Nuclear Size Regulation Research Articles

Nuclear Structure, Size Regulation, and Role in Cell Migration.

The nucleus serves as a pivotal regulatory and control hub in the cell, governing numerous aspects of cellular functions, including DNA replication, transcription, and RNA processing. Therefore, any deviations in nuclear morphology, structure, or organization can strongly affect cellular activities. In this review, we provide an updated perspective on the structure and function of nuclear components, focusing on the linker of nucleoskeleton and cytoskeleton complex, the nuclear envelope, the nuclear lamina, and chromatin. Additionally, nuclear size should be considered a fundamental parameter for the cellular state. Its regulation is tightly linked to environmental changes, development, and various diseases, including cancer. Hence, we also provide a concise overview of different mechanisms by which nuclear size is determined, the emerging role of the nucleus as a mechanical sensor, and the implications of altered nuclear morphology on the physiology of diseased cells.

STEM-06. NUCLEAR STRUCTURE MAINTAINS GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma stem cells (GSCs) contribute to therapeutic resistance, promote tumor angiogenesis, and evade anti-tumor immune responses. We and others have shown that GSCs display differential regulation of their epigenetic landscapes and metabolic profiles and contribute to the striking cellular heterogeneity and complex tumor microenvironments. Here, we find that GSCs undergo dramatic changes in chromatin organization and nuclear morphology upon differentiation. Leveraging unbiased RNA sequencing and protein profiling screens comparing GSCs and differentiated glioma cells, we identified nuclear membrane molecular regulation that defines nuclear size and lipid regulation. Genetic or pharmacologic targeting of nuclear envelope function reduced cancer stem cell proliferation, self-renewal, and in vivo tumor growth. Collectively, we define a novel role of the nuclear envelope in maintaining chromatin state and cancer stem cell self-renewing, informing the development of novel potential therapeutic paradigms for glioblastoma.

Volume measurement reveals the mechano-mediated regulation of nuclear size

Eukaryotic cells finely control their size and, typically, nuclear volume scales with cell volume. The corresponding nuclear to cell volume ratio (N/C ratio) is narrowly distributed for a given cell-type and deviation from this distribution is a well-known hallmark of senescence or cancer. However, mainly for the lack of methods to simultaneously measure both cellular and nuclear volumes, the mechanisms underlying their size regulation and coupling remain unclear.To overcome this limitation, we developed an imaging method for measuring in real time both cytoplasmic and nuclear volumes of living cells.

High-content imaging-based pooled CRISPR screens in mammalian cells.

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

Nucleoplasmin is a limiting component in the scaling of nuclear size with cytoplasmic volume.

How nuclear size is regulated relative to cell size is a fundamental cell biological question. Reductions in both cell and nuclear sizes during Xenopus laevis embryogenesis provide a robust scaling system to study mechanisms of nuclear size regulation. To test if the volume of embryonic cytoplasm is limiting for nuclear growth, we encapsulated gastrula-stage embryonic cytoplasm and nuclei in droplets of defined volume using microfluidics. Nuclei grew and reached new steady-state sizes as a function of cytoplasmic volume, supporting a limiting component mechanism of nuclear size control. Through biochemical fractionation, we identified the histone chaperone nucleoplasmin (Npm2) as a putative nuclear size effector. Cellular amounts of Npm2 decrease over development, and nuclear size was sensitive to Npm2 levels both in vitro and in vivo, affecting nuclear histone levels and chromatin organization. We propose that reductions in cell volume and the amounts of limiting components, such as Npm2, contribute to developmental nuclear size scaling.

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/β-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.

Nuclear speckle fusion via long-range directional motion regulates speckle morphology after transcriptional inhibition.

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significantly increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 µm and with velocities between 0.2 µm/min and 1.5 µm/min. Frequently, three or even four speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer, but larger, speckles after transcriptional inhibition. These speckle movements are not actin dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle marker protein SON. Similar long-range speckle movements and fusion events were observed after heat shock or heavy metal stress, and during late G2 and early prophase. Our observations suggest a mechanism for long-range, directional nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization. This article has an associated First Person interview with the first author of the paper.

The Multiple Ways Nuclei Scale on a Multinucleated Muscle Cell Scale

In mononucleated cells, nuclear size scales with cell size, but does this relationship extend to multinucleated cells? In this issue of Developmental Cell,Windner etal. (2019) examine scaling of nuclei in multinucleated Drosophila muscle fibers and identify global and local cellular inputs that contribute to nuclear size regulation.

PP2A-B55/SUR-6 collaborates with the nuclear lamina for centrosome separation during mitotic entry.

Across most sexually reproducing animals, centrosomes are provided to the oocyte through fertilization and must be positioned properly to establish the zygotic mitotic spindle. How centrosomes are positioned in space and time through the concerted action of key mitotic entry biochemical regulators, including protein phosphatase 2A (PP2A-B55/SUR-6), biophysical regulators, including dynein, and the nuclear lamina is unclear. Here, we uncover a role for PP2A-B55/SUR-6 in regulating centrosome separation. Mechanistically, PP2A-B55/SUR-6 regulates nuclear size before mitotic entry, in turn affecting nuclear envelope–based dynein density and motor capacity. Computational simulations predicted the requirement of PP2A-B55/SUR-6 regulation of nuclear size and nuclear-envelope dynein density for proper centrosome separation. Conversely, compromising nuclear lamina integrity led to centrosome detachment from the nuclear envelope and migration defects. Removal of PP2A-B55/SUR-6 and the nuclear lamina simultaneously further disrupted centrosome separation, leading to unseparated centrosome pairs dissociated from the nuclear envelope. Taking these combined results into consideration, we propose a model in which centrosomes migrate and are positioned through the concerted action of PP2A-B55/SUR-6–regulated nuclear envelope–based dynein pulling forces and centrosome–nuclear envelope tethering. Our results add critical precision to models of centrosome separation relative to the nucleus during spindle formation in cell division.

Cytoplasmic Volume and Limiting Nucleoplasmin Scale Nuclear Size During <i>Xenopus Laevis</i> Development

How nuclear size is regulated relative to cell size is a fundamental cell biological question. Reductions in both cell and nuclear sizes during Xenopus laevis embryogenesis provide a robust scaling system to study mechanisms of nuclear size regulation. To test if the volume of embryonic cytoplasm is limiting for nuclear growth, we encapsulated gastrula stage embryonic cytoplasm and nuclei in droplets of defined volume using microfluidics. Nuclei grew and reached new steady-state sizes as a function of cytoplasmic volume, supporting a limiting component mechanism of nuclear size control. Through biochemical fractionation, we identified the histone chaperone nucleoplasmin (Npm2) as a putative nuclear size-scaling factor. Cellular amounts of Npm2 decrease over development, and nuclear size was sensitive to Npm2 levels both in vitro and in vivo, affecting nuclear histone levels and chromatin organization. Thus, reductions in cell volume with concomitant decreases in Npm2 amounts represent a developmental mechanism of nuclear size-scaling that may also be relevant to cancers with increased nuclear size.

Abstract 4456: Increased NTF2 levels in melanoma cell lines affect nuclear size and cancer cell characteristics

Abstract Xenopus studies have implicated two nuclear transport factors in the regulation of nuclear size: NTF2 and importin α. Importin α, a positive regulator of nuclear size, has been suggested as a biomarker in breast cancer and non-small cell lung carcinoma. We have focused our studies on NTF2. In different staged melanoma cell lines and tissue samples, we observed an inverse correlation between NTF2 levels and nuclear size. Compared to cancer cells, normal melanocytes exhibited the smallest nuclei and highest NTF2 levels. Based on transient transfection experiments in HeLa cells, MRC5 cells, and different melanoma cell lines, we found that increasing NTF2 expression levels reduces nuclear size. The largest effect was observed in WM3211 melanoma cells in which NTF2 expression reduced nuclear cross-sectional area by 40%. We next generated a stably transfected NTF2-inducible version of the metastatic melanoma cell line WM983B. By altering the doxycycline concentration, we can titrate the level of NTF2 protein, and we found that 20 ng/ml of doxycycline increases NTF2 levels by 5-fold and decreases the nuclear cross-sectional area by about 10%. Doxycycline-treated cells exhibited 3-fold higher rates of apoptosis and a 40% reduction in the rate of cell migration in a wound-healing assay. In vivo experiments were performed on RAG2 and NSG mice where NTF2-inducible cells were injected intravenously in order to examine metastatic potential. The number of lung metastases was reduced by 7-fold in doxycycline-treated mice compared to those that were not treated, suggesting that NTF2 overexpression suppresses metastatic potential. We are now testing the hypothesis that altering nuclear size by NTF2 expression leads to chromatin remodeling and altered gene expression. We performed RNAseq on doxycycline-treated and non-treated cells. Differentially expressed genes include those involved in cell migration (e.g. PRKX, LAMA5, ADAMTS15, ANK3, CERS6) and cell survival (e.g. RAMP1, WNK3, BMP5, TP63). Performing DNA FISH will show if the nuclear positioning of these genes is changed when nuclear size is reduced. We are now planning to test a combination therapy in mice in which we reduce nuclear size and treat with the well-established melanoma drug vemurafenib. Most melanoma patients with BRAF mutations show a good response to this drug, however relapse often occurs with drug-resistant melanoma cells expressing high levels of the cancer stem cell markers JARID1B, CD271, and fibronectin. According to our RNAseq data, cells expressing higher levels of NTF2 show reduced gene expression of JARID1B and fibronectin. Thus increasing NTF2 expression in drug-resistant tumors may improve prognosis and survival time. Citation Format: Lidija D. Vukovic, Bradley A. Stohr, Dan L. Levy. Increased NTF2 levels in melanoma cell lines affect nuclear size and cancer cell characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4456.

SIRT1 regulates nuclear number and domain size in skeletal muscle fibers.

Skeletal muscle fibers are giant multinucleated cells wherein individual nuclei govern the protein synthesis in a finite volume of cytoplasm; this is termed the myonuclear domain (MND). The factors that control MND size remain to be defined. In the present study, we studied the contribution of the NAD+‐dependent deacetylase, sirtuin 1 (SIRT1), to the regulation of nuclear number and MND size. For this, we isolated myofibers from mice with tissue‐specific inactivation (mKO) or inducible overexpression (imOX) of SIRT1 and analyzed the 3D organisation of myonuclei. In imOX mice, the number of nuclei was increased whilst the average MND size was decreased as compared to littermate controls. Our findings were the opposite in mKO mice. Muscle stem cell (satellite cell) numbers were reduced in mKO muscles, a possible explanation for the lower density of myonuclei in these mice; however, no change was observed in imOX mice, suggesting that other factors might also be involved, such as the functional regulation of stem cells/muscle precursors. Interestingly, however, the changes in the MND volume did not impact the force‐generating capacity of muscle fibers. Taken together, our results demonstrate that SIRT1 is a key regulator of MND sizes, although the underlying molecular mechanisms and the cause‐effect relationship between MND and muscle function remain to be fully defined.

Nucleus Assembly and Import in Xenopus laevis Egg Extract.

Xenopus egg extract represents a powerful cell-free biochemical tool for studying organelle assembly and function. Large quantities of cytoplasm can be isolated, and biochemical manipulation of extract composition and cell cycle state is relatively straightforward. In this protocol, we describe the reconstitution of nuclear assembly by adding a chromatin source to interphasic X. laevis egg extract. Intact nuclei assemble within 30-45 min of initiating the reaction, followed by nuclear growth. We also describe methods for imaging and quantifying nuclear import kinetics. Recombinant proteins or small molecules of interest can be added to the extract before or after nuclear assembly, and immunodepletion allows for removal of specific proteins from the extract. This approach will continue to inform mechanisms of nuclear assembly, nuclear pore complex assembly and function, nucleocytoplasmic transport, DNA replication, nuclear envelope breakdown, and nuclear size and shape regulation.

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.

Characterization of the lamin analogue NMCP2 in the monocot Allium cepa.

Nuclear lamina organization is similar in metazoan and plants though the latter lack orthologs of lamins, the main components of the metazoan lamina. Current evidence suggests that Nuclear Matrix Constituent Proteins (NMCPs) are the lamin analogues in plants as these proteins share several key features: higher-order secondary structure and domain layout, subnuclear distribution, and involvement in the regulation of nuclear shape and size, as well as in higher-order chromatin organization. Previously, we studied the NMCP family in flowering plants (angiosperms), in which it comprises two phylogenetic groups: NMCP1 and NMCP2. At present, in silico information about NMCP proteins in embryophytes is relatively advanced, though very few proteins, most of them of the NMCP1 type, have been extensively studied in vivo. We previously characterized the NCMP1 protein in the monocot Allium cepa. Here, we report the key features of a second protein of this species NMCP2, which presents a conserved sequence and domain layout. Immunofluorescence and immunoelectronmicroscopy evidence co-localization of endogenous AcNMCP2 and AcNMCP1 in the lamina, while Western blotting and immunoconfocal microscopy reveal a similar pattern of expression and distribution of both NMCP proteins in different root tissues. Our results provide novel insight about endogenous NMCP2-type proteins and complete the characterization of the NMCP family in A. cepa, thus advancing the current understanding of these structural proteins constituting the plant lamina.

PKC-mediated phosphorylation of nuclear lamins at a single serine residue regulates interphase nuclear size in Xenopus and mammalian cells.

How nuclear size is regulated is a fundamental cell-biological question with relevance to cancers, which often exhibit enlarged nuclei. We previously reported that conventional protein kinase C (cPKC) contributes to nuclear size reductions that occur during early Xenopus development. Here we report that PKC-mediated phosphorylation of lamin B3 (LB3) contributes to this mechanism of nuclear size regulation. By mapping PKC phosphorylation sites on LB3 and testing the effects of phosphomutants in Xenopus laevis embryos, we identify the novel site S267 as being an important determinant of nuclear size. Furthermore, FRAP studies demonstrate that phosphorylation at this site increases lamina dynamics, providing a mechanistic explanation for how PKC activity influences nuclear size. We subsequently map this X. laevis LB3 phosphorylation site to a conserved site in mammalian lamin A (LA), S268. Manipulating PKC activity in cultured mammalian cells alters nuclear size, as does expression of LA-S268 phosphomutants. Taken together, these data demonstrate that PKC-mediated lamin phosphorylation is a conserved mechanism of nuclear size regulation.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation.

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

Abstract 475: NTF2 regulates nuclear size in mammalian cells and may contribute to altered nuclear size in melanoma

Abstract Nuclear size and morphology are distinct cancer features that pathologists use for diagnostic purposes. Proteins that regulate nuclear size have been identified in Xenopus, where nuclear import rates correlate with nuclear size. The levels of two nuclear import factors, importin α and NTF2, were shown to be particularly important in nuclear size regulation. It has also been shown that importin α2 is a potential biomarker in non-small cell lung cancer, as well as importin α1 in breast cancer. Our data implicate that NTF2 is a possible cancer biomarker. Our main goal is to analyze how these nuclear scaling factors affect nuclear size during carcinogenesis and to test how nuclear size impacts cancer growth characteristics. We find that manipulating the levels of importin α2 and NTF2 in HeLa and MRC-5 cells leads to altered nuclear size, demonstrating a conserved function for these factors in nuclear size regulation. To place these results in a cancer context, we are focusing on melanoma cell lines, including radial and vertical growth primary melanomas and metastatic melanomas. We find that, compared to normal melanocytes, the nuclear-to-cytoplasmic (N/C) volume ratio is increased in the cancer cell lines. Western blot analysis revealed an inverse correlation between NTF2 protein levels and nuclear size. We confirmed these results using a melanoma tissue microarray, finding similar differences in nuclear size and NTF2 expression levels between benign nevi and melanomas. We next performed transient transfection experiments with our melanoma cell lines. Importin α2 knock down led to smaller nuclear size, while knocking down the levels of NTF2 increased nuclear size, consistent with our Xenopus results. We also observed reduced nuclear size when NTF2 was ectopically expressed. Currently we are generating stable, inducible melanoma cell lines where we can precisely alter NTF2 and importin α expression levels using doxycycline. We will use these inducible cell lines to examine how altering nuclear size impacts cell proliferation rates, invasiveness, and apoptosis. We will also investigate how nuclear size affects gene positioning using FISH, focusing on genes involved in melanoma formation and metastasis. In the long term, we plan to move these studies into a mouse model where we can test how nuclear size affects in vivo metastatic capacity. Citation Format: Lidija D. Vukovic, Bradley A. Stohr, Dan L. Levy. NTF2 regulates nuclear size in mammalian cells and may contribute to altered nuclear size in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 475.

Nucleus downscaling in mouse embryos is regulated by cooperative developmental and geometric programs.

Maintaining appropriate nucleus size is important for cell health, but the mechanisms by which this is achieved are poorly understood. Controlling nucleus size is a particular challenge in early development, where the nucleus must downscale in size with progressive reductive cell divisions. Here we use live and fixed imaging, micromanipulation approaches, and small molecule analyses during preimplantation mouse development to probe the mechanisms by which nucleus size is determined. We find a close correlation between cell and nuclear size at any given developmental stage, and show that experimental cytoplasmic reduction can alter nuclear size, together indicating that cell size helps dictate nuclear proportions. Additionally, however, by creating embryos with over-sized blastomeres we present evidence of a developmental program that drives nuclear downscaling independently of cell size. We show that this developmental program does not correspond with nuclear import rates, but provide evidence that PKC activity may contribute to this mechanism. We propose a model in which nuclear size regulation during early development is a multi-mode process wherein nucleus size is set by cytoplasmic factors, and fine-tuned on a cell-by-cell basis according to cell size.

Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation.