Types of cell death

Programmed cell death (PCD) Necrosis

PCD Type I
(Apoptosis)

PCD Type II
(Autophagic cell death)
 Coining the term ‘apoptosis’

•  Greek word (apo + ptosis)

–  Falling off of petals or leaves

•  First appeared in 1972 paper

–  Kerr, Wyllie, and Currie

Reserved for video

Kerr, J.F.R. et al. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in
tissue kinetics. Br J Cancer, 26(4), 239-257.
http://www.nature.com/bjc/journal/v26/n4/abs/bjc197233a.html
Coining the term ‘apoptosis’
 A history of apoptosis

•  1842 – Carl Vogt observes apoptosis

•  1885 – Walther Flemming adds observations

•  1965 – Kerr and colleagues rediscover apoptosis

 A history of apoptosis

•  1972 – Kerr, Wyllie and Currie

–  comprehensive characterization of apoptosis
–  electron-microscopic level

Reserved for video

Kerr, J.F.R. et al. (1972)
 Apoptosis: early observations

•  Morphological changes condensation

–  separation of individual cells separation

–  cell shrinkage
fragmentation
–  condensation (cytoplasm, chromatin)
–  fragmentation (apoptotic bodies)
–  phagocytosis (engulfment) phagocytosis

–  degradation lysosomal
degradation

Kerr, J.F.R. et al. (1972)

 Movies

Plasma membrane blebbing during apoptosis Apoptosis under the microscope

1m 33s 0m 18s

Garland Science Publishing The Human Body - The End of Life (BBC)

http://www.youtube.com/watch?v=xkASer3gEnc http://www.youtube.com/watch?v=NU0M3uqGCuw
 Apoptosis: early observations
condensation

•  Stages of apoptosis separation

–  cell death specification live or die?

fragmentation

–  commitment to cell death point of no return

–  killing, engulfment and degradation
phagocytosis

lysosomal
degradation

Kerr, J.F.R. et al. (1972)

 Necrosis vs. Apoptosis
Necrosis Apoptosis
–  large numbers of cells affected –  separation of individual cells
–  cell swelling –  cell shrinkage
–  rupture of organelles –  condensation (cytoplasm, chromatin)
–  rupture of plasma membrane –  fragmentation (apoptotic bodies)
–  phagocytosis (engulfment)
–  degradation

Immune response No immune response

(inflammation)

Kerr, J.F.R. (1995)

What are the molecular mechanisms?
 First molecular clue
DNA fragmentation

•  1978 – Zakharyan and Pogosyan

–  chromatin is cleaved
–  inter-nucleosomal fragments
–  multiples of ~180 base pairs
 First molecular clue
DNA fragmentation

•  DNA fragmentation
–  cleavage of chromatin into inter-nucleosomal
fragments (~180 bp and multiples thereof)

.
.
.

~180 bp

~180 bp
 First molecular clue
DNA fragmentation

•  DNA fragmentation as an assay

–  isolate DNA from cells, analyze on gel, check for DNA ladder
•  TUNEL assay
–  also indicates the presence of DNA fragmentation
–  used for individual cells in situ

First clue: DNA fragmentation

Assays: DNA laddering, TUNEL assay

Implication: endonucleases are being activated
 Second molecular clue
PS flipping
“eat me”
•  Macrophages (phagocytes)
–  recognize apoptotic signals and bind to them
–  engulf apoptotic cells and degrade them

How do phagocytes recognize

apoptotic cells?

“eat me” signals

 Second molecular clue
PS flipping

•  The plasma membrane

–  lipid bilayer (inner/outer leaflets) composed of different phospholipids
•  phosphatidylcholine (PC)
•  phosphatidylethanolamine (PE)
•  phosphatidylglycerol (PG)
•  phosphatidylserine (PS)

outer
inner
 Second molecular clue
PS flipping

•  Phospholipid asymmetry
–  Some predominately found in the outer leaflet (e.g. PC)
–  Some predominately found in the inner leaflet (e.g. PS)

phosphatidylserine
(inner leaflet)
Healthy cell
 Second molecular clue
PS flipping

•  Macrophages bind to, and engulf, vesicles with (–) charge on outer leaflet
–  phosphatidylserine (PS) is negatively-charged
–  Hypothesis: · PS asymmetry is lost in apoptotic cells
· macrophages recognize PS as an “eat me” signal

– – – – – – –
outer
inner
 Second molecular clue
PS flipping
phosphatidylserine “eat me”
(outer leaflet)
•  1992 – Henson and coworkers find PS
asymmetry in apoptotic cells
–  phosphatidylserine (PS) is externalized
–  appears on outer leaflet of cells
–  generation of the ‘eat-me-signal’

In apoptotic cells, externalization of

phosphatidylserine (PS) to the outer leaflet of
the plasma membrane is an “eat me” signal

Apoptotic cell
 Second molecular clue
PS flipping

•  Annexin V assay AV
–  protein binds to negatively-charged phospholipids, such as PS
–  protein is coupled to a fluorophore, which gives off fluorescence
–  this fluorescence can be visualized using microscopy

AV
 Second molecular clue
PS flipping

•  Flippases
–  transfer phospholipids from
inner to outer leaflet

Second clue: PS Flipping

flippase
Assay: Annexin V labeling
Implication: involvement of flippases