Apoptosis Flashcards
What is apoptosis?
- naturally occuring cell death –> unwanted cells are eliminated
- programmed
- active and regulated
What is the difference between apoptosis and necrosis?
- apoptosis: programmed –> no release of cellular conentent, no inflammation
- necrosis: accidental –> release of cellular contents, inflammation
What is necrosis? When does it occur?
pathological process which occurs when cells are exposed to physical or chemical noxious agents (e.g. UV, hypoxia, extreme temperatures, infections, toxic substances)
name the steps of apoptosis
- cell shrinkage
- condensation and fragmentation of nuclear DNA
- formation of bleds and apoptotic bodies
- no release of cellular contents
- phagocytosis, no inflammation, individual cell death
name the steps of necrosis
- cell swelling
- clumping and random degradation of nuclear DNA
- cell rupture
- release of cellular contents
- inflammation and damage of neighboring tissue
When does apoptosis occur throughout life? What is the function?
during embryogenesis and adulthood
- morphogenesis in child development
- maintain of homeostasis of tissues, organs and organ systems
- elimination of cells with damaging potential
What is the role of apoptosis in embryogenesis? Give examples
morphogenesis
- shaping developinmg structuresd
- tissue and organ development
- removal of transient structures
examples
- formation of interdigital spaces of fingers and toes
- elimination of half of all neurons
. sexual differentiation
What happens if too little or too much apoptosis occurs?
too little: autoimmune disease and cancer
too much: Neurodegenerative disease, AIDS
What are the hallmarks of apoptosis (kr!!!)
- decrease of the mitochondrial transmembrane potential
- activation of specific proteins (caspases, nucleaeses)
- exposure of phosphatidylserine on the cell’s surface
- chromatin condensation, association of chromatin bodies to the nuclear membrane, nucleus fragmentation as well as some fragmentation of DNA
- cytoskleton colapse, disruption of cell-cell adhesion, cell-matrix interaction and cell shrinkage
- membrane blebbing and formation of apoptotic bodies
- phagocytosis by neighboring cells or macrophages without inflammation
- elimination of a single cell
- if apoptotic bodies are not disposed of, they rupture and undergo secondary necrosis
name methods to detect apoptosis
biochemical features: JC1 (mitochondrial potential) dye, Annexin V
morphological features: DNA laddering, TUNEL, DAPI staining
How can JC1 dye be used for analysis of the mitochondrial transmembrane potential?
normal potenital: dye aggregation –> red fluorescent
depolarization: monomeric dye –> green
How can Anexin V be used to detect exposure of PS?
Annexin V binds with high affinity to PS –> Annexin labelled with flourescent dye can be used for analysis –> also use DNA bindind dye to distinguish necrosis and apoptosis
–> analysis via flow cytometry or microscopy
describe the morphology of apoptotic nuclei
- chromatin condensation
- association of chromatin bodies to the nuclear membrane
- nucleus fragmentation
How can DNA laddering be used to visualize apoptosis inducing DNA fragmentation?
- linker DNA (DNA between histones) is easily accesible to endonucleases
- cleavage preferentially in the linker DNA
- generation of a DNA ladder containing nucleosomal and oligonucleosomal DNA fragments
–> detection of middle/late apoptotic cells
What is the principle behind the TUNEL assay?
during apoptosis double-strand breaks occur which lead to free 3’ OH termini –> add terminal deoxynucleotidyl transferase –> TdT recognizes free 3’ OH termini –> attachment of labeled nucleotides to all 3’ OH ends via template independent DNA polymerase function of TdT
–> measure via flow cytomerty or fluorescence microscopy
How can the collapse of the cytokeratin network be detected in apoptotic cells?
use of monoclonal antibody against cytokeratin –> fluorescence mircroscopy
describe the steps of disassembly of an apoptotic cell
- apoptotic membrane blebbing
- apoptotic mebrane protrusion formation
- cell fragmentation
How is chromosomal DNA cleaved during phagocytosis?
cleavage by phagocyte specific endodeoxyribonucleases
- hydrolytic cleavage of phosphodiester linkage
- peak activity at acidic pH (lysosomes)
Name apoptosis inducing signals
- withdrawk of cytokines and grwoth factors
- loss of adhesion
- endogenous clock
- DNA damage
- Infection
- cytotoxins
- stimulation of cytokines and growth factors
Why is c. elegans a good animal model? (kr)
- simple but many similarities with higher animals (same basic tissues as other animals)
- organized with mouth and brain at the anterior end and anus at the posterior end
- easy to handle
- development is invariant
- constant number of cells
- the lineage of each cell is known
- short life cycle and short life span
What does the form of programmed cell death in c. elegans development look like? Where does it tkae place?
wave form
–> in somatic cells and gemale germ cells
How is apoptosis regulated in c.elegans
regulation through a series of direct protein-protein interactions:
EGL1 inhibits CED9 (anti apoptotic) –> CED4 activated –> CED3 activated –> substrate cleavage and detah
Describe the extrinsic (receptor dependent) and intrinsic (mitochondria dependent) pathway of apoptosis
extrinsic:
- binding of ligands to specific death receptors
- activation of caspases
intrinsic:
- activation of pro-apoptotic proteins
- release of pro-apoptotic factors from mitochondria
- formation of apoptosome
- activation of caspases
describe the effectors of the intrinsic pathway of apoptosis in mammals
pro-apoptotic proteins of the BCL-2 family inhibt anti-apoptotic proteins of the BCL-2 family –> activator Apaf1 activated –> proteases /caspases) activated –> substrate cleavage and cell death
how is the apoptosome formed? How is initiator caspase 9 formed?
caspase has closed conformation, ADP bound and is a compact monomer. The caspase is autoinhibited –> binding of cytochrome c and exchange from adp to atp –> open configuration of caspase and extenden apaf1 subunit –> oligomerization –> formation of apoptosome complex mediated by NOD –> recruitment of initiator pro-caspases-9 –> close proximity leads to autoactivation of caspase 9 –> activated initiator caspase 9 remains bound to the apoptosome where it activates affector caspases
What is the effect of caspases in apoptosis?
destruction of protein function:
- Paxillin
- AKT
activation of protein function
- Ca2+ independent phospholipase A2
- Pannexin
- Helicard
Name mitochondrial pro-apoptotic factors
- cytochrome c: apoptosome
- smac/Diabolo: inhibit caspas einhibitors
- Omi/HtrA2: inhibit caspase inhibitors
- endonuclease G: DNA fragmentation
- AIF: chromatin condensation
