Identification of epigenetic modulators as determinants of nuclear size and shape.

The shape and size of the human cell nucleus is highly variable among cell types and tissues. Changes in nuclear morphology are associated with disease, including cancer, as well as with premature and normal aging. Despite the very fundamental nature of nuclear morphology, the cellular factors that determine nuclear shape and size are not well understood. To identify regulators of nuclear architecture in a systematic and unbiased fashion, we performed a high-throughput imaging-based siRNA screen targeting 867 nuclear proteins including chromatin-associated proteins, epigenetic regulators, and nuclear envelope components. Using multiple morphometric parameters, and eliminating cell cycle effectors, we identified a set of novel determinants of nuclear size and shape. Interestingly, most identified factors altered nuclear morphology without affecting the levels of lamin proteins, which are known prominent regulators of nuclear shape. In contrast, a major group of nuclear shape regulators were modifiers of repressive heterochromatin. Biochemical and molecular analysis uncovered a direct physical interaction of histone H3 with lamin A mediated via combinatorial histone modifications. Furthermore, disease-causing lamin A mutations that result in disruption of nuclear shape inhibited lamin A-histone H3 interactions. Oncogenic histone H3.3 mutants defective for H3K27 methylation resulted in nuclear morphology abnormalities. Altogether, our results represent a systematic exploration of cellular factors involved in determining nuclear morphology and they identify the interaction of lamin A with histone H3 as an important contributor to nuclear morphology in human cells.

Quality control of protein complex composition.

Eukaryotic cells possess hundreds of protein complexes that contain multiple subunits and must be formed at the correct time and place during development. Despite specific assembly pathways, cells frequently encounter complexes with missing or aberrant subunits that can disrupt important signaling events. Cells, therefore, employ several ubiquitin-dependent quality control pathways that can prevent, correct, or degrade flawed complexes. In this review, we will discuss our emerging understanding of such quality control of protein complex composition.

An insect antifreeze protein from Anatolica polita enhances the cryoprotection of Xenopus laevis eggs and embryos.

Cryoprotection is of interest in many fields of research, necessitating a greater understanding of different cryoprotective agents. Antifreeze proteins have been identified that have the ability to confer cryoprotection in certain organisms. Antifreeze proteins are an evolutionary adaptation that contributes to the freeze resistance of certain fish, insects, bacteria and plants. These proteins adsorb to an ice crystal's surface and restrict its growth within a certain temperature range. We investigated the ability of an antifreeze protein from the desert beetle Anatolica polita, ApAFP752, to confer cryoprotection in the frog Xenopus laevis. Xenopus laevis eggs and embryos microinjected with ApAFP752 exhibited reduced damage and increased survival after a freeze-thaw cycle in a concentration-dependent manner. We also demonstrate that ApAFP752 localizes to the plasma membrane in eggs and embryonic blastomeres and is not toxic for early development. These studies show the potential of an insect antifreeze protein to confer cryoprotection in amphibian eggs and embryos.

An E3 ligase guide to the galaxy of small-molecule-induced protein degradation.

Induced protein degradation accomplishes elimination, rather than inhibition, of pathological proteins. Key to the success of this novel therapeutic modality is the modification of proteins with ubiquitin chains, which is brought about by molecular glues or bivalent compounds that induce proximity between the target protein and an E3 ligase. The human genome encodes ∼600 E3 ligases that differ widely in their structures, catalytic mechanisms, modes of regulation, and physiological roles. While many of these enzymes hold great promise for drug discovery, few have been successfully engaged by small-molecule degraders. Here, we review E3 ligases that are being used for induced protein degradation. Based on these prior successes and our growing understanding of the biology and biochemistry of E3 ligases, we propose new ubiquitylation enzymes that can be harnessed for drug discovery to firmly establish induced protein degradation as a specific and efficient therapeutic approach.

Structural basis for dimerization quality control.

Most quality control pathways target misfolded proteins to prevent toxic aggregation and neurodegeneration1. Dimerization quality control further improves proteostasis by eliminating complexes of aberrant composition2, but how it detects incorrect subunits remains unknown. Here we provide structural insight into target selection by SCF-FBXL17, a dimerization-quality-control E3 ligase that ubiquitylates and helps to degrade inactive heterodimers of BTB proteins while sparing functional homodimers. We find that SCF-FBXL17 disrupts aberrant BTB dimers that fail to stabilize an intermolecular ß-sheet around a highly divergent ß-strand of the BTB domain. Complex dissociation allows SCF-FBXL17 to wrap around a single BTB domain, resulting in robust ubiquitylation. SCF-FBXL17 therefore probes both shape and complementarity of BTB domains, a mechanism that is well suited to establish quality control of complex composition for recurrent interaction modules.

The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity.

How intracellular organelles acquire their characteristic sizes is a fundamental question in cell biology. Given stereotypical changes in nuclear size in cancer, it is important to understand the mechanisms that control nuclear size in human cells. Using a high-throughput imaging RNAi screen, we identify and mechanistically characterize ELYS, a nucleoporin required for post-mitotic nuclear pore complex (NPC) assembly, as a determinant of nuclear size in mammalian cells. ELYS knockdown results in small nuclei, reduced nuclear lamin B2 localization, lower NPC density, and decreased nuclear import. Increasing nuclear import by importin α overexpression rescues nuclear size and lamin B2 import, while inhibiting importin α/ß-mediated nuclear import decreases nuclear size. Conversely, ELYS overexpression increases nuclear size, enriches nuclear lamin B2 at the nuclear periphery, and elevates NPC density and nuclear import. Consistent with these observations, knockdown or inhibition of exportin 1 increases nuclear size. Thus, we identify ELYS as a novel positive effector of mammalian nuclear size and propose that nuclear size is sensitive to NPC density and nuclear import capacity.

Altering the levels of nuclear import factors in early Xenopus laevis embryos affects later development.

More than just a container for DNA, the nuclear envelope carries out a wide variety of critical and highly regulated cellular functions. One of these functions is nuclear import, and in this study we investigate how altering the levels of nuclear transport factors impacts developmental progression and organismal size. During early Xenopus laevis embryogenesis, the timing of a key developmental event, the midblastula transition (MBT), is sensitive to nuclear import factor levels. How might altering nuclear import factors and MBT timing in the early embryo affect downstream development of the organism? We microinjected X. laevis two-cell embryos with mRNA to increase levels of importin α or NTF2, resulting in differential amounts of nuclear import factors in the two halves of the embryo. Compared to controls, these embryos exhibited delayed gastrulation, curved neural plates, and bent tadpoles with different sized eyes. Furthermore, embryos microinjected with NTF2 developed into smaller froglets compared to control microinjected embryos. We propose that altering nuclear import factors and nuclear size affects MBT timing, cell size, and cell number, subsequently disrupting later development. Thus, altering nuclear import factors early in development can affect function and size at the organismal level.

In vivo mitotic spindle scaling can be modulated by changing the levels of a single protein: the microtubule polymerase XMAP215.

In many organisms, early embryonic development is characterized by a series of reductive cell divisions that result in rapid increases in cell number and concomitant decreases in cell size. Intracellular organelles, such as the nucleus and mitotic spindle, also become progressively smaller during this developmental window, but the molecular and mechanistic underpinnings of these scaling relationships are not fully understood. For the mitotic spindle, changes in cytoplasmic volume are sufficient to account for size scaling during early development in certain organisms. This observation is consistent with models that evoke a limiting component, whereby the smaller absolute number of spindle components in smaller cells limits spindle size. Here we investigate the role of a candidate factor for developmental spindle scaling, the microtubule polymerase XMAP215. Microinjection of additional XMAP215 protein into Xenopus laevis embryos was sufficient to induce the assembly of larger spindles during developmental stages 6.5, 7, and 8, whereas addition of a polymerase-incompetent XMAP215 mutant resulted in a downward shift in the in vivo spindle scaling curve. In sum, these results indicate that even small cells are able to produce larger spindles if microtubule growth rates are increased and suggest that structural components are not limiting.

Both Nuclear Size and DNA Amount Contribute to Midblastula Transition Timing in Xenopus laevis.

During early Xenopus laevis embryogenesis both nuclear and cell volumes decrease with the nuclear-to-cytoplasmic (N/C) volume ratio reaching a maximum at the midblastula transition (MBT). At the MBT, embryonic transcription is upregulated and cell cycles lengthen. Early studies demonstrated a role for the DNA-to-cytoplasmic ratio in the control of MBT timing. By altering nuclear size, we previously showed that the N/C volume ratio also contributes to proper MBT timing. Here we examine the relative contributions of nuclear size and DNA amount to MBT timing by simultaneously altering nuclear size and ploidy in X. laevis embryos. Compared to diploid embryos, haploids exhibited a delay in both zygotic gene expression and cell cycle lengthening, while diploid embryos with increased N/C volume ratios showed early expression of zygotic genes and premature lengthening of cell cycles. Interestingly, haploids with increased N/C volume ratios exhibited an intermediate effect on the timing of zygotic gene expression and cell cycle lengthening. Decreasing nuclear size in post-MBT haploid embryos caused a further delay in cell cycle lengthening and the expression of some zygotic genes. Our data suggest that both the N/C volume ratio and DNA amount contribute to the regulation of MBT timing with neither parameter being dominant.

More Cytoplasm, More Problems.

In this issue of Developmental Cell, Kyogoku and Kitajima (2017) investigate the effect of cytoplasmic volume on the fidelity of chromosome segregation during meiosis in mouse oocytes. The authors find that large cytoplasmic volume affects spindle pole morphology, chromosome alignment, and stringency of checkpoint signaling, resulting in error-prone chromosome segregation.

Use of Xenopus cell-free extracts to study size regulation of subcellular structures.

Striking size variations are prominent throughout biology, at the organismal, cellular, and subcellular levels. Important fundamental questions concern organelle size regulation and how organelle size is regulated relative to cell size, also known as scaling. Uncovering mechanisms of organelle size regulation will inform the functional significance of size as well as the implications of misregulated size, for instance in the case of nuclear enlargement in cancer. Xenopus egg and embryo extracts are powerful cell-free systems that have been utilized extensively for mechanistic and functional studies of various organelles and subcellular structures. The open biochemical nature of the extract permits facile manipulation of its composition, and in recent years extract approaches have illuminated mechanisms of organelle size regulation. This review largely focuses on in vitro Xenopus studies that have identified regulators of nuclear and spindle size. We also discuss potential relationships between size scaling of the nucleus and spindle, size regulation of other subcellular structures, and extract experiments that have clarified developmental timing mechanisms. We conclude by offering some future prospects, notably the integration of Xenopus extract with microfluidic technology.

New Insights into Mechanisms and Functions of Nuclear Size Regulation.

Nuclear size is generally maintained within a defined range in a given cell type. Changes in cell size that occur during cell growth, development, and differentiation are accompanied by dynamic nuclear size adjustments in order to establish appropriate nuclear-to-cytoplasmic volume relationships. It has long been recognized that aberrations in nuclear size are associated with certain disease states, most notably cancer. Nuclear size and morphology must impact nuclear and cellular functions. Understanding these functional implications requires an understanding of the mechanisms that control nuclear size. In this review, we first provide a general overview of the diverse cellular structures and activities that contribute to nuclear size control, including structural components of the nucleus, effects of DNA amount and chromatin compaction, signaling, and transport pathways that impinge on the nucleus, extranuclear structures, and cell cycle state. We then detail some of the key mechanistic findings about nuclear size regulation that have been gleaned from a variety of model organisms. Lastly, we review studies that have implicated nuclear size in the regulation of cell and nuclear function and speculate on the potential functional significance of nuclear size in chromatin organization, gene expression, nuclear mechanics, and disease. With many fundamental cell biological questions remaining to be answered, the field of nuclear size regulation is still wide open.

Nuclear size is sensitive to NTF2 protein levels in a manner dependent on Ran binding.

Altered nuclear size is associated with many cancers, and determining whether cancer-associated changes in nuclear size contribute to carcinogenesis necessitates an understanding of mechanisms of nuclear size regulation. Although nuclear import rates generally positively correlate with nuclear size, NTF2 levels negatively affect nuclear size, despite the role of NTF2 (also known as NUTF2) in nuclear recycling of the import factor Ran. We show that binding of Ran to NTF2 is required for NTF2 to inhibit nuclear expansion and import of large cargo molecules in Xenopus laevis egg and embryo extracts, consistent with our observation that NTF2 reduces the diameter of the nuclear pore complex (NPC) in a Ran-binding-dependent manner. Furthermore, we demonstrate that ectopic NTF2 expression in Xenopus embryos and mammalian tissue culture cells alters nuclear size. Finally, we show that increases in nuclear size during melanoma progression correlate with reduced NTF2 expression, and increasing NTF2 levels in melanoma cells is sufficient to reduce nuclear size. These results show a conserved capacity for NTF2 to impact on nuclear size, and we propose that NTF2 might be a new cancer biomarker.

Concentration-dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells.

A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change.

Nuclear size scaling during Xenopus early development contributes to midblastula transition timing.

Early Xenopus laevis embryogenesis is a robust system for investigating mechanisms of developmental timing. After a series of rapid cell divisions with concomitant reductions in cell size, the first major developmental transition is the midblastula transition (MBT), when zygotic transcription begins and cell cycles elongate. Whereas the maintenance of a constant nuclear-to-cytoplasmic (N/C) volume ratio is a conserved cellular property, it has long been recognized that the N/C volume ratio changes dramatically during early Xenopus development. We investigated how changes in nuclear size and the N/C volume ratio during early development contribute to the regulation of MBT timing. Whereas previous studies suggested a role for the N/C volume ratio in MBT timing, none directly tested the effects of altering nuclear size. In this study, we first quantify blastomere and nuclear sizes in X. laevis embryos, demonstrating that the N/C volume ratio increases prior to the MBT. We then manipulate nuclear volume in embryos by microinjecting different nuclear scaling factors, including import proteins, lamins, and reticulons. Using this approach, we show that increasing the N/C volume ratio in pre-MBT embryos leads to premature activation of zygotic gene transcription and early onset of longer cell cycles. Conversely, decreasing the N/C volume ratio delays zygotic transcription and leads to additional rapid cell divisions. Whereas the DNA-to-cytoplasmic ratio has been implicated in MBT timing, our data show that nuclear size also contributes to the regulation of MBT timing, demonstrating the functional significance of nuclear size during development.

Mechanisms of nuclear size regulation in model systems and cancer.

Changes in nuclear size have long been used by cytopathologists as an important parameter to diagnose, stage, and prognose many cancers. Mechanisms underlying these changes and functional links between nuclear size and malignancy are largely unknown. Understanding mechanisms of nuclear size regulation and the physiological significance of proper nuclear size control will inform the interplay between altered nuclear size and oncogenesis. In this chapter we review what is known about molecular mechanisms of nuclear size control based on research in model experimental systems including yeast, Xenopus, Tetrahymena, Drosophila, plants, mice, and mammalian cell culture. We discuss how nuclear size is influenced by DNA ploidy, nuclear structural components, cytoplasmic factors and nucleocytoplasmic transport, the cytoskeleton, and the extracellular matrix. Based on these mechanistic insights, we speculate about how nuclear size might impact cell physiology and whether altered nuclear size could contribute to cancer development and progression. We end with some outstanding questions about mechanisms and functions of nuclear size regulation.

Sizing and shaping the nucleus: mechanisms and significance.

The size and shape of the nucleus are tightly regulated, indicating the physiological significance of proper nuclear morphology, yet the mechanisms and functions of nuclear size and shape regulation remain poorly understood. Correlations between altered nuclear morphology and certain disease states have long been observed, most notably many cancers are diagnosed and staged based on graded increases in nuclear size. Here we review recent studies investigating the mechanisms regulating nuclear size and shape, how mitotic events influence nuclear morphology, and the role of nuclear size and shape in subnuclear chromatin organization and cancer progression.

Nuclear size regulation: from single cells to development and disease.

Cell size varies greatly among different cell types and organisms, especially during early development when cell division is rapid with little overall growth. A fundamental question is how organelle size is regulated relative to cell size. The nucleus exhibits exquisite size scaling during development and between species, and nuclear size is often altered in cancer cells. Recent studies have elucidated mechanisms of nuclear size regulation in a variety of experimental systems, opening the door to future research on how nuclear size impacts upon cell and nuclear function and subnuclear organization. In this review we discuss studies that have clarified nuclear size control mechanisms and how these results have or will contribute to our understanding of the functional significance of nuclear size.