apoptosis - exam Flashcards by Anya James

WHAT
cellular response to…

environmental cues

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What defines programmed cell death vs accidental cell death?

Programmed is controlled and there are cues and steps

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what is apoptosis

a major form a programmed cell death

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other forms of programmed cell death

o Autophagy (self-eating) – degradation of cytoplasmic materials via lysosomes
o Necroptosis – programme necrosis (it can accidental)
o Many more types as well

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major cell death subroutines

RDC: regulated cell death
ICD: immunogenic cell death
ADCD: autophagy-dependent cell death
LDCD: lysosome-dependent cell death
MPT: mitochondrial permeability transition

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APOPTOSIS:
morphological features

cell shrinks, chromatin condenses

budding

apoptotic bodies are phagozytosed; no inflammation

Through this process membrane is intact and cellular contents are not released

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necrosis morphological features

necrosis (cell swells)

cells becomes leaky, blebbing

cellular and nuclear lysis causes inflammation

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APOPTOSIS:
more to discover
early and late

Early apoptotic events: apical actin accumulation, actin bundles connecting nuclei, apical migration of nuclei

Late apoptotic events: basal cell delamination, chromatin condensation, DNA fragmentation

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APOPTOSIS:
why is it important

How many cells die each day due to apoptosis in a human adult?
o 50-70 billion cells
How much weight of dead skin cells do you lose in a year
o 8 pounds

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APOPTOSIS:
when does it occur
developmentally programmed

o Patterning/morphogenesis
 Sculpturing structures; deleting unwanted tissues; regulating cell numbers

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APOPTOSIS:
when does it occur
stress-induced

eliminates damaged cells

stress e.g. environmental toxins

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APOPTOSIS:
when does it occur
degenerative disease

Defective tissue homeostasis: degenerative diseases

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APOPTOSIS:
what does it inhibit

cancer

Evasion of apoptosis is a hallmark of cancer

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APOPTOSIS:
how does cancer evade it

sustaining proliferative signalling

evading growth supressors

activating invasion and metatasis

enabling replicative immortality

inducing angiogenesis

resisting cell death

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HOW DOES APOPTOSIS OCCUR

Intrinsic/extrinsic pathways mainly through mitochondria/death receptors and caspases

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HOW DOES APOPTOSIS OCCUR
conserved?

Evolutionary conserved core apoptotic machinery

caspases

HOW DOES APOPTOSIS OCCUR
apoptotic caspases

A family of cysteine proteases that are synthesized as inactive precursor enzymes or proenzymes (cut up proteins)
Pro-capases are activated by cleavage events and formation of a heterotetramer of two large (p20) and two small (p10) subunits
Two groups of caspases: prodomain and function
Two classes – separated due to structure and function

HOW DOES APOPTOSIS OCCUR
apoptotic caspases
effector caspases

shorter prodomain

HOW DOES APOPTOSIS OCCUR
apoptotic caspases
initiator caspase

long prodomain
- DED: death effector domain
- CARD: caspase-recruitment domain
once activated prodomain chopped off

HOW DOES APOPTOSIS OCCUR
mechanisms

Release of mitochondrial cytochrome c and other pro-apoptotic proteins

Then (two pathways):
1. Inhibition of IAPs
2. Formation of apoptosome (important structure in initiating caspase)

Intrinsic vs extrinsic pathways

HOW DOES APOPTOSIS OCCUR
mechanisms
Release of mitochondrial cytochrome c and other pro-apoptotic proteins

There is caspase independent apoptosis
* Caspase-independent apoptosis, also known as caspase-independent cell death (CICD), is a type of cell death that occurs when caspases are not activated. It’s a programmed cell death that can occur in response to most intrinsic apoptotic cues.
- The major ones goes through caspases

HOW DOES APOPTOSIS OCCUR
mechanisms
two pathways

Inhibition of IAPs
- IAP antagonists (e.g. Smac, HtrA2) stops action of inhibitor of apoptosis proteins (e.g. XIAP)
Formation of apoptosome (important structure in initiating caspase)

HOW DOES APOPTOSIS OCCUR
mechanisms
extrinsic pathway

Extrinsic (death receptor)
o Surface cell death signal (e.g. TNFa/TNFR, FasL/FasR)
o –> death-inducing signalling complex (DISC)
o –> caspase 8 activtation
o –> caspase 3/7 activation
o –> apoptosis

HOW DOES APOPTOSIS OCCUR
mechanisms
intrinsic pathway

Intrinsic (mitochondrial)
o DNA damage signals (or cell stress)
o –> p53
o –> Bcl-2 family proteins (e.g. Bax, Bak, Bcl-2) [Bax and Bak activate, Bcl-2 inhibits]
o –> mitochondrial Cyt C release
o –> caspase 3/7 activation
o –> apoptosis

HOW DO WE DETECT APOPTOSIS effector capases act on: 1. Inactivation of DNA repair enzymes

* poly (ADP-ribose) polymerase (PARP) * When damage is too much to be repaired cell needed to be killed (checkpoints + cell cycle)

HOW DO WE DETECT APOPTOSIS effector capases act on: 2. Inactivation of anti-apoptotic proteins

* Bcl-2 and Bcl-XL * In response to stimuli, allows balance to shift towards apoptosis

HOW DO WE DETECT APOPTOSIS effector capases act on: 3. Breakdown of structural nuclear proteins

* Degradation of Lamins by Caspase 6 * DNA needs to be fragmentated at the end of apoptosis, allows DNA to be exposed and cleaved

HOW DO WE DETECT APOPTOSIS effector capases act on: 4. Endonuclease activation and chromosomal DNA fragmentation

* ICAD (inhibitor of caspase activated DNase) and CAD * Need to be inactivated most the time otherwise DNA would be continually fragmented

HOW DO WE DETECT APOPTOSIS effector capases act on: 5. Phosphatidylserine (PtdSer or PS) exposure: “eat-me” signal for macrophages

* Caspase(3/6/7)-mediated cleavage of phospholipid flippase * Exposure of PS on membranes (stops it from going back inside)

HOW DO WE DETECT APOPTOSIS

morphological features cellular markers apoptosis pathway components

HOW DO WE DETECT APOPTOSIS - Morphological features:

o Apoptotic bodies o Chromatin condensation: Hoechst 33258 staining

HOW DO WE DETECT APOPTOSIS - Cellular markers

Vital dyes e.g. acridine orange DNA fragmentation - Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) or DNA laddering Nuclear breakdown - Loss of lamin B from nuclear envelope Caspase activity - Cleaved caspase 3 antibody or cleavage reporters Annexin V labelling of phosphatidylserine

HOW DO WE DETECT APOPTOSIS - Apoptosis pathways components:

p53, Bax, Cyt c and IAPs

WHY DO WE STUDY APOPTOSIS

- Context-dependent roles of apoptosis and caspases

WHY DO WE STUDY APOPTOSIS cancer

Apoptosis inhibits cancer - Evasion of apoptosis is a hallmark of cancer - Therefore apoptosis inducing therapies can be anti-cancer therapies Apoptosis promotes cancer - Apoptosis-induced proliferation Apoptosis promotes carcinogenesis: context dependent

WHY DO WE STUDY APOPTOSIS non-apoptotic function of caspases

- Caspase activation o Cell death o Dendritic pruning o Learning/memory o Sperm individualisation o Compensatory proliferation o Immunity o Differentiation o Enucleation