MUNI SCI Cell pathology RNDr. Jan Škoda, Ph.D. Department of Experimental Biology Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Outline - Cellular responses to stress conditions Physical stress (temperature, radiation) - Chemical stress (xenobiotics) - Biological stress (intracellular parasites) -Cell death - Necrosis - Necroptosis Apoptosis - Autophagic cell death - Ferroptosis 2 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cellular responses to stress conditions 3 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Stress factors Non-specific stress factors Significant increase of temperature, heavy metals, aldehydes -> denaturation of proteins Specific (targeted) stress conditions - Radiation of a specific wavelength -> absorption by target molecules or specific response at the cellular level -Specific inhibitors/activators (antibiotics, toxins etc.) -> target specific process in the cell 4 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cellular stress response - Programmed adaptive processes of the cell -Aimed to protect against the damaging consequences of the stress factors and restore homeostasis -Various stress-signaling pathways -> changes in the gene expression -> upregulation of stress (heat shock) proteins and other genes required to restore the balance - Prolonged activation of stress-signaling induces expression of proteins involved in activation of cell death 5 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Physical stress 6 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Heat stress - Irreversible damage of cells under heat shock - 40-50°C - Changes in the tertiary structure of proteins -> defects in their function - Disrupted coordination of metabolic pathways (optimal temperature for enzymes) - Disorganization of biomemebranes Disrupted cytoskeleton (F-actin stabilization and depolymerization of microtubules) - Heat shock proteins - Upregulated in response to various stressors (not only increased temperature) - Evolutionarily conserved molecular chaperones ■ Refolding of misfolded proteins: restore correct conformation or target proteins for degradation (cochaperones with ubiquitin ligase activity) 7 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Hyperthermophilic bacteria -Tolerate extremely high temperatures -Optimal temperature ~90°C -Common in domain Archea -Thermostable enzymes (proteins) - Isolation of commercially useful enzymes (e.g., polymerases): fast reactions, hot start reactions etc. Bacteria Mostly mesophiles with some thermophiles Temperature I'CI Archaea Many thermophiles Eukaryotes No thermophiles LUCA Environmental temperature unknown 8 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Hot springs (e.g., Grand Prismatic Spring) Deep-sea hydrothermal vents 9 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cold stress - Irreversible damage in temperatures around 0°C - Mechanical damage of biomembranes/organelles caused by ice crystals, desiccation of the cell, vitrification (glass-like form; prevents biologically relevant movement of molecules) - Animal cells are highly prone to this type of stress - Mechanisms of cell resistance - Reduced water content: seeds, spores - Different composition of lipids in membranes (lower melting point), specific antifreezing proteins (prevent ice nucleation and growth), cold-active enzymes: psychrofilic bacteria, algae, fungi, insects 10 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) fl 11 Bil 700en Cell Biology /10 - Cell pathology (11 May 2022) Electromagnetic radiation: Visible light - Weakly absorbed by the cytoplasm -Generally effective only when strong intensities / laser beams applied - Presence of pigments increases the effects Photosensitizers - Molecules increasing sensitivity of cells to light -Absorb light -> activation of photosensitizer -> oxidative stress -Eosin, fluorescein, acridine, chlorophyll, porfyrins... 12 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) fl Electromagnetic radiation: Ultraviolet light - Effects correlate with the energy (shorter wavelength): UVC (200-280 nm) > UVB (280-315 nm) > UVA (315-400 nm) Direct effect: UVC induces cyclobutan pyrimidine dimers and other pyrimidine dimer photoproducts o o HN NH Indirect effects: production of reactive oxygen n n^o species (ROS) -> oxidative damage (DNA, proteins, lipids) of cell structures 13 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Short-term and long-term effects - Induction of apoptosis -> photoaging Mutations -> e.g., skin cancer (a) Normal DNA exposec to UV radiation (b) DNA absorbs radialion and becomes damaged Cell nucleus Cell-repair enzymes (c) Damaged DNA torms pynmidine dinners which prevent normal cell lunctioning _____ One of throe things then occurs 1. Enzymes accurately repair damaged DNA 3. Cell cannot repair damage and dies Cell later divces 2. Damaged DNA s rarely ^accurately repaired: this -ii mutation is then retained when the cell civ ides How sunlight causes shon- and long-term skirt damage. 2h ■ ' 1 'i 24 h 48 h Apoptotic cells (cleaved caspase 3) in UV-irradiated (100 mJ/cm2) epidermis 14 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Electromagnetic radiation: Ionizing radiation -Gamma rays, X-rays, (high energy UV light) - Higher frequency (y > X-rays) = lower ionization density -> less damage Gamma knife radioactive cobalt converging gamma Low ionization density High ionization (rv^ density -Cells a 15 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) 11 Electromagnetic radiation: Ionizing radiation - Direct effects: DNA breaks -Indirect effects: ROS production (a) Ionizing radiation (b) Non-ionizing radiation Radiosensitivity - Cells of higher organisms Cells (species) with high DNA content, gametes - Rapidly proliferating cells (bone marrow, lymph nodes, germ cells, epithelial cells, embryonal cells...) 16 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Mechanical stress - Cells with the cell wall are highly resistant - Intermediary filaments in animal cells crucial for protection from mechanical stress (tissue integrity) -Mechanical forces (tension, compression...) may cause critical damage of membranes - Actin filaments reorganization - Transmission of the mechanical stress - signaling - Compensate local mechanical stress (micromanipulation studies) - Recovery of membrane after perforation (e.g., after microinjection) 17 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Actin remodeling after mechanical stress induced by stretching the cell culture surface Control p Control Intracytoplasmic sperm injection Unstretched D Stretch (60 min) Chemical stress (xenobiotics) 19 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Xenobiotics -Chemical substances not naturally occurring in the cell (organism) -Often toxic - accumulation, interaction with target molecules - Specific and non-specific effects Classification of xenobiotics -Chemical composition (heavy metals, acids, alkaloids...) -Origin (chemical toxins, biological toxins) - Mechanism of action (DNA damage, cytoskeleton defects, inhibition of protein synthesis, inhibition of respiratory chain...) 20 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) fl Specific inhibitors: Synthesis of biopolymers -Crosslinking of DNA: mitomycin C -Nucleotide synthesis inhibitors: azaserine, C-mercaptopurine - Blockage of RNA polymerase movement: actinomycin D - Binding to RNA polymerase: a-amanitin, ethionin, lomofungin - Peptidyl transferase inhibition: cycloheximide - Blocking AA-tRNA binding: tetracycline - Blocking mRNA translocation: chloramphenicol Polypeptide release from ribosome: puromycin - Release of ribosomes from ER: aflatoxins 21 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Specific inhibitors: Membrane function -Changes in phospholipids -> formation of micelles: phospholipases - Interactions with cholesterol -> membrane break down: saponins (digitonin), nystatin, amphotericin - Increasing ion permeability: valinomycin, gramicidin A - Blocking sodium-potassium ion pump (N+/K+ ATPase): ouabain 22 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Specific inhibitors: Energetic metabolism - Uncoupling oxidative phosphorylation from ATP synthesis in mitochondria (allowing protons to bypass ATP synthase): benzimidazoles, 2,4-dinitrophenol - Blocking electron transport chain complexes: cyanides, metformin/phenformin - Inhibition of dehydrogenases: urethane, disulfiram, barbiturates 23 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Specific inhibitors: Cytoskeleton dynamics - Microtubule polymerization inhibition: colchicine, vinca alkaloids (vincristine, vinblastine) -Microtubule depolymerization inhibition: taxanes, paclitaxel - Inhibition of actin filament polymerization: cytochalasins, latrunculins - Inhibition of actin filament depolymerization: phalloidin, jasplakinolide 24 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Phalloidin - Mouse fibroblasts F-actin (Phalloidin), DNA 25 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cytochalasin B - Human skin fibroblasts F-actin 26 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Biological stress factors 27 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Intracellular parasitism - Viruses (cell lysis or integration into the genome) - Mycoplasma - lack cell wall - Rickettsia, Chlamydia, Listeria monocytogenes - Plasmodium malariae, Toxoplasma gondii Changes of the infected cells - Metabolism (exploit protein synthesis apparatus; affect growth rate) - Cell morphology (cytoskeleton, plasma membrane) - Behavior of the infected cell, whole organism - Modulation of apoptosis (pro- & anti-apoptotic effects, stress signaling) 28 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) 29 Bil 700en Cell Biology /10 - Cell pathology (11 May 2022) - Mycoplasma hyorhinis DNA (nuclei and mycoplasma DNA) 30 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cell death Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Types of cell death - Necrosis = catastrophic cell death caused by non-specific stress factors and/or extreme damage - Necroptosis = programmed form of necrosis; regulated by specific factors - Apoptosis = programmed cell death -Autophagic cell death - Ferroptosis = iron-dependent programmed cell death 32 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cell death: Necrosis 0 - Cell pathology (11 May 2022) Induction of necrosis - Rapid and extensive damage - mechanisms of programmed cell death cannot be activated - Effects of extreme non-specific stress factors -Changes in concentration of ions, in pH... - Depletion of energy sources - Extremely high or low temperatures - Mechanical trauma - damaging cells / tissues 34 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Progression and hallmarks of necrosis -Overloading of mitochondria with Ca2+ - Loss of mitochondrial membrane potential: jATP -Generation of ROS - mitochondrial damage -Swelling and rupture of mitochondria -Swelling of the cell and lysis - Release of cell contents -> inflammation -At the tissue level: formation of a necrotic lesion —► invasion of macrophages —► inflammation Normal cell Heat, trauma, anoxia and pathogens Necrosis 35 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cell becomes leaky membrane blebbing - res? ví-. Lysis and inflammaUon Reversible injury Myelin figure Swelling of endoplasmic reticulum and mitochondria Membrane blebs Progressive injury Myelin figures Breakdown of plasma membrane, organelles and nucleus; leakage of contents NECROSIS Amorphous densities in mitochondria viable cell B primary necrosis 2nm 36 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) 37 Bil 700en Cell Biology /10 - Cell pathology (11 May 2022) fl Cell death: Necroptosis 0 - Cell pathology (11 May 2022) Necroptosis Regulated cell death with features of apoptosis and necrosis Triggered by similar stimuli as extrinsic apoptotic pathway Specific proteins (RIPK3, MLKL etc.) shift extrinsic apoptosis to necrosis ^^=^=^. mode of cell death TNFa FASL TRAIL APP řTNFR .-TAS IItRAILR WDR6 / /Viral nfeclion Endpoint: same as necrosis Immunogenic: favored in defense against certain pathogens c mtDNA/RNA___ RIPK1 RIPK3) ----------.------------_— ( trif ) > (ripio) (mlkl-) _____ 39 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cell death: Apoptosis 0 - Cell pathology (11 May 2022) Br. J. Cancer (1972) 26, 239 Apoptosis APOPTOSIS: A BASIC BIOLOGICAL PHENOMENON WITH WIDE-RANGING IMPLICATIONS IN TISSUE KINETICS j. F. R. KERR*, A. H. WYLLIE and A. R. CURRIEf From the Department of Pathology, University of Aberdeen R&oeived for publication April 1872 Summary.—The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations. Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological. The structural changes take place In two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments. In the second stage these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the Ingesting cells. Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development. It occurs spontaneously in untreated malignant neoplasms, and participates In at least some types of therapeutically induced tumour regression. It Is implicated in both physiological involution and atrophy of various tissues and organs. It can also be triggered by noxious agents, both in the embryo and adult animal. -1972: Kerr, Wyllie and Currie coined the term aTTOTTTwai^: dropping/falling off 41 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) 2002 Nobel Prize in Physiology or Medicine Sydney Brenner H. Robert Horvitz John E. Sulston -for their discoveries concerning genetic regulation of organ development and programmed cell death 42 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) fl Caenorhabditis elegans - Hermaphrodite adult: 959 somatic cells -131 cells eliminated by apoptosis during development: predictability, easy to observe -Genes involved in regulation of apoptosis - 14 Ced genes: - Ced-3, Ced-4 - induction of apoptosis - Ced-9 - anti-apoptotic role 43 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Hallmarks of apoptosis -Chromatin condensation - Nuclear fragmentation - Plasma membrane integrity retained -Cellular fragmentation into apoptotic bodies 44 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) A Apoptosis im- mitochondrial morphology preserved » DNA lfh(( ■ fragmentsJ*V>\ V. nuclear changes intact membram (^) apoptot m % c bodies normal condensation (coll blebbing) fragmentation secondary necrosis C Necrosis mitochondrial morphological changes^ Uf % \ • kr\%\ -\jJ«sJ>J chromatin pattern conserved membrane breakdown normal reversible swelling irreversible swelling disintegration 46 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) The role of apoptosis - Regulated elimination of unwanted cells 1. Indispensable for proper development of multicellular organisms - Elimination of tissues between digits during embryogenesis: sculpting hands/paws - Resorption of the tail during tadpole metamorphosis into a frog -Adjusting the number of developing nerve cells to the number of target cells 47 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) fl nerve cells • ••• target cells apoptotic nerve cells CELL DEATH ADJUSTS NUMBER OF \ NERVE CELLS A TO SIZE OF mK \ TARGET survival factor released by target cells 48 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Bil 700en Cell Biology /10 - Cell pathology (11 May 2022) Syndactyly: defects in apoptosis 50 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) The role of apoptosis - Regulated elimination of unwanted cells 2. Quality-control mechanism for eliminating abnormal, misplaced, defective cells dangerous for the organism integrity -Cells infected with viruses (parasitic bacteria can inhibit apoptosis) - Effector cells of the immune system after the immune response -Cells with damaged DNA (checkpoint kinases -> p53 downstream targets: DNA repair or induction of apoptosis) vs. cellular senescence - Precancerous cells and transformed cells 51 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) III Apoptosis Healthy cell Genetic errors result from injury 3o errors remain Cell unable to repair errors Injury to cell Cell attempts to repair errors All errors repaired Additional injury to cell 0 Cell programs * O itself to die via j apoptosis DNA damage Genome instability e.g. oncogene activation, mutations in p53 and disruption of apoptosis signalling DNA repair Apoptosis Nature Reviews I Cancer Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) The level of cellular stress matters - Nature and severity of the stress dictates whether a cell becomes senescent or undergoes apoptosis -Senescent cells - Prolonged and generally irreversible cell cycle arrest - induced by less sever damage - Active prosurvival pathways = enhanced resistance to apoptosis 53 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Tissue homeostasis - apoptosis vs. proliferation - Failure of apoptosis -> development of neoplasms (benign -> malignant) Phases of Neoplasia Precursors to Neoplasia Metastasis Apoptosis and Cell Proliferation in Tissue Homeostasis Proliferation « Apoptosis 1 Proliferation | Apoptosis i Proliferation Apoptosis Proliferation » Apoptosis Proliferation 1 Apoptosis 1 proliferation of cells neoplasia regeneration of cells nooplasia equilibrium dystrophy dystrophy 54 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Induction of apoptosis Withdrawal of antiapoptotic (survival) stimuli - Growth factors - various cell types including neurons - Interleukin 2 (IL-2), IL-3 - lymphocytes, hematopoietic stem cells Receiving apoptotic stimuli -Internal signals Prolonged or severe stress: upregulation of ROS-damaged molecules, DNA damage, ER stress, viral infection, amino acid starvation, glucose deprivation... - External signals - Death ligands binding to death receptors 55 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) III Phases of apoptosis Initiation phase = induction of apoptosis - Intrinsic pathway: mitochondrial outer membrane permeabilization & release of cytochrome - Extrinsic pathway: activation of death receptors - Activation of initiator caspases Execution phase Activation of executioner caspases - Cell shrinkage, plasma membrane blebbing - Changes in the plasma membrane composition ■ Chromatin condensation, degradation and fragmentation Proteolytic cleavage of intracellular proteins Fragmentation into apoptotic bodies (followed by phagocytosis) 56 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Caspases - proteolytic cleavage Cysteine-aspartic proteases: cleave proteins at the carboxy group of aspartic acid residue Inter-subunit linker 13 human caspases: Initiator (2,8, 9, 10) Executioner (3, 6, 7) Synthesized as inactive zymogens (pro-caspases) Activated by cleavage Inflammation (Group I) Initiator caspases (Group II) Executioner caspases (Group III) Group I Caspase-2 (h,m) Caspase-8 (h,m) Caspase-9 (h,m) Caspase-10(h) MH. -3 (h.m) CaspasG-6 (h,m) -7 (h,m) Caspase-12 (m) Caspase-14 (h,m) Alternate caspase functions Pyroptosis, apo ptosis, metabolism E R stress, apoptosis Not determined Pyroptosis, apoptosis, leukocyte migration Cell cycle, autophagy, genomic stability, aging, tumorigenesis and tumour suppression, pentose phosphate and lipid metabolism Inflammasome, necroptosis, lens cell differentiation, terminal keratinocyte differentiation, erythrocyte and platelet formation, T- and B-cell proliferation Synaptic loss, erythropoiesis, embryonic development and leukemic and muscle differentiation Not determined Apoptosis, embryonic and hematopoietic stem cell differentiation, osteoblasts and neuronal differentiation, myotube formation, synaptic plasticity, autophagy Cell cycle, synaptic loss and disfunction, B-cell proliferation Bone development, dentinogenesis E R stress Keratinocyte differentiation Small catalytic domain Large catalytic domain 57 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Proteolytic cleavage Caspases -Cleavage activated by apoptotic stimuli (intrinsic or extrinsic) - Initiator pro-caspases dimerization after binding to specific protein complexes -> autocatalytic cleavage of the pro-domain -> active caspases - Executioner pro-caspases cleaved by initiator caspases = caspase cascade caspase cascade u /1 \ one molecule of active initiator caspase '//n^ '//iws '//it\s many molecules of executioner caspases / \ cleavage of cleavage of cytosolic protein nuclear lamin 58 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Proteolytic cleavage Caspases might be activated by other proteases - Granzyme B - produced by cytotoxic T cells and NK cells -Cathepsins - released from ER; direct (cleavage) and indirect activation of caspases; positive feedback loop -Calpains - activated by Ca2+ (e.g., ER stress) Cytotoxic T lymphocyte • Granzyme B • Perforin Target cell Bid Caspase 8 Caspase 3 Caspases-» Apoptosis -All these protases also target (activate) other proapoptotic proteins & facilitate proteolytic cleavage 59 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Intrinsic (mitochondrial) apoptotic pathway - Balance of anti-apoptotic and pro-apoptotic BCL-2 proteins (1) Apoptotic stimuli • Nutrient deprivation • Growth factor deprivation • Ionizing radiation • Reactive oxygen species • Chemotherapy (cytotoxic or targeted) • Excessive mitogenic stimulation T Upstream signalling • p53 • ER stress • MYC • Kinase inhibition Pro-apoptotic BH3-only 'activators' (BIM.tBID, PUMA) Pro-survival (BCL-2, BCL-W, BCL-X BFL1, MCL1) Pro-apoptotic pore-forming (BAX, BAK) ' BH3-only sensitizers' inhibition of anti-apoptotic BCL-2 proteins -> pro-apoptotic BAX & BAK oligomerization = formation of pores in the mitochondrial outer membrane - Release of cytochrome c from mitochondria into the cytosol - Formation of apoptosome 62 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) fl Apoptosome -Cytosolic Apaf-1 + (d)ATP - Cytochrome c Active apoptosome: -Apaf-1 CARD domain binds pro-caspase 9 -Cleavage into active initiation caspase 9: activates executioner caspases a Apaf-1 (card (■ ( AAA* ATPase r//|} Helical ^ ': Winged [ Super helical Locked form Cytochrome c wd40 (b Cytochrome c dATP/ATP -J dADP/ADP executioner caspases Death ligand Plasma membrane Cytoplasm Death-inducing signaling complex FADD I Death receptor S B w A i Cellular stress (ca>p-»3,6,7) 1 —n I Mitochondria J Cell 66 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Extrinsic vs. intrinsic Extrinsic - Death receptors -Caspase 8 (and 2,10) Intrinsic - MOMP and cytochrome c release - Caspase 9 Executioner caspases shared Extrinsic pathway Death receptors (e.g. TRAILR and FAS) Pro-caspase-8 and pro-caspase-10 Intrinsic pathway Intrinsic lethal stimuli: DNA damage, ER stress, hypoxia and metabolic stress Caspase-8 and caspase-10 — Caspase-3 and caspase-7 Caspase-3 and caspase-7 67 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) □Plasma membrane blebbing - Defects and degradation of cortical cytoskeleton Changes in the plasma membrane - Externalization of phosphatidylserine: signal for phagocytosis 68 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Nuclear collapse -Chromatin condensation and degradation of nuclear lamina - Internucleoasomal cleavage of DNA: DNA ladder - Nuclear fragmentation Lane A: Control Lane B: Apoptosis Lane C: Necrosis Ladder pattern of DNA 70 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cell fragmentation into apoptotic bodies F-actin, DNA - Actin/non-muscle myosin II contraction - blebbing and apoptotic bodies formation - Plasma membrane integrity remains intact - Apoptotic bodies comprise nuclear fragments, organelles, cytoplasm 72 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Phagocytosis of apoptotic bodies - Engulfment of apoptotic cells/apoptotic bodies by neighboring phagocytes (macrophages and dendritic cells) - Phagocytes secretes anti-inflammatory cytokines 73 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Apoptosis Intrinsic UV, osmotic stress, growth factor withdrawal Extrinsic TNF-a/lymphotoxin-a A FAS ligand/FasL A TFtAIL A ©seed DNA shredding Cell shrinking membrane blebbing 4 74 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cell death: Autophagic cell death 75 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) fl Autophagy - Regulated process of targeting and delivery of cellular components and organelles into lysosomes for degradation (and recycling) - Macroautophagy - best described, autophagosome formation; selective/non-selective - Microautophagy - direct invagination of lysosomes; selective/non-selective -Chaperone-mediated autophagy - selective 76 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) fl 77 Bil 700en Cell Biology /10 - Cell pathology (11 May 2022) Macroautophagy process PI3K III nucleation complex AMPK T (D Autophagy initiation ^2) Membrane nucleation and phagophore formation (3) Phagophore expansion f " ^"(ATGIO ©ULK1 initiation complex Delivery of lipids in ATG9-containing vesicles (4) Fusion with Autolysosome the lysosome Membrane-bound PI3P-binding complex I LIR (^Sj^ Autophagy receptor LC3 conjugation system 78 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) 2016 Nobel Prize in Physiology or Medicine -for his discoveries of mechanisms for autophagy 79 Bil 700en Cell Biology /10 - Cell pathology (11 May 2022) II Autophagic cell death - Autophagy-associated cell death - Excessive autophagy that accompanies apoptosis - Autophagy-dependent cell death Inhibition of autophagy prevents cell death, apoptosis or necrosis not involved ■ Context dependent: e.g., midgut of Drosophila larvae; hippocampal neural stem cells after insulin withdrawal... Autophagy-associated Signal / X 1 Autophagy Apoptosis 1 Cell death Autophagy-dependent cell death Autophagy 80 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Normal cell Opening of plasma membrane channels and extracellular fluid influx Nuclear compaction (pyknosis; Loss of mitochondrial membrane potential Further cell shrinkage Nuclear • fragmentation (karyorrhexis) Vacuoles with membrane Apoptosis Massive cytoplasmic vacuolization and loss w*K>r'5 Autophagy-associated ° 0,8ane"es cell death Necrosis Plasma membrane rupture 81 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Cell death: Ferroptosis 82 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) Ferroptosis -Iron-dependent programmed cell death Characterized by large amount of iron accumulation and iron-catalyzed lipid peroxidation during the cell death process - Dysregulation of iron metabolism, glutathione (GSH, antioxidant) depletion... - Excessive ROS production -> rupture of mitochondrial outer membrane 83 Bi1700en Cell Biology /10 - Cell pathology (11 May 2022) 84 Bil 700en Cell Biology /10 - Cell pathology (11 May 2022)