The organization and number of microtubules (MTs) in a cell depend on the proper regulation of MT nucleation. Currently, the mechanism of nucleation is the most poorly understood aspect of MT ...dynamics. XMAP215/chTOG/Alp14/Stu2 proteins are MT polymerases that stimulate MT polymerization at MT plus ends by binding and releasing tubulin dimers. Although these proteins also localize to MT organizing centers and have nucleating activity in vitro, it is not yet clear whether these proteins participate in MT nucleation in vivo. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, the XMAP215 ortholog Alp14 is critical for efficient MT nucleation in vivo. In multiple assays, loss of Alp14 function led to reduced nucleation rate and numbers of interphase MT bundles. Conversely, activation of Alp14 led to increased nucleation frequency. Alp14 associated with Mto1 and γ-tubulin complex components, and artificially targeting Alp14 to the γ-tubulin ring complexes (γ-TuRCs) stimulated nucleation. In imaging individual nucleation events, we found that Alp14 transiently associated with a γ-tubulin particle shortly before the appearance of a new MT. The transforming acidic coiled-coil (TACC) ortholog Alp7 mediated the localization of Alp14 at nucleation sites but not plus ends, and was required for efficient nucleation but not for MT polymerization. Our findings provide the strongest evidence to date that Alp14 serves as a critical MT nucleation factor in vivo. We suggest a model in which Alp14 associates with the γ-tubulin complex in an Alp7-dependent manner to facilitate the assembly or stabilization of the nascent MT.
•Alp14 (XMAP215) and Alp7 (TACC) promote microtubule nucleation in vivo•Alp14 interaction with Mto1 and the γ-tubulin complex is dependent on Alp7•Increasing the affinity of Alp14 for Mto1 increases nucleation frequency•Timed appearance of Alp14 at the nucleation site suggests a nucleation timeline
Microtubules are dynamic polymers that help to organize cellular contents and divide the cell. New microtubules arise by a process of nucleation, in which tubulin subunits are stitched together to begin forming a hollow tube. Flor-Parra et al. identify XMAP215/Alp14 as a nucleation factor that facilitates the assembly of the microtubule.
Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve ...transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress.
We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20
during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20
N-terminus through a canonical MAPK docking site. Most important, the Cdc20
pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit.
Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20
turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.
The aim of this study was to evaluate the robustness of infra-red thermometry to estimate stomatal conductance (gs) in grapefruit trees. For this purpose, the ability of several thermal indices to ...determine gs was tested for different: (i) irrigation strategies: full irrigation (FI) and regulated deficit irrigation (RDI), (ii) water sources: transfer water (TW) -optimal for agricultural uses- and saline reclaimed water (RW) and (iii) phenological stages: flowering-sprouting (FS) and fruit growth (FG). During two growing seasons, measurements of gs and canopy temperature (Tc) were taken in 14-year-old ´Star Ruby´ grapefruit trees (Citrus paradisi Macf.,). Air temperature (Ta) was also recorded and then the derived thermal indices: Tc-Ta, and CWSI (crop water stress index) were calculated. Subsequently, thermal-gs correlations were established at different averaged thermal data intervals, and VPD (vapour pressure deficit) and PAR (photosynthetically active radiation) conditions. The results indicated that both, water quality and deficit irrigation, affected negatively gs and Tc during FG. In addition, the highest r2 values in thermal indices-gs correlations were found when i) the thermal data was averaged 60 min before the gs measurement, ii) gs was measured over a wide range of VPD (between 0 and 3.5 kPa), and iii) PAR was 1200 µmol m−2 s−1. The ability of the thermal indices to estimate gs improved under RDI and RW conditions, when compared to the control treatment (TW-FI). Moreover, the thermal indices that more accurately estimated gs were Tc-Ta and CWSI in FS (P < 0.001), and the absolute values of Tc in FG (P < 0.001). Overall, thermal indices obtained by infra-red sensors could be a useful tool to estimate gs in grapefruit trees. However, irrigation treatment, water quality, tree phenological stage and environmental conditions (VPD and PAR) must be considered when interpreting thermal indices-gs correlations.
•Infra-red sensors allowed for estimating the stomatal conductance of grapefruit trees.•Environmental conditions affected the estimation of stomatal conductance.•Crop phenological stage affected thermal indices-stomatal conductance correlations.
Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid ...chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5′-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.
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•An unbiased LC-MS screen reveals FAMIN as a purine nucleoside enzyme•FAMIN combines adenosine phosphorylase with ADA-, PNP-, and MTAP-like activities•FAMIN enables a purine nucleotide cycle (PNC) preventing cytoplasmic acidification•The FAMIN-dependent PNC balances the glycolysis-mitochondrial redox interface
Disease-linked, orphan FAMIN is an evolutionarily conserved, multifunctional purine nucleoside enzyme, with not only ADA-, PNP-, and MTAP-like activities but also adenosine phosphorylase activity. FAMIN enables a purine nucleotide cycle that balances the cytoplasmic-mitochondrial redox interface and prevents cytoplasmic acidification.
In metazoans, the nuclear envelope (NE) breakdown (NEBD) occurs during “open” mitosis and meiosis. In the fission yeast Schizosaccharomyces pombe, the mitosis and the first meiotic division (MI) are ...“closed,” during which the NE is maintained. Intriguingly, during the second meiotic division (MII), the NE is also maintained, but nuclear and cytoplasmic molecules are mixed similarly to open mitosis, a phenomenon of unknown biological significance called “virtual” NEBD (vNEBD). Here, we show that importin-α-dependent nucleocytoplasmic transport regulates spindle disassembly late in anaphase B at MI, as previously reported for mitosis. At MII, however, spindle dissolution is triggered by vNEBD early in anaphase B, a mechanism that short-circuits the nucleocytoplasmic transport system. We demonstrate that the sequential action of these two spindle disassembly systems regulates the spatiotemporal order and ploidy of the meiotic products.
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•Imp1-dependent transport drives spindle dissolution in S. pombe meiosis I•vNEBD short-circuits the Imp1-dependent disassembly, shortening maximum spindle length•Imp1-dependent and vNEBD spindle disassembly systems are distinct but exchangeable•Sequential Imp1- and vNEBD-driven spindle disassembly controls spore order and ploidy
vNEBD is a phenomenon described in the fission yeast second meiotic division (MII), which resembles the nuclear envelope breakdown that characterizes the mitotic phase in eukaryotes. Flor-Parra et al. describe a biological role of vNEBD in spindle disassembly.
Still’s disease, the paradigm of autoinflammation-cum-autoimmunity, predisposes for a cytokine storm with excessive T lymphocyte activation upon viral infection. Loss of function of the purine ...nucleoside enzyme FAMIN is the sole known cause for monogenic Still’s disease. Here we discovered that a FAMIN-enabled purine metabolon in dendritic cells (DCs) restrains CD4+ and CD8+ T cell priming. DCs with absent FAMIN activity prime for enhanced antigen-specific cytotoxicity, IFNγ secretion, and T cell expansion, resulting in excessive influenza A virus-specific responses. Enhanced priming is already manifest with hypomorphic FAMIN-I254V, for which ∼6% of mankind is homozygous. FAMIN controls membrane trafficking and restrains antigen presentation in an NADH/NAD+-dependent manner by balancing flux through adenine-guanine nucleotide interconversion cycles. FAMIN additionally converts hypoxanthine into inosine, which DCs release to dampen T cell activation. Compromised FAMIN consequently enhances immunosurveillance of syngeneic tumors. FAMIN is a biochemical checkpoint that protects against excessive antiviral T cell responses, autoimmunity, and autoinflammation.
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•A FAMIN-enabled purine metabolon in dendritic cells restrains T cell priming•FAMIN prevents cytoplasmic NADH/NAD+ reductive stress that enhances priming•Inosine generated from hypoxanthine by FAMIN inhibits T cell priming•FAMIN ameliorates immunopathology in influenza but dampens tumor surveillance
Saveljeva et al. identify a biochemical mechanism in dendritic cells that restrains T cell priming and prevents immunopathology but dampens tumor surveillance. FAMIN enables a purine nucleotide cycle, which prevents cytoplasmic NADH/NAD+ reductive stress that augments antigen presentation, and it generates inosine, which inhibits T cell activation.
Awake prone positioning (awake-PP) in non-intubated coronavirus disease 2019 (COVID-19) patients could avoid endotracheal intubation, reduce the use of critical care resources, and improve survival. ...We aimed to examine whether the combination of high-flow nasal oxygen therapy (HFNO) with awake-PP prevents the need for intubation when compared to HFNO alone.
Prospective, multicenter, adjusted observational cohort study in consecutive COVID-19 patients with acute respiratory failure (ARF) receiving respiratory support with HFNO from 12 March to 9 June 2020. Patients were classified as HFNO with or without awake-PP. Logistic models were fitted to predict treatment at baseline using the following variables: age, sex, obesity, non-respiratory Sequential Organ Failure Assessment score, APACHE-II, C-reactive protein, days from symptoms onset to HFNO initiation, respiratory rate, and peripheral oxyhemoglobin saturation. We compared data on demographics, vital signs, laboratory markers, need for invasive mechanical ventilation, days to intubation, ICU length of stay, and ICU mortality between HFNO patients with and without awake-PP.
A total of 1076 patients with COVID-19 ARF were admitted, of which 199 patients received HFNO and were analyzed. Fifty-five (27.6%) were pronated during HFNO; 60 (41%) and 22 (40%) patients from the HFNO and HFNO + awake-PP groups were intubated. The use of awake-PP as an adjunctive therapy to HFNO did not reduce the risk of intubation RR 0.87 (95% CI 0.53-1.43), p = 0.60. Patients treated with HFNO + awake-PP showed a trend for delay in intubation compared to HFNO alone median 1 (interquartile range, IQR 1.0-2.5) vs 2 IQR 1.0-3.0 days (p = 0.055), but awake-PP did not affect 28-day mortality RR 1.04 (95% CI 0.40-2.72), p = 0.92.
In patients with COVID-19 ARF treated with HFNO, the use of awake-PP did not reduce the need for intubation or affect mortality.
Critically ill patients with coronavirus disease 19 (COVID-19) have a high fatality rate likely due to a dysregulated immune response. Corticosteroids could attenuate this inappropriate response, ...although there are still some concerns regarding its use, timing, and dose.
This is a nationwide, prospective, multicenter, observational, cohort study in critically ill adult patients with COVID-19 admitted into Intensive Care Units (ICU) in Spain from 12th March to 29th June 2020. Using a multivariable Cox model with inverse probability weighting, we compared relevant outcomes between patients treated with early corticosteroids (before or within the first 48 h of ICU admission) with those who did not receive early corticosteroids (delayed group) or any corticosteroids at all (never group). Primary endpoint was ICU mortality. Secondary endpoints included 7-day mortality, ventilator-free days, and complications.
A total of 691 patients out of 882 (78.3%) received corticosteroid during their hospital stay. Patients treated with early-corticosteroids (n = 485) had lower ICU mortality (30.3% vs. never 36.6% and delayed 44.2%) and lower 7-day mortality (7.2% vs. never 15.2%) compared to non-early treated patients. They also had higher number of ventilator-free days, less length of ICU stay, and less secondary infections than delayed treated patients. There were no differences in medical complications between groups. Of note, early use of moderate-to-high doses was associated with better outcomes than low dose regimens.
Early use of corticosteroids in critically ill patients with COVID-19 is associated with lower mortality than no or delayed use, and fewer complications than delayed use.
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