Disordered region of nuclear membrane protein Bqt4 recruits phosphatidic acid to the nuclear envelope to maintain its structural integrity.

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE.

Ceramide synthase homolog Tlc4 maintains nuclear envelope integrity via its Golgi translocation.

Maintaining the integrity of the nuclear envelope (NE) is essential for preventing genomic DNA damage. Recent studies have shown that enzymes that catalyze lipid synthesis are involved in NE maintenance, but the underlying mechanism remains unclear. Here, we found that the ceramide synthase (CerS) homolog in the fission yeast Schizosaccharomyces pombe Tlc4 (SPAC17A2.02c) suppressed NE defects in cells lacking the NE proteins Lem2 and Bqt4. Tlc4 possesses a TRAM/LAG1/CLN8 domain that is conserved in CerS proteins and functions through its non-catalytic activity. Tlc4 was localized at the NE and endoplasmic reticulum, similar to CerS proteins, and also showed unique additional localization at the cis- and medial-Golgi cisternae. Growth and mutation analyses revealed that Golgi localization of Tlc4 was tightly linked to its activity of suppressing the defects in the double-deletion mutant of Lem2 and Bqt4. Our results suggest that Lem2 and Bqt4 control the translocation of Tlc4 from the NE to the Golgi, which is necessary for maintaining NE integrity.

A ubiquitin-proteasome pathway degrades the inner nuclear membrane protein Bqt4 to maintain nuclear membrane homeostasis.

Aberrant accumulation of inner nuclear membrane (INM) proteins is associated with deformed nuclear morphology and mammalian diseases. However, the mechanisms underlying the maintenance of INM homeostasis remain poorly understood. In this study, we explored the degradation mechanisms of the INM protein Bqt4 in the fission yeast Schizosaccharomyces pombe. We have previously shown that Bqt4 interacts with the transmembrane protein Bqt3 at the INM and is degraded in the absence of Bqt3. Here, we reveal that excess Bqt4, unassociated with Bqt3, is targeted for degradation by the ubiquitin-proteasome system localized in the nucleus and Bqt3 antagonizes this process. The degradation process involves the Doa10 E3 ligase complex at the INM. Bqt4 is a tail-anchored protein and the Cdc48 complex is required for its degradation. The C-terminal transmembrane domain of Bqt4 was necessary and sufficient for proteasome-dependent protein degradation. Accumulation of Bqt4 at the INM impaired cell viability with nuclear envelope deformation, suggesting that quantity control of Bqt4 plays an important role in nuclear membrane homeostasis.

Noonan syndrome-like phenotype associated with an ERF frameshift variant.

Noonan syndrome is a so-called "RASopathy," that is characterized by short stature, distinctive facial features, congenital heart defects, and developmental delay. Of individuals with a clinical diagnosis of Noonan syndrome, 80%-90% have pathogenic variants in the known genes implicated in the disorder, but the molecular mechanism is unknown in the remaining cases. Heterozygous pathogenic variants of ETS2 repressor factor (ERF), which functions as a repressor in the RAS/MAPK signaling pathway, cause syndromic craniosynostosis. Here, we report an ERF frameshift variant cosegregating with a Noonan syndrome-like phenotype in a family. The proband was a 3-year-old female who presented with dysmorphic facial features, including proptosis, hypertelorism, slightly down slanted palpebral fissures, low-set posteriorly rotated ears, depressed nasal bridge, short stature, and developmental delay. Exome sequencing of the proband identified a heterozygous ERF variant [NM_006494.4: c.185del p.(Glu62Glyfs*15)]. Her mother and sister showed a similar phenotype and had the same heterozygous ERF variant. A large proportion of the previously reported patients with syndromic craniosynostosis and pathogenic ERF variants also showed characteristic features that overlap with those of Noonan syndrome. The present finding supports an association between heterozygous ERF variants and a Noonan syndrome-like phenotype.

Fission yeast Ish1 and Les1 interact with each other in the lumen of the nuclear envelope.

The nuclear envelope (NE) provides a permeable barrier that separates the eukaryotic genome from the cytoplasm. NE is a double membrane composed of inner and outer nuclear membranes. Ish1 is a stress-responsive NE protein in the fission yeast, Schizosaccharomyces pombe. Les1 is another NE protein that shares several similar domains with Ish1, but the relationship between them remains unknown. In this study, using fluorescence and electron microscopy, we found that most regions of these proteins were localized within the NE lumen. We also found that Ish1 interacted with Les1 via its C-terminal region in the NE lumen and that the NE localization of Ish1 depended on the C-terminal region of Les1. Ish1 and Les1 were co-localized at the NE in interphase cells, but when the nucleus divided at the end of mitosis (closed mitosis), they showed distinguishable localization at the midzone membrane domain. These results suggest the regulated interaction between Ish1 and Les1 in the NE lumen, although this interaction does not appear to be essential for cell survival.

Mobility of kinetochore proteins measured by FRAP analysis in living cells.

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry.

DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.

Impacts of the STING-IFNAR1-STAT1-IRF1 pathway on the cellular immune reaction induced by fractionated irradiation.

Radiotherapy (RT) combined with immune checkpoint inhibitors has recently produced outstanding results and is expected to be adaptable for various cancers. However, the precise molecular mechanism by which immune reactions are induced by fractionated RT is still controversial. We aimed to investigate the mechanism of the immune response regarding multifractionated, long-term radiation, which is most often combined with immunotherapy. Two human esophageal cancer cell lines, KYSE-450 and OE-21, were irradiated by fractionated irradiation (FIR) daily at a dose of 3 Gy in 5 d/wk for 2 weeks. Western blot analysis and RNA sequencing identified type I interferon (IFN) and the stimulator of IFN genes (STING) pathway as candidates that regulate immune response by FIR. We inhibited STING, IFNAR1, STAT1, and IFN regulatory factor 1 (IRF1) and investigated the effects on the immune response in cancer cells and the invasion of surrounding immune cells. We herein revealed type I IFN-dependent immune reactions and the positive feedback of STING, IRF1, and phosphorylated STAT1 induced by FIR. Knocking out STING, IFNAR1, STAT1, and IRF1 resulted in a poorer immunological response than that in WT cells. The STING-KO KYSE-450 cell line showed significantly less invasion of PBMCs than the WT cell line under FIR. In the analysis of STING-KO cells and migrated PBMCs, we confirmed the occurrence of STING-dependent immune activation under FIR. In conclusion, we identified that the STING-IFNAR1-STAT1-IRF1 axis regulates immune reactions in cancer cells triggered by FIR and that the STING pathway also contributes to immune cell invasion of cancer cells.

Comprehensive screening for drugs that modify radiation-induced immune responses.

BACKGROUND: Combination therapy based on radiotherapy and immune checkpoint inhibitors (ICIs) was recently reported as effective for various cancers. The radiation-induced immune response (RIIR) is an essential feature in ICI-combined radiotherapy; however, the effects of drugs used concomitantly with RIIR remain unclear. We screened for drugs that can modify RIIR to understand the mutual relationship between radiotherapy and combined drugs in ICI-combined radiotherapy. METHODS: We established a high-throughput system with reporter gene assays for evaluating RIIR, focusing on factors acting downstream of the STING-IRF pathway, which can stimulate cancer cells, T cells, and dendritic cells. We further quantified the effects of 2595 drugs, including those approved by the Food and Drug Administration, on RIIR in vitro. RESULTS: The reporter assay results correlated well with the expression of immune response proteins such as programmed death-ligand 1. This high-throughput system enabled the identification of drugs including cytotoxic agents, molecular-targeted agents, and other agents that activate or suppress RIIR. CONCLUSIONS: Our study provides an encyclopedic catalogue of clinically approved drugs based on their effect on RIIR. In ICIs combined radiotherapy, activation of STING-IFN may improve the therapeutic effect and our result could form a biological basis for further clinical trials combining radiotherapy with ICIs.

Treatment response after palliative radiotherapy for bleeding gastric cancer: a multicenter prospective observational study (JROSG 17-3).

BACKGROUND: Palliative radiotherapy seems to be rarely performed for incurable gastric cancer. In this first multicenter study, we examined the effectiveness of palliative radiotherapy and investigated whether biologically effective dose (BED) is associated with survival, response, or re-bleeding. METHODS: Eligibility criteria included blood transfusion or hemoglobin levels < 8.0 g/dL. The primary endpoint was the intention-to-treat (ITT) bleeding response rate at 4 weeks. Response entailed all of the following criteria: (i) hemoglobin levels ≥ 8.0 g/dL; (ii) 7 consecutive days without blood transfusion anytime between enrollment and blood sampling; and (iii) no salvage treatment (surgery, endoscopic treatment, transcatheter embolization, or re-irradiation) for bleeding gastric cancer. Re-bleeding was defined as the need for blood transfusion or salvage treatment. RESULTS: We enrolled 55 patients from 15 institutions. The ITT response rates were 47%, 53%, and 49% at 2, 4, and 8 weeks, respectively. The per-protocol response rates were 56%, 78%, and 90% at 2, 4, and 8 weeks, respectively. Neither response nor BED (α/ß = 10) predicted overall survival. Multivariable Fine-Gray model showed that BED was not a significant predictor of response. Univariable Cox model showed that BED was not significantly associated with re-bleeding. Grades 1, 2, 3, and, ≥ 4 radiation-related adverse events were reported in 11, 9, 1, and 0 patients, respectively. CONCLUSIONS: The per-protocol response rate increased to 90% during the 8-week follow-up. The frequent occurrence of death starting shortly after enrollment lowered the ITT response rate. BED was not associated with survival, bleeding response, or re-bleeding.

The very-long-chain fatty acid elongase Elo2 rescues lethal defects associated with loss of the nuclear barrier function in fission yeast cells.

In eukaryotic cells, chromosomes are confined to the nucleus, which is compartmentalized by the nuclear membranes; these are continuous with the endoplasmic reticulum membranes. Maintaining the homeostasis of these membranes is an important cellular activity performed by lipid metabolic enzymes. However, how lipid metabolic enzymes affect nuclear membrane functions remains to be elucidated. We found that the very-long-chain fatty acid elongase Elo2 is located in the nuclear membrane and prevents lethal defects associated with nuclear membrane ruptures in mutants of the nuclear membrane proteins Lem2 and Bqt4 in the fission yeast Schizosaccharomyces pombe. Lipid composition analysis shows that t20:0/24:0 phytoceramide (a conjugate of C20:0 phytosphingosine and C24:0 fatty acid) is a major ceramide species in S. pombe The quantity of this ceramide is reduced in the absence of Lem2, and restored by increased expression of Elo2. Furthermore, loss of S. pombe Elo2 can be rescued by its human orthologs. These results suggest that the conserved very-long-chain fatty acid elongase producing the ceramide component is essential for nuclear membrane integrity and cell viability in eukaryotes.This article has an associated First Person interview with the first author of the paper.

Comparative analysis of the immune responses in cancer cells irradiated with X-ray, proton and carbon-ion beams.

Radiotherapy (RT) is an effective treatment option for cancer; however, its efficacy remains less than optimal in locally advanced cancer. Immune checkpoint inhibitor-based therapy, including the administration of anti-PD-L1 antibodies, is a promising approach that works synergistically with RT. Proton beam therapy and carbon-ion therapy are common options for patients with cancer. Proton and carbon ions are reported to induce an immune reaction in cancer cells; however, the underlying mechanisms remain unclear. Here, we aimed to compare the immune responses after irradiation (IR) with X-ray, protons, and carbon ions in an oesophageal cancer cell line and the underlying mechanisms. An oesophageal cancer cell line, KYSE450, was irradiated with 1 fraction/15 GyE (Gy equivalent) of X-ray, proton, or carbon-ion beams, and then, the cells were harvested for RNA sequencing and gene enrichment analysis. We also knocked out STING and STAT1 in the quest for mechanistic insights. RNA sequencing data revealed that gene expression signatures and biological processes were different in KYSE450 irradiated with X-ray, proton, and carbon-ion beams 6-24 h after IR. However, after 3 days, a common gene expression signature was detected, associated with biological pathways involved in innate immune responses. Gene knock-out experiments revealed that the STING-STAT1 axis underlies the immune reactions after IR. X-Ray, proton, and carbon-ion IRs induced similar immune responses, regulated by the STING-STAT1 axis.

Intracellular ATP levels influence cell fates in Dictyostelium discoideum differentiation.

Multicellular organisms contain various differentiated cells. Fate determination of these cells remains a fundamental issue. The cellular slime mold Dictyostelium discoideum is a useful model organism for studying differentiation; it proliferates as single cells in nutrient-rich conditions, which aggregate into a multicellular body upon starvation, subsequently differentiating into stalk cells or spores. The fates of these cells can be predicted in the vegetative phase: Cells expressing higher and lower levels of omt12 differentiate into stalk cells and spores, respectively. However, omt12 is merely a marker gene and changes in its expression do not influence the cell fate, and determinant factors remain unknown. In this study, we analyzed cell fate determinants in the stalk-destined and spore-destined cells that were sorted based on omt12 expression. Luciferase assay demonstrated higher levels of intracellular ATP in the stalk-destined cells than in the spore-destined cells. Live-cell observation during development using ATP sensor probes revealed that cells with higher ATP levels differentiated into stalk cells. Furthermore, reducing the ATP level by treating with an inhibitor of ATP production changed the differentiation fates of the stalk-destined cells to spores. These results suggest that intracellular ATP levels influence cell fates in D. discoideum differentiation.

Human Ebp1 rescues the synthetic lethal growth of fission yeast cells lacking Cdb4 and Nup184.

Cdb4 is a protein with unknown functions that binds to curved DNA in vitro in the fission yeast Schizosaccharomyces pombe. Homologues of Cdb4 were identified in a wide range of eukaryotes, including human Ebp1. Both S. pombe Cdb4 and human Ebp1 are nonpeptidase members of the methionine aminopeptidase family. It has been reported that Ebp1 homologues are involved in cell growth regulation and differentiation. However, opposing functions have also been considered and debated upon, and the precise biological functions of this conserved protein are largely unknown. S. pombe cdb4 is a nonessential gene, and no obvious phenotypes have been detected in cells with cdb4 gene deletion. In this study, we identified nup184, encoding a component of the nuclear pore complex, as a gene responsible for the synthetic lethal phenotype associated with cdb4. Furthermore, the synthetic lethal phenotype of Cdb4 was suppressed by over-expression of human Ebp1, suggesting that it has conserved crucial functions in S. pombe Cdb4 and human Ebp1. This synthetic lethal phenotype associated with Cdb4 and Nup184 provides a molecular genetics tool to study the functions of S. pombe Cdb4 and its conserved members of proteins, including human Ebp1.

Utility of whole brain radiation therapy for leptomeningeal carcinomatosis.

BACKGROUND: Although whole brain radiation therapy (WBRT) is commonly used as first-line treatment for leptomeningeal carcinomatosis, the prognosis is uncertain despite treatment. Moreover, the benefit of WBRT for leptomeningeal carcinomatosis has not been adequately evaluated. Therefore, this study aimed to clarify the utility of WBRT for leptomeningeal carcinomatosis. METHODS: Consecutive patients who received WBRT for leptomeningeal carcinomatosis or brain metastasis from solid tumors between January 2008 and July 2017 were retrospectively evaluated. The overall survival, symptom relief, and adverse events were compared between patients with leptomeningeal carcinomatosis and those with brain metastasis after WBRT. RESULTS: Of the 277 treated patients, 204 patients (22 with leptomeningeal carcinomatosis and 182 with brain metastasis) were included in the study. The median overall survival was 440 days (95% confidence interval [CI] 0-931 days) for patients with leptomeningeal carcinomatosis and 322 days (95% CI 196-448 days) for those with brain metastasis (p = 0.972 on the log-rank test). On evaluating the overall survival of patients with leptomeningeal carcinomatosis, the prognostic factors of performance status 0-1, no extracranial metastasis, and no symptoms at the time of WBRT showed a significant survival advantage on univariate analysis. Among patients with leptomeningeal carcinomatosis, those with headache and nausea often showed improvement while those with depressed levels of consciousness and seizures did not. On comparing all-grade adverse events, vomiting and seizures were more frequent in patients with leptomeningeal carcinomatosis than in those with brain metastasis. CONCLUSIONS: WBRT was generally well tolerated and effective for treating patients with leptomeningeal carcinomatosis.

DNA Methylation Status of SHOX-Flanking CpG Islands in Healthy Individuals and Short Stature Patients with Pseudoautosomal Copy Number Variations.

SHOX resides in the short arm pseudoautosomal region (PAR1) of the sex chromosomes and escapes X inactivation. SHOX haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). A substantial percentage of cases with SHOX haploinsufficiency arise from pseudoautosomal copy number variations (CNVs) involving putative enhancer regions of SHOX. Our previous study using peripheral blood samples showed that some CpG dinucleotides adjacent to SHOX exon 1 were hypomethylated in a healthy woman and methylated in a woman with gross X chromosomal rearrangements. However, it remains unknown whether submicroscopic pseudoautosomal CNVs cause aberrant DNA methylation of SHOX-flanking CpG islands. In this study, we examined the DNA methylation status of SHOX-flanking CpG islands in 50 healthy individuals and 10 ISS/LWD patients with pseudoautosomal CNVs. In silico analysis detected 3 CpG islands within the 20-kb region from the translation start site of SHOX. Pyrosequencing and bisulfite sequencing of genomic DNA samples revealed that these CpG islands were barely methylated in peripheral blood cells and cultured chondrocytes of healthy individuals, as well as in peripheral blood cells of ISS/LWD patients with pseudoautosomal CNVs. These results, in conjunction with our previous findings, indicate that the DNA methylation status of SHOX-flanking CpG islands can be affected by gross X-chromosomal abnormalities, but not by submicroscopic CNVs in PAR1. Such CNVs likely disturb SHOX expression through DNA methylation-independent mechanisms, which need to be determined in future studies.

Lem2 is retained at the nuclear envelope through its interaction with Bqt4 in fission yeast.

Inner nuclear membrane (INM) proteins are thought to play important roles in modulating nuclear organization and function through their interactions with chromatin. However, these INM proteins share redundant functions in metazoans that pose difficulties for functional studies. The fission yeast Schizosaccharomyces pombe exhibits a relatively small number of INM proteins, and molecular genetic tools are available to separate their redundant functions. In S. pombe, it has been reported that among potentially redundant INM proteins, Lem2 displays a unique genetic interaction with another INM protein, Bqt4, which is involved in anchoring telomeres to the nuclear envelope. Double mutations in the lem2 and bqt4 genes confer synthetic lethality during vegetative growth. Here, we show that Lem2 is retained at the nuclear envelope through its interaction with Bqt4, as the loss of Bqt4 results in the exclusive accumulation of Lem2 to the spindle pole body (SPB). An N-terminal nucleoplasmic region of Lem2 bears affinity to both Bqt4 and the SPB in a competitive manner. In contrast, the synthetic lethality of the lem2 bqt4 double mutant is suppressed by the C-terminal region of Lem2. These results indicate that the N-terminal and C-terminal domains of Lem2 show independent functions with respect to Bqt4.

Reconstruction of root systems in Cryptomeria japonica using root point coordinates and diameters.

MAIN CONCLUSION: We developed simple algorithms for reconstructing tree root system architecture using only the root point coordinate and diameter, which can be systematically obtained without digging up the root systems. Root system architecture (RSA) is strongly related to various root functions of the tree. The aim of this study was to develop a three-dimensional (3D) RSA model using systematically obtained information on root locations and root diameters at the locations. We excavated root systems of Cryptomeria japonica and systematically obtained XYZ coordinates and root diameters using a 10-cm grid. We clarified the patterns of the root point connections and developed a reconstructed root system model. We found that the root diameters farther from the stump centre are smaller. Additionally, we found that the root lengths of the segments running between the base and the connected root point were smaller than those of other root segments, and the inner angle between the base and the stump and between the base and the connected root point was narrower than for the other pairs. The new RSA model developed according to these results had average accuracies of 0.64 and 0.80 for estimates of total volume and length, respectively. The developed model can estimate 3D RSA using only root point data, which can be obtained without digging up root systems. This suggests a wide applicability of this model in root function evaluation.

Identification of Conserved MEL-28/ELYS Domains with Essential Roles in Nuclear Assembly and Chromosome Segregation.

Nucleoporins are the constituents of nuclear pore complexes (NPCs) and are essential regulators of nucleocytoplasmic transport, gene expression and genome stability. The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes. Here we present a systematic functional and structural analysis of MEL-28 in C. elegans early development and human ELYS in cultured cells. We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation. Surprisingly, we found that perturbations to MEL-28's conserved AT-hook domain do not affect MEL-28 localization although they disrupt MEL-28 function and delay cell cycle progression in a DNA damage checkpoint-dependent manner. Our analyses also uncover a novel meiotic role of MEL-28. Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.