Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in <em>Saccharomyces cerevisiae</em>

The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism

SARS-CoV-2 RNA levels in Scotland’s wastewater

Nationwide, wastewater-based monitoring was newly established in Scotland to track the levels of SARS-CoV-2 viral RNA shed into the sewage network, during the COVID-19 pandemic. We present a curated, reference dataset produced by this national programme, from May 2020 to February 2022. Viral levels were analysed by RT-qPCR assays of the N1 gene, on RNA extracted from wastewater sampled at 162 locations. Locations were sampled up to four times per week, typically once or twice per week, and in response to local needs. We report sampling site locations with geographical coordinates, the total population in the catchment for each site, and the information necessary for data normalisation, such as the incoming wastewater flow values and ammonia concentration, when these were available. The methodology for viral quantification and data analysis is briefly described, with links to detailed protocols online. These wastewater data are contributing to estimates of disease prevalence and the viral reproduction number (R) in Scotland and in the UK

Analysis of the genetic phylogeny of multifocal prostate cancer identifies multiple independent clonal expansions in neoplastic and morphologically normal prostate tissue.

Genome-wide DNA sequencing was used to decrypt the phylogeny of multiple samples from distinct areas of cancer and morphologically normal tissue taken from the prostates of three men. Mutations were present at high levels in morphologically normal tissue distant from the cancer, reflecting clonal expansions, and the underlying mutational processes at work in morphologically normal tissue were also at work in cancer. Our observations demonstrate the existence of ongoing abnormal mutational processes, consistent with field effects, underlying carcinogenesis. This mechanism gives rise to extensive branching evolution and cancer clone mixing, as exemplified by the coexistence of multiple cancer lineages harboring distinct ERG fusions within a single cancer nodule. Subsets of mutations were shared either by morphologically normal and malignant tissues or between different ERG lineages, indicating earlier or separate clonal cell expansions. Our observations inform on the origin of multifocal disease and have implications for prostate cancer therapy in individual cases

Growth with 17-Beta-estradiol results in enhanced mitogenic activity of medium from preadipocytes of massively obese persons

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Ipl1-dependent Sli15 phosphorylation is dispensable for chromosome bi-orientation.

<p>Wild type <i>SLI15</i> (VMY316), <i>sli15-20A</i> (VMY318) and <i>sli15-20D</i> (VMY320) cells containing <i>CEN5</i>-(<i>tetO)₃₃₆, tetR</i>-GFP, Venus-<i>TUB1</i> and p<i>MET3-CDC20</i> were arrested in G1 with α-factor at 26°C and then released to a metaphase block in rich medium (containing 2 mM methionine to deplete Cdc20) for 2.5 h. (<b>A</b>) Representative stills from time-lapse images of live cells. Bi-oriented chromosomes show dynamic splitting and reassociation of sister <i>CEN5s</i>. Green, <i>CEN5</i> labeled with <i>tetR</i>-GFP; red, Venus-tubulin. (<b>B</b>) Quantification of chromosome bi-orientation in metaphase-arrested cells from multiple time-lapse fields (n = number of cells scored in each category).</p

Characterization of the <i>sli15-20A</i> and <i>sli15-20D</i> alleles.

<p>(<b>A</b>) Schematic representation of Sli15 showing the 14 phosphorylation sites mapped <i>in vitro</i> (black) and the six additional sites discussed in the text (blue) that were mutated in <i>sli15-20A</i> and <i>sli15-20D</i>. The microtubule-binding region (residues 227–559) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089399#pone.0089399-Kang1" target="_blank">[23]</a> is shaded green and the conserved IN box (residues 626–698) is shaded blue. (<b>B</b>) Equivalent 10-fold dilutions of a wild-type strain (K699) and of <i>sli15Δ::KanMX6</i> strains with either wild-type <i>SLI15</i> (VMY30), <i>sli15-20A</i> (VMY148) or <i>sli15-20D</i> (VMY187) integrated at the <i>his3</i> locus were spotted onto YPAD agar in the presence of absence of benomyl at 11 µg/ml and grown for two days at 26°C or 37°C as indicated.</p

Yeast Strains.

a<p>All strains are in the W303 background: <i>ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2</i> Gal⁺.</p

Identification of Ipl1 phosphorylation sites on Sli15 following <i>in vitro</i> phosphorylation.

a<p>L, linear mode; R, reflector mode.</p>b<p>m/z values are average [M+H]⁺ for linear mode and monoisotopic [M+H]⁺ for reflector mode.</p>c<p><b>s</b>, phosphoserine; <b>t</b>, phosphothreonine; <b>m</b>, oxidized methionine; (K), (R), residue preceding trypsin/Lys-C cleavage site.</p

Mimicking constitutive Ipl1-dependent phosphorylation of Sli15 interferes with the checkpoint response to reduced cohesion.

<p>(A) Wild-type <i>SLI15</i> (VMY194), <i>sli15-20A</i> (VMY162), and <i>sli15-20D</i> (VMY191) strains expressing Pds1-<i>myc</i>₁₈ were arrested in G₁ with α-factor and synchronously released into YPD medium in the presence (+NOC) or absence (−NOC) of 30 µg/ml nocodazole. Samples were collected at the indicated times. Levels of Pds1-myc₁₈ (Pds1) and Cdc28 (loading control) were monitored by immunoblotting using anti-<i>myc</i> and anti-Cdc28 antibodies, respectively. (B) Wild-type p<i>GAL-SCC1 SLI15</i> (VMY222), p<i>GAL-SCC1 sli15-20A</i> (VMY166) and p<i>GAL-SCC1 sli15-20D</i> (VMY356) cells expressing Pds1-<i>myc</i>₁₈ were arrested with α-factor for 2 h in medium containing galactose and then released in medium containing glucose to repress p<i>GAL-SCC1</i>. Pds1 and Cdc28 were monitored as described for panel A.</p