Selective autophagy of mitochondria on a ubiquitin-endoplasmic reticulum platform

Correction: Developmental Cell, Volume 55, Issue 2 https://doi.org/10.1016/j.devcel.2020.10.002The dynamics and co-ordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes, or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required earliest followed by autophosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps whereas ULK1/2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way suggesting multiple initiation events. Targeted ubiquitinated mitochondrial are cradled by endoplasmic reticulum strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands providing platforms for formation of the mitophagosomes.Peer reviewe

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.Fil: Renna, Felipe Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Herrera Lopez, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Manifava, Maria. The Babraham Institute; Reino UnidoFil: Ktistakis, Nicholas. The Babraham Institute; Reino UnidoFil: Vaccaro, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

Interaction of phospholipase D1 with a casein-kinase-2-like serine kinase

Phospholipase D (PLD)1 was phosphorylated in vivo and by an associated kinase in vitro following immunoprecipitation. Both phosphorylation events were greatly reduced in a catalytically inactive point mutant in which the serine residue at position 911 was converted into alanine (S911A). The kinase could be enriched from detergent-extracted brain membranes and bind and phosphorylate PLD1 that was immunoprecipitated from COS-7 cells. Using in-gel kinase assays we determined that the size of the kinase is approximately 40 kDa and that PLD1 is more effective than S911A in binding the kinase. Preliminary analysis of the phosphorylation sites on PLD1 suggested that the kinase belongs to the casein kinase 2 (CK2) family. Consistent with this, we found that the kinase could utilize GTP, and could be inhibited by heparin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Membrane fractions from Chinese hamster ovary (CHO) cell lines that inducibly express PLD1 contained an endogenous kinase activity that phosphorylated PLD1 using GTP and was inhibited by DRB. Direct evidence that the kinase is CK2 came from observations that immunoprecipitates using PLD1 antibodies contained immunoreactive CK2alpha, and immunoprecipitates using CK2alpha antibodies contained immunoreactive PLD1. Co-expression of PLD1 in COS-7 cells with the two recombinant CK2 subunits, alpha or beta, suggests that the association of PLD1 with the kinase is through the beta subunit. Supporting this, phosphorylation of PLD1 by purified recombinant CK2alpha was enhanced by purified recombinant CK2beta. Assays measuring PLD1 catalytic activity following phosphorylation by CK2 suggest that this phosphorylation event does not influence PLD1-mediated hydrolysis of phosphatidylcholine in vitro

Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity

The Mycobacterium ulcerans exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of Sqstm1 reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in eif2ak3−/-/perk−/- cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease

Gender impact on children's knowledge and perceptions regarding cardiovascular disease risk factors: A school-based survey in Greece

BACKGROUND: Cardiovascular disease (CVD) risk factors are adopted during childhood and adolescence. Health literacy at these ages remains the cornerstone of a healthy adult life. The aim of the study was to examine the role of gender regarding CVD risk factors' awareness and to develop an evaluation tool for the assessment of CVD risk factors' knowledge and perception among children. METHODS: During the school years 2014–2015 and 2015–2016, 1728 students aged 10–12 years (5th and 6th grade), from 5 Greek cities (including Athens metropolitan area), were enrolled; nearly 45% were boys (participation rate varied from 95% to 100% from school to school). Students and their parents completed an anonymous questionnaire; students' somatometric characteristics were also recorded. Schools were randomly selected. Linear regression models were applied to evaluate the impact of children's gender on knowledge and perceptions about CVD risk factors. RESULTS: Significant higher percentage of correct answers, among girls compared to boys, was revealed regarding the weekly consumption of legumes, the breakfast weekly consumption, and the effects of soft drinks on health (all P < 0.05). As far as CVD risk factors' knowledge, significantly higher percentage of girls than boys also answered that high blood pressure and television viewing are bad for health and particularly for heart-related problems (all P < 0.05). Girls had a significantly higher mean score of 0.304 than boys, after adjusting for several confounders (P = 0.029). CONCLUSION: Health education programs should take into account gender differences in children's perception and attitudes toward CVD risk factors, in order to increase awareness of children and eventually reduce CVD risk during adulthood