Nuclear size and shape control.

The nucleus displays a wide range of sizes and shapes in different species and cell types, yet its size scaling and many of the key structural constituents that determine its shape are highly conserved. In this review, we discuss the cellular properties and processes that contribute to nuclear size and shape control, drawing examples from across eukaryotes and highlighting conserved themes and pathways. We then outline physiological roles that have been uncovered for specific nuclear morphologies and disease pathologies associated with aberrant nuclear morphology. We argue that a comparative approach, assessing and integrating observations from different systems, will be a powerful way to help us address the open questions surrounding functional roles of nuclear size and shape in cell physiology.

Cell division geometries as central organizers of early embryo development.

Early cellular patterning is a critical step of embryonic development that determines the proper progression of morphogenesis in all metazoans. It relies on a series of rapid reductive divisions occurring simultaneously with the specification of the fate of different subsets of cells. Multiple species developmental strategies emerged in the form of a unique cleavage pattern with stereotyped division geometries. Cleavage geometries have long been associated to the emergence of canonical developmental features such as cell cycle asynchrony, zygotic genome activation and fate specification. Yet, the direct causal role of division positioning on blastomere cell behavior remain partially understood. Oriented and/or asymmetric divisions define blastomere cell sizes, contacts and positions, with potential immediate impact on cellular decisions, lineage specification and morphogenesis. Division positions also instruct daughter cells polarity, mechanics and geometries, thereby influencing subsequent division events, in an emergent interplay that may pattern early embryos independently of firm deterministic genetic programs. We here review the recent literature which helped to delineate mechanisms and functions of division positioning in early embryos.

Fluid shear stress regulates the survival of circulating tumor cells via nuclear expansion.

Distant metastasis mainly occurs through hematogenous dissemination, where suspended circulating tumor cells (CTCs) experience a considerable level of fluid shear stress. We recently reported that shear flow induced substantial apoptosis of CTCs, although a small subpopulation could still persist. However, how suspended tumor cells survive in shear flow remains poorly understood. This study finds that fluid shear stress eliminates the majority of suspended CTCs and increases nuclear size, whereas it has no effect on the viability of adherent tumor cells and decreases their nuclear size. Shear flow promotes histone acetylation in suspended tumor cells, the inhibition of which using one drug suppresses shear-induced nuclear expansion, suggesting that shear stress might increase nuclear size through histone acetylation. Suppressing histone acetylation-mediated nuclear expansion enhances shear-induced apoptosis of CTCs. These findings suggest that suspended tumor cells respond to shear stress through histone acetylation-mediated nuclear expansion, which protects CTCs from shear-induced destruction. Our study elucidates a unique mechanism underlying the mechanotransduction of suspended CTCs to shear flow, which might hold therapeutic promise for CTC eradication.

The nuclear lamina is required for proper development and nuclear shape distortion in tomato.

The nuclear lamina in plant cells is composed of plant-specific proteins, including nuclear matrix constituent proteins (NMCPs), which have been postulated to be functional analogs of lamin proteins that provide structural integrity to the organelle and help stabilize the three-dimensional organization of the genome. Using genomic editing, we generated alleles for the three genes encoding NMCPs in cultivated tomato (Solanum lycopersicum) to determine if the consequences of perturbing the nuclear lamina in this crop species were similar to or distinct from those observed in the model Arabidopsis thaliana. Loss of the sole NMCP2-class protein was lethal in tomato but is tolerated in Arabidopsis. Moreover, depletion of NMCP1-type nuclear lamina proteins leads to distinct developmental phenotypes in tomato, including leaf morphology defects and reduced root growth rate (in nmcp1b mutants), compared with cognate mutants in Arabidopsis. These findings suggest that the nuclear lamina interfaces with different developmental and signaling pathways in tomato compared with Arabidopsis. At the subcellular level, however, tomato nmcp mutants resembled their Arabidopsis counterparts in displaying smaller and more spherical nuclei in differentiated cells. This result argues that the plant nuclear lamina facilitates nuclear shape distortion in response to forces exerted on the organelle within the cell.

Tumour cell characteristics and microenvironment composition correspond to clinical presentation in newly diagnosed nodular lymphocyte-predominant Hodgkin lymphoma.

Different studies have characterized the microenvironment and its prognostic impact in classic Hodgkin lymphoma whereas such analyses are pending for nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). We thus investigated characteristics of tumour cells and microenvironment in NLPHL and evaluated possible correlations with the clinical presentation. Lymph node samples from 152 NLPHL patients who had first-line treatment within the randomized German Hodgkin Study Group HD16-HD18 trials were available and analysed with regard to IgD status and nuclear size of the tumour cells as well as presence of PD1-positive follicular T helper cells and CD163-positive macrophages in the microenvironment. While large tumour cell nuclei and high numbers of PD1-positive follicular T helper cells in the microenvironment were more common in patients presenting with early/intermediate stages than in patients with advanced-stage disease (p < 0.0001, unpaired t-test; p = 0.0022, Mann-Whitney test), no differences between risk groups were observed in terms of the IgD status of the tumour cells and the content of CD163-positive macrophages in the microenvironment. PD1-positive follicular T helper cells were present in both cases with typical and variant growth patterns and rosetting around the tumour cells was observed in 96% of patients, indicating an important role of PD1-positive follicular T helper cells in NLPHL.

Specialization of nuclear membrane in eukaryotes.

The size of the intracellular structure that encloses genomic DNA - known as the nucleus in eukaryotes and nucleoid in prokaryotes - is believed to scale according to cell size and genomic content inside them across the tree of life. However, an actual scaling relationship remains largely unexplored across eukaryotic species. Here, I collected a large dataset of nuclear and cell volumes in diverse species across different phyla, including some prokaryotes, from the published literature and assessed the scaling relationship. Although entire inter-species data showed that nuclear volume correlates with cell volume, the quantitative scaling property exhibited differences among prokaryotes, unicellular eukaryotes and multicellular eukaryotes. Additionally, the nuclear volume correlates with genomic content inside the nucleus of multicellular eukaryotes but not of prokaryotes and unicellular eukaryotes. In this Hypothesis, I, thus, propose that the basic concept of nuclear-size scaling is conserved across eukaryotes; however, structural and mechanical properties of nuclear membranes and chromatin can result in different scaling relationships of nuclear volume to cell volume and genomic content among species. In particular, eukaryote-specific properties of the nuclear membrane may contribute to the extreme flexibility of nuclear size with regard to DNA density inside the nucleus.

Nuclear morphology in breast lesions: refining its assessment to improve diagnostic concordance.

AIMS: Although evaluation of nuclear morphology is important for the diagnosis and categorisation of breast lesions, the criteria used to assess nuclear atypia rely upon the subjective evaluation of several features that may result in inter- and intraobserver variation. This study aims to refine the definitions of cytonuclear features in various breast lesions. METHODS AND RESULTS: ImageJ was used to assess the nuclear morphological features including nuclear diameter, axis length, perimeter, area, circularity and roundness in 160 breast lesions comprising ductal carcinoma in situ (DCIS), invasive breast carcinoma of no special type (IBC-NST), tubular carcinoma, usual ductal hyperplasia (UDH), columnar cell change (CCC) and flat epithelial atypia (FEA). Reference cells included normal epithelial cells, red blood cells (RBCs) and lymphocytes. Reference cells showed size differences not only between normal epithelial cells and RBCs but also between RBCs in varied-sized blood vessels. Nottingham grade nuclear pleomorphism scores 1 and 3 cut-offs in IBC-NST, compared to normal epithelial cells, were < ×1.2 and > ×1.4 that of mean maximum Feret's diameter and < ×1.6 and > ×2.4 that of mean nuclear area, respectively. Nuclear morphometrics were significantly different in low-grade IBC-NST versus tubular carcinoma, low-grade DCIS versus UDH and CCC versus FEA. No differences in the nuclear features between grade-matched DCIS and IBC-NST were identified. CONCLUSION: This study provides a guide for the assessment of nuclear atypia in breast lesions, refines the comparison with reference cells and highlights the potential diagnostic value of image analysis tools in the era of digital pathology.

Differential contribution of nuclear size scaling mechanisms between Xenopus species.

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.

A homeostatic mechanism rapidly corrects aberrant nucleocytoplasmic ratios maintaining nuclear size in fission yeast.

Nuclear size scales with cell size across a wide range of cell types. The mechanism by which this scaling is maintained in growing cells remains unclear. Here, we investigate the mechanism of nuclear size homeostasis in the simple eukaryote fission yeast, by monitoring the recovery of aberrant nuclear volume to cell volume (N/C) ratios following perturbation. We demonstrate that both high and low N/C ratios correct rapidly, maintaining nuclear size homeostasis. We assess the kinetics of nuclear and cellular growth and of N/C ratio correction, and demonstrate that nuclear and cellular growth rates are not directly coupled. We propose that the mechanism underlying nuclear size homeostasis involves multiple limiting factors implicated in processes including nucleocytoplasmic transport, lipid biogenesis and RNA processing. We speculate that these link cellular size increases to changes in nuclear contents, which in turn lead to changes in nuclear membrane surface area. Our study reveals that there is rapid nuclear size homeostasis in cells, informing understanding of nuclear size control and size homeostasis of other membrane-bound organelles.

The challenge of staying in shape: nuclear size matters.

Cellular organelles have unique morphology and the organelle size to cell size ratio is regulated. Nucleus is one of the most prominent, usually round in shape, organelle of a eukaryotic cell that occupies 8-10% of cellular volume. The shape and size of nucleus is known to undergo remodeling during processes such as cell growth, division and certain stresses. Regulation of protein and lipid distribution at the nuclear envelope is crucial for preserving the nuclear morphology and size. As size and morphology are interlinked, altering one influences the other. In this perspective, we discuss the relationship between size and shape regulation of the nucleus.

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.

Control of cellular organization and its coordination with the cell cycle.

Cells organize themselves to maintain proper shape, structure, and size during growth and division for their cellular functions. However, how these cellular organizations coordinate with the cell cycle is not well understood. This review focuses on cell morphogenesis and size of the membrane-bound nucleus in the fission yeast Schizosaccharomyces pombe. Growth polarity, an important factor for cell morphogenesis, in rod-shaped fission yeast is restricted to the cell tips and dynamically changes depending on the cell cycle stage. Furthermore, nuclear size in fission yeast is proportional to the cell size, resulting in a constant ratio between nuclear volume and cellular volume (N/C ratio). This review summarizes the signaling pathway(s) involved in growth polarity control and key factors involved in N/C ratio control and provides their roles in coordination between cell organization and the cell cycle.

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.

Variation in nuclear size and PD-L2 positivity correlate with aggressive chromophobe renal cell carcinoma.

Chromophobe renal cell carcinoma (CRCC) is not amenable to International Society for Urologic Pathology-endorsed nucleolar grading. Novel grading approaches were proposed, but the rarity of adverse pathology hampers their discriminatory value. We investigate simple linear micrometer measurements and a proposed immunostain in CRCCs. 32 patients' CRCCs were studied: 12 adverse cases (stage pT3, recurrence, or metastasis), 15 controls (stage ≤pT2, no recurrence or metastasis after >3 years), and 8 metastases (3 were paired with primary adverse cases). The ratio of greatest dimensions of largest and smallest nuclei, in each of 5 "worst" high-power fields, excluding those with degenerative features, was designated variation in nuclear size (VNS). Percent multinucleate cells (PMC) were also counted. Mouse anti PD-L2 monoclonal antibody immunostaining was performed. Mean VNS measured in adverse primary and control primary tumors were 3.7 ±â€¯0.5 and 2.4 ±â€¯0.4 respectively (P < .001), and 3.4 ±â€¯0.4 for metastases (P < .001). Optimal VNS cut-off was 2.5, with sensitivity and specificity 0.85 and 0.81, respectively. PMCs were 6.0 ±â€¯3.0 for adverse group, 5.7 ±â€¯2.7 for controls, and 4.1 ±â€¯1.6 for metastases (P = NS). PD-L2 could not discriminate adverse versus good primary tumors (χ21.6, P = .2), but was higher in metastases (χ2 6.9, P < .01), or metastases plus adverse primary tumors (χ2 4.8, P = .03), compared to good-pathology primary tumors. In conclusion, VNS is an easily obtained measurement that can predict adverse behavior of chromophobe RCC, and may impart value for needle biopsy reporting and the choice of active surveillance. PD-L2 was elevated in metastases but was less useful for primary tumors.

Breaking the scale: how disrupting the karyoplasmic ratio gives cancer cells an advantage for metastatic invasion.

Nuclear size normally scales with the size of the cell, but in cancer this 'karyoplasmic ratio' is disrupted. This is particularly so in more metastatic tumors where changes in the karyoplasmic ratio are used in both diagnosis and prognosis for several tumor types. However, the direction of nuclear size changes differs for particular tumor types: for example in breast cancer, larger nuclear size correlates with increased metastasis, while for lung cancer smaller nuclear size correlates with increased metastasis. Thus, there must be tissue-specific drivers of the nuclear size changes, but proteins thus far linked to nuclear size regulation are widely expressed. Notably, for these tumor types, ploidy changes have been excluded as the basis for nuclear size changes, and so, the increased metastasis is more likely to have a basis in the nuclear morphology change itself. We review what is known about nuclear size regulation and postulate how such nuclear size changes can increase metastasis and why the directionality can differ for particular tumor types.

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.

Subclassification of prostate cancer circulating tumor cells by nuclear size reveals very small nuclear circulating tumor cells in patients with visceral metastases.

BACKGROUND: Although enumeration of circulating tumor cells (CTCs) has shown some clinical value, the pool of CTCs contains a mixture of cells that contains additional information that can be extracted. The authors subclassified CTCs by shape features focusing on nuclear size and related this with clinical information. METHODS: A total of 148 blood samples were obtained from 57 patients with prostate cancer across the spectrum of metastatic states: no metastasis, nonvisceral metastasis, and visceral metastasis. CTCs captured and enumerated on NanoVelcro Chips (CytoLumina, Los Angeles, Calif) were subjected to pathologic review including nuclear size. The distribution of nuclear size was analyzed using a Gaussian mixture model. Correlations were made between CTC subpopulations and metastatic status. RESULTS: Statistical modeling of nuclear size distribution revealed 3 distinct subpopulations: large nuclear CTCs, small nuclear CTCs, and very small nuclear CTCs (vsnCTCs). Small nuclear CTCs and vsnCTC identified those patients with metastatic disease. However, vsnCTC counts alone were found to be elevated in patients with visceral metastases when compared with those without (0.36 ± 0.69 vs 1.95 ± 3.77 cells/mL blood; P<.001). Serial enumeration studies suggested the emergence of vsnCTCs occurred before the detection of visceral metastases. CONCLUSIONS: There are morphologic subsets of CTCs that can be identified by fundamental pathologic approaches, such as nuclear size measurement. The results of this observational study strongly suggest that CTCs contain relevant information regarding disease status. In particular, the detection of vsnCTCs was found to be correlated with the presence of visceral metastases and should be formally explored as a putative blood-borne biomarker to identify patients at risk of developing this clinical evolution of prostate cancer.

A new approach for deducing rms proton radii from charge-changing reactions of neutron-rich nuclei and the reaction-target dependence.

We report the charge-changing cross sections (σcc) of 24 p-shell nuclides on both hydrogen and carbon at about 900A MeV, of which 8,9Li, 10-12Be, 10,14,15B, 14,15,17-22N and 16O on hydrogen and 8,9Li on carbon are for the first time. Benefiting from the data set, we found a new and robust relationship between the scaling factor of the Glauber model calculations and the separation energies of the nuclei of interest on both targets. This allows us to deduce proton radii (Rp) for the first time from the cross sections on hydrogen. Nearly identical Rp values are deduced from both target data for the neutron-rich carbon isotopes; however, the Rp from the hydrogen target is systematically smaller in the neutron-rich nitrogen isotopes. This calls for further experimental and theoretical investigations.

Nuclear Softness Promotes the Metastatic Potential of Large-Nucleated Colorectal Cancer Cells via the ErbB4-Akt1-Lamin A/C Signaling Pathway.

Abnormal nuclear enlargement is a diagnostic and physical hallmark of malignant tumors. Large nuclei are positively associated with an increased risk of developing metastasis; however, a large nucleus is inevitably more resistant to cell migration due to its size. The present study demonstrated that the nuclear size of primary colorectal cancer (CRC) cells at an advanced stage was larger than cells at an early stage. In addition, the nuclei of CRC liver metastases were larger than those of the corresponding primary CRC tissues. CRC cells were sorted into large-nucleated cells (LNCs) and small-nucleated cells (SNCs). Purified LNCs exhibited greater constricted migratory and metastatic capacity than SNCs in vitro and in vivo. Mechanistically, ErbB4 was highly expressed in LNCs, which phosphorylated lamin A/C at serine 22 via the ErbB4-Akt1 signaling pathway. Furthermore, the level of phosphorylated lamin A/C was a negative determinant of nuclear stiffness. Taken together, CRC LNCs possessed greater constricted migratory and metastatic potential than SNCs due to ErbB4-Akt1-mediated lamin A/C phosphorylation and nuclear softening. These results may provide a potential treatment strategy for tumor metastasis by targeting nuclear stiffness in patients with cancer, particularly CRC.

Postsynaptic BMP signaling regulates myonuclear properties in Drosophila larval muscles.

The syncytial mammalian muscle fiber contains a heterogeneous population of (myo)nuclei. At the neuromuscular junction (NMJ), myonuclei have specialized positioning and gene expression. However, it remains unclear how myonuclei are recruited and what regulates myonuclear output at the NMJ. Here, we identify specific properties of myonuclei located near the Drosophila larval NMJ. These synaptic myonuclei have increased size in relation to their surrounding cytoplasmic domain (scaling), increased DNA content (ploidy), and increased levels of transcription factor pMad, a readout for BMP signaling activity. Our genetic manipulations show local BMP signaling affects muscle size, nuclear size, ploidy, and NMJ size and function. In support, RNA sequencing analysis reveals that pMad regulates genes involved in muscle growth, ploidy (i.e., E2f1), and neurotransmission. Our data suggest that muscle BMP signaling instructs synaptic myonuclear output that then positively shapes the NMJ synapse. This study deepens our understanding of how myonuclear heterogeneity supports local signaling demands to fine tune cellular function and NMJ activity.