Nuclear Segregation Research Articles

Isolation of Cardiomyocytes Undergoing Mitosis With Complete Cytokinesis.

Adult human cardiomyocytes do not complete cytokinesis despite passing through the S-phase of the cell cycle. As a result, polyploidization and multinucleation occur. To get a deeper understanding of the mechanisms surrounding division of cardiomyocytes, there is a crucial need for a technique to isolate cardiomyocytes that complete cell division/cytokinesis. Markers of cell cycle progression based on DNA content cannot distinguish between mitotic cardiomyocytes that fail to complete cytokinesis from those cells that undergo true cell division. With the use of molecular beacons (MBs) targeting specific mRNAs, we aimed to identify truly proliferative cardiomyocytes derived from human induced pluripotent stem cells. Fluorescence-activated cell sorting combined with MBs was performed to sort cardiomyocyte populations enriched for mitotic cells. Expressions of cell cycle specific genes were confirmed by means of reverse transcription-quantitative polymerase chain reaction and single-cell RNA sequencing (scRNA-seq) combined with gene signatures of cell cycle progression. We characterized the sorted groups by proliferation assays and time-lapse microscopy which confirmed the proliferative advantage of MB-positive cell populations relative to MB-negative and G2/M populations. Gene expression analysis revealed that the MB-positive cardiomyocyte subpopulation exhibited patterns consistent with the processes of nuclear division, chromosome segregation, and transition from M to G1 phase. The use of dual-MBs targeting CDC20 and SPG20 mRNAs enabled the enrichment of cytokinetic events (CDC20highSPG20high). Interestingly, cells that did not complete cytokinesis and remained binucleated were found to be CDC20lowSPG20high while polyploid cardiomyocytes that replicated DNA but failed to complete karyokinesis were found to be CDC20lowSPG20low. This study demonstrates a novel alternative to existing DNA content-based approaches for sorting cardiomyocytes with true mitotic potential that can be used to study the unique dynamics of cardiomyocyte nuclei during mitosis. Our technique for sorting live cardiomyocytes undergoing cytokinesis would provide a basis for future studies to uncover mechanisms underlying the development and regeneration of heart tissue.

DNA sequence-dependent chromatin architecture and nuclear hubs formation

In this study, by exploring chromatin conformation capture data, we show that the nuclear segregation of Topologically Associated Domains (TADs) is contributed by DNA sequence composition. GC-peaks and valleys of TADs strongly influence interchromosomal interactions and chromatin 3D structure. To gain insight on the compositional and functional constraints associated with chromatin interactions and TADs formation, we analysed intra-TAD and intra-loop GC variations. This led to the identification of clear GC-gradients, along which, the density of genes, super-enhancers, transcriptional activity, and CTCF binding sites occupancy co-vary non-randomly. Further, the analysis of DNA base composition of nucleolar aggregates and nuclear speckles showed strong sequence-dependant effects. We conjecture that dynamic DNA binding affinity and flexibility underlay the emergence of chromatin condensates, their growth is likely promoted in mechanically soft regions (GC-rich) of the lowest chromatin and nucleosome densities. As a practical perspective, the strong linear association between sequence composition and interchromosomal contacts can help define consensus chromatin interactions, which in turn may be used to study alternative states of chromatin architecture.

The role of CSE1L expression in cervical lymph node metastasis of larynx tumors

IntroductionAccording to international reports, 30–40% of all head and neck cancers are larynx cancers, comprising 1–2.5% of all cancer types. Cervical nodal involvement has been reported to be 40% and 65% in T3 and T4 cases, respectively. Five-year survival in patients with cervical lymph node metastasis has been demonstrated to be 50% lower compared to patients with no metastasis. Chromosome segregation like 1 protein; is a DNA fragment isolated by Brinkmann et al. in 1995 that corresponds to yeast chromosome segregation protein. Studies on the effect of chromosome segregation like 1 protein expression in head and neck tumors are rare and it has been shown that nuclear chromosome segregation like 1 protein is over-expressed in these studies where gastrointestinal and breast tumors over-expressed cytoplasmic chromosome segregation like 1 protein. ObjectiveChromosome segregation like 1 protein may regulate the proliferation and metastasis of T3–T4 glottic larynx cancer. The aim of this study is to show the relationship between chromosome segregation like 1 protein expression and cervical lymph node metastasis of T3–T4 glottic larynx cancer. MethodsA total of 57 male patients who were operated for T3–T4 glottic cancer in a tertiary referral hospital was included in this study. There were 28 patients with cervical lymph node metastasis and 29 patients without lymph node metastasis. Immunohistochemistry was carried out on formalin-fixed, paraffin-embedded archival glottic larynx tumour tissue. According to the percentage of immunoreactive cells, chromosome segregation like 1 protein status was analyzed. ResultsAmong the patients, who had no cervical lymph node metastasis, 15 patients showed weak nuclear staining, 12 patients showed moderate nuclear staining and only 2 patients showed high nuclear staining for chromosome segregation like 1 protein. Among the patients who had cervical lymph node metastasis, 18 patients showed high nuclear staining, 9 patients showed moderate staining and only one patient showed weak staining for chromosome segregation like 1 protein. None of the metastatic patients showed cytoplasmic staining and only one patient in the non-metastatic group showed cytoplasmic staining for chromosome segregation like 1 protein. There was a positive correlation between nuclear chromosome segregation like 1 protein expression and cervical lymph node metastasis (r = 0,668) and it was statistically significant (p < 0,001). ConclusionChromosome segregation like 1 protein expression is correlated with lymph node metastasis in T3–T4 glottic cancers. This may change the approach to cervical node treatment in patients with glottic cancers in future.

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Four novel biomarkers for bladder cancer identified by weighted gene coexpression network analysis.

Bladder cancer (BC) is one of the most malignancies in terms of incidence and recurrence worldwide. The aim of this study is to find out novel and prognostic biomarkers for patients with BC. First, we identified 258 differentially expressed genes by using GSE19915 from Gene Expression Omnibus database. Second, a total of 33 modules were identified by constructing a coexpression network by using weighted gene coexpression network analysis and yellow module was regarded as the key module. Furthermore, by constructing protein-protein interaction networks, we preliminarily picked out 13 genes. Among them, four hubgenes (CCNB1, KIF4A, TPX2, and TRIP13) were eventually identified by using five different methods (survival analysis, one-way analysis of variance, the Spearman correlation analysis, receiver operating characteristic curve, and expression value comparison), which were significantly correlated with the prognosis of BC. The validation of transcriptional and translational levels made sense (based on Oncomine and The Human Protein Atlas database). Moreover, functional enrichment analysis suggested that all the hub genes played crucial roles in chromosome segregation, sister chromatid segregation, nuclear chromosome segregation, mitotic nuclear division, nuclear division, and organelle fission during cell mitosis. In addition, three of the hub genes (KIF4A, TPX2, and TRIP13) might be potential targets of cancer drugs according to the results of the genetical alteration. In conclusion, this study indicates that four hub genes have great predictive value for the prognosis of BC, and may contribute to the exploration of the further and more in-depth research of BC.

Asymmetric distribution of glucose transporter mRNA provides a growth advantage in yeast.

Asymmetric localization of mRNA is important for cell fate decisions in eukaryotes and provides the means for localized protein synthesis in a variety of cell types. Here, we show that hexose transporter mRNAs are retained in the mother cell of S.cerevisiae until metaphase-anaphase transition (MAT) and then are released into the bud. The retained mRNA was translationally less active but bound to ribosomes before MAT Importantly, when cells were shifted from starvation to glucose-rich conditions, HXT2 mRNA, but none of the other HXT mRNAs, was enriched in the bud after MAT This enrichment was dependent on the Ras/cAMP/PKA pathway, the APC ortholog Kar9, and nuclear segregation into the bud. Competition experiments between strains that only expressed one hexose transporter at a time revealed that HXT2 only cells grow faster than their counterparts when released from starvation. Therefore, asymmetric distribution of HXT2 mRNA provides a growth advantage for daughters, who are better prepared for nutritional changes in the environment. Our data provide evidence that asymmetric mRNA localization is an important factor in determining cellular fitness.

Cell cycle and cleavage events during in vitro cultivation of bloodstream forms of Trypanosoma lewisi, a zoonotic pathogen

ABSTRACT Trypanosoma (Herpetosoma) lewisi is a globally distributed rat trypanosome, currently considered as a zoonotic pathogen; however, a detailed understanding of the morphological events occurring during the cell cycle is lacking. This study aimed to investigate the cell cycle morphology and cleavage events of Trypanosoma lewisi (T. lewisi) during in vitro cultivation. By establishing in vitro cultivation of T. lewisi at 37°C, various cell morphologies and stages could be observed. We have provided a quantitative analysis of the morphological events during T. lewisi proliferation. We confirmed a generation time of 12.14 ± 0.79 hours, which is similar to that in vivo (12.21 ± 0.14 hours). We also found that there are two distinct cell cycles, with a two-way transformation connection in the developmental status of this parasite, which was contrasted with the previous model of multiple division patterns seen in T. lewisi. We quantified the timing of cell cycle phases (G1n, 0.56 U; Sn, 0.14 U; G2n, 0.16 U; M, 0.06 U; C, 0.08 U; G1k, 0.65 U; Sk, 0.10 U; G2k, 0.17 U; D, 0.03 U; A, 0.05 U) and their morphological characteristics, particularly with respect to the position of kinetoplast(s) and nucleus/nuclei. Interestingly, we found that both nuclear synthesis initiation and segregation in T. lewisi occurred prior to kinetoplast, different to the order of replication found in Trypanosoma brucei and Trypanosoma cruzi, implicating a distinct cell cycle control mechanism in T. lewisi. We characterized the morphological events during the T. lewisi cell cycle and presented evidence to support the existence of two distinct cell cycles with two-way transformation between them. These results provide insights into the differentiation and evolution of this parasite and its related species.

Dual role for fungal-specific outer kinetochore proteins during cell cycle and development in Magnaporthe oryzae.

The outer kinetochore DASH complex (also known as the Dam1 complex) ensures proper spindle structure and chromosome segregation. While DASH complex protein requirement diverges among different yeasts, its role in filamentous fungi has not yet been investigated. We studied the dynamics and role of middle (Mis12) and outer (Dam1 and Ask1) kinetochore proteins in the filamentous fungal pathogen, Magnaporthe oryzae, which undergoes multiple cell cycle-linked developmental transitions. While Mis12 was constitutively present in the nucleus, Dam1 and Ask1 were recruited only during mitosis. Although Dam1 was not required for viability, loss of its function (dam1Δ) delayed mitotic progression, resulting in impaired conidial and hyphal development. Both Dam1 and Ask1 also localised to the hyphal tips, in the form of punctae oscillating back and forth from the growing ends, suggesting that Magnaporthe DASH complex proteins may play a non-canonical role in polarised growth during interphase, in addition to their function in nuclear segregation during mitosis. Impaired appressorial (infection structure) development and host penetration in the dam1Δ mutant suggest that fungus-specific Dam1 complex proteins could be an attractive target for a novel anti-fungal strategy.This article has an associated First Person interview with the first author of the paper.

A Novel Actin Binding Drug with In Vivo Efficacy.

Occidiofungin is produced by the soil bacterium Burkolderia contaminans MS14 and is structurally similar or identical to the burkholdines, xylocandins, and cepacidines. This study identified the primary cellular target of occidiofungin, which was determined to be actin. The modification of occidiofungin with a functional alkyne group enabled affinity purification assays and localization studies in yeast. Occidiofungin has a subtle effect on actin dynamics that triggers apoptotic cell death. We demonstrate the highly specific localization of occidiofungin to cellular regions rich in actin in yeast and the binding of occidiofungin to purified actin in vitro Furthermore, a disruption of actin-mediated cellular processes, such as endocytosis, nuclear segregation, and hyphal formation, was observed. All of these processes require the formation of stable actin cables, which are disrupted following the addition of a subinhibitory concentration of occidiofungin. We were also able to demonstrate the effectiveness of occidiofungin in treating a vulvovaginal yeast infection in a murine model. The results of this study are important for the development of an efficacious novel class of actin binding drugs that may fill the existing gap in treatment options for fungal infections or different types of cancer.

Mms21: A Putative SUMO E3 Ligase in Candida albicans That Negatively Regulates Invasiveness and Filamentation, and Is Required for the Genotoxic and Cellular Stress Response.

In the life cycle of the fungal pathogen Candida albicans, the formation of filamentous cells is a differentiation process that is critically involved in host tissue invasion, and in adaptation to host cell and environmental stresses. Here, we have used the Gene Replacement And Conditional Expression library to identify genes controlling invasiveness and filamentation; conditional repression of the library revealed 69 mutants that triggered these processes. Intriguingly, the genes encoding the small ubiquitin-like modifier (SUMO) E3 ligase Mms21, and all other tested members of the sumoylation pathway, were both nonessential and capable of triggering filamentation upon repression, suggesting an important role for sumoylation in controlling filamentation in C. albicans We have investigated Mms21 in detail. Both Mms21 nulls (mms21Δ/Δ) and SP [Siz/Pias (protein inhibitor of activated signal transducer and activator of transcription)] domain (SUMO E3 ligase domain)-deleted mutants displayed invasiveness, filamentation, and abnormal nuclear segregation; filament formation occurred even in the absence of the hyphal transcription factor Efg1. Transcriptional analysis of mms21Δ/Δ showed an increase in expression from two- to eightfold above that of the wild-type for hyphal-specific genes, including ECE1, PGA13, PGA26, HWP1, ALS1, ALS3, SOD4, SOD5, UME6, and HGC1 The Mms21-deleted mutants were unable to recover from DNA-damaging agents like methyl methane sulfonate, hydroxyurea, hydrogen peroxide, and UV radiation, suggesting that the protein is important for genotoxic stress responses. In addition, the mms21Δ/Δ mutant displayed sensitivity to cell wall and thermal stresses, and to different antifungal drugs. All these findings suggest that Mms21 plays important roles in cellular differentiation, DNA damage and cellular stress responses, and in response to antifungal drugs.

The anillin-related Int1 protein and the Sep7 septin collaborate to maintain cellular ploidy in Candida albicans

Variation in cell ploidy is a common feature of Candida albicans clinical isolates that are resistant to the antifungal drug fluconazole. Here, we report that the anillin-related protein Int1 interacts with septins for coupling cytokinesis with nuclear segregation. Loss of Int1 results in a rapid disassembly of duplicated septin rings from the bud neck at the onset of actomyosin ring contraction. Strikingly, this has no major impact on cytokinesis and septum formation. However, Int1 genetically interacts with the Sep7 septin, maintaining the diffusion barrier at the bud neck and guarantying a faithful nuclear segregation. Indeed, int1ΔΔ sep7ΔΔ mutant cells, in contrast to int1ΔΔ cdc10ΔΔ, undergo a premature activation of mitotic exit prior to the alignment of the mitotic spindle with the division axis, producing large multinucleated cells. Some of these multinucleated cells arise from trimeras similar to those observed upon fluconazole exposure. Finally, the defects in nuclear segregation could be in part due to the inability to maintain the Lte1 mitotic exit activator at the cortex of the daughter cell. These results suggest that Int1 and Sep7 play a role in maintaining genome stability by acting as a diffusion barrier for Lte1.

Genome-Scale Genetic Interactions and Cell Imaging Confirm Cytokinesis as Deleterious to Transient Topoisomerase II Deficiency in Saccharomyces cerevisiae.

Topoisomerase II (Top2) is an essential protein that resolves DNA catenations. When Top2 is inactivated, mitotic catastrophe results from massive entanglement of chromosomes. Top2 is also the target of many first-line anticancer drugs, the so-called Top2 poisons. Often, tumors become resistant to these drugs by acquiring hypomorphic mutations in the genes encoding Top2. Here, we have compared the cell cycle and nuclear segregation of two coisogenic Saccharomyces cerevisiae strains carrying top2 thermosensitive alleles that differ in their resistance to Top2 poisons: the broadly-used poison-sensitive top2-4 and the poison-resistant top2-5. Furthermore, we have performed genome-scale synthetic genetic array (SGA) analyses for both alleles under permissive conditions, chronic sublethal Top2 downregulation, and acute, yet transient, Top2 inactivation. We find that slowing down mitotic progression, especially at the time of execution of the mitotic exit network (MEN), protects against Top2 deficiency. In all conditions, genetic protection was stronger in top2-5; this correlated with cell biology experiments in this mutant, whereby we observed destabilization of both chromatin and ultrafine anaphase bridges by execution of MEN and cytokinesis. Interestingly, whereas transient inactivation of the critical MEN driver Cdc15 partly suppressed top2-5 lethality, this was not the case when earlier steps within anaphase were disrupted; i.e., top2-5 cdc14-1. We discuss the basis of this difference and suggest that accelerated progression through mitosis may be a therapeutic strategy to hypersensitize cancer cells carrying hypomorphic mutations in TOP2.

Mitotic nuclear pore complex segregation involves Nup2 in Aspergillus nidulans.

Transport through nuclear pore complexes (NPCs) during interphase is facilitated by the nucleoporin Nup2 via its importin α- and Ran-binding domains. However, Aspergillus nidulans and vertebrate Nup2 also locate to chromatin during mitosis, suggestive of mitotic functions. In this study, we report that Nup2 is required for mitotic NPC inheritance in A. nidulans Interestingly, the role of Nup2 during mitotic NPC segregation is independent of its importin α- and Ran-binding domains but relies on a central targeting domain that is necessary for localization and viability. To test whether mitotic chromatin-associated Nup2 might function to bridge NPCs with chromatin during segregation, we provided an artificial link between NPCs and chromatin via Nup133 and histone H1. Using this approach, we bypassed the requirement of Nup2 for NPC segregation. This indicates that A. nidulans cells ensure accurate mitotic NPC segregation to daughter nuclei by linking mitotic DNA and NPC segregation via the mitotic specific chromatin association of Nup2.

Elastic-Fluid Model for DNA Damage and Mutation from Nuclear Fluid Segregation Due to Cell Migration

When cells migrate through constricting pores, they incur DNA damage and develop genomic variation. Experiments show that this damage is not due to DNA breakage from mechanical stress on chromatin in the deformed nucleus. Here we propose a model for a mechanism by which nuclear deformation can lead to DNA damage. We treat the nucleus as an elastic-fluid system with an elastic component (chromatin) and fluid component that can be squeezed out when the nucleus is deformed. We couple the elastic-fluid model to the kinetics of DNA breakage and repair by assuming that the local volume fraction of the elastic component controls the rate of damage per unit volume due to naturally occurring DNA breaks, whereas the volume fraction of the fluid component controls the rate of repair of DNA breaks per unit volume by repair factors, which are soluble in the fluid. By comparing our results to a number of experiments on controlled migration through pores, we show that squeeze-out of the fluid, and hence of the mobile repair factors, is sufficient to account for the extent of DNA damage and genomic variation observed experimentally. We also use our model for migration through a cylindrical pore to estimate the variation with tissue stiffness of the mutation rate in tumors.

Contributions of β-tubulin to cellular morphology, sporulation and virulence in the insect-fungal pathogen, Metarhizium acridum

β-tubulin is an elementary subunit of microtubules that form the cytoskeleton, participating in a wide range of cellular processes. The contributions of the single β-tubulin gene in affecting cell morphology, sporulation and virulence were examined in the entomopathogenic fungus Metarhizium acridum. Targeted gene knockout of β-tubulin resulted in resistance to benomyl but impaired proper nuclear segregation, lipid droplet transport, and deposition of chitin to the cell wall. M. acridum β-tubulin mutants displayed wavy hyphal growth and densely packed, wrinkled colonies. Decreases in the rate of phialides formation and conidial yield were observed for the β-tubulin mutant, which was also impaired in virulence towards locust hosts as compared to wild type and complemented strains. Morphological analyses of infection structures revealed development of bifurcated germ tubes, with reduced appressoria formation seen in the β-tubulin mutant. M. acridum β-tubulin mutant appressoria were aberrant in morphology and displayed decreased turgor pressure. The ability of the M. acridum β-tubulin mutant to proliferate in the insect hemolymph both in vitro and in vivo was also significantly reduced. Our results indicate that in M. acridum, β-tubulin is not essential for survival but that it contributes to cellular transport of organelles and cell wall materials, impacting growth, appressorial differentiation, virulence, and sporulation.

Orchestrating cellular signaling pathways-the cellular "conductor" protein tyrosine phosphatase interacting protein 51 (PTPIP51).

The protein tyrosine phosphatase interacting protein 51 (PTPIP51) is thought to regulate crucial cellular functions such as mitosis, apoptosis, migration, differentiation and communication between organelles as a scaffold protein. These diverse functions are modulated by the tyrosine/serine phosphorylation status of PTPIP51. This review interconnects the insights obtained about the action of PTPIP51 in mitogen-activated protein kinase signaling, nuclear factor kB signaling, calcium homeostasis and chromosomal segregation and identifies important signaling hubs. The interference of PTPIP51 in such multiprotein complexes and their PTPIP51-modulated cross-talk makes PTPIP51 an ideal target for novel drugs such as the small molecule LDC-3. Graphical Abstract ᅟ.

TRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast.

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct6A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct6A37 or Ψ38/39 and mcm5U34 or s2U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNALysUUU or tRNAGlnUUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm5U34 or s2U34, and this defect can be rescued by overexpression of tRNAGlnUUG. Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

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.

Intracellular delivery of poly(I:C) induces apoptosis of fibroblast-like synoviocytes via an unknown dsRNA sensor

Fibroblast-like synoviocytes (FLS) express functional membranous and cytoplasmic sensors for double-stranded (ds)RNA. Notably, FLS undergo apoptosis upon transfection with the synthetic dsRNA analog poly(I:C). We here studied the mechanism of intracellular poly(I:C) recognition and subsequent cell death in FLS. FLS responded similarly to poly(I:C) or 3pRNA transfection; however, only intracellular delivery of poly(I:C) induced significant cell death, accompanied by upregulation of pro-apoptotic proteins Puma and Noxa, caspase 3 cleavage, and nuclear segregation. Knockdown of the DExD/H-box helicase MDA5 did not affect the response to intracellular poly(I:C); in contrast, knockdown of RIG-I abrogated the response to 3pRNA. Knockdown of the downstream adaptor proteins IPS, STING, and TRIF or inhibition of TBK1 did not affect the response to intracellular poly(I:C), while knockdown of IFNAR blocked intracellular poly(I:C)-mediated signaling and cell death. We conclude that a so far unknown intracellular sensor recognizes linear dsRNA and induces apoptosis in FLS.

Anillin-related protein Mid1 regulates timely formation of the contractile ring in the fission yeast Schizosaccharomyces japonicus.

In the fission yeast Schizosaccharomyces pombe (Sp), Mid1/Dmf1 plays an important role in positioning the division site by inducing formation of the contractile ring (CR). Mid1, emanating from the nucleus located in the cell center, forms a dozen of nodes in the middle cell cortex ahead of mitosis, and actin filaments and myosin II accumulated at each node interact and assemble the CR in metaphase. Curiously, in another fission yeast S. japonicus (Sj), CR formation begins after nuclear segregation in late anaphase. Here, we investigated the role of S. japonicus Mid1 during mitosis to compare the molecular mechanisms that determine the cell division site in Schizosaccharomyces. Similar to Sp Mid1, Sj Mid1 often accumulated in the nucleus of interphase cells. Moreover, Sj Mid1 localized to cortical dots with myosin II in the future division site and formed a medial ring in mitotic cells. However, S. japonicus cells without Mid1 function still carried out symmetrical binary division. Therefore, the Mid1 dependency for positional control of the cell division site is possibly different between the two species. Meanwhile, we found that Sj Mid1 enhanced CR formation, in a manner possibly similar to that by Sp Mid1.