Chromatin phase separation and nuclear shape fluctuations are correlated in a polymer model of the nucleus.

Abnormal cell nuclear shapes are hallmarks of diseases, including progeria, muscular dystrophy, and many cancers. Experiments have shown that disruption of heterochromatin and increases in euchromatin lead to nuclear deformations, such as blebs and ruptures. However, the physical mechanisms through which chromatin governs nuclear shape are poorly understood. To investigate how heterochromatin and euchromatin might govern nuclear morphology, we studied chromatin microphase separation in a composite coarse-grained polymer and elastic shell simulation model. By varying chromatin density, heterochromatin composition, and heterochromatin-lamina interactions, we show how the chromatin phase organization may perturb nuclear shape. Increasing chromatin density stabilizes the lamina against large fluctuations. However, increasing heterochromatin levels or heterochromatin-lamina interactions enhances nuclear shape fluctuations by a "wetting"-like interaction. In contrast, fluctuations are insensitive to heterochromatin's internal structure. Our simulations suggest that peripheral heterochromatin accumulation could perturb nuclear morphology, while nuclear shape stabilization likely occurs through mechanisms other than chromatin microphase organization.

Nuclear functions regulated by the VRK1 kinase.

In the nucleus, the VRK1 Ser-Thr kinase is distributed in nucleoplasm and chromatin, where it has different roles. VRK1 expression increases in response to mitogenic signals. VRK1 regulates cyclin D1 expression at G0 exit and facilitates chromosome condensation at the end of G2 and G2/M progression to mitosis. These effects are mediated by the phosphorylation of histone H3 at Thr3 by VRK1, and later in mitosis by haspin. VRK1 regulates the apigenetic patterns of histones in processes requiring chromating remodeling, such as transcription, replication and DNA repair. VRK1 is overexpressed in tumors, facilitating tumor progression and resistance to genotoxic treatments. VRK1 also regulates the organization of Cajal bodies assembled on coilin, which are necessary for the assembly of different types of RNP complexes. VRK1 pathogenic variants cuase defects in Cajal bodies, functionally altering neurons with long axons and leading to neurological diseases, such as amyotrophic laterla sclerosis, spinal muscular atrophy, distal hereditay motor neuropathies and Charcot-Marie-Tooth.

Research progress on the relationship between AURKA and tumorigenesis: the neglected nuclear function of AURKA.

AURKA is a threonine or serine kinase that needs to be activated by TPX2, Bora and other factors. AURKA is located on chromosome 20 and is amplified or overexpressed in many human cancers, such as breast cancer. AURKA regulates some basic cellular processes, and this regulation is realized via the phosphorylation of downstream substrates. AURKA can function in either the cytoplasm or the nucleus. It can promote the transcription and expression of oncogenes together with other transcription factors in the nucleus, including FoxM1, C-Myc, and NF-κB. In addition, it also sustains carcinogenic signaling, such as N-Myc and Wnt signaling. This article will focus on the role of AURKA in the nucleus and its carcinogenic characteristics that are independent of its kinase activity to provide a theoretical explanation for mechanisms of resistance to kinase inhibitors and a reference for future research on targeted inhibitors.

AURKA plays an important role in the control of the proliferation, invasion, cell cycle regulation and self-renewal of cancer stem cells.Small molecule kinase inhibitors targeting AURKA have been developed, but the overall response rate of patients in clinical trials is not ideal, prompting us to pay attention to the non-kinase activity of AURKA.This review focuses on the nuclear function of AURKA and its oncogenic properties independent of kinase activity, demonstrating that the nuclear substrate of AURKA and the remote allosteric site of the kinase may be targets of anticancer therapy.

CLK2 mediates IκBα-independent early termination of NF-κB activation by inducing cytoplasmic redistribution and degradation.

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.

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.

Use of topical methylene blue to image nuclear morphometry with a low-cost scanning darkfield microendoscope.

Significance: Fiber-optic microendoscopy is a promising approach to noninvasively visualize epithelial nuclear morphometry for early cancer and precancer detection. However, the broader clinical application of this approach is limited by a lack of topical contrast agents available for in vivo use. Aim: The aim of this study was to evaluate the ability to image nuclear morphometry in vivo with a novel fiber-optic microendoscope used together with topical application of methylene blue (MB), a dye with FDA approval for use in chromoendoscopy in the gastrointestinal tract. Approach: The low-cost, high-resolution microendoscope implements scanning darkfield imaging without complex optomechanical components by leveraging programmable illumination and the rolling shutter of the image sensor. We validate the integration of our system and MB staining for visualizing epithelial cell nuclei by performing ex vivo imaging on fresh animal specimens and in vivo imaging on healthy volunteers. Results: The results indicate that scanning darkfield imaging significantly reduces specular reflection and resolves epithelial nuclei with enhanced image contrast and spatial resolution compared to non-scanning widefield imaging. The image quality of darkfield images with MB staining is comparable to that of fluorescence images with proflavine staining. Conclusions: Our approach enables real-time microscopic evaluation of nuclear patterns and has the potential to be a powerful noninvasive tool for early cancer detection.

Parental experiences orchestrate locust egg hatching synchrony by regulating nuclear export of precursor miRNA.

Parental experiences can affect the phenotypic plasticity of offspring. In locusts, the population density that adults experience regulates the number and hatching synchrony of their eggs, contributing to locust outbreaks. However, the pathway of signal transmission from parents to offspring remains unclear. Here, we find that transcription factor Forkhead box protein N1 (FOXN1) responds to high population density and activates the polypyrimidine tract-binding protein 1 (Ptbp1) in locusts. FOXN1-PTBP1 serves as an upstream regulator of miR-276, a miRNA to control egg-hatching synchrony. PTBP1 boosts the nucleo-cytoplasmic transport of pre-miR-276 in a "CU motif"-dependent manner, by collaborating with the primary exportin protein exportin 5 (XPO5). Enhanced nuclear export of pre-miR-276 elevates miR-276 expression in terminal oocytes, where FOXN1 activates Ptbp1 and leads to egg-hatching synchrony in response to high population density. Additionally, PTBP1-prompted nuclear export of pre-miR-276 is conserved in insects, implying a ubiquitous mechanism to mediate transgenerational effects.

VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

The HSV-1 pUL37 protein promotes cell invasion by regulating the kinesin-1 motor.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), recruit microtubule motor proteins to invade cells. The incoming viral particle traffics to nuclei in a two-step process. First, the particle uses the dynein-dynactin motor to sustain transport to the centrosome. In neurons, this step is responsible for long-distance retrograde axonal transport and is an important component of the neuroinvasive property shared by these viruses. Second, a kinesin-dependent mechanism redirects the particle from the centrosome to the nucleus. We have reported that the kinesin motor used during the second step of invasion is assimilated into nascent virions during the previous round of infection. Here, we report that the HSV-1 pUL37 tegument protein suppresses the assimilated kinesin-1 motor during retrograde axonal transport. Region 2 (R2) of pUL37 was required for suppression and functioned independently of the autoinhibitory mechanism native to kinesin-1. Furthermore, the motor domain and proximal coiled coil of kinesin-1 were sufficient for HSV-1 assimilation, pUL37 suppression, and nuclear trafficking. pUL37 localized to the centrosome, the site of assimilated kinesin-1 activation during infection, when expressed in cells in the absence of other viral proteins; however, pUL37 did not suppress kinesin-1 in this context. These results indicate that the pUL37 tegument protein spatially and temporally regulates kinesin-1 via the amino-terminal motor region in the context of the incoming viral particle.

Pentamethylquercetin attenuates angiotensin II-induced abdominal aortic aneurysm formation by blocking nuclear translocation of C/EBPß at Lys253.

BACKGROUND: Pentamethylquercetin (PMQ) is a natural polymethyl flavonoid that possesses anti-apoptotic and other biological properties. Abdominal aortic aneurysm (AAA), a fatal vascular disease with a high risk of rupture, is associated with phenotypic switching and apoptosis of medial vascular smooth muscle cells (VSMCs). This study aimed to investigate the protective effects of PMQ on the development of AAA and the underlying mechanism. METHODS: ApoE-/- mice were continuously infused with angiotensin II (Ang II) for 4 weeks to develop the AAA model. Intragastric administration of PMQ was initiated 5 days before Ang II infusion and continued for 4 weeks. In vitro, VSMCs were cultured and pretreated with PMQ, stimulated with Ang II. Real-time PCR, western blotting, and immunofluorescence staining were used to examine the roles and mechanisms of PMQ on the phenotypic switching and apoptosis of VSMCs. RESULTS: PMQ dose-dependently reduced the incidence of Ang II-induced AAA, aneurysm diameter enlargement, elastin degradation, VSMCs phenotypic switching and apoptosis. Furthermore, PMQ also inhibited phenotypic switching and apoptosis in Ang II-stimulated VSMCs. PMQ exerted protective effects by regulating the C/EBPß/PTEN/AKT/GSK-3ß axis. AAV-mediated overexpression of PTEN reduced the therapeutic effects of PMQ in the AAA model mice, suggesting that the effects of PMQ on Ang II-mediated AAA formation were related to the PTEN/AKT/GSK-3ß axis. PMQ inhibited VSMCs phenotypic switching and apoptosis by bounding to C/EBPß at Lys253 with hydrogen bond to regulate C/EBPß nuclear translocation and PTEN/AKT/GSK-3ß axis, thereby inhibiting Ang II-induced AAA formation. CONCLUSIONS: Pentamethylquercetin inhibits angiotensin II-induced abdominal aortic aneurysm formation by bounding to C/EBPß at Lys253. Therefore, PMQ prevents the formation of AAA and reduces the incidence of AAA.

Nuclear mTOR Signaling Orchestrates Transcriptional Programs Underlying Cellular Growth and Metabolism.

mTOR is a central regulator of cell growth and metabolism in response to mitogenic and nutrient signals. Notably, mTOR is not only found in the cytoplasm but also in the nucleus. This review highlights direct involvement of nuclear mTOR in regulating transcription factors, orchestrating epigenetic modifications, and facilitating chromatin remodeling. These effects intricately modulate gene expression programs associated with growth and metabolic processes. Furthermore, the review underscores the importance of nuclear mTOR in mediating the interplay between metabolism and epigenetic modifications. By integrating its functions in nutrient signaling and gene expression related to growth and metabolism, nuclear mTOR emerges as a central hub governing cellular homeostasis, malignant transformation, and cancer progression. Better understanding of nuclear mTOR signaling has the potential to lead to novel therapies against cancer and other growth-related diseases.

p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol.

The AAA+-ATPase valosin-containing protein (VCP; also called p97 or Cdc48), a major protein unfolding machinery with a variety of essential functions, localizes to different subcellular compartments where it has different functions. However, the processes regulating the distribution of VCP between the cytosol and nucleus are not understood. Here, we identified p37 (also called UBXN2B) as a major factor regulating VCP nucleocytoplasmic shuttling. p37-dependent VCP localization was crucial for local cytosolic VCP functions, such as autophagy, and nuclear functions in DNA damage repair. Mutations in VCP causing multisystem proteinopathy enhanced its association with p37, leading to decreased nuclear localization of VCP, which enhanced susceptibility to DNA damage accumulation. Both VCP localization and DNA damage susceptibility in cells with such mutations were normalized by lowering p37 levels. Thus, we uncovered a mechanism by which VCP nucleocytoplasmic distribution is fine-tuned, providing a means for VCP to respond appropriately to local needs.

Nuclear actin structure regulates chromatin accessibility.

Polymerized ß-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.

Single-cell mtDNA dynamics in tumors is driven by coregulation of nuclear and mitochondrial genomes.

The extent of cell-to-cell variation in tumor mitochondrial DNA (mtDNA) copy number and genotype, and the phenotypic and evolutionary consequences of such variation, are poorly characterized. Here we use amplification-free single-cell whole-genome sequencing (Direct Library Prep (DLP+)) to simultaneously assay mtDNA copy number and nuclear DNA (nuDNA) in 72,275 single cells derived from immortalized cell lines, patient-derived xenografts and primary human tumors. Cells typically contained thousands of mtDNA copies, but variation in mtDNA copy number was extensive and strongly associated with cell size. Pervasive whole-genome doubling events in nuDNA associated with stoichiometrically balanced adaptations in mtDNA copy number, implying that mtDNA-to-nuDNA ratio, rather than mtDNA copy number itself, mediated downstream phenotypes. Finally, multimodal analysis of DLP+ and single-cell RNA sequencing identified both somatic loss-of-function and germline noncoding variants in mtDNA linked to heteroplasmy-dependent changes in mtDNA copy number and mitochondrial transcription, revealing phenotypic adaptations to disrupted nuclear/mitochondrial balance.

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing.

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and is associated with metabolic and muscle degenerative diseases; however, an understanding of the biological properties of IMAT and its interplay with the surrounding muscle fibers is severely lacking. In recent years, single-cell and nuclei RNA sequencing have provided us with cell type-specific atlases of several human tissues. However, the cellular composition of human IMAT remains largely unexplored due to the inherent challenges of its accessibility from biopsy collection in humans. In addition to the limited amount of tissue collected, the processing of human IMAT is complicated due to its proximity to skeletal muscle tissue and fascia. The lipid-laden nature of the adipocytes makes it incompatible with single-cell isolation. Hence, single nuclei RNA sequencing is optimal for obtaining high-dimensional transcriptomics at single-cell resolution and provides the potential to uncover the biology of this depot, including the exact cellular composition of IMAT. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing. This protocol allows for the profiling of thousands of nuclei using a droplet-based approach, thus providing the capacity to detect rare and low-abundant cell types.

mPPTMP195 nanoparticles enhance fracture recovery through HDAC4 nuclear translocation inhibition.

Delayed repair of fractures seriously impacts patients' health and significantly increases financial burdens. Consequently, there is a growing clinical demand for effective fracture treatment. While current materials used for fracture repair have partially addressed bone integrity issues, they still possess limitations. These challenges include issues associated with autologous material donor sites, intricate preparation procedures for artificial biomaterials, suboptimal biocompatibility, and extended degradation cycles, all of which are detrimental to bone regeneration. Hence, there is an urgent need to design a novel material with a straightforward preparation method that can substantially enhance bone regeneration. In this context, we developed a novel nanoparticle, mPPTMP195, to enhance the bioavailability of TMP195 for fracture treatment. Our results demonstrate that mPPTMP195 effectively promotes the differentiation of bone marrow mesenchymal stem cells into osteoblasts while inhibiting the differentiation of bone marrow mononuclear macrophages into osteoclasts. Moreover, in a mouse femur fracture model, mPPTMP195 nanoparticles exhibited superior therapeutic effects compared to free TMP195. Ultimately, our study highlights that mPPTMP195 accelerates fracture repair by preventing HDAC4 translocation from the cytoplasm to the nucleus, thereby activating the NRF2/HO-1 signaling pathway. In conclusion, our study not only proposes a new strategy for fracture treatment but also provides an efficient nano-delivery system for the widespread application of TMP195 in various other diseases.

PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis.

Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron-sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron-sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

Light-Driven Mitochondrion-to-Nucleus DNA Cascade Fluorescence Imaging and Enhanced Cancer Cell Photoablation.

Nucleic acids are mainly found in the mitochondria and nuclei of cells. Detecting nucleic acids in the mitochondrion and nucleus in cascade mode is crucial for understanding diverse biological processes. This study introduces a novel nucleic acid-based fluorescent styrene dye (SPP) that exhibits light-driven cascade migration from the mitochondrion to the nucleus. By introducing N-arylpyridine on one side of the styrene dye skeleton and a bis(2-ethylsulfanyl-ethy)-amino unit on the other side, we found that SPP exhibits excellent DNA specificity (16-fold, FDNA/Ffree) and a stronger binding force to nuclear DNA (-5.09 kcal/mol) than to mitochondrial DNA (-2.59 kcal/mol). SPP initially accumulates in the mitochondrion and then migrates to the nucleus within 10 s under light irradiation. By tracking the damage to nucleic acids in apoptotic cells, SPP allows the successful visualization of the differences between apoptosis and ferroptosis. Finally, a triphenylamine segment with photodynamic effects was incorporated into SPP to form a photosensitizer (MTPA-SPP), which targets the mitochondria for photosensitization and then migrates to the nucleus under light irradiation for enhanced photodynamic cancer cell treatment. This innovative nucleic acid-based fluorescent molecule with light-triggered mitochondrion-to-nucleus migration ability provides a feasible approach for the in situ identification of nucleic acids, monitoring of subcellular physiological events, and efficient photodynamic therapy.

Heme oxygenase activates calcium release from the endoplasmic reticulum of bovine mammary epithelial cells to promote TFEB entry into the nucleus to reduce the intracellular load of Mycoplasma bovis.

Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca2+- transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca2+) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca2+-TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections.

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.