Lecture 12: Apoptosis

Give examples of external factors that can cause necrotic cell death

Infection or trauma

Necrotic cell death is [a passive/an active] process associated with progressive ______ of cell structure

Passive, disintegration

Necrosis usually elicits an acute ____ response in cells wherein neutrophils are present

inflammatory

Apoptotic cell death is mediated by ___ products

gene

Apoptosis is a normal series of events. What is the result of these events?

Removal of damaged cells, cells that are no longer needed, or cells that pose risk

Apoptosis is an orchestrated sequence of events characterized by ___ and ____ fragmentation that leads to clean cell death

Nuclear, DNA

Apoptosis involves mechanisms for killing cells by recruiting _____ and presenting a signal to them for __ ____

Macrophages, cell engulfment

Describe apoptosis during development

During embryogenesis and fetal development, physiological apoptosis contributes to shaping digits, removal of excessive neuronal connections, and removal of cytotoxic T cells

Physiological apoptosis can be initiated by _____ to eliminate cells when their function is complete, when they are ____-_____ lymphocytes (in the case of autoimmunity), or the loss of cells during _____

Hormones, self-reactive, proliferation

Apoptosis can be induced by pathology. Give examples of cases of this.

DNA damage due to radiation and/or chemotherapy, accumulation of misfiled proteins in the ER (ER stress), viral infections (e.g. HIV, adenovirus, and SARS-Cov-2), duct obstruction causing organ atrophy (e.g. kidney stones)

Several diseases are linked to reduced or elevated apoptosis. Give examples

Cancer, Parkinson's, Alzheimer's, and Huntington's diseases, T1D

Apoptosis usually affects scattered ____ cells, with control of intracellular environment ____ in early stages, leading to the cell _____

Individual, maintained, contracting

Necrosis usually affected ____ areas of _____ cells, with control of intracellular environment ___ in early stages, leading to organelles and cells _____

Large, contiguous, lost, swelling

Describe the chromatin morphology in necrosis vs apoptosis

In necrosis, nuclear chromatin marginates early while the injury is still reversible and the DNA is cleaved into random sizes (shown as a smear in electrophoresis). In apoptosis, nuclear chromatin marginates and chromatin condenses, becoming very compact, and DNA is cleaved into multi-200 bp units (shown as a ladder in electrophoresis)

What happens in late stage apoptosis?

The cell membrane blebs and apoptotic bodies containing nuclear fragments are shed. The intact apoptotic bodies are phagocytosed without chemotactic signals

What happens in late stage necrosis?

The cell membrane ruptures as terminal event and the cell content is released, acting as a chemotactic signal, leading to neutrophil infiltration for degradation of cellular debris

Apoptosis is: A. Caused by inflammation B. A process to reduce the size of an organ C. Changes cell types in response to stress D. A beneficial process to eliminate damaged cells

D. A beneficial process to eliminate damaged cells

How does the extrinsic pathway of apoptosis get activated?

Through death receptors

How does the intrinsic pathway of apoptosis get activated?

Through mitochondrial mechanisms

Describe the initiation of the extrinsic pathway of apoptosis

Extracellular stimuli activate intracellular signalling through death receptors which are domains on members of the TNF-α family and related protein Fas

What is TNFR1?

A death receptor that is preassembled at the cell membrane as a trimer

Binding of TNF-α causes ______ change of the death domains, leading to an ______ signalling cascade that leads to the execution phase

Conformational, intracellular

Describe the execution phase of extrinsic apoptosis

Bound TNF receptors recruit procaspases to the intracellular domain of the receptor, where they confer other procaspases to caspases, which activate executioner caspases, leading to apoptosis

Describe the initiation of the intrinsic pathway of apoptosis

Signalling is initiated by intracellular stimuli (e.g. irreparable genetic damage or severe oxidative stress), which induces increased mitochondrial permeability, causing leakage of pro-apoptotic caspase-activating molecules into the cytoplasm (cytochrome C), where they form part of a multiprotein complex called the apoptosome that irreversibly commits the cell to apoptosis

Within the apoptosome Apaf-1 exists in ______ form in complex with ADP. when this binds to cytochrome c, ____ is replaced with ____ and that forms a _____ complex with cytochrome c

Autoinhibitory, ADP, dATP, heptameric

The apoptosome complex recruits monomeric ______ __ and helps it to form a dimer which undergoes _____ _____ to form an active heterotetrametic complex that cleaves and activates _____ ___, which leads to _____ caspases and apoptosis

Procaspase 9, autolytic cleavage, caspase 3, executioner

Both the intrinsic and extrinsic apoptosis pathways converge onto a ____ activation cascade

caspase

Caspases targets protein _____, including FAK, PKB, PKC, Raf1, to alter cell ____, cleaving ____ proteins, leading to nuclear breakdown, and activating CAD, which is a ____ _____ that causes characteristic internucleosomal DNA cleavage (giving the ladder)

Kinases, adhesion, structural, cytoplasmic endonuclease

Which of the following is involved in the intrinsic pathway of apoptosis? A. Cytochrome a B. Cytochrome b C. Cytochrome c D. Cytochrome D

C. Cytochrome c

What are the three biochemical hallmarks of apoptosis

Fragmented chromosomal DNA, PtdSer expression on the cell surface, loss of mitochondrial potential

Describe what happens with PtdSer during apoptosis

It starts on the inside of the cells (in live cells) and diffuses through the broken nuclear membrane onto the outside of cells during death

What is propidium iodide? What does it do?

It is a marker that sticks to DNA. It can access the DNA in dead cells through the broken nuclear membrane, causing it to accumulate

What is annexin V?

It is a protein that binds to PtdSer on the outside of cells

What does a TUNEL assay do?

It detects late-stage apoptosis

Why is imaging better than something like flow cytometry?

Imaging shows the whole story (e.g. proximity to other things)

Why should one never put a fluorescent overlay with a phase contrast?

The depth of imaging and diffusion in the phase contrast is not compatible with overlay, causing lose of contrast and sharpness

Mitochondrial membrane potential is a key indicator of mitochondrial activity, as it reflects what?

The process of electron transport and oxidative phosphorylation (driving ATP production)

Mitochondria with a collapse in membrane potential exhibit what structural changes?

Rounding and ballooning

How does a collapse in mitochondrial membrane potential show the using JC-1?

There is reduced red emission and diffusion of green fluorescence into the cytosol

Describe the process of clearing dead cells

The dying cells secrete factors that recruit phagocytes without inflammation

Why do dead cells need to be cleared out?

They need to be cleared before cells undergo secondary necrosis

What happens during apoptosis that results phagocyte recruitment?

A phospholipid scramblase moves PtdSer to the outer leaflet of the plasma membrane, which are recognized as eat me signals by specialized macrophages

Describe the inflammatory process of necrosis vs apoptosis

Necrosed cells cause inflammation, while apoptosed cells disappear without initiating inflammation

In cell culture, how is necrosis distinguished from apoptosis?

Using cell morphology and timeline of death

What are the 3 types of lipid translocators?

ATP, flippase, floppase

What do flippases do?

They translocate specific lipids from outside to inside

What do floppases do?

They translocate specific lipids from inside to outside

____ maintain the asymmetrical distribution of PtdSer

Flippases

____ are driven by the existing lipid gradient to non-specifically and bi-directionally transport lipids between leaflets (move PtdSer to outer membrane)

Scramblases

What is the unusual characteristic of cell membrane seen in apoptotic cells? A. Cell membrane exposes PtdSer on the outer side B. Cell membrane exposes cholesterol and lipid rafts on the outer side C. Cell membrane ruptures D. Cell membrane channels stop working

A. Cell membrane exposes PtdSer on the outer side

What is necroptosis?

A form of programmed necrosis

What is ferroptosis?

A form of programmed cell death dependent upon iron, which is accompanied by a large amount of iron accumulation and lipid peroxidation during the cell death process

What signals do apoptotic cells display for macrophages?

Find me and eat me

What is a hallmark of DNA cleavage in apoptosis?

Multi-200 bp units

What activates the extrinsic pathway of apoptosis?

TNF

What is the receptor in the extrinsic pathway of apoptosis?

TNFR1

What is TNFR1?

A death receptor preassembled at the cell membrane as a trimer

What is a death domain?

Intracellular domain of TNFR1

Describe the signal cascade of the extrinsic pathway

TNF binds to TNFR1 and causes conformational change in the death domains which recruit procaspases to convert other procaspases to caspases which activate executioner caspases, leading to apoptosis

What triggers the intrinsic pathway of apoptosis?

Intracellular stimuli that increase mitochondrial permeability, releasing caspase-activating molecules into the cytoplasm

What is cytochrome C?

A caspase-activating molecule

What is the apoptosome?

A multiprotein complex in the cytosol that includes cytochrome c, procaspase-9, and Apaf-1

At what point is the cell irreversibly committed to apoptosis by the intrinsic pathway?

After the release of apoptotic mitochondrial proteins

How does Apaf-1 get activated in the apoptosome?

Cytochrome c binds and replaces the ADP with dATP forming a heptameric complex with cytochrome c

What is caspase 3?

Executioner capsase

How does caspase 9 get activated?

Procaspase 9 gets recruited by the apoptosome and undergoes autocatalytic cleavage to form an active heterotetrameric complex

How does caspase 3 get activated by the intrinsic pathway?

Caspase 9 cleaves and activates caspase 3

What does caspase 3 do?

Coordinates destruction of DNA and cytoskeletal proteins

What is Bcl-2?

An anti-apoptotic protein family

What is Bad protein?

A pro-apoptotic protein

What is Bax protein

A pro-apoptotic protein that forms a protein-lined channel in the outer mitochondrial membrane to increase permeability and leakage of cytochrome c

What does Bcl-2 do to Bax?

It prevents Bax oligomerization and inactivates it by binding to it

How do the intrinsic and extrinsic pathways of apoptosis converge?

They converge on a caspase activation cascade on caspase 3 which cleaves DNA and cytoskeletal components

What does caspase target?

Structural proteins to induce nuclear breakdown, protein kinases involved in cell adhesion, and activation of CAD to cleave DNA

What is CAD?

Cytoplasmic endonuclease that cleaves in multi-200 bp segments

What is PtdSer?

An anionic phospholipid that is moved to the outside of cells by flippases during apoptosis acting as an eat me signal

What are three biochemical hallmarks of apoptosis?

Fragmented chromosomal DNA, PtdSer expression on the cell surface, and loss of mitochondrial potential

What is propidium iodide and what does it do?

A stain that sticks to DNA, allowing detection of free DNA during apoptosis

Why is PtdSer used for detecting apoptosis?

It is on the inside of living cells but the outside of dead cells, meaning if it detected, the cell is apoptotic

What is annexing V?

A protein that binds to PtdSer and relays the eat me signal for apoptosis

What can a TUNEL assay be used for?

Late-stage detection of apoptosis

What is JC-1 able to show?

A dye that changes from green to orange-red to reflect mitochondrial membrane potential showing mitochondrial activity

What does scramblase do during apoptosis?

Moves PtdSer molecules to the outer leaflet of the plasma membrane

What role do specialized macrophages play in apoptosis?

They phagocytose the apoptotic cells before they undergo secondary necrosis

What do flippases do in the context of apoptosis regulation?

They use ATP to move PtdSer inside cells maintaining an asymmetrical distribution

What do floppases do?

They use ATP to move PtdSer outside of cells

What causes inhibition of flippase activity during apoptosis?

Cleavage by caspase 3

What causes increases PtdSer exposure during apoptosis?

C-terminal cleavage of XK proteins by caspase 3 causing dimerization and scramblase formation and activity

What is TIM4?

PtdSer receptor on macrophages

What are TAM receptors?

RTKs on macrophages that causes phagocytic cupping and engulfing of the apoptotic cell

What is the unusual characteristic of cell membrane seen in apoptotic cells?

Cell membrane exposes PtdSer on the outer side

How does DNA damage activate apoptosis?

p53 accumulates and arrests cell cycle leading to apoptosis via the intrinsic pathway

How do diseases such as Alzheimer's Parkinson's and Huntington's cause apoptosis?

Misfolded protein triggers the unfolded protein response and ER stress leading to apoptosis via the intrinsic pathway

How do cytotoxic T lymphocytes activate apoptosis?

Activation of extrinsic apoptotic pathway to activate preformed endogenous molecules