Nuclear Pores Assemble from Nucleoporin Condensates During Oogenesis.

The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. Here, we show that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. We delineate a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.

MCM complexes are barriers that restrict cohesin-mediated loop extrusion.

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1-3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6-12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.

Assembly of endogenous oskar mRNA particles for motor-dependent transport in the Drosophila oocyte.

oskar mRNA localization at the oocyte posterior pole is essential for correct patterning of the Drosophila embryo. Here we show at the ultrastructural level that endogenous oskar ribonucleoprotein complexes (RNPs) assemble sequentially with initial recruitment of Hrp48 and the exon junction complex (EJC) to oskar transcripts in the nurse cell nuclei, and subsequent recruitment of Staufen and microtubule motors, following transport to the cytoplasm. oskar particles are non-membrane-bound structures that coalesce as they move from the oocyte anterior to the posterior pole. Our analysis uncovers a role for the EJC component Barentsz in recruiting Tropomyosin II (TmII) to oskar particles in the ooplasm and reveals that TmII is required for kinesin binding to the RNPs. Finally, we show that both kinesin and dynein associate with oskar particles and are the primary microtubule motors responsible for transport of the RNPs within the oocyte.

An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs.

Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport-competent mRNPs. We show that the posterior-targeting kinesin-1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein-dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin-1 recruitment requires the DmTropomyosin1-I/C isoform, an atypical RNA-binding tropomyosin that binds directly to dimerizing oskar 3'UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin-1 and that the motor is activated during mid-oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo-dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo-transport processes and between the cargo-associated dynein and kinesin-1.

One-step enzymatic modification of RNA 3' termini using polymerase θ.

Site-specific modification of synthetic and cellular RNA such as with specific nucleobases, fluorophores and attachment chemistries is important for a variety of basic and applied research applications. However, simple and efficient methods to modify RNA such as at the 3' terminus with specific nucleobases or nucleotide analogs conjugated to various chemical moieties are lacking. Here, we develop and characterize a one-step enzymatic method to modify RNA 3' termini using recombinant human polymerase theta (Polθ). We demonstrate that Polθ efficiently adds 30-50 2'-deoxyribonucleotides to the 3' terminus of RNA molecules of various lengths and sequences, and extends RNA 3' termini with an assortment of 2'-deoxy and 2',3'-dideoxy ribonucleotide analogs containing functional chemistries, such as high affinity attachment moieties and fluorophores. In contrast to Polθ, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polθ shows a strong preference for adding deoxyribonucleotides to RNA, but can also add ribonucleotides with relatively high efficiency in particular sequence contexts. We anticipate that this unique activity of Polθ will become invaluable for applications requiring 3' terminal modification of RNA and potentially enzymatic synthesis of RNA.

Enzymatic production of single-molecule FISH and RNA capture probes.

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.

[Recent advances in pediatric non-Hodgkin lymphoma. Report on a retrospective single-center cohort and review of the literature]. / Újdonságok a gyermekkori non-Hodgkin-limfóma kezelésében és kivizsgálásában. Szakirodalmi áttekintés és a debreceni gyermekhematológiai központ adatai.

Classification, staging and treatment response criteria of pediatric NHL have been revised. Long-term survival reaches ~90% at the expense of severe acute toxicities. The outcome of refractory and relapsed cases is poor. The small number of patients hinders introduction of targeted therapies. Here we summarize principles and perspectives of pediatric NHL supported by results of a retrospective clinical survey. Twenty-five patients (21 boys, 4 girls; mean age: 11.9 years) were registered between 2009 and 2018: 11 Burkitt lymphomas, 4 diffuse large B-cell lymphomas, 5 T-cell lymphoblastic lymphomas, and 1-1 grey-zone lymphoma, anaplastic large-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic lymphoma, and Castleman disease. Remission rate was 22/25, 20/25 patients survived (mean follow-up time: 3.9 years). Chemotherapies according to NHL-BFM 95, CHOP, FAB/LMB96, Inter-B-NHL Ritux 2010, Euro-LB02, and ALCL99 were applied. Adjuvant immunotherapy was applied in patients with mature B-cell NHL (rituximab in 7 cases, obinutuzumab in 2 relapsed cases). In Castleman disease siltuximab was applied.

'Poking' microtubules bring about nuclear wriggling to position nuclei.

Nuclei wriggle in the cells of the follicle epithelium of the Drosophila pre-vitellogenic egg primordia. Although similar phenomena have been reported for a number of cultured cell types and some neurons in the zebrafish embryo, the mechanism and importance of the process have remained unexplained. Wriggling involves successive sudden and random minor turns of the nuclei, approximately three twists per minute with roughly 12° per twist, one of which lasts typically for 14 seconds. Wriggling is generated by the growing microtubules seeded throughout the cell cortex, which, while poking the nuclei, buckle and exert 5-40 piconewtons over ∼16 seconds. While wriggling, the nuclei drift ∼5 µm in a day in the immensely growing follicle cells along the apical-basal axis from the apical to the basal cell region. A >2-fold excess of the microtubules nucleated in the apical cell region, as compared with those seeded in the basal cell cortex, makes the nuclei drift along the apical-basal axis. Nuclear wriggling and positioning appear to be tightly related processes: they cease simultaneously when the nuclei become anchored by the actin cytoskeleton; moreover, colchicine or taxol treatment eliminates both nuclear wriggling and positioning. We propose that the wriggling nuclei reveal a thus far undescribed nuclear positioning mechanism.

Decreased VEGF Level Is Associated with Elevated Ferritin Concentration in Bronchoalveolar Lavage Fluid of Children with Interstitial Lung Diseases.

BACKGROUND: A decreased level of vascular endothelial growth factor (VEGF) was previously described in bronchoalveolar lavage fluid (BALF) of adults with interstitial lung diseases (ILD) due to bronchial epithelial cell apoptosis and its proteolytic degradation. Elevated intrapulmonary ferritin was produced by alveolar cells that promoted oxidative injury in such patients. OBJECTIVES: In this study, we analyzed the concentrations of VEGF and ferritin in BALF samples of ILD children and studied the relationship between their levels and the degree of inflammation. METHODS: BALF and serum concentration of VEGF as well as ferritin and albumin in BALF samples were measured using enzyme-linked immunosorbent assay in children with idiopathic interstitial pneumonia (n = 16), hypersensitivity pneumonitis (n = 11) and idiopathic pulmonary hemosiderosis (n = 3). Twenty-four age- and gender-matched subjects with suspicious foreign body aspiration served as a control group. RESULTS: VEGF per albumin levels in BALF were significantly decreased in ILD children compared to controls (1,075 [784-1,415] pg/mg albumin vs. 2,741 [1,131-4,660] pg/mg albumin, p = 0.0008). These values showed a significant negative correlation with inflammatory markers of total immune cell count in BALF (r = -0.411, p = 0.002) and serum C-reactive protein (r = -0.367, p = 0.006). Although serum VEGF was augmented in ILD children versus controls, no difference was observed among the ILD groups. In addition, BALF ferritin/albumin level (688 [188-1,571] ng/mg albumin vs. 256 [178-350] ng/mg albumin, p = 0.022) was significantly higher than normal in ILD individuals, especially in idiopathic pulmonary hemosiderosis. CONCLUSION: Depressed VEGF and increased ferritin in BALF may reflect the severity of chronic pulmonary inflammation in altered respiratory epithelium of childhood ILD.

Brightness through local constraint--LNA-enhanced FIT hybridization probes for in vivo ribonucleotide particle tracking.

Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe-target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild-type living Drosophila melanogaster oocytes.

Brightness enhanced DNA FIT-probes for wash-free RNA imaging in tissue.

Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fluorescence is required when unbound probes cannot be washed away. Furthermore, emission should be bright in order to enable detection against autofluorescent background. The development of fluorescence-quenched hybridization probes has led to remarkable improvement of fluorescence responsiveness. Yet, comparably little attention has been paid to the brightness of smart probes. We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness the highly responsive TO nucleoside was combined with the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside appears to serve as a light collector that increases the extinction coefficient and transfers excitation energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL mol(-1) cm(-1) at λex = 516 nm). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid RNA imaging in tissue without the need for cutting-edge equipment, time-consuming washing, or signal amplification.

An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport.

Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). Here, we show that orchestration of oskar RNP transport in the Drosophila germline relies on interplay between two double-stranded RNA-binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). We find that Staufen antagonizes Egl-mediated transport of oskar mRNA by dynein both in vitro and in vivo. Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. We additionally show that Egl associates with staufen (stau) mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. Our observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation of stau mRNA, and thus protein, in the oocyte enables motor switching on oskar RNPs by downregulating dynein activity.

Childhood Acute Lymphoblastic Leukemia: Results of the Randomized Acute Lymphoblastic Leukemia Intercontinental-Berlin-Frankfurt-Münster 2009 Trial.

PURPOSE: The International Berlin-Frankfurt-Münster (BFM) study group conducted a study on pediatric acute lymphoblastic leukemia (ALL). Minimal residual disease (MRD) was assessed using flow cytometry (FCM), and the impact of early intensification and methotrexate (MTX) dose on survival was evaluated. PATIENTS AND METHODS: We included 6,187 patients younger than 19 years. MRD by FCM refined the risk group definition previously used in the ALL intercontinental-BFM 2002 study on the basis of age, WBC count, unfavorable genetic aberrations, and treatment response measured morphologically. Patients at intermediate risk (IR) and high risk (HR) were randomly assigned to protocol augmented protocol I phase B (IB) versus IB regimen. MTX doses of 2 versus 5 g/m2 every 2 weeks, four times, were evaluated in precursor B-cell-ALL (pcB-ALL) IR. RESULTS: The 5-year event-free survival (EFS ± SE) and overall survival (OS ± SE) rates were 75.2% ± 0.6% and 82.6% ± 0.5%, respectively. Their values in risk groups were standard risk (n = 624), 90.7% ± 1.4% and 94.7% ± 1.1%; IR (n = 4,111), 77.9% ± 0.7% and 85.7% ± 0.6%; and HR (n = 1,452), 60.8% ± 1.5% and 68.4% ± 1.4%, respectively. MRD by FCM was available in 82.6% of cases. The 5-year EFS rates in patients randomly assigned to protocol IB (n = 1,669) and augmented IB (n = 1,620) were 73.6% ± 1.2% and 72.8% ± 1.2%, respectively (P = .55), while those in patients receiving MTX doses of 2 g/m2 (n = 1,056) and MTX 5 g/m2 (n = 1,027) were 78.8% ± 1.4% and 78.9% ± 1.4%, respectively (P = .84). CONCLUSION: The MRDs were successfully assessed using FCM. An MTX dose of 2 g/m2 was effective in preventing relapse in non-HR pcB-ALL. Augmented IB showed no advantages over the standard IB.[Media: see text].

Zygotic genome activation by the totipotency pioneer factor Nr5a2.

Life begins with a switch in genetic control from the maternal to the embryonic genome during zygotic genome activation (ZGA). Despite its importance, the essential regulators of ZGA remain largely unknown in mammals. On the basis of de novo motif searches, we identified the orphan nuclear receptor Nr5a2 as a key activator of major ZGA in mouse two-cell embryos. Nr5a2 is required for progression beyond the two-cell stage. It binds to its motif within SINE B1/Alu retrotransposable elements found in cis-regulatory regions of ZGA genes. Chemical inhibition suggests that 72% of ZGA genes are regulated by Nr5a2 and potentially other orphan nuclear receptors. Nr5a2 promotes chromatin accessibility during ZGA and binds nucleosomal DNA in vitro. We conclude that Nr5a2 is an essential pioneer factor that regulates ZGA.

Microtubule-based motor-mediated mRNA localization in Drosophila oocytes and embryos.

RNA localization coupled to translational repression is a general mechanism for creating structural and functional asymmetry within the cell. While there are many possible ways to target an mRNA to its destination, a large fraction of the studied transcripts undertake active transport mediated by cytoskeletal elements (microtubules and actin filaments) and associated mechanoenzymes. Among the best-studied model systems of RNA localization are the oocyte and the early embryo of Drosophila melanogaster, for which many well-characterized tools have been developed to study this cell biological phenomenon in a dynamic, developing system in its in vivo context. In the present paper, we review the current evidence and models explaining the different modes of RNA localization that depend on active transport within cells.

Profiling cellular diversity in sponges informs animal cell type and nervous system evolution.

The evolutionary origin of metazoan cell types such as neurons and muscles is not known. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identified 18 distinct cell types. These include nitric oxide­sensitive contractile pinacocytes, amoeboid phagocytes, and secretory neuroid cells that reside in close contact with digestive choanocytes that express scaffolding and receptor proteins. Visualizing neuroid cells by correlative x-ray and electron microscopy revealed secretory vesicles and cellular projections enwrapping choanocyte microvilli and cilia. Our data show a communication system that is organized around sponge digestive chambers, using conserved modules that became incorporated into the pre- and postsynapse in the nervous systems of other animals.

HorkaD, a chromosome instability-causing mutation in Drosophila, is a dominant-negative allele of Lodestar.

Correct segregation of chromosomes is particularly challenging during the rapid nuclear divisions of early embryogenesis. This process is disrupted by Horka(D), a dominant-negative mutation in Drosophila melanogaster that causes female sterility due to chromosome tangling and nondisjunction during oogenesis and early embryogenesis. Horka(D) also renders chromosomes unstable during spermatogenesis, which leads to the formation of diplo//haplo mosaics, including the gynandromorphs. Complete loss of gene function brings about maternal-effect lethality: embryos of the females without the Horka(D)-identified gene perish due to disrupted centrosome function, defective spindle assembly, formation of chromatin bridges, and abnormal chromosome segregation during the cleavage divisions. These defects are indicators of mitotic catastrophe and suggest that the gene product acts during the meiotic and the cleavage divisions, an idea that is supported by the observation that germ-line chimeras exhibit excessive germ-line and cleavage function. The gene affected by the Horka(D) mutation is lodestar, a member of the helicase-related genes. The Horka(D) mutation results in replacement of Ala777 with Thr, which we suggest causes chromosome instability by increasing the affinity of Lodestar for chromatin.

In vivo analysis of MT-based vesicle transport by confocal reflection microscopy.

The use of confocal reflection microscopy (CRM) for the in vivo analysis of microtubule (MT) mediated transport of lipid droplets in the developing Drosophila egg primordia is described here. The developing Drosophila oocytes are ideal objects to study MT-mediated transport in vivo: transport of e.g. the lipid droplets can be conveniently, selectively and sensitively monitored through CRM and the egg primordia are readily available for physical, chemical and/or genetic manipulations. CRM is a non-destructive way to follow vesicle movement and allows high frame rate image recording. When combined with fluorescence imaging, CRM offers simultaneous visualization of the cargo and the protein(s) of interest, i.e. a motor or a cargo adapter, thus allowing a better understanding of MT-mediated transport and spatiotemporal coordination of the transport machinery.

Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena.

Autophagy ensures the turnover of cytoplasm and requires the coordinated action of Atg proteins, some of which also have moonlighting functions in higher eukaryotes. Here we show that the transmembrane protein Atg9 is required for female fertility, and its loss leads to defects in actin cytoskeleton organization in the ovary and enhances filopodia formation in neurons in Drosophila. Atg9 localizes to the plasma membrane anchor points of actin cables and is also important for the integrity of the cortical actin network. Of note, such phenotypes are not seen in other Atg mutants, suggesting that these are independent of autophagy defects. Mechanistically, we identify the known actin regulators profilin and Ena/VASP as novel binding partners of Atg9 based on microscopy, biochemical, and genetic interactions. Accordingly, the localization of both profilin and Ena depends on Atg9. Taken together, our data identify a new and unexpected role for Atg9 in actin cytoskeleton regulation.