arms resulting in X-shaped mitotic chromosomes in both stage 3 and stage 8 mitotic embryo extracts (Figure 2A) was not observed when stage 8 embryo nuclei were added to egg extracts as chromosome arms remained tightly associated along their lengths (Figure 3C). Taken together, these results indicate that the factors determining mitotic chromosome size remain associated with chromatin in G2-arrested embryo nuclei even when introduced into metaphase egg extracts, while other factors that determine mitotic chromosome morphology do not. Chromosome scaling correlates with differential recruitment of condensin I, topo II, and linker histone H1.8 Robust recapitulation of chromosome scaling in metaphase-arrested egg extracts enabled molecular-level analysis of potential scaling factors, which was not technically feasible in embryo extracts that cannot transit the cell cycle in vitro. We examined three proteins known to influence chromosome size and morphology in Xenopus, condensin I (the predominant condensin in Xenopus eggs and embryos), topoisomerase II (topo II), and the maternal linker histone, termed H1.8 (Maresca et al., 2005; Nielsen et al., 2020; Shintomi and Hirano, 2011). After performing immunostaining of short embryo chromosomes or long sperm chromosomes formed in the same egg extracts, the abundance of each factor was calculated by normalizing immunofluorescence signal to DNA dye intensity (Figure 3C, see ‘Materials and methods’). We found that short embryo chromosomes contained less condensin I and topo II, but more histone H1.8 relative to long replicated sperm chromosomes (Figure 3D, Figure 3—figure supplement 3). We also examined condensin II, which is 5-fold less abundant than condensin I in Xenopus egg extract and plays a minor role in setting mitotic chromosome size (Ono et al., 2003; Shintomi and Hirano, 2011), and similarly observed lower levels on embryo chromosomes relative to sperm chromosomes (Figure 3—figure supplement 4). These results are consistent with a study showing that depletion of histone H1.8 from egg extracts lengthens mitotic chromosomes (Maresca et al., 2005) and more recent work showing that histone H1.8 inhibits binding of condensin I and topo II to mitotic chromosomes (Choppakatla et al., 2021). Therefore, differential recruitment of condensins, topo II, or histone H1.8 may contribute to mitotic chromosome scaling during embryogenesis. Our previous work showed that short embryo chromosomes could be reset to lengths observed in replicated sperm samples by cycling the mitotic chromosomes through an additional interphase in egg extracts (Figure 3—figure supplement 5A and B; Kieserman and Heald, 2011). To test whether the abundance of candidate scaling factors was affected, we performed immunofluorescence on mitotic embryo chromosomes formed during the first or second metaphase and found that the abundance of all three factors increased in the second metaphase (Figure 3—figure supplement 5C–F, Figure 3—figure supplement 6). Of the three factors, condensin I levels increased the most (2-fold), returning to levels found on replicated sperm chromosomes (Figure 3—figure supplement 5C and D, Figure 3—figure supplement 6A). Our observation that histone H1.8 levels increased slightly after the second metaphase suggests that condensin I recruitment is not necessarily regulated by H1.8, and that condensin I can override the DNA compaction activity of the linker histone to lengthen embryo chromosomes. Mitotic chromosomes scale through extensive remodeling of DNA loop architecture Condensins shape mitotic chromosomes through their ability to form and extrude loops from the central axis (Ganji et al., 2018; Goloborodko et al., 2016). In silico models of loop extrusion activity suggested that tuning the abundance of condensin could dramatically alter DNA loop architecture and thus chromosome dimensions (Goloborodko et al., 2016). However, these models have not been tested under physiological conditions that relate to chromosome size changes in vivo. To assess how DNA loop size and arrangement are altered in the context of mitotic chromosome scaling, we performed Hi-C on long sperm chromosomes and short embryo chromosomes formed in egg extracts. Hi-C contact maps indicated that short embryo chromosomes had increased longer-range genomic contacts along their entire length, as evidenced by thickening of the diagonal (Figure 4A). To quantify this effect, we plotted the decay of contact frequencies (P) as a function of genomic distance in base pairs (s) (Figure 4B). The shape of P(s) was similar to that observed in earlier work on mitotic chromosomes from human, chicken, and Xenopus (Choppakatla et al., 2021; Gibcus et al., 2018; Naumova et al., 2013), and for rod-shaped dinoflagellate chromosomes (Nand et al., 2021), indicating the same overall organization of mitotic chromosomes across diverse species. Changes in the slope of P(s) have been informative for modeling the underlying organization of DNA into layers of loops (Gibcus et al., 2018) and are more easily visualized by plotting the derivative of P(s) (Figure 4C). Based on this previous work, the amount of DNA contained within a layer is estimated by the genomic distance at which the derivative is at its minimum, which was 106 bp for sperm chromosomes compared to ~107 bp for embryo chromosomes (Figure 4C, gray bar). Within a layer, DNA loop size can be estimated from where the derivative peaks at smaller genomic distances, which was between 104–105 bp for sperm chromosomes vs. 105–106 bp for embryo chromosomes (Figure 4C, orange bar). Combined with our immunofluorescence results from Figure 3, these data are consistent with a model in which mitotic chromosomes scale smaller during development through decreased recruitment of condensin I, resulting in larger DNA loops and more DNA per layer, thus accommodating more DNA on a shorter chromosome axis (Figure 4D). Figure 4 Download asset Open asset Mitotic chromosomes scale through extensive remodeling of DNA loop architecture. (A) Hi-C maps of chromosome 4S from replicated sperm or stage 8 embryo chromosomes formed in metaphase egg extracts. (B) Plots comparing how contact frequency (P) genome-wide decays as a function of genomic distance (s) for replicated sperm (yellow) or stage 8 mitotic chromosomes (blue). (C) Derivative of contact frequencies from (B). Based on previous models, peaks at 104–106 bp show differences in loop size (orange bar), while inflection points at 106–107 bp reveal differences in DNA amount per layer (gray bar). (D) Model depicting how lower condensin I occupancy on short embryo chromosomes results in an increase in DNA loop size and DNA per layer. Plots display average values from two biological replicates. Source data available as Figure 4—source data 1. Figure 4—source data 1 This file contains all of the source data for Figure 4 and related supplemental figures. https://cdn.elifesciences.org/articles/84360/elife-84360-fig4-data1-v2.zip Download elife-84360-fig4-data1-v2.zip Nuclear-cytoplasmic ratio regulates mitotic chromosome scaling, but not nuclear or spindle scaling We next investigated possible mechanisms that could decrease the abundance of condensin I on mitotic chromosomes as they scale smaller during development. Characteristic features of cleavage divisions during early embryogenesis include a lack of cell growth and minimal gene expression, which results in exponentially increasing copies of the genome within the same total volume of cytoplasm. The increase in N/C ratio, defined here as the number of nuclei per volume of cytoplasm, titrates a finite maternal pool of DNA binding factors so that they are distributed to more and more genome copies with each subsequent cell cycle, thus lowering their abundance per genome. This effect is thought to underlie activation of zygotic transcription at the mid-blastula transition (Amodeo et al., 2015; Collart et al., 2013), and titration of the histone chaperone Npm2 was shown to play a role in nuclear scaling (Chen et al., 2019). To test whether N/C ratio could play a role in mitotic chromosome scaling, we tested two different concentrations of sperm nuclei corresponding to either early (~78 sperm nuclei/μL) or late (~1250 nuclei/μL) blastula stage embryos (Figure 5A). After allowing the nuclei to replicate in interphase egg extracts, we added back metaphase-arrested egg extracts, isolated mitotic chromosomes for length measurements and performed immunofluorescence for condensin I, topo II, and H1.8. We found that mimicking increased N/C ratio by adding a higher concentration of sperm nuclei into egg extract decreased mitotic chromosome length ~1.3-fold (Figure 5B), consistent with the decrease in metaphase plate size observed in vivo at the stage of development corresponding to the N/C ratios tested (stages 6–7, Figure 1D). This size change was accompanied by an ~1.6-fold decrease in condensin I abundance on mitotic chromosomes (Figure 5C), with less significant changes for histone H1.8 and topo II (Figure 5—figure supplements 1 and 2). Interestingly, we found that the increased N/C ratio mimicked in this experiment did not significantly affect nuclear size and increased spindle size (Figure 5D and E), suggesting that the N/C ratio is only capable of scaling mitotic chromosome size, but not these other subcellular structures. Previous work using lipid droplets to encapsulate spindles and nuclei in different sized compartments showed that both structures scale to compartment volume through a limiting-component mechanism (Chen et al., 2019; Good et al., 2013; Leech et al., 2022). However, the range of N/C ratios (defined again as genome copies per volume cytoplasm) tested here was significantly lower (approximately 10-fold) than in those studies, suggesting that maternal components do not become size-limiting for spindles and nuclei until later developmental stages. Figure 5 with 4 supplements see all Down