Caractérisation et méthodes d études de la mort cellulaire par cytométrie en flux

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Caractérisation et méthodes d études de la mort cellulaire par cytométrie en flux Université de Bourgogne Gérard LIZARD - Inserm EA7270 - Equipe Biochimie du Peroxysome, inflammation et Métabolisme Lipidique Faculté des Sciences Gabriel 6, Bd Gabriel, 21000 Dijon - FRANCE Rabat, 29 novembre 2018 CytoMagh 2018 Gérard Lizard

Two pathways of cell death leading to necrosis and apoptosis. At the top is shown a normal cell. 1A: Swelling. 1B: Vacuolization, blebbing, and increased permeability. 1C: Necrotic changes. ie, coagulation, shrinkage, and karyolysis. 2A: Shrinkage and pyknosis. 2B: Budding and karyorhexis. 2C: Necrotic changes, ie, breakup into a cluster of apoptotic bodies. Majno G & Joris I Am J Pathol 1995, 146: 3-15.

ORGANELLES INVOLVED IN CELL DEATH Organelles Apoptosis Autophagy Necrosis / Necroptosis - Mitochondria + + (mitophagy) + - Lysosomes + / - + + / - - Endoplasmicreticulum (ER) + / - + + / - - peroxisome? + (pexophagy)? Bröker LE et al. Clin Cancer Res 2005, 11: 3155-3162.

Relations peroxysome / mitochondrie Implication dans le contrôle de l équilibre RedOx et l activation de la mort cellulaire Lismont C, Nordgren M, Van Veldhoven PP, Fransen M. Front Cell Dev Biol. 2015, 27;3:35. 4

Cell Death (initial concept) Apoptosis apoptotic morphology Necrosis necrotic morphology Active programmed cell death Caspase-dependent cell death Mitochondrial pathway (Associated or not with reticulum stress) Death receptor pathway Passive unprogrammed cell death Classical /Canonical Necrosis Cell Death Independent of Caspases Kitanaka C & Kuchino Y Cell Death Differ 1999, 6: 508-515.

Cell death Apoptosis Autophagy Necrosis Active programm cell death Caspase-independent Type-2 cell death Active programm cell death Caspase-dependent Type-1 cell death Active programm cell death Caspase-independent cell death Necroptosis (RIP1, RIP3) Secondary necrosis Passive programm cell death Caspase-independent cell death Classical necrosis (primary necrosis) Type-3 cell death

AVAILABLE METHODS ALLOWING THE CHARACTERIZATION OF APOPTOSIS, NECROSIS / NECROPOTOSIS, AND AUTOPHAGY

FUNCTIONNAL CRITERIA ASSOCIATED WITH APOPTOSIS, NECROSIS, AND NECROPTOSIS Microscopy, flow cytometry, biochemistry - enhanced permeability of cytoplasmic membrane (trypan blue, fluorescent probes, LDH) - externalization of phosphatidylserine double staining with AnnexinV /propidium iodide (PI) (or aminoactinomycine D (AAD)) to distinguish between normal (AnnexinV - /PI - ), necrotic (AnnexinV + /PI + ), and apoptotic (AnnexinV + /PI - ) cells - Coloration SYTO16 - IP - Sub-G1 peak (not present in normal and necrotic cells) - loss of transmembrane mitochondrial potential: numerous fluorescent probes available. Currently, DioC 6 (3) and JC1 are the most reliable. - FLICA (fluorochromes labeled inhibitors of caspases): in situ identification of activated caspases (does not permit to distinguish between necrosis and caspase-independent cell death) - TUNEL (Terminal deoxynucleotidyl transferase dutp nick end labeling): in situ identification of internucleosomal DNA fragmentation

APOPTOSIS / NECROSIS: MORPHOLOGICAL CRITERIA Electron microscopy A: Control. B: Cells treated by VP-16; apoptotic cells with condensed and perinuclear chromatin are observed, cytoplasmic and nuclear membrane integrity are preserved as well as morphology of mitochondria (arrow). C: Cells treated by NaN3; necrotic cells are characterized by a loss of integrity of cytoplasmic and nuclear membranes, degradation of cytoplasm and chromatin. Fluorescence microscopy after staining with Hoechst 33342 D: Control cells. Nuclei show regular contours. E: Cells treated by VP-16; cell with fragmented (f) nucleus. F: Cells treated by VP-16; cell with condensed (c) nucleus. G: Necrotic (n) cells observed under treatment by NaN3; the nucleus is diffuse and irregular. (Lizard G et al. Cytometry 1995, 21: 275-283)

MORPHOLOGICAL CRITERIA ASSOCIATED WITH APOPTOSIS, NECROSIS AND NECROPTOSIS * Phase contrast microscopy: loss of refringence; loss or not of cell adhesion? * Brightfield microscopy: after staining with GIEMSA, * Fluorescence microscopy: Hoechst staining allows to easily distinguish between normal, necrotic and apoptotic cells (nuclear criteria) U937 G Control 7-keto 40 µg/ml * Transmission electron microscopy (nuclear criteria) * Flow cytometry: changes of light scatter properties on non-fixed cells (FSC ; SSC more or less )

Annexin V-FITC / PI (Amnis technology) http://www.amnis.com Henery S et al. Apoptosis 2008, 13: 1054-1063

PI (or 7AAD) Phosphatidylserine externalization : Annexin V-FITC / PI test Apoptosis Necrosis (phosphatidylserines) Cells Viable (V) Apoptotic (A) Necrotic (N) Annexine V PI Control N Treated V A PI: propidium iodide 7 AAD: 7 amino actinomycin D Annexin V - FITC

Towards an Understanding of Apoptosis Detection by SYTO Dyes Donald Wlodkowic,1* Joanna Skommer,1 and Jukka Pelkonen1,2 1Institute of Clinical Sciences, Department of Clinical Microbiology, University of Kuopio, Kuopio, Finland 2Department of Clinical Microbiology, Kuopio University Hospital, Kuopio, Finland Cytometry Part A 71A:61 72 (2007) Necrotic cells Viable cells Apoptotic cells

Number of cells Sub-G1 (FCM) HUVECs primary or secondary necrosis Sub-G1 apoptosis control cholesterol 7-ketocholesterol 7ß-hydroxycholesterol Lizard G et al. Am J Pathol 1996, 148: 1625-1638

% cells with depolarized mitochondria Mitochondrial potential (FCM) Control 7-ketocholesterol 7 -hydroxycholesterol 100 80 control 7-ketocholesterol (40 µg/ml) 7b-hydroxycholesterol (20 µg/ml) 60 40 20 0 0 6 12 18 24 Time of treatment (hours)

FLICA / FLISP (Microscopy, Flow Cytometry) Grabarek J & Darzynkiewicz Z Exp Hematol 2002, 30: 982-989

TUNEL (Flow Cytometry)

TUNEL (Amnis technology)

Phase contrast microscopy, Hoechst 33342, GIEMSA, TUNEL Apoptosis = TUNEL positive cells Necrosis = TUNEL negative cells Lizard G et al., Arterioscler Thromb Vasc Biol 1999, 19: 1190-2000

BIOCHEMICAL CRITERIA ASSOCIATED WITH APOPTOSIS, NECROSIS, AND NECROPTOSIS * Analysis of the DNA fragmentation pattern on agarose gel - Apoptosis: DNA ladder, multiple of 150-200 base pairs (internucleosomal DNA fragmentation) - Necrosis: no DNA ladder, smears * Enzymatic activities (LDH) * Electrophoresis on acrylamide gel and Western Blotting

Stimuli apoptotique : génération de céramides (détection par immunofluorescence) Expression de céramides Anticorps anti-céramides Cellule normale Hoechst Cellule pré-apoptotique Cellule apoptotique Huang WC, Chen CL, Lin YS, Lin CF. Apoptotic sphingolipid ceramide in cancer therapy.

MCF-7 MCF-7/c3 Caspase-3/7 Caspase-8 Caspase-6 DYm without caspase-3 Oncosis t-bid Bid cytochrome-c Caspase-9 Caspase-3 AIF, EndoG PARP ICAD CAD Necrosis/Oncosis = no caspase activation Caspase-8 Caspase-7 Apoptosis Prunet C et al. Cell Death Differ, 2001; J Biochem Mol Toxicol, 2005

CHARACTERIZATION OF AUTOPHAGY: VISUAL CRITERIA

CHARACTERIZATION OF AUTOPHAGY: ULTRASTRUCTURAL CRITERIA Transmission Electron Microscopy 7KC (50 µm, 24 h) Control A C F 1 µm 1 µm 1 µm EtOH (0.1%) B D G apl ap 1 µm 0.1 µm E H apl 1 µm ap 0.5 µm 0.1 µm ap: autophagosome; apl: autophagolysosome Gérard Lizard

Control EtOH (0.1%) EtOH (1%) EtOH (1.1%) α-cyclodextrin (125 µm) α-cyclodextrin + EtOH (0.1 %) α-cyclodextrin + EtOH (1.1 %) α-tocopherol (400 µm) DHA (50 µm) 7KC (50 µm) 7KC + α-toco 7KC + DHA 7KC +α-toco+ DHA 7β-OHC (50 µm) 7β-OHC +α-toco 7β-OHC +DHA 7β-OHC + α-toco+dha 24S-OHC (50µM) 24S-OHC +α-toco 24S-OHC +DHA 24S-OHC+ α-toco +DHA CHARACTERIZATION OF AUTOPHAGY: BIOCHEMICAL CRITERIA Murine oligodendrocytes (158N) - autophagy and apoptosis (oxiapoptophagy) Caspase -3 (35 kda) Cleaved Caspase -3 (17 kda) PARP (116 kda) Cleaved PARP (89 kda) Bcl-2 (26 kda) LC3-I (18 kda) LC3-II (16 kda) Ratio LC3-II/LC3-I 0.6 0.6 0.6 0.6 0.7 0.5 0.4 0.6 0.5 3.8 0.8 0.6 0.7 1.7 1.3 1.2 1.3 3.4 1.7 1.2 0.9 β-actin (42 kda)

Apoptosis Necrosis Necroptosis Autophagy Oxiapoptophagy Necroapoptophagy Phosphatidyl serine externalization (Annexin V positive + PI negative) Mitochondrial depolarization Non Random DNA degradation (DNA ladder) Phosphatidyl serine externalization (Annexin V positive + PI positive) Mitochondrial depolarization Random DNA degradation Caspases activation LC3 conversion: LC3I to LC3-II RIP1 (and RIP3) expression

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