Impact of the interplay between stemness features, p53 and pol iota on replication pathway choices

Using human embryonic, adult and cancer stem cells/stem cell-like cells (SCs), we demonstrate that DNA replication speed differs in SCs and their differentiated counterparts. While SCs decelerate DNA replication, differentiated cells synthesize DNA faster and accumulate DNA damage. Notably, both replication phenotypes depend on p53 and polymerase iota (POL$_{ι}$). By exploring protein interactions and newly synthesized DNA, we show that SCs promote complex formation of p53 and POL$_{ι}$ at replication sites. Intriguingly, in SCs the translocase ZRANB3 is recruited to POL$_{ι}$ and required for slow-down of DNA replication. The known role of ZRANB3 in fork reversal suggests that the p53–POL$_{ι}$ complex mediates slow but safe bypass of replication barriers in SCs. In differentiated cells, POL$_{ι}$ localizes more transiently to sites of DNA synthesis and no longer interacts with p53 facilitating fast POL$_{ι}$-dependent DNA replication. In this alternative scenario, POL$_{ι}$ associates with the p53 target p21, which antagonizes PCNA poly-ubiquitination and, thereby potentially disfavors the recruitment of translocases. Altogether, we provide evidence for diametrically opposed DNA replication phenotypes in SCs and their differentiated counterparts putting DNA replication-based strategies in the spotlight for the creation of therapeutic opportunities targeting SCs

dOCRL maintains immune cell quiescence in Drosophila by regulating endosomal traffic

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome

Metatalk in American academic talk: The cases of "point" and "thing"

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/88138/1/swales-metatalk_american_academic.pd

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Multiple biochemical properties of the p53 molecule contribute to activation of polymerase iota-dependent DNA damage tolerance

Abstract We have previously reported that p53 decelerates nascent DNA elongation in complex with the translesion synthesis (TLS) polymerase ι (POLι) which triggers a homology-directed DNA damage tolerance (DDT) pathway to bypass obstacles during DNA replication. Here, we demonstrate that this DDT pathway relies on multiple p53 activities, which can be disrupted by TP53 mutations including those frequently found in cancer tissues. We show that the p53-mediated DDT pathway depends on its oligomerization domain (OD), while its regulatory C-terminus is not involved. Mutation of residues S315 and D48/D49, which abrogate p53 interactions with the DNA repair and replication proteins topoisomerase I and RPA, respectively, and residues L22/W23, which disrupt formation of p53-POLι complexes, all prevent this DDT pathway. Our results demonstrate that the p53-mediated DDT requires the formation of a DNA binding-proficient p53 tetramer, recruitment of such tetramer to RPA-coated forks and p53 complex formation with POLι. Importantly, our mutational analysis demonstrates that transcriptional transactivation is dispensable for the POLι-mediated DDT pathway, which we show protects against DNA replication damage from endogenous and exogenous sources

OCRL regulates PIP₂ homeostasis in diverse endosomal compartments.

<p>(A) Endogenously tagged dOCRL (magenta) co-localizes strongly with Clc-GFP (green), single channel confocal slices shown below in gray. (B) Pearson correlations between endogenously tagged dOCRL and trafficking compartments in live primary hemocytes. dOCRL localizes only moderately with other endosomal compartments. (C-D) <i>docrl</i>^Δ3 hemocytes exhibit increased PH-PLC-Cherry marker of PIP₂ (magenta) in live primary hemocytes in all membrane compartments examined (green). (E) The phosphatase activity of dOCRL is necessary but not sufficient to restrict hemocyte number. Scale bars in A,C are 10 μm. Data are presented as mean +/- SEM. Individual values in B,D represent single cells. <b>Associated with</b> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007052#pgen.1007052.s004" target="_blank">S4 Fig</a>.</p

Aberrant endosomal compartment function in <i>docrl</i> mutants.

<p>(A) <i>docrl</i> mutant hemocytes exhibit defective scavenger receptor mediated endocytosis of mBSA (magenta). (B) <i>docrl</i> mutant hemocytes are competent for phagocytosis. Images show 2D maximum intensity projection of confocal stacks, showing myr-mRFP-expressing hemocytes incubated with Alexa-488 labeled <i>E</i>. <i>coli</i>. Lower panels show ZX projections taken at the plane marked by arrows in the respective projections. Z sections show internalized <i>E</i>. <i>coli</i>. (Middle) Quantification of mean number of <i>E</i>. <i>coli</i> particles. (Left) Fraction of particles completely engulfed by hemocytes. Data are presented as mean +/- SEM. (C) The structure and abundance of Lysotracker-positive (acidified) compartments is altered in <i>docrl</i> mutants, and rescued cell-autonomously by re-expression of <i>docrl</i>. Image shows 2D maximum intensity projection of confocal stacks. (D) <i>docrl</i> mutants exhibit defective accumulation of autophagic compartments, marked by GFP-mCherry-Atg8a (GC::Atg8A). Quantification shows Pearson’s Correlation Coefficient per cell +/- s.e.m. for GFP and mCherry (left) and mean fluorescence +/- s.e.m. for each fluorophore/cell (right). All panels are shown with the same brightness and contrast. Individual values in graphs represent single cells. Scale bars are 10 μm. <b>Associated with</b> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007052#pgen.1007052.s006" target="_blank">S6 Fig</a>.</p

Aberrant endosomal compartment structure in <i>docrl</i> mutants.

<p>(A-C) Loss of <i>docrl</i> disrupts the structure of multiple endosomal compartments, including fragmentation and perinuclear accumulation of Clc (A) and Rab5 (B), and expansion of Rab7 (C). Images show single confocal slices at the plasma membrane and an internal Z plane. Scale bar is 10 μm. (D) Quantification of endosomal compartment levels in <i>docrl</i>^Δ3 mutants. Rab7 levels are increased ~3 fold, while Clc and Rab11 levels are slightly increased. N in panel D represent individual cells equally pooled from three independent sets of larvae. (E) Hemocytes from injured wild-type larvae exhibit defective Rab7-positive endosome structure. Scale bar is 5 μm. All panels are representative single confocal slices from live primary hemocytes. <b>Associated with</b> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007052#pgen.1007052.s005" target="_blank">S5 Fig</a>.</p

Loss of <i>docrl</i> causes increased actin assembly in hemocytes.

<p>(A) <i>docrl</i>^Δ3 hemocytes exhibit greater F-actin assembly (phalloidin, green) and a spikier morphology than control cells. (B-C) Quantification of F-actin intensity (B) and ‘spikiness’ (C). Both phenotypes are rescued by hemocyte-autonomous re-expression of dOCRL-GFP. (D) Infection of larvae with <i>M. luteus</i> promotes assembly of F-actin in hemocytes. (E) Sparse re-expression in hemocytes with HH-LT-GAL4 reduces actin assembly in GFP+ rescue hemocytes, but not GFP- hemocytes that do not express the rescue construct. (F) Expression of phosphatase alone moderately rescues actin accumulation (phalloidin staining, gray), though actin accumulation at ruffles (arrowhead) and intracellular puncta (arrows) are still observed. Data are presented as mean +/- SEM. Sample N in panels B-F represent individual cells pooled equally from three independent samples of 2–4 larvae. Scale bars are 10 μm.</p