Mechanical stress confers nuclear and functional changes in derived leukemia cells from persistent confined migration.

Nuclear deformability plays a critical role in cell migration. During this process, the remodeling of internal components of the nucleus has a direct impact on DNA damage and cell behavior; however, how persistent migration promotes nuclear changes leading to phenotypical and functional consequences remains poorly understood. Here, we described that the persistent migration through physical barriers was sufficient to promote permanent modifications in migratory-altered cells. We found that derived cells from confined migration showed changes in lamin B1 localization, cell morphology and transcription. Further analysis confirmed that migratory-altered cells showed functional differences in DNA repair, cell response to chemotherapy and cell migration in vivo homing experiments. Experimental modulation of actin polymerization affected the redistribution of lamin B1, and the basal levels of DNA damage in migratory-altered cells. Finally, since major nuclear changes were present in migratory-altered cells, we applied a multidisciplinary biochemical and biophysical approach to identify that confined conditions promoted a different biomechanical response of the nucleus in migratory-altered cells. Our observations suggest that mechanical compression during persistent cell migration has a role in stable nuclear and genomic alterations that might handle the genetic instability and cellular heterogeneity in aging diseases and cancer.

Maternal SMC2 is essential for embryonic development via participating chromosome condensation in mice.

During the oocyte growth, maturation and zygote development, chromatin structure keeps changing to regulate different nuclear activities. Here, we reported the role of SMC2, a core component of condensin complex, in oocyte and embryo development. Oocyte-specific conditional knockout of SMC2 caused female infertility. In the absence of SMC2, oocyte meiotic maturation and ovulation occurred normally, but chromosome condensation showed defects and DNA damages were accumulated in oocytes. The pronuclei were abnormally organized and micronuclei were frequently observed in fertilized eggs, their activity was impaired, and embryo development was arrested at the one-cell stage, suggesting that maternal SMC2 is essential for embryonic development.

Scaling of nuclear numbers and their spatial arrangement in skeletal muscle cell size regulation.

Many cells display considerable functional plasticity and depend on the regulation of numerous organelles and macromolecules for their maintenance. In large cells, organelles also need to be carefully distributed to supply the cell with essential resources and regulate intracellular activities. Having multiple copies of the largest eukaryotic organelle, the nucleus, epitomizes the importance of scaling gene products to large cytoplasmic volumes in skeletal muscle fibers. Scaling of intracellular constituents within mammalian muscle fibers is, however, poorly understood, but according to the myonuclear domain hypothesis, a single nucleus supports a finite amount of cytoplasm and is thus postulated to act autonomously, causing the nuclear number to be commensurate with fiber volume. In addition, the orderly peripheral distribution of myonuclei is a hallmark of normal cell physiology, as nuclear mispositioning is associated with impaired muscle function. Because underlying structures of complex cell behaviors are commonly formalized by scaling laws and thus emphasize emerging principles of size regulation, the work presented herein offers more of a unified conceptual platform based on principles from physics, chemistry, geometry, and biology to explore cell size-dependent correlations of the largest mammalian cell by means of scaling.

Single-chromosome dynamics reveals locus-dependent dynamics and chromosome territory orientation.

Dynamic chromatin organization instantly influences DNA accessibility through modulating local macromolecular density and interactions, driving changes in transcription activities. Chromatin dynamics have been reported to be locally confined but contribute to coherent chromatin motion across the entire nucleus. However, the regulation of dynamics, nuclear orientation and compaction of subregions along a single chromosome are not well-understood. We used CRISPR-based real-time single-particle tracking and polymer models to characterize the dynamics of specific genomic loci and determine compaction levels of large human chromosomal domains. Our studies showed that chromosome compaction changed during interphase and that compactions of two arms on chromosome 19 were different. The dynamics of genomic loci were subdiffusive and dependent on chromosome regions and transcription states. Surprisingly, the correlation between locus-dependent nuclear localization and mobility was negligible. Strong tethering interactions detected at the pericentromeric region implies local condensation or associations with organelles within local nuclear microenvironments, such as chromatin-nuclear body association. Based on our findings, we propose a 'guided radial model' for the nuclear orientation of the long arm of chromosome 19.

Omne vivum ex ovo: the oocyte reprogramming and remodeling activities.

In brief: Understanding the establishment of post-fertilization totipotency has broad implications for modern biotechnologies. This review summarizes the current knowledge of putative egg components governing this process following natural fertilization and after somatic cell nuclear transfer. Abstract: The mammalian oocyte is a unique cell, and comprehending its physiology and biology is essential for understanding fertilization, totipotency and early events of embryogenesis. Consequently, research in these areas influences the outcomes of various technologies, for example, the production and conservation of laboratory and large animals with rare and valuable genotypes, the rescue of the species near extinction, as well as success in human assisted reproduction. Nevertheless, even the most advanced and sophisticated reproductive technologies of today do not always guarantee a favorable outcome. Elucidating the interactions of oocyte components with its natural partner cell - the sperm or an 'unnatural' somatic nucleus, when the somatic cell nucleus transfer is used is essential for understanding how totipotency is established and thus defining the requirements for normal development. One of the crucial aspects is the stoichiometry of different reprogramming and remodeling factors present in the oocyte and their balance. Here, we discuss how these factors, in combination, may lead to the formation of a new organism. We focus on the laboratory mouse and its genetic models, as this species has been instrumental in shaping our understanding of early post-fertilization events.

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.

Sculpting Rupture-Free Nuclear Shapes in Fibrous Environments.

Cytoskeleton-mediated force transmission regulates nucleus morphology. How nuclei shaping occurs in fibrous in vivo environments remains poorly understood. Here suspended nanofiber networks of precisely tunable (nm-µm) diameters are used to quantify nucleus plasticity in fibrous environments mimicking the natural extracellular matrix. Contrary to the apical cap over the nucleus in cells on 2-dimensional surfaces, the cytoskeleton of cells on fibers displays a uniform actin network caging the nucleus. The role of contractility-driven caging in sculpting nuclear shapes is investigated as cells spread on aligned single fibers, doublets, and multiple fibers of varying diameters. Cell contractility increases with fiber diameter due to increased focal adhesion clustering and density of actin stress fibers, which correlates with increased mechanosensitive transcription factor Yes-associated protein (YAP) translocation to the nucleus. Unexpectedly, large- and small-diameter fiber combinations lead to teardrop-shaped nuclei due to stress fiber anisotropy across the cell. As cells spread on fibers, diameter-dependent nuclear envelope invaginations that run the nucleus's length are formed at fiber contact sites. The sharpest invaginations enriched with heterochromatin clustering and sites of DNA repair are insufficient to trigger nucleus rupture. Overall, the authors quantitate the previously unknown sculpting and adaptability of nuclei to fibrous environments with pathophysiological implications.

Suppression of PI3K signaling is linked to autophagy activation and the spatiotemporal induction of the lens organelle free zone.

The terminal steps of lens cell differentiation require elimination of all organelles to create a central Organelle Free Zone (OFZ) that is required for lens function of focusing images on the retina. Previous studies show that the spatiotemporal elimination of these organelles during development is autophagy-dependent. We now show that the inhibition of PI3K signaling in lens organ culture results in the premature induction of autophagy within 24 h, including a significant increase in LAMP1+ lysosomes, and the removal of lens organelles from the center of the lens. Specific inhibition of just the PI3K/Akt signaling axis was directly linked to the elimination of mitochondria and ER, while pan-PI3K inhibitors that block all PI3K downstream signaling removed all organelles, including nuclei. Therefore, blocking the PI3K/Akt pathway was alone insufficient to remove nuclei. RNAseq analysis revealed increased mRNA levels of the endogenous inhibitor of PI3K activation, PIK3IP1, in differentiating lens fiber cells preceding the induction of OFZ formation. Co-immunoprecipitation confirmed that PIK3IP1 associates with multiple PI3K p110 isoforms just prior to formation of the OFZ, providing a likely endogenous mechanism for blocking all PI3K signaling and activating the autophagy pathway required to form the OFZ during lens development.

Screening of Differentially Expressed Genes and Localization Analysis of Female Gametophyte at the Free Nuclear Mitosis Stage in Pinus tabuliformis Carr.

Female sterility is a common phenomenon in the plant world, and systematic research has not been carried out in gymnosperms. In this study, the ovules of No. 28 sterile line and No. 15 fertile line Pinus tabuliformis were used as materials, and a total of 18 cDNA libraries were sequenced by the HiSeqTM 4000 platform to analyze the differentially expressed genes (DEGs) and simple sequence repeats (SSRs) between the two lines. In addition, this study further analyzed the DEGs involved in the signal transduction of plant hormones, revealing that the signal pathways related to auxin, cytokinin, and gibberellin were blocked in the sterile ovule. Additionally, real-time fluorescent quantitative PCR verified that the expression trend of DEGs related to plant hormones was consistent with the results of high-throughput sequencing. Frozen sections and fluorescence in situ hybridization (FISH) were used to study the temporal and spatial expression patterns of PtRab in the ovules of P. tabuliformis. It was found that PtRab was significantly expressed in female gametophytes and rarely expressed in the surrounding diploid tissues. This study further explained the molecular regulation mechanism of female sterility in P. tabuliformis, preliminarily mining the key factors of ovule abortion in gymnosperms at the transcriptional level.

SPRITE: a genome-wide method for mapping higher-order 3D interactions in the nucleus using combinatorial split-and-pool barcoding.

A fundamental question in gene regulation is how cell-type-specific gene expression is influenced by the packaging of DNA within the nucleus of each cell. We recently developed Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which enables mapping of higher-order interactions within the nucleus. SPRITE works by cross-linking interacting DNA, RNA and protein molecules and then mapping DNA-DNA spatial arrangements through an iterative split-and-pool barcoding method. All DNA molecules within a cross-linked complex are barcoded by repeatedly splitting complexes across a 96-well plate, ligating molecules with a unique tag sequence, and pooling all complexes into a single well before repeating the tagging. Because all molecules in a cross-linked complex are covalently attached, they will sort together throughout each round of split-and-pool and will obtain the same series of SPRITE tags, which we refer to as a barcode. The DNA fragments and their associated barcodes are sequenced, and all reads sharing identical barcodes are matched to reconstruct interactions. SPRITE accurately maps pairwise DNA interactions within the nucleus and measures higher-order spatial contacts occurring among up to thousands of simultaneously interacting molecules. Here, we provide a detailed protocol for the experimental steps of SPRITE, including a video ( https://youtu.be/6SdWkBxQGlg ). Furthermore, we provide an automated computational pipeline available on GitHub that allows experimenters to seamlessly generate SPRITE interaction matrices starting with raw fastq files. The protocol takes ~5 d from cell cross-linking to high-throughput sequencing for the experimental steps and 1 d for data processing.

Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase.

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

A deep generative model of 3D single-cell organization.

We introduce a framework for end-to-end integrative modeling of 3D single-cell multi-channel fluorescent image data of diverse subcellular structures. We employ stacked conditional ß-variational autoencoders to first learn a latent representation of cell morphology, and then learn a latent representation of subcellular structure localization which is conditioned on the learned cell morphology. Our model is flexible and can be trained on images of arbitrary subcellular structures and at varying degrees of sparsity and reconstruction fidelity. We train our full model on 3D cell image data and explore design trade-offs in the 2D setting. Once trained, our model can be used to predict plausible locations of structures in cells where these structures were not imaged. The trained model can also be used to quantify the variation in the location of subcellular structures by generating plausible instantiations of each structure in arbitrary cell geometries. We apply our trained model to a small drug perturbation screen to demonstrate its applicability to new data. We show how the latent representations of drugged cells differ from unperturbed cells as expected by on-target effects of the drugs.

Yolk platelets impede nuclear expansion in Xenopus embryos.

During metazoan early embryogenesis, the intracellular properties of proteins and organelles change dynamically through rapid cleavage. In particular, a change in the nucleus size is known to contribute to embryonic development-dependent cell cycle and gene expression regulation. Here, we compared the nuclear sizes of various blastomeres from developing Xenopus embryos and analyzed the mechanisms that control the nuclear expansion dynamics by manipulating the amount of intracellular components in a cell-free system. Nuclear expansion was slower in blastomeres from vegetal hemispheres during a longer interphase than in those from animal hemispheres. Furthermore, upon recapitulating interphase events by manipulating the concentration of yolk platelets, which are originally rich in the vegetal blastomeres, in cell-free cytoplasmic extracts, nuclear expansion and DNA replication became slower than that in normal yolk-free conditions. Under these conditions, the supplemented yolk platelets accumulated around the nucleus in a microtubule-dependent manner and impeded the organization of the endoplasmic reticulum network. Overall, we propose that yolk platelets around the nucleus reduce membrane supply from the endoplasmic reticulum to the nucleus, resulting in slower nuclear expansion and cell cycle progression in the yolk-rich vegetal blastomeres.

Asymmetry is defined during meiosis in the oocyte of the parthenogenetic nematode Diploscapter pachys.

Asymmetric cell division is an essential feature of normal development and certain pathologies. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo, particularly how symmetry of the actomyosin cortical cytoskeleton is broken by a sperm-derived signal at fertilization, upstream of polarity establishment. Diploscapter pachys is the closest parthenogenetic relative to C. elegans, and D. pachys one-cell embryos also divide asymmetrically. However how polarity is triggered in the absence of sperm remains unknown. In post-meiotic embryos, we find that the nucleus inhabits principally one embryo hemisphere, the future posterior pole. When forced to one pole by centrifugation, the nucleus returns to its preferred pole, although poles appear identical as concerns cortical ruffling and actin cytoskeleton. The location of the meiotic spindle also correlates with the future posterior pole and slight actin enrichment is observed at that pole in some early embryos along with microtubule structures emanating from the meiotic spindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachys oocytes. All together our results are consistent with the idea that polarity of the D. pachys embryo is attained during meiosis, seemingly based on the location of the meiotic spindle, by a mechanism that may be present but suppressed in C. elegans.

Nucleus and chloroplast: A necessary understanding to overcome heat stress in Pinus radiata.

The recovery and maintenance of plant homeostasis under stressful environments are complex processes involving organelle crosstalk for a coordinated cellular response. Here, we revealed through nuclear and chloroplast subcellular proteomics, biochemical cell profiles and targeted transcriptomics how chloroplasts and nuclei developed their responses under increased temperatures in a long-lived species (Pinus radiata). Parallel to photosynthetic impairment and reactive oxygen species production in the chloroplast, a DNA damage response was triggered in the nucleus followed by an altered chromatin conformation. In addition, in the nuclei, we found several proteins, such as HEMERA or WHIRLY, which change their locations from the chloroplasts to the nuclei carrying the stress message. Additionally, our data showed a deep rearrangement of RNA metabolism in both organelles, revealing microRNAs and AGO1 as potential regulators of the acclimation mechanisms. Altogether, our study highlights the synchronisation among the different stages required for thermotolerance acquisition in P. radiata, pointing out the role of chromatin conformation and posttranscriptional gene regulation in overcoming heat stress and assuring plant survival for the following years.

Mitochondrial-nuclear cross-talk in the human brain is modulated by cell type and perturbed in neurodegenerative disease.

Mitochondrial dysfunction contributes to the pathogenesis of many neurodegenerative diseases. The mitochondrial genome encodes core respiratory chain proteins, but the vast majority of mitochondrial proteins are nuclear-encoded, making interactions between the two genomes vital for cell function. Here, we examine these relationships by comparing mitochondrial and nuclear gene expression across different regions of the human brain in healthy and disease cohorts. We find strong regional patterns that are modulated by cell-type and reflect functional specialisation. Nuclear genes causally implicated in sporadic Parkinson's and Alzheimer's disease (AD) show much stronger relationships with the mitochondrial genome than expected by chance, and mitochondrial-nuclear relationships are highly perturbed in AD cases, particularly through synaptic and lysosomal pathways, potentially implicating the regulation of energy balance and removal of dysfunction mitochondria in the etiology or progression of the disease. Finally, we present MitoNuclearCOEXPlorer, a tool to interrogate key mitochondria-nuclear relationships in multi-dimensional brain data.

The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair.

Transient nuclear envelope ruptures during interphase (NERDI) occur due to cytoskeletal compressive forces at sites of weakened lamina, and delayed NERDI repair results in genomic instability. Nuclear envelope (NE) sealing is completed by endosomal sorting complex required for transport (ESCRT) machinery. A key unanswered question is how local compressive forces are counteracted to allow efficient membrane resealing. Here, we identify the ESCRT-associated protein BROX as a crucial factor required to accelerate repair of the NE. Critically, BROX binds Nesprin-2G, a component of the linker of nucleoskeleton and cytoskeleton complex (LINC). This interaction promotes Nesprin-2G ubiquitination and facilitates the relaxation of mechanical stress imposed by compressive actin fibers at the rupture site. Thus, BROX rebalances excessive cytoskeletal forces in cells experiencing NE instability to promote effective NERDI repair. Our results demonstrate that BROX coordinates mechanoregulation with membrane remodeling to ensure the maintenance of nuclear-cytoplasmic compartmentalization and genomic stability.

Mechanotransduction at the Plasma Membrane-Cytoskeleton Interface.

Mechanical cues are crucial for survival, adaptation, and normal homeostasis in virtually every cell type. The transduction of mechanical messages into intracellular biochemical messages is termed mechanotransduction. While significant advances in biochemical signaling have been made in the last few decades, the role of mechanotransduction in physiological and pathological processes has been largely overlooked until recently. In this review, the role of interactions between the cytoskeleton and cell-cell/cell-matrix adhesions in transducing mechanical signals is discussed. In addition, mechanosensors that reside in the cell membrane and the transduction of mechanical signals to the nucleus are discussed. Finally, we describe two examples in which mechanotransduction plays a significant role in normal physiology and disease development. The first example is the role of mechanotransduction in the proliferation and metastasis of cancerous cells. In this system, the role of mechanotransduction in cellular processes, including proliferation, differentiation, and motility, is described. In the second example, the role of mechanotransduction in a mechanically active organ, the gastrointestinal tract, is described. In the gut, mechanotransduction contributes to normal physiology and the development of motility disorders.

Courtship Ritual of Male and Female Nuclei during Fertilization in Neurospora crassa.

Sexual reproduction is a key process influencing the evolution and adaptation of animals, plants, and many eukaryotic microorganisms, such as fungi. However, the sequential cell biology of fertilization and the associated nuclear dynamics after plasmogamy are poorly understood in filamentous fungi. Using histone-fluorescent parental isolates, we tracked male and female nuclei during fertilization in the model ascomycete Neurospora crassa using live-cell imaging. This study unravels the behavior of trichogyne resident female nuclei and the extraordinary manner in which male nuclei migrate up the trichogyne to the protoperithecium. Our observations raise new fundamental questions about the modus operandi of nucleus movements during sexual reproduction, male and female nuclear identity, guidance of nuclei within the trichogyne and, unexpectedly, the avoidance of "polyspermy" in fungi. The spatiotemporal dynamics of male nuclei within the trichogyne following plasmogamy are also described, where the speed and the deformation of male nuclei are of the most dramatic observed to date in a living organism. IMPORTANCE Using live-cell fluorescence imaging, for the first time we have observed live male and female nuclei during sexual reproduction in the model fungus Neurospora crassa. This study reveals the specific behavior of resident female nuclei within the trichogyne (the female organ) after fertilization and the extraordinary manner in which male nuclei migrate across the trichogyne toward their final destination, the protoperithecium, where karyogamy takes place. Importantly, the speed and deformation of male nuclei were found to be among the most dramatic ever observed in a living organism. Furthermore, we observed that entry of male nuclei into protoperithecia may block the entry of other male nuclei, suggesting that a process analogous to polyspermy avoidance could exist in fungi. Our live-cell imaging approach opens new opportunities for novel research on cell-signaling during sexual reproduction in fungi and, on a broader scale, nuclear dynamics in eukaryotes.

Muscle repair after physiological damage relies on nuclear migration for cellular reconstruction.

Regeneration of skeletal muscle is a highly synchronized process that requires muscle stem cells (satellite cells). We found that localized injuries, as experienced through exercise, activate a myofiber self-repair mechanism that is independent of satellite cells in mice and humans. Mouse muscle injury triggers a signaling cascade involving calcium, Cdc42, and phosphokinase C that attracts myonuclei to the damaged site via microtubules and dynein. These nuclear movements accelerate sarcomere repair and locally deliver messenger RNA (mRNA) for cellular reconstruction. Myofiber self-repair is a cell-autonomous protective mechanism and represents an alternative model for understanding the restoration of muscle architecture in health and disease.