Cell death and Autophagy

Tryptan Blue

Dye taken up by dead/dying cells with ruptured plasma membranes. (necrosis/secondary necrosis)

Calcein AM

cleaved to fluorigenic product via intracellular esterases for viable cells (used with propidium iodide) (general viability)

Propidium idodide

intercalates DNA of cells with ruptured plasma membranes (used with calcein AM) (necrosis/secondary necrosis)

LDH Assay

A cell will release a bunch of LDH when damaged, and you can measure the products of LDH reactions to see how the cell is doing (Necrosis/secondary necorsis)

What is a way to measure Apoptosis?

DNA fragmentation/Nuclear condensation

What is a way to measure Necrosis

Cell swelling

What is a way to measure autophagic stress?

The accumulation of autophagosomes

Apoptotic cell death

Active/regulated cell death

Shrinkage and nuclear condensation and fragmentation

clevagage of DNA into 200bp

Capsases cleave important proteins, these bodies are formed and phagocytosed

No inflammation and plasma membrane is intact. Requires ATP

Extrinsic stimulus pathway

Fas ligand → Fas (trimerize) → FADD → Caspase-8 → Caspase-3 (activated) then death!

Intrinsic stimulus pathway

Stimuli → stress signal → distruption of Bcl-2 and Bax (higher Bax) → Apoptosome forms → activates caspase-3 → death

What are capases?

Cysteine proteases that cleave after aspartate residues. very important for apoptotic morphology

Have three clases: Initiators, effectors, and cytokine activators

What is Bcl-2?

It is the B-cell leukemia/lymphoma-2

Pro-survival oncogene, prevelant in their name sake

What is the Bcl-2-Bax Rheostat?

Bax-Bcl-2 heterodimer is the baseline

Bax homodimers from pro-apoptotic signals

Bcl-2 homodimers from anti-apoptotic signals

What does BH3 lead to?

Bcl02 has a BH domain, and can be inhibited this way which leads to pro-apoptotic signals

What is Bid and what does it do?

It is cleaved by caspase 8, then it can accelerate Bax/Bax-dependent mitochondrial apoptosis

What are IAPs?

They are inhibitors of apoptosis proteins

Capsases are inhibited when IAPs bind to them

MOMP causes release of Smac, Smac inhibits and promotes degredation of Smac allows for caspase signaling.

How do you measure Apoptosis?

1. Caspase activation

2. Annexin V labeling

3. Bax translocation of mitocondria

4. Mitocondrial release of cytochome c or AIF

5. Clevage of caspase substrates

How do you measure Apoptosis/Necrosis

1. DNA fragmentation

2. Nicked DNA

3. Annexin V-PI uptake

What do apoptosis and necrosis look like on a DNA laddering Gel?

Apoptosis: ~1-200bp fragments

Necrosis: smear like appearance

Fluorescence-based activity assays

Similar to QT-PCR, fluorescence donor with a quenching acceptor, eventually degreaded by an enzyme and will flouresce afterwards

Is TUNEL appropriate to tell if something is JUST going apoptosis?

No! it detects DNA damage from both apoptosis and necrosis

Caspase-independent, regulated cell death

1. Apoptosis-inducing factor (AIF). A mitocondrial protein, released upon stimuli and translocates into nucleus

2. Calpains, proteases activated by intracellular rises in calcium, role in many injury and disease models of cell death, linked to apoptosis, necorsis, autophagic cell death

Necrosis

Passive cell death

Vacuolation of cytoplasm

Cell swelling

Rupture.breakdown of plasma membrane

Induction of inflammation around dying cell from release of cell contents

Often triggered by ATP deplection (so energy independent)

Necroptosis

Death receptor activation in the presence of apoptosis inhibition

REquires RIP1 activity

RIP-containing “Necrosome”

PARP1 over-activation-ATP/NAD+ depletion

Morphology of necrosis

Necrostatins: small molecule inhibitors of necroptosis

Autophagy

Lysosomal degredation pathway for all cytoplasmic material

Occurs constitutively in all cells

Also induced in responses to nutrient status

Provides long-term organellar quality control

Three types: macroautophagy, chaperone-mediated autophagy, mirocautophagy

Chaperone-mediated Autophagy

Constitutive and inducible

Direct transport

Lysosome membrane receptor enables protein internalization

Transport and degredation of proteins

Selective

Macroautophagy

Constitutive or inducible formation of autophagosomes

Vesicle-mediated fusion events transport cargo to lysosomes for degredation

Responds to nutrient demands, organellar quality control

PI3-K/ATG gene-regulated

What does mTOR do?

It is a inhibitor of autophagy and is regualted upstream by TSC ½ which has a whole host of things it is regulated by. Active TSC means inactive mTOR.

What does p62 do?

Targets ubiquitinated proteins for autophagic degredation by binding to LC3-II

What is the difference between Ubiqutin proteasome system vs macroautophagy for protein degredation?

Mainly size, only macrophagy can degreade larger protein aggregates

How do you measure macrophagy?

EM

MAP-LC3-II

p62

Autophagic flux using inhibitors/knockdown

autophagic flux using tfLC3 Assay

Up-regulation/interaction of Atg proteins

Degredation of radioactively labeled. long-lived proteins

How do you measure chaperone-mediated autophagy?

Levels of hsc70, Lamp2a

Degredation of radioactively-labeled proteins

How can you tell if something is an autophagosome?

It has a double membrane!

what does LC3-II detect?

The accumulation of autophagosomes

What does cleaved caspase-3 indicate?

Apoptosis!

What is Arg 5 invovled with?

Development of vesciles or something like that

What does Rapamycin do?

Inhbits mTOR

What do 3MA and wortmannin do?

It inhibits macroautophagy induction

Autophagic stress

Any alteration in autophagic flux

May promote survival and/or regulate cell death

What is autophagic flux?

general activity of the autophagy-lysosome pathway

How can you tell Autophagic cell death?

Accumulation of autophagic vacuoles

Membranous whorls

Osmiophilic debris

Altered numbers'/size of mitocondria

Concurrent with apoptosis/necrosis

What is the difference between autophagy and autophagic cell death?

Autophagy is a survival-promoting to cope with stress

Autophagic cell death: usually related to aberrant alterations. by removing autophagy, you can prevent cell death

How does apoptosis and autophagy talk?

Bcl-2/Bcl-X interactions with beclin inhibit beclin-dependent autophagy

BH3 domain-only Bcl-2 family members bind to beclin, disrupt Bcl-2.Bcl-X - beclin interaction and inhibition of autophagy, resultant autophagy induction