Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Isolation of Phytochrome B Photobodies.

Phytochrome B (phyB), a plant photoreceptor, forms a membraneless organelle known as a photobody. Here, we present a protocol for the isolation of phyB photobodies through fluorescence-activated particle sorting from mature transgenic Arabidopsis leaves expressing phyB-GFP. This protocol involves the isolation of nuclei from frozen ground leaves using sucrose gradient centrifugation, the disruption of nuclear envelopes by sonication, and the subsequent isolation of phyB photobodies through fluorescence-activated particle sorting. We include experimental tips and notes for each step.

Nuclear actin dynamics and functions at a glance.

Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.

Crafty mimicry grants nuclear pore entry to HIV.

The size of the nuclear pore should, in principle, prevent HIV-1 entry. However, HIV-1 capsid is able to gain nuclear pore entry. In a recent issue of Nature, Fu et al. and Dickson et al. demonstrate that the HIV-1 capsid mimics the nuclear transport protein karyopherins to access host nuclei.

Size-dependent steady state saturation limit in biomolecular transport through nuclear membranes.

The nucleus preserves the genomic DNA of eukaryotic organisms and maintains the integrity of the cell by regulating the transport of molecules across the nuclear membrane. It is hitherto assumed that small molecules having a size below the passive permeability limit are allowed to diffuse freely to the nucleus while the transport of larger molecules is regulated via an active mechanism involving energy. Here we report on the kinetics of nuclear import and export of dextran molecules having a size below the passive permeability limit. The studies carried out using time-lapse confocal fluorescence microscopy show a clear deviation from the passive diffusion model. In particular, it is observed that the steady-state concentration of dextran molecules inside the nucleus is consistently less than the concentration outside, in contradiction to the predictions of the passive diffusion model. Detailed analysis and modeling of the transport show that the nuclear export rates significantly differ from the import rates, and the difference in rates is dependent on the size of the molecules. The nuclear export rates are further confirmed by an independent experimental study where we observe the diffusion of dextran molecules from the nucleus directly. Our experiments and transport model would suggest that the nucleus actively rejects exogenous macromolecules even below the passive permeability limit. This result can have a significant impact on biomedical research, especially in areas related to targeted drug delivery and gene therapy.

Mechanobiology of the nucleus during the G2-M transition.

Cellular behavior is continuously influenced by mechanical forces. These forces span the cytoskeleton and reach the nucleus, where they trigger mechanotransduction pathways that regulate downstream biochemical events. Therefore, the nucleus has emerged as a regulator of cellular response to mechanical stimuli. Cell cycle progression is regulated by cyclin-CDK complexes. Recent studies demonstrated these biochemical pathways are influenced by mechanical signals, highlighting the interdependence of cellular mechanics and cell cycle regulation. In particular, the transition from G2 to mitosis (G2-M) shows significant changes in nuclear structure and organization, ranging from nuclear pore complex (NPC) and nuclear lamina disassembly to chromosome condensation. The remodeling of these mechanically active nuclear components indicates that mitotic entry is particularly sensitive to forces. Here, we address how mechanical forces crosstalk with the nucleus to determine the timing and efficiency of the G2-M transition. Finally, we discuss how the deregulation of nuclear mechanics has consequences for mitosis.

Reverse Mechanotransduction: Driving Chromatin Compaction to Decompaction Increases Cell Adhesion Strength and Contractility.

Mechanical extracellular signals elicit chromatin remodeling via the mechanotransduction pathway, thus determining cellular function. However, the reverse pathway is an open question: does chromatin remodeling shape cells, regulating their adhesion strength? With fluidic force microscopy, we can directly measure the adhesion strength of epithelial cells by driving chromatin compaction to decompaction with chromatin remodelers. We observe that chromatin compaction, induced by performing histone acetyltransferase inhibition or ATP depletion, leads to a reduction in nuclear volume, disrupting actin cytoskeleton and focal adhesion assembly, and ultimately decreases in cell adhesion strength and traction force. Conversely, when chromatin decompaction is drived by removing the remodelers, cells recover their original shape, adhesion strength, and traction force. During chromatin decompaction, cells use depolymerized proteins to restore focal adhesion assemblies rather than neo-synthesized cytoskeletal proteins. We conclude that chromatin remodeling shapes cells, regulating adhesion strength through a reverse mechanotransduction pathway from the nucleus to the cell surface involving RhoA activation.

'Getting Better'-Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

The herpesviral nuclear egress represents an essential step of viral replication efficiency in host cells, as it defines the nucleocytoplasmic release of viral capsids. Due to the size limitation of the nuclear pores, viral nuclear capsids are unable to traverse the nuclear envelope without a destabilization of this natural host-specific barrier. To this end, herpesviruses evolved the regulatory nuclear egress complex (NEC), composed of a heterodimer unit of two conserved viral NEC proteins (core NEC) and a large-size extension of this complex including various viral and cellular NEC-associated proteins (multicomponent NEC). Notably, the NEC harbors the pronounced ability to oligomerize (core NEC hexamers and lattices), to multimerize into higher-order complexes, and, ultimately, to closely interact with the migrating nuclear capsids. Moreover, most, if not all, of these NEC proteins comprise regulatory modifications by phosphorylation, so that the responsible kinases, and additional enzymatic activities, are part of the multicomponent NEC. This sophisticated basis of NEC-specific structural and functional interactions offers a variety of different modes of antiviral interference by pharmacological or nonconventional inhibitors. Since the multifaceted combination of NEC activities represents a highly conserved key regulatory stage of herpesviral replication, it may provide a unique opportunity towards a broad, pan-antiherpesviral mechanism of drug targeting. This review presents an update on chances, challenges, and current achievements in the development of NEC-directed antiherpesviral strategies.

Lamin A/C and PI(4,5)P2-A Novel Complex in the Cell Nucleus.

Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.

The Role of ß-Dystroglycan in Nuclear Dynamics.

Dystroglycan is a ubiquitously expressed heterodimeric cell-surface laminin receptor with roles in cell adhesion, signalling, and membrane stabilisation. More recently, the transmembrane ß-subunit of dystroglycan has been shown to localise to both the nuclear envelope and the nucleoplasm. This has led to the hypothesis that dystroglycan may have a structural role at the nuclear envelope analogous to its role at the plasma membrane. The biochemical fraction of myoblast cells clearly supports the presence of dystroglycan in the nucleus. Deletion of the dystroglycan protein by disruption of the DAG1 locus using CRISPR/Cas9 leads to changes in nuclear size but not overall morphology; moreover, the Young's modulus of dystroglycan-deleted nuclei, as determined by atomic force microscopy, is unaltered. Dystroglycan-disrupted myoblasts are also no more susceptible to nuclear stresses including chemical and mechanical, than normal myoblasts. Re-expression of dystroglycan in DAG1-disrupted myoblasts restores nuclear size without affecting other nuclear parameters.

Two ancient membrane pores mediate mitochondrial-nucleus membrane contact sites.

Coordination between nucleus and mitochondria is essential for cell survival, and thus numerous communication routes have been established between these two organelles over eukaryotic cell evolution. One route for organelle communication is via membrane contact sites, functional appositions formed by molecular tethers. We describe a novel nuclear-mitochondrial membrane contact site in the protozoan Toxoplasma gondii. We have identified specific contacts occurring at the nuclear pore and demonstrated an interaction between components of the nuclear pore and the mitochondrial protein translocon, highlighting them as molecular tethers. Genetic disruption of the nuclear pore or the TOM translocon components, TgNup503 or TgTom40, respectively, result in contact site reduction, supporting their potential involvement in this tether. TgNup503 depletion further leads to specific mitochondrial morphology and functional defects, supporting a role for nuclear-mitochondrial contacts in mediating their communication. The discovery of a contact formed through interaction between two ancient mitochondrial and nuclear complexes sets the ground for better understanding of mitochondrial-nuclear crosstalk in eukaryotes.

ANP32e Binds Histone H2A.Z in a Cell Cycle-Dependent Manner and Regulates Its Protein Stability in the Cytoplasm.

ANP32e, a chaperone of H2A.Z, is receiving increasing attention because of its association with cancer growth and progression. An unanswered question is whether ANP32e regulates H2A.Z dynamics during the cell cycle; this could have clear implications for the proliferation of cancer cells. We confirmed that ANP32e regulates the growth of human U2OS cancer cells and preferentially interacts with H2A.Z during the G1 phase of the cell cycle. Unexpectedly, ANP32e does not mediate the removal of H2A.Z from chromatin, is not a stable component of the p400 remodeling complex and is not strongly associated with chromatin. Instead, most ANP32e is in the cytoplasm. Here, ANP32e preferentially interacts with H2A.Z in the G1 phase in response to an increase in H2A.Z protein abundance and regulates its protein stability. This G1-specific interaction was also observed in the nucleoplasm but was unrelated to any change in H2A.Z abundance. These results challenge the idea that ANP32e regulates the abundance of H2A.Z in chromatin as part of a chromatin remodeling complex. We propose that ANP32e is a molecular chaperone that maintains the soluble pool of H2A.Z by regulating its protein stability and acting as a buffer in response to cell cycle-dependent changes in H2A.Z abundance.

A high-content screen reveals new regulators of nuclear membrane stability.

Nuclear membrane rupture is a physiological response to multiple in vivo processes, such as cell migration, that can cause extensive genome instability and upregulate invasive and inflammatory pathways. However, the underlying molecular mechanisms of rupture are unclear and few regulators have been identified. In this study, we developed a reporter that is size excluded from re-compartmentalization following nuclear rupture events. This allows for robust detection of factors influencing nuclear integrity in fixed cells. We combined this with an automated image analysis pipeline in a high-content siRNA screen to identify new proteins that both increase and decrease nuclear rupture frequency in cancer cells. Pathway analysis identified an enrichment of nuclear membrane and ER factors in our hits and we demonstrate that one of these, the protein phosphatase CTDNEP1, is required for nuclear stability. Analysis of known rupture determinants, including an automated quantitative analysis of nuclear lamina gaps, are consistent with CTDNEP1 acting independently of actin and nuclear lamina organization. Our findings provide new insights into the molecular mechanism of nuclear rupture and define a highly adaptable program for rupture analysis that removes a substantial barrier to new discoveries in the field.

WW domains form a folded type of nuclear localization signal to guide YAP1 nuclear import.

The nuclear translocation of YAP1 is significantly implicated in the proliferation, stemness, and metastasis of cancer cells. Although the molecular basis underlying YAP1 subcellular distribution has been extensively explored, it remains to be elucidated how the nuclear localization signal guides YAP1 to pass through the nuclear pore complex. Here, we define a globular type of nuclear localization signal composed of folded WW domains, named as WW-NLS. It directs YAP1 nuclear import through the heterodimeric nuclear transport receptors KPNA-KPNB1, bypassing the canonical nuclear localization signal that has been well documented in KPNA/KPNB1-mediated nuclear import. Strikingly, competitive interference with the function of the WW-NLS significantly attenuates YAP1 nuclear translocation and damages stemness gene activation and sphere formation in malignant breast cancer cells. Our findings elucidate a novel globular type of nuclear localization signal to facilitate nuclear entry of WW-containing proteins including YAP1.

Quantitative Analysis of Epigenetic Modifications in Fagopyrum Nuclei with Confocal Microscope, ImageJ, and R Studio.

Epigenetic programming plays a vital role in regulating pluripotency genes, which become activated or inactivated during the processes of dedifferentiation and differentiation during an organism's development. The analysis of epigenetic modifications has become possible through the technique of immunostaining, where specific antibodies allow the identification of a single target protein. This chapter describes a detailed protocol for the analysis of the epigenetic modifications with the use of confocal microscopy, subsequent image, and statistical analysis on the example of Fagopyrum calli with the use of nine antibodies raised against histone H3 and H4 methylation and acetylation on several lysines as well as DNA methylation.

Nuclear localization of heparanase 2 (Hpa2) attenuates breast carcinoma growth and metastasis.

Unlike the intense research effort devoted to exploring the significance of heparanase in cancer, very little attention was given to Hpa2, a close homolog of heparanase. Here, we explored the role of Hpa2 in breast cancer. Unexpectedly, we found that patients endowed with high levels of Hpa2 exhibited a higher incidence of tumor metastasis and survived less than patients with low levels of Hpa2. Immunohistochemical examination revealed that in normal breast tissue, Hpa2 localizes primarily in the cell nucleus. In striking contrast, in breast carcinoma, Hpa2 expression is not only decreased but also loses its nuclear localization and appears diffuse in the cell cytoplasm. Importantly, breast cancer patients in which nuclear localization of Hpa2 is retained exhibited reduced lymph-node metastasis, suggesting that nuclear localization of Hpa2 plays a protective role in breast cancer progression. To examine this possibility, we engineered a gene construct that directs Hpa2 to the cell nucleus (Hpa2-Nuc). Notably, overexpression of Hpa2 in breast carcinoma cells resulted in bigger tumors, whereas targeting Hpa2 to the cell nucleus attenuated tumor growth and tumor metastasis. RNAseq analysis was performed to reveal differentially expressed genes (DEG) in Hpa2-Nuc tumors vs. control. The analysis revealed, among others, decreased expression of genes associated with the hallmark of Kras, beta-catenin, and TNF-alpha (via NFkB) signaling. Our results imply that nuclear localization of Hpa2 prominently regulates gene transcription, resulting in attenuation of breast tumorigenesis. Thus, nuclear Hpa2 may be used as a predictive parameter in personalized medicine for breast cancer patients.

Tyr352 as a Predominant Phosphosite in the Understudied Kinase and Molecular Target, HIPK1: Implications for Cancer Therapy.

Homeodomain-interacting protein kinase 1 (HIPK1) is majorly found in the nucleoplasm. HIPK1 is associated with cell proliferation, tumor necrosis factor-mediated cellular apoptosis, transcription regulation, and DNA damage response, and thought to play significant roles in health and common diseases such as cancer. Despite this, HIPK1 remains an understudied molecular target. In the present study, based on a systematic screening and mapping approach, we assembled 424 qualitative and 44 quantitative phosphoproteome datasets with 15 phosphosites in HIPK1 reported across multiple studies. These HIPK1 phosphosites were not currently attributed to any functions. Among them, Tyr352 within the kinase domain was identified as the predominant phosphosite modulated in 22 differential datasets. To analyze the functional association of HIPK1 Tyr352, we first employed a stringent criterion to derive its positively and negatively correlated protein phosphosites. Subsequently, we categorized the correlated phosphosites in known interactors, known/predicted kinases, and substrates of HIPK1, for their prioritized validation. Bioinformatics analysis identified their significant association with biological processes such as the regulation of RNA splicing, DNA-templated transcription, and cellular metabolic processes. HIPK1 Tyr352 was also identified to be upregulated in Her2+ cell lines and a subset of pancreatic and cholangiocarcinoma tissues. These data and the systems biology approach undertaken in the present study serve as a platform to explore the functional role of other phosphosites in HIPK1, and by extension, inform cancer drug discovery and oncotherapy innovation. In all, this study highlights the comprehensive phosphosite map of HIPK1 kinase and the first of its kind phosphosite-centric analysis of HIPK1 kinase based on global-level phosphoproteomics datasets derived from human cellular differential experiments across distinct experimental conditions.

The exon junction complex is required for DMD gene splicing fidelity and myogenic differentiation.

Deposition of the exon junction complex (EJC) upstream of exon-exon junctions helps maintain transcriptome integrity by preventing spurious re-splicing events in already spliced mRNAs. Here we investigate the importance of EJC for the correct splicing of the 2.2-megabase-long human DMD pre-mRNA, which encodes dystrophin, an essential protein involved in cytoskeletal organization and cell signaling. Using targeted RNA-seq, we show that knock-down of the eIF4A3 and Y14 core components of EJC in a human muscle cell line causes an accumulation of mis-splicing events clustered towards the 3' end of the DMD transcript (Dp427m). This deregulation is conserved in the short Dp71 isoform expressed ubiquitously except in adult skeletal muscle and is rescued with wild-type eIF4A3 and Y14 proteins but not with an EJC assembly-defective mutant eIF4A3. MLN51 protein and EJC-associated ASAP/PSAP complexes independently modulate the inclusion of the regulated exons 71 and 78. Our data confirm the protective role of EJC in maintaining splicing fidelity, which in the DMD gene is necessary to preserve the function of the critical C-terminal protein-protein interaction domain of dystrophin present in all tissue-specific isoforms. Given the role of the EJC in maintaining the integrity of dystrophin, we asked whether the EJC could also be involved in the regulation of a mechanism as complex as skeletal muscle differentiation. We found that eIF4A3 knockdown impairs myogenic differentiation by blocking myotube formation. Collectively, our data provide new insights into the functional roles of EJC in human skeletal muscle.

Unveiling the epigenetic landscape of plants using flow cytometry approach.

Plants are sessile creatures that have to adapt constantly changing environmental circumstances. Plants are subjected to a range of abiotic stressors as a result of unpredictable climate change. Understanding how stress-responsive genes are regulated can help us better understand how plants can adapt to changing environmental conditions. Epigenetic markers that dynamically change in response to stimuli, such as DNA methylation and histone modifications are known to regulate gene expression. Individual cells or particles' physical and/or chemical properties can be measured using the method known as flow cytometry. It may therefore be used to evaluate changes in DNA methylation, histone modifications, and other epigenetic markers, making it a potent tool for researching epigenetics in plants. We explore the use of flow cytometry as a technique for examining epigenetic traits in this thorough discussion. The separation of cell nuclei and their subsequent labeling with fluorescent antibodies, offering information on the epigenetic mechanisms in plants when utilizing flow cytometry. We also go through the use of high-throughput data analysis methods to unravel the complex epigenetic processes occurring inside plant systems.

Mitigating transcription noise via protein sharing in syncytial cells.

Bursty transcription allows nuclei to concentrate the work of transcribing mRNA into short, intermittent intervals, potentially reducing transcriptional interference. However, bursts of mRNA production can increase noise in protein abundances. Here, we formulate models for gene expression in syncytia, or multinucleate cells, showing that protein abundance noise may be mitigated locally via spatial averaging of diffuse proteins. Our modeling shows a universal reduction in protein noise, which increases with the average number of nuclei per cell and persists even when the number of nuclei is itself a random variable. Experimental data comparing distributions of a cyclin mRNA that is conserved between brewer's yeast and a closely related filamentous fungus Ashbya gossypii confirm that syncytism is permissive of greater levels of transcriptional noise. Our findings suggest that division of transcriptional labor between nuclei allows syncytia to sidestep tradeoffs between efficiency and precision of gene expression.