Download Topic Report Cell Death: From Morphological to Molecular Definitions

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Introduction

Cell death classification:
 Morphological appearance
○ Apoptotic, Necrotic, Autophagic, Mitotic catastrophe
 Molecular (enzymological) criteria
○ Involvement of nucleases or proteases.
 Functional aspects
○ Programmed / accidental
○ Physiological / pathological
 Immunological characteristics
○ Immunogenic or non-immunogenic
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Morphological Aspects of
Different Modes of Cell Death
Cell death mode
Morphological
characteristics
Apoptosis
Reduction of cellular and nuclear
volume (pyknosis).
Nuclear fragmentation (karyorrhexis).
Plasma membrane blebbing.
Autophagy
Lack of chromatin condensation.
Massive vacuolization of the
cytoplasm.
Necrosis
Cytoplasmic swelling.
Swelling of cytoplasmic organelles.
Rupture of plasma membrane.
Mitotic
catastrophe
Micro-nucleation
Multi-nucleation
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Morphological vs. Biochemical
Classification of Cell Death

Morphological classifications dominate the cell death
research scene even after the introduction of
biochemical assays into the laboratory routine.

Biochemical methods for assessing cell death are
quantitative, and hence less prone to operatordependent misinterpretations.
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The Biochemical Processes
Underlying Cell Death
Programmed cell death =\= apoptosis =\= caspase activation =\= non-immunogenic cell death
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When is a Cell ‘Dead’ ?
• Dying cells are engaged in a process that is reversible until a first irreversible
phase or ‘point-of-no-return’ is trespassed.
Definition
Notes
Methods of detection
Massive activation of
caspases
Caspases execute the classic apoptotic
program, yet in several instances,
caspase-independent death occurs.
Immunoblotting.
FACS quantification by means
of fluorogenic substrates or
specific antibodies.
Δψm dissipation
Protracted Δψm loss usually precedes MMP
and cell death.
FACS quantification with Δψm sensitive probes.
Calcein-cobalt technique.
MMP
Complete MMP results in the liberation of
lethal catabolic enzymes or activators of
such enzymes.
IF colocalization studies.
Immunoblotting after
subcellular fractionation.
PS exposure
PS exposure on the outer leaflet of the
plasma membrane often is an early event
of apoptosis, but may be reversible.
FACS quantification of Annexin
V binding
•
•
•
•
•
Δψm,, : mitochondrial transmembrane potnetial
FACS : fluorescence-activated cell sorter
IF : immunofluorescence
MMP : mitochondrial membrane permeabilization
PS : phosphatidylserine
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Definitions of Cell Death

A cell should be considered dead when any one of
molecular-morphological criteria is met:
Definition
Notes
Methods of detection
Loss of plasma
membrane integrity
Plasma membrane has broken down,
resulting in the loss of cell’s identity.
(IF) Microscopy and/or FACS
to assess the exclusion of vital
dyes.
Cell fragmentation
The cell has undergone complete
fragmentation into discrete bodies (usually
referred to as apoptotic bodies).
(IF) Microscopy.
FACS quantification of
hypodiploid events (sub-G1
peak).
Engulfment by adjacent
cells
The corpse or its fragments have been
phagocytosed by neighboring cells.
(IF) Microscopy.
FACS colocalization studies.
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Conclusion


Until now, the field of cell death research has been
dominated by morphological definitions that ignore
relentlessly increasing knowledge of the
biochemical features of distinct cell death
subroutines.
It would be a desideratum to replace
morphological aspects with biochemical/functional
criteria to classify cell death modalities.
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