Gastric-type glandular lesions of the female genital tract excluding the cervix: emerging pathological entities.

In the last two decades or so, a spectrum of benign, premalignant and malignant cervical glandular lesions exhibiting gastric differentiation has been described, with gastric-type adenocarcinoma representing the most common human papillomavirus (HPV)-independent cervical adenocarcinoma. More recently, limited literature has reported a variety of gastric-type glandular lesions at other sites within the female genital tract and, as in the cervix (the most common site for these lesions), a spectrum of benign, premalignant and malignant lesions has been proposed. We provide an update and review of the emerging spectrum of gastric-type glandular lesions at female genital tract sites other than the cervix. In the endometrium, putative gastric-type glandular lesions include mucinous metaplasia of gastric-type, atypical mucinous proliferation of gastric-type and gastric-type adenocarcinoma. Similarly in the vagina, gastric-type adenosis, atypical adenosis and adenocarcinoma have been described. There have also been occasional reports of gastric-type lesions involving the ovary and fallopian tube. We provide guidance on how to recognise gastric-type lesions morphologically and immunophenotypically and stress that sometimes these lesions occur at more than one site within the female genital tract (synchronous/multifocal gastric-type lesions of the female genital tract), sometimes in association with Peutz-Jeghers syndrome.

Nanoscopic Assessment of Anti-SARS-CoV-2 Spike Neutralizing Antibody Using High-Speed AFM.

Anti-spike neutralizing antibodies (S NAbs) have been developed for prevention and treatment against COVID-19. The nanoscopic characterization of the dynamic interaction between spike proteins and S NAbs remains difficult. By using high-speed atomic force microscopy (HS-AFM), we elucidate the molecular property of an S NAb and its interaction with spike proteins. The S NAb appeared as monomers with a Y conformation at low density and formed hexameric oligomers at high density. The dynamic S NAb-spike protein interaction at RBD induces neither RBD opening nor S1 subunit shedding. Furthermore, the interaction was stable at endosomal pH. These findings indicated that the S NAb could have a negligible risk of antibody-dependent enhancement. Dynamic movement of spike proteins on small extracellular vesicles (S sEV) resembled that on SARS-CoV-2. The sensitivity of variant S sEVs to S NAb could be evaluated using HS-AFM. Altogether, we demonstrate a nanoscopic assessment platform for evaluating the binding property of S NAbs.

p53-Abnormal "Fields of Dysplasia" in Human Papillomavirus-Independent Vulvar Squamous Cell Carcinoma Impacts Margins and Recurrence Risk.

Abnormal p53 (p53abn) immunohistochemical (IHC) staining patterns can be found in vulvar squamous cell carcinoma (VSCC) and differentiated vulvar intraepithelial neoplasia (dVIN). They can also be found in the adjacent skin that shows morphology that falls short of the traditional diagnostic threshold for dVIN. Vulvectomy specimens containing human papillomavirus-independent p53abn VSCC with margins originally reported as negative for invasive and in situ disease were identified. Sections showing the closest approach by invasive or in situ neoplasia to margins were stained with p53 IHC stains. We evaluated the following: (1) detection of morphologically occult p53abn in situ neoplasia, (2) rates of margin status change after p53 IHC staining, and (3) effect of p53abn IHC staining at margins on the 2-year local recurrence rates. Seventy-three human papillomavirus-independent p53abn VSCCs were included. Half (35/73, 48%) had documented an in situ lesion in the original report. The use of p53 IHC staining identified 21 additional cases (29%) with the p53abn in situ lesions that were originally unrecognized. The histology of in situ lesions in the p53abn "field" varied and became more subtle (morphologically occult) farther away from the VSCC. Fifteen (21%) cases had a morphologically occult and previously unrecognized p53abn in situ lesion present at a resection margin, which conferred an increased risk of local recurrence (5/7 [71.4%] vs 6/22 [27.3%], P = .036). The p53abn in situ lesions at a margin were confirmed to have TP53 mutations by sequencing. p53 IHC staining identified morphologically occult p53abn in situ lesions surrounding human papillomavirus-independent VSCC. p53abn IHC staining at a margin was associated with a 3-fold increased risk of local recurrence.

Inhibition of Canonical Wnt Signaling Promotes Ex Vivo Maintenance and Proliferation of Hematopoietic Stem Cells in Zebrafish.

The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow; however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Coculture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared with candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (>2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.

NSP9 of SARS-CoV-2 attenuates nuclear transport by hampering nucleoporin 62 dynamics and functions in host cells.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

Significance of p53 and presence of differentiated vulvar intra-epithelial neoplasia (dVIN) at resection margin in early stage human papillomavirus-independent vulvar squamous cell carcinoma.

OBJECTIVE: Vulvar squamous cell carcinoma and in situ lesions can be stratified by human papillomavirus (HPV) and TP53 status into prognostic risk groups using p16 and p53 immunohistochemistry. We assessed the significance of vulvar squamous cell carcinoma resection margin positivity for either differentiated vulvar intra-epithelial neoplasia (dVIN) or abnormal p53 immunohistochemistry, and other pathologic variables, in a cohort of patients with HPV-independent (HPV-I) p53 abnormal (p53abn) vulvar squamous cell carcinomas. METHODS: Patients with stage I-II HPV-I p53abn vulvar squamous cell carcinoma with negative invasive margins who did not receive adjuvant radiation from a single institution were included. Tumors underwent margin reassessment using p53 immunohistochemistry. Cases were segregated into (1) morphologic dVIN at margin; or (2) abnormal p53 immunohistochemistry staining at margin without morphologic dVIN (p53abn immunohistochemistry); or (3) margins negative by morphology and p53 immunohistochemistry. Clinicopathologic/outcome data were collected. RESULTS: A total of 51 patients were evaluated: (1) 12 with dVIN on margin; (2) 12 with p53abn immunohistochemistry on margin without morphologic dVIN; and (3) 27 with margins negative for morphologic dVIN and p53abn immunohistochemistry. The recurrence rate for patients with dVIN or p53abn immunohistochemistry on the margin was equally high at 75% each, compared with 33% with margins negative for morphologic dVIN and p53abn immunohistochemistry (p=0.009). On multivariate analysis, positive in situ margins maintained an association with disease recurrence (p=0.03) whereas invasive margin distance (radial and deep), lymphovascular invasion, and tumor size did not. CONCLUSIONS: Patients with stage I-II HPV-I vulvar squamous cell carcinoma with margins positive for either dVIN or p53abn immunohistochemistry without morphologic dVIN showed increased disease recurrence, regardless of invasive margin distance. These findings show that p53 immunohistochemistry is a useful adjunct for evaluating margin status in HPV-I vulvar squamous cell carcinoma and may support repeat excision for positive in situ margins (dVIN or p53abn immunohistochemistry).

Inflammatory and mitogenic signals drive interleukin 23 subunit alpha (IL23A) secretion independent of IL12B in intestinal epithelial cells.

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11-7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.

Overexpression of SARS-CoV-2 protein ORF6 dislocates RAE1 and NUP98 from the nuclear pore complex.

The novel human betacoronavirus SARS-CoV-2 has caused an unprecedented pandemic in the 21st century. Several studies have revealed interactions between SARS-CoV-2 viral proteins and host nucleoporins, yet their functions are largely unknown. Here, we demonstrate that the open-reading frame 6 (ORF6) of SARS-CoV-2 can directly manipulate localization and functions of nucleoporins. We found that ORF6 protein disrupted nuclear rim staining of nucleoporins RAE1 and NUP98. Consequently, this disruption caused aberrant nucleocytoplasmic trafficking and led to nuclear accumulation of mRNA transporters such as hnRNPA1. Ultimately, host cell nucleus size was reduced and cell growth was halted.

High-Speed AFM Reveals Molecular Dynamics of Human Influenza A Hemagglutinin and Its Interaction with Exosomes.

Influenza A hemagglutinin (HA) is one of the crucial virulence factors that mediate host tropism and viral infectivity. Presently, the mechanism of the fusogenic transition of HA remains elusive. Here, we used high-speed atomic force microscopy (HS-AFM) to decipher the molecular dynamics of HA and its interaction with exosomes. Our data reveal that the native conformation of HA in the neutral buffer is ellipsoidal, and HA undergoes a conformational change in an acidic buffer. Real-time visualization of the fusogenic transition by HS-AFM suggests that the mechanism is possibly fit to the "uncaging" model, and HA intermediate appears as Y-shaped. A firm interaction between the HA and exosome in an acidic buffer indicates the insertion of a fusion peptide into the exosomal layer and subsequently destabilizes the layer, resulting in the deformation or rupture of exosomes, releasing exosomal contents. In contrast, the HA-exosome interaction is weak in a neutral buffer because the interaction is mediated by weak bonds between the HA receptor-binding site and receptors on the exosome.

Predictive biomarkers and tumor microenvironment in female genital melanomas: a multi-institutional study of 55 cases.

Female genital melanomas are rare. At diagnosis, most affected patients have advanced disease. Surgery remains the primary treatment, and adjuvant therapy is largely ineffective. Recently, immune checkpoints and the mitogen-activated protein kinase pathway have been explored as treatment targets. However, evaluation of these biomarkers in genital melanomas is limited. We evaluated the clinicopathological features of 20 vulvar, 32 vaginal, and three cervical melanomas and assessed programmed cell death ligand 1 (PD-L1) expression, CD8 tumor-infiltrating lymphocyte density, mismatch repair proteins, VE1 immunohistochemistry, and KIT and BRAF mutations. The median age of the patients was 66 years, and median tumor sizes were 25, 30, and 20 mm for vulvar, vaginal, and cervical tumors, respectively. Mean mitotic figures were 18, 19, and 30 per mm2. Thirty-seven patients (67%) had operable tumors. After a median follow-up of 15 months, only nine patients (16%) were alive. Eight of the nine survivors did not have lymph node metastasis. Using 5% as the threshold, PD-L1 expression was observed in 55%, 50%, and 33% of vulvar, vaginal, and cervical tumors, respectively, when the Roche SP263 antibody was used and 20%, 53%, and 0%, respectively, when the Dako 28-8 antibody was used. The median CD8 tumor-infiltrating lymphocyte density was significantly higher in vulvar/vaginal than cervical melanomas and correlated with PD-L1 expression. No cases exhibited loss of mismatch repair proteins. Five cases harbored KIT mutations, three of which were hotspots. BRAF V600E mutation was not detected. Univariable analysis showed that tumor size greater than or equal to 33 mm, mitotic figures of greater than or equal to 10 per mm2, lymph node metastasis, and low CD8+ tumor-infiltrating lymphocyte density were adverse prognostic factors. Thus, patients with genital melanomas have a poor prognosis, and evaluation of multiple biomarkers is necessary to identify patients who may benefit from immunotherapy or targeted therapy.

ROCK-dependent phosphorylation of NUP62 regulates p63 nuclear transport and squamous cell carcinoma proliferation.

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type-specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation-inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine-glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC.

The mitosis-specific marker phosphohistone-H3 (PHH3) is an independent prognosticator in uterine smooth muscle tumours: an outcome-based study.

AIMS: Accurate mitosis counting, which is important in the diagnosis of uterine smooth muscle tumours (USMTs), is often difficult and subjective. The mitosis-specific immunohistochemical marker phosphohistone-H3 (PHH3) has been shown to be diagnostically useful, but its expression, in relation to outcome, has not been thoroughly investigated. The aim of this study is to evaluate PHH3 as a diagnostic and prognostic marker in USMTs. METHODS AND RESULTS: PHH3 expression was evaluated in 55 leiomyosarcomas (LMSs), 26 smooth muscle tumours of uncertain malignant potential (STUMPs), 18 leiomyomas with bizarre nuclei (LBN), and 12 leiomyomas (LMs). Scores were expressed as counts per 10 high-power fields (HPFs). Median follow-up durations of patients with LMS, STUMP, LBN and LM were, respectively, 39, 78, 65.5 and 49.5 months. Twenty-eight patients with LMSs (50.9%) died, and two (7.7%) patients with STUMPs experienced recurrence. The median PHH3 scores for LMSs were significantly higher than those for other categories of tumour. A score of ≥29/10 HPFs was also independently associated with a poor outcome. To test whether the PHH3 score could distinguish between benign USMTs with atypical histology and those that were clinically malignant, two biological groups were further delineated. Patients in group 1 (18 LBNs and 24 STUMPs) all had an uneventful outcome, whereas patients in group 2 (two recurrent STUMPs and 32 LMSs) all had a recurrence or tumour-related death. Median PHH3 scores for the two groups were, respectively, 2/10 HPFs and 27/10 HPFs. A PHH3 score of ≥7/10 HPFs was highly associated with malignancy. CONCLUSION: PHH3 is useful in evaluation of the biological behaviour of USMTs, and may serve as a prognostic indicator for LMSs.

Impact of Nucleoporin-Mediated Chromatin Localization and Nuclear Architecture on HIV Integration Site Selection.

It has been known for a number of years that integration sites of human immunodeficiency virus type 1 (HIV-1) DNA show a preference for actively expressed chromosomal locations. A number of viral and cellular proteins are implicated in this process, but the underlying mechanism is not clear. Two recent breakthrough publications advance our understanding of HIV integration site selection by focusing on the localization of the preferred target genes of integration. These studies reveal that knockdown of certain nucleoporins and components of nucleocytoplasmic trafficking alter integration site preference, not by altering the trafficking of the viral genome but by altering the chromatin subtype localization relative to the structure of the nucleus. Here, we describe the link between the nuclear basket nucleoporins (Tpr and Nup153) and chromatin organization and how altering the host environment by manipulating nuclear structure may have important implications for the preferential integration of HIV into actively transcribed genes, facilitating efficient viral replication.

FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling.

Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts.

Erythrocyte and Biochemical Abnormalities as Diagnostic Markers in Dogs With Hemangiosarcoma Related Hemoabdomen.

OBJECTIVE: To investigate: 1) acanthocytosis and presence of acanthocytes in peritoneal fluid as a diagnostic marker for hemangiosarcoma (HSA) in dogs with non-traumatic hemoabdomen; and 2) the association between other erythrocyte, biochemical, and hematologic abnormalities as a mean of differentiating HSA from other disease. STUDY DESIGN: Prospective double-blinded cohort study. ANIMALS: Dogs (n = 40) with non-traumatic hemoabdomen. METHODS: Dogs diagnosed with hemoabdomen (January 2012 to May 2013) had cytologic evaluation of abdominal effusion and peripheral blood smears. Peripheral blood CBC, PT, and aPTT, as well as blood and effusion acanthocytes, keratocytes, schistocytes, lactate, glucose, PCV, and TP results were compared using the paired t-test or Fisher's exact test. Based on histologic confirmation of HSA, dogs were divided into 2 groups (HSA, non-HSA) and variables compared. RESULTS: There was no significant difference in erythrocyte morphology in abdominal effusion or peripheral blood between dogs with HSA or non-HSA related hemoabdomen. Platelet concentration and peripheral blood PCV were significantly lower in the HSA group. CONCLUSIONS: A reliable preoperative biochemical or cytologic test to differentiate between HSA and non-HSA related hemoabdomen was not identified.

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.

Optimization of Zwitterionic Polymers for Cell Cryopreservation.

Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3 C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion ) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3 C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3 C)_DSion(100%) /1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect.

Phase-separated super-enhancers confer an innate radioresistance on genomic DNA.

Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.

An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

Super-enhancer trapping by the nuclear pore via intrinsically disordered regions of proteins in squamous cell carcinoma cells.

Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level.