Diverse model systems reveal common principles of meiosis

Abstract A meeting report on the 14th Gordon Research Conference on Meiosis, held at Colby Sawyer College, New London, NH, USA, 9–15 June 2018, chaired by Monica Colaiacovo, Harvard Medical School

Activation of the γ-Tubulin Complex by the Mto1/2 Complex

SummaryThe multisubunit γ-tubulin complex (γ-TuC) is critical for microtubule nucleation in eukaryotic cells [1, 2], but it remains unclear how the γ-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm [3, 4]. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the γ-TuC and recruits it to several different types of cytoplasmic MTOC sites [5–10]. Here, we show that the Mto1/2 complex activates γ-TuC-dependent microtubule nucleation independently of localizing the γ-TuC. This was achieved through the construction of a “minimal” version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto1, Mto2, and γ-TuC subunits Alp4 (GCP2) and Alp6 (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing ∼13 copies each of Mto1 and Mto2 per active complex and likely equimolar amounts of γ-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast γ-tubulin small complex and that multimerization of Mto2 in particular may underlie assembly of active microtubule nucleation complexes

A persistent neutrophil-associated immune signature characterizes post-COVID-19 pulmonary sequelae

Interstitial lung disease and associated fibrosis occur in a proportion of individuals who have recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through unknown mechanisms. We studied individuals with severe coronavirus disease 2019 (COVID-19) after recovery from acute illness. Individuals with evidence of interstitial lung changes at 3 to 6 months after recovery had an up-regulated neutrophil-associated immune signature including increased chemokines, proteases, and markers of neutrophil extracellular traps that were detectable in the blood. Similar pathways were enriched in the upper airway with a concomitant increase in antiviral type I interferon signaling. Interaction analysis of the peripheral phosphoproteome identified enriched kinases critical for neutrophil inflammatory pathways. Evaluation of these individuals at 12 months after recovery indicated that a subset of the individuals had not yet achieved full normalization of radiological and functional changes. These data provide insight into mechanisms driving development of pulmonary sequelae during and after COVID-19 and provide a rational basis for development of targeted approaches to prevent long-term complications

Deletion of Genes Encoding Arginase Improves Use of "Heavy" Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast

<div><p>The use of “heavy” isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry in the fission yeast <i>Schizosaccharomyces pombe</i> is hindered by the fact that under normal conditions, arginine is extensively catabolized <i>in vivo</i>, resulting in the appearance of “heavy”-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This “arginine conversion problem” significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when ¹³C₆-arginine (Arg-6) is used for labeling, it is less successful when ¹³C₆¹⁵N₄-arginine (Arg-10), a theoretically preferable label, is used. In particular, we find that with this method, “heavy”-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of ¹³C₅¹⁵N₂-arginine (Arg-7) in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes <i>car1+</i> and <i>aru1+</i> prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC.</p></div

Pom1 regulates the assembly of Cdr2-Mid1 cortical nodes for robust spatial control of cytokinesis

Proper division plane positioning is essential to achieve faithful DNA segregation and to control daughter cell size, positioning, or fate within tissues. In Schizosaccharomyces pombe, division plane positioning is controlled positively by export of the division plane positioning factor Mid1/anillin from the nucleus and negatively by the Pom1/DYRK (dual-specificity tyrosine-regulated kinase) gradients emanating from cell tips. Pom1 restricts to the cell middle cortical cytokinetic ring precursor nodes organized by the SAD-like kinase Cdr2 and Mid1/anillin through an unknown mechanism. In this study, we show that Pom1 modulates Cdr2 association with membranes by phosphorylation of a basic region cooperating with the lipid-binding KA-1 domain. Pom1 also inhibits Cdr2 interaction with Mid1, reducing its clustering ability, possibly by down-regulation of Cdr2 kinase activity. We propose that the dual regulation exerted by Pom1 on Cdr2 prevents Cdr2 assembly into stable nodes in the cell tip region where Pom1 concentration is high, which ensures proper positioning of cytokinetic ring precursors at the cell geometrical center and robust and accurate division plane positioning

“Heavy”-isotope label from ¹³C₆¹⁵N₄-arginine (Arg-10) is converted into other amino acids in fission yeast.

<p>Mass spectra of tryptic peptides (TIFFKDDGNYK and GIDFKEDGNILGHK in (A) and (B), respectively) from <i>S</i>. <i>pombe</i> Mto2[17A]-GFP fusion protein isolated from <i>car2∆ arg1-230 lys3-37</i> cells grown in either (i) unlabeled arginine (Arg-0) and unlabeled lysine (Lys-0), (ii) ¹³C₆-arginine (Arg-6) and ¹³C¹⁵N₂-lysine (Lys-8), or (iii) ¹³C₆¹⁵N₄-arginine (Arg-10) and ¹³C₆¹⁵N₂-lysine (Lys-8), as indicated. In peptides from cells grown in Arg-10, additional higher-molecular-mass peaks are observed (iii), indicating conversion of “heavy”-isotope label into other amino acids. Such peaks are not observed from cells grown in Arg-6. To simplify comparison, peptides shown here do not contain arginine residues, so the masses of monoisotopic peaks of peptides from cells grown in Arg-6 and Arg-10 are identical (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129548#pone.0129548.g002" target="_blank">Fig 2</a>). Mass-to-charge (m/z) ratios of monoisotopic peaks and inferred peptide charge-states are indicated in magenta.</p

Arg-10 conversion is prevented in arginase-deficient cells.

<p>Mass spectra of tryptic peptides (DAEGMSHIWQLR and AIQLELENLSSQAFR in (A) and (B), respectively) from <i>S</i>. <i>pombe</i> Mto1 protein (SPCC417.07c) isolated from (i, ii) <i>car2∆ arg1-230 lys3-37</i> or (iii) <i>car1∆ aru1∆ arg1-230 lys3-37</i> cells, grown in either (i) unlabeled arginine (Arg-0) and unlabeled lysine (Lys-0), or (ii, iii) ¹³C₆¹⁵N₄-arginine (Arg-10) and ¹³C₆¹⁵N₂-lysine (Lys-8), as indicated. In peptides isolated from <i>car2∆</i> cells grown in Arg-10, extensive conversion is observed, resulting in both higher-molecular-mass peaks and lower-molecular-mass “pre-peaks” (ii; see also Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129548#pone.0129548.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129548#pone.0129548.g002" target="_blank">2</a>). These are not observed in peptides from <i>car1∆ aru1∆</i> cells grown in Arg-10 (iii). Mass-to-charge (m/z) ratios of monoisotopic peaks for each growth condition (i.e., assuming no conversion of labeled arginine) and inferred peptide charge-states are indicated in magenta. Pre-peaks are indicated in blue.</p

Anabolic and catabolic reactions from ¹³C₆¹⁵N₄-arginine (Arg-10) leading to formation of “heavy” ammonium and “heavy” bicarbonate, and resynthesis of ¹³C₅¹⁵N₂-arginine (Arg-7).

<p>Two S. pombe arginases, Car1 and Aru1, catalyze the conversion of Arg-10 into ¹³C₅¹⁵N₂-ornithine (“heavy” ornithine; “heavy” atoms are shown in red). This also produces “heavy” urea, which can be hydrolyzed by urease Ure1 to produce “heavy” ammonia and “heavy” carbon dioxide (shown here as ammonium and bicarbonate ions, respectively), which can subsequently be incorporated into additional amino acids. Ornithine can be converted back into arginine through citrulline and arginino-succinate intermediates, ultimately leading to formation of Arg-7.</p