Buněčná smrt aneb mnoho způsobů, jak zemřít.
Dr. Jan Balvan
Department of Pathological Physiology
Cell Death: Many Ways to Die.
Brief History of Cell Death Research
1842 - Karl Vogt noticed dying cells in toads (formation of
vertebrae). The first scientific observation of regulated cell death
(RCD).
1965 - Lockshin and Williams - specific cells die during the
metamorphosis of the silkworm, this type of cell death is
'programmed' because these cells were destined to die according
to a 'construction manual' for the insect.
1972 - Kerr et al. - specific type of cell death in human tissues in
which the cells and nuclei became condensed and fragmented,
and they called this cell death process 'apoptosis'. They proposed
that apoptosis is crucial for regulating cell populations during
tissue development and turnover
1973 – Schweichel and Merker originally described three forms of
programmed cell death which they called types I (apoptosis), II
(autophagy) and III (necrosis).
2005 – present, NCCD (Nomenclature Committee on Cell
Death) publication (Guido Kroemer et.al.).
Carroll M. Williams Richard A. Lockshin
John Kerr Andrew Wyllie
Karl Vogt
Guido Kroemer
Lockshin, R. Programmed cell death 50 (and beyond). Cell Death Differ 23, 10–17
(2016). https://doi.org/10.1038/cdd.2015.126
Tang, D., Kang, R., Berghe, T.V. et al. The molecular machinery of regulated cell
death. Cell Res 29, 347–364 (2019). https://doi.org/10.1038/s41422-019-0164-5
Brief History of Cell Death
Lockshin, R. Programmed cell death 50 (and beyond). Cell Death Differ 23, 10–17 (2016).
https://doi.org/10.1038/cdd.2015.126
• Cell death plays a central role in all aspects of life. It is involved in the development of
multicellular organisms and tissue homeostasis where cell death depletes dispensable
cells.
• Cells may die from accidental cell death (ACD) or regulated cell death (RCD).
• ACD is a biologically uncontrolled process, whereas RCD involves tightly structured
signaling cascades and molecularly defined effector mechanisms.
• Cell death is critical for fighting off infections and is associated with multiple diseases
that are caused by deregulated or dysfunctional cell death signaling.
Accidental vs. Regulated Cell Death
Galluzzi, L., Bravo-San Pedro, J., Vitale, I. et al. Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ 22, 58–73 (2015). https://doi.org/10.1038/cdd.2014.137
Regulated Cell Death
Galluzzi L, Vitale I, Aaronson SA, et al. Molecular
mechanisms of cell death: recommendations of
the Nomenclature Committee on Cell Death
2018. Cell Death and Differentiation.
2018;25(3):486-541.
• Apoptosis is the first described form of programmed cell death, and it plays a critical role in tissue
homeostasis.
• It contributes to cell turnover, the proper functioning of the immune system, and embryonic development.
• There are several key characteristics of apoptosis:
cellular, organelle, and DNA fragmentation and formation of apoptotic bodies
active, energy consuming process executed by a subset of cellular proteins
Even though, in general, this process is immunological silent, apoptosis has been shown to be involved in
inflammatory pathologies as well.
Apoptosis
Apoptosis
There are two (or 3) major pathways that mediate apoptosis: intrinsic and extrinsic pathways.
The third modality of apoptosis induction is
cell‐based. Cytotoxic T cells can engage cells that
present non‐self‐antigens leading to cell death
induction by proteases called granzymes.
All apoptotic pathways converge on the central
proteases of this pathway: caspases, which are
either playing a role in transmitting cell death
stimulus (initiator caspases) or in the execution
(effector caspases).
Intrinsic apoptosis is controlled by the equilibrium
of the different Bcl‐2 (B‐cell lymphoma 2) family
members which can be disrupted by various stimuli
leading to cell death.
During extrinsic apoptosis, TNF (tumor necrosis factor) superfamily (TNFSF)
can induce cell death by binding to their cell surface receptors and
activating a deathly signaling cascade causing extrinsic apoptosis.
Caspases
• Caspases (cysteine-aspartate proteases) are proteolytic
enzymes generally known for their role in controlling cell
death and inflammation.
• Their role in cell death was described more than 20 years ago
with the discovery of ced-3 as a trigger for cell death during
the development of Caenorhabditis elegans.
• Caspases are involved in cell death by apoptosis, necroptosis
and pyroptosis. Caspase function is not just about cell death.
• Non-apoptotic roles of caspases include proliferation, tumor
suppression, differentiation, nervous system development
and axon navigation, aging and angiogenesis.
Shalini S, Dorstyn L, Dawar S, Kumar S. Old, new and emerging functions of caspases. Cell Death & Differentiation. 2015;22(4):526-539.
https://hopes.stanford.edu/caspase-6-inhibition/
Caspases
Domain structure and functional
classification of placental mammalian
caspases.
Caspase-1, -4, -5, -11 and -12 are
inflammatory caspases.
Apoptotic caspase-2, -8, -9 and -10 are
initiators
Caspase-3, -6 and -7 are key
executioner caspases.
CARD, caspase recruitment domain;
DED, death effector domain;
L, large subunit;
S, small subunit;
S*, short form;
L*, long form
Shalini S, Dorstyn L, Dawar S, Kumar S. Old, new and emerging functions of caspases. Cell Death & Differentiation. 2015;22(4):526-539.
Intrinsic Apoptosis
Bedoui, S., Herold, M.J. & Strasser, A. Emerging connectivity of programmed cell death
pathways and its physiological implications. Nat Rev Mol Cell Biol 21, 678–695 (2020).
https://doi.org/10.1038/s41580-020-0270-8
• Involves increases in the expression or activity of proapoptotic
BH3-only proteins that bind with high affinity to
members of the pro-survival BCL-2 protein family, which in healthy
cells keep the effectors of apoptosis, BAX, and BAK, in inactive
states.
• When all pro-survival BCL-2 proteins within a cell are functionally
neutralized by BH3-only proteins, BAK and BAX are unleashed in
order to oligomerize and assemble into structures that cause a
breach of the outer mitochondrial membrane, thereby
inducing mitochondrial outer membrane
permeabilization (MOMP).
• MOMP causes the release of mitochondrial proteins.
• Cytochrome c binds to APAF-1 promoting formation of the
apoptosome.
• Pro-forms of the initiator caspase 9 are recruited into the
apoptosome, resulting in caspase 9 activation promoting the
downstream proteolytic activation of the effector caspases 3 and 7.
• Activation of caspase 3 and 7 cascade can be attenuated by XIAP,
one of the inhibitor of apoptosis proteins (IAPs). MOMP also causes
the release of SMAC (also known as DIABLO) and HTR2, which both
can block XIAP and thereby prevent it from inhibiting caspases.
Extrinsic Apoptosis
• Triggered by TNF family ligand‐receptor interactions, most
prominently by TNF family ligands: TNF, FasL, TRAIL, and TL1A.
• The receptor complexes either recruit FADD (Fas‐associated protein
with death domain) or TRADD (TNFRSF1A‐associated via death
domain) to the oligomerized complex.
• FasL binds to its transmembrane receptor Fas, which recruits FADD
via death domain (DD) interactions.
• FADD contains a DD and also a death effector domain (DED), which
allows the recruitment of caspase‐8 forming the death inducing
signaling complex—DISC.
• The proximity of multiple caspase‐8 molecules induces the
transactivation by proteolytic cleavage.
• Cleavage results in the p18 and p10 fragments which activate
caspase‐3 and caspase‐7 (type I apoptosis).
• Insufficient activation of caspase‐3 leads to type II apoptosis in
which caspase‐8 cleaves the BH3‐only protein BID to generate its
activated form: truncated BID (tBID).
• tBID stimulates intrinsic apoptotic pathway by directly binding to
Bax/Bak inducing MOMP (type II apoptosis).
• The two pathways are cell line dependent, and their activation is
differentially regulated by XIAP expression.
The EMBO Journal (2021) 40: e106700.
Bedoui, S., Herold, M.J. & Strasser, A. Emerging connectivity of programmed cell death
pathways and its physiological implications. Nat Rev Mol Cell Biol 21, 678–695 (2020).
https://doi.org/10.1038/s41580-020-0270-8
Apoptosis sensitivity during development
Apoptosis is differently and dynamically regulated across the mammalian lifespan.
Tissues that are highly proliferative (developing tissues, adult haematopoietic system) are typically primed for apoptosis (red).
High apoptotic priming in these tissues makes them highly sensitive to various insults .
Tissues that are largely postmitotic are apoptosis refractory (green), whereas tissues that are characterized as unprimed (yellow)
contain highly heterogeneous cell types that differ in apoptosis sensitivity.
The level of priming within cells or tissues is dependent on the expression of BCL-2 family proteins BAX and/or BAK.
Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nature Reviews Molecular Cell Biology. 2019;20(3):175-193.
Regulated Necrosis vs Apoptosis - Lytic vs Non-lytic cell death
Galluzzi L, Vitale I, Aaronson SA, et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee
on Cell Death 2018. Cell Death and Differentiation. 2018;25(3):486-541.
• Non-lytic cell death, apoptosis
(the integrity of plasma
membrane is sustained).
• Plasma membrane rupture
(PMR) is the final cataclysmic
event in lytic cell death
(regulated or accidental
necrosis).
• PMR releases intracellular
molecules known as damageassociated
molecular patterns
(DAMPs) that propagate the
inflammatory response.
Bedoui, S., Herold, M.J. & Strasser, A. Emerging connectivity of programmed cell death pathways and its physiological implications. Nat Rev Mol Cell Biol 21, 678–695 (2020). https://doi.org/10.1038/s41580-020-0270-8
Pyroptosis
Pyroptosis is a potent inflammatory mode of lytic cell death triggered by diverse infectious and sterile insults.
It is driven by the pore-forming fragment of gasdermin D (GSDMD) and releases two exemplar proteins:
pro-inflammatory cytokine IL-1β, and LDH, a standard marker of PMR and lytic cell death.
Two sequential steps for pyroptosis:
• initial formation of a small plasma membrane pore that causes the release of IL-1β and non-selective ionic fluxes
• subsequent PMR attributable to oncotic cell swelling with final PMR by NINJ1 protein.
Caspase 1 and caspase 11 (caspase 4 and
caspase 5 are the human homologues of
mouse caspase 11) have important roles
in pyroptosis, that is widely considered to
be involved in defending the organism
against pathogens
Bedoui, S., Herold, M.J. & Strasser, A. Emerging connectivity of programmed cell
death pathways and its physiological implications. Nat Rev Mol Cell Biol 21, 678–
695 (2020). https://doi.org/10.1038/s41580-020-0270-8
Necroptosis
Necroptosis is a pathway for genetically programmed lytic cell death that is thought to have a role in the killing of pathogeninfected
cells and/or damaged cells during certain degenerative or inflammatory disorders.
Necroptosis can be induced by multiple innate immune signaling pathways.
These pathways all lead to the phosphorylation and activation of the necroptotic kinase RIPK3, which in the case of deathreceptor-induced
necroptosis also requires RIPK1 activity. RIPK3 activates MLKL through phosphorylation and allows
trafficking of MLKL to the plasma membrane, where it induces membrane permeabilization.
Bedoui, S., Herold, M.J. & Strasser, A.
Emerging connectivity of programmed cell
death pathways and its physiological
implications. Nat Rev Mol Cell Biol 21, 678–
695 (2020). https://doi.org/10.1038/s41580-
020-0270-8
The role of cell death in host responses to infection.
Intracellular pathogens released from dying cells can be engulfed
by nearby macrophages and neutrophils whose subsequent
activation results in the secretion of cytokines and chemokines
that support the immune response (for example, via recruitment
of cells involved in adaptive immunity).
DAMPs, PAMPs and antigens released from dying cells are also
sensed and engulfed by dendritic cells, and this allows these
potent antigen-presenting cells to prime naive T lymphocytes,
which enables them to find and destroy additional infected cells,
as well as aiding in the differentiation of B cells into plasma cells
that produce pathogen-specific antibodies
Bedoui, S., Herold, M.J. & Strasser, A. Emerging connectivity of programmed cell death pathways and its
physiological implications. Nat Rev Mol Cell Biol 21, 678–695 (2020). https://doi.org/10.1038/s41580-020-0270-8
The role of cell death in host responses to infection.
The role of cell death in host responses to infection.
Although necrosis and pyroptosis are important barriers against microbial pathogens, disruption of their regulation
causes numerous autoimmune and inflammatory conditions leading to various diseases.
Regulated Cell Death
Necroptosis, pyroptosis, ferroptosis, and NETosis are types of
programmed necrosis where lytic cell death is mediated by an
activatable genetic program.
Accidental and programmed necrosis share morphological
features: Swelling of the cell and permeabilization of the cell
membrane associated with the release of potentially
dangerous contents of the dying cell (DAMPs) - induction of
inflammation.
Inflammation associated with necrosis is caused by
inflammatory cytokines and DAMPs (cell molecules released
into the environment with loss of membrane integrity) from
cells subject to necrotic cell death.
Defects in programmed necrosis and efferocytosis are
associated with the development of inflammation and
autoimmune diseases.
Ahmed A, Tait SWG. Targeting immunogenic cell death in cancer. Molecular Oncology. 2020;14(12):2994-3006.
Other forms of regulated cell death
Autophagy
The process of recycling cellular material, adaptation and maintenance of homeostasis of the internal environment of the cell.
Under physiological conditions, it contributes to genome stability by regulating damaged proteins and organelles.
An important process in the differentiation of cells of the immune system and other tissues.
An important role in the adaptation of the newborn to oral food.
Disruption of this process is associated with many human pathologies: Neurodegenerative diseases (Alzheimer, Parkinson,…) insufficient
degradation of proteins by autophagy (eg. beta amyloid in the plaques of NS cells) is the cause of these diseases.
Cancer diseases:
An important mechanism of
resistance (including MDR) and
tumor cell metastasis (testing of
inhibitors and inducers of
autophagy in clinical trials).
https://www.youtube.com/watch?v=Hqs1WzTwBEU&ab_channel=WallStreetJournal
Autophagy‐dependent cell death (ADCD) relies exclusively on the autophagic pathway components, which is an important
distinction given that autophagy can also coincide with other forms of cell death.
ADCD can proceed by two different pathways:
• Cell death induced by extensive degradation of organelles which is dependent on the autophagic flux.
• Autosis, does not depend on the fusion of autophagosomes and lysosomes.
In both cases, vacuole formation in the cytoplasm can be detected. Treatment of cancer cells with resveratrol triggers the
autophagic flux‐dependent ADCD, without activating apoptosis or necroptosis.
The massive degradation by lysosome fusion leads to a breakdown of the cytoplasmic organization with loss of organelles such
as endoplasmic reticulum or mitochondria.
Autosis can be induced by starvation or hypoxia, which leads to cell swelling and eventually rupture of the plasma membrane.
Autotic cells were also identified in samples of patients with severe anorexia nervosa.
ADCD has been shown in association with physiological process as well as various pathologies including reperfusion injuries
and various forms of cancer.
Autophagy‐dependent cell death (ADCD)
Other forms of regulated cell death
Mitochondrial permeability transition pore (MPTP)‐mediated necrosis
MPTP can mediate necrosis based on changes in the intracellular microenvironment. Two factors that can induce opening of
the pores are oxidative stress and cytosolic/ mitochondrial Ca2+ accumulation. The pores allow the flux of molecules leading
to breakdown of the H+ gradient and subsequently halting the ATP synthesis.
Parthanatos
Parthanatos is a form of regulated cell death dependent on poly(ADP) ribose polymerase 1 (PARP1). PARP1 is part of the
DNA repair machinery which binds DNA. Severe DNA damage by prolonged generation of reactive oxygen species or reactive
nitrogen species (RNS) induces recruitment and activation of PARP1 to the leading to the formation of PAR polymers and
depletion of NAD+ and ATP, which might be fatal for the cell.
NETosis
Neutrophils are part of the innate immune system, and their main task is to neutralize pathogens by phagocytosis or
degranulation. Another form of host defense is the formation of NET (neutrophil extracellular traps). NETosis describes the
process of neutrophil DNA release into the extracellular space. The release of neutrophil DNA containing different proteins
with anti‐pathogenic activity can be associated with cell death but can be independent of it as well.
Ferroptosis
Ferroptosis is a form of regulated cell death that depends on iron (Fe2+)‐mediated lipid peroxidation induced by ROS.
Entosis and Cannibalism
Digestion of engulfed homotypic or heterotypic cell.
Holographic Microscopy and Quantitative Phase Imaging (QPI)
• Long-term monitoring of the cell population
• Analysis of morphological and dynamic
parameters in time
Holographic Microscopy and Quantitative Phase Imaging (QPI)
▪ Beams from both arms
are focused onto the CCD
camera
▪ The beams interfere and
form a hologram
▪ The hologram is recorded
and further processed on
PC to produce quantitative
phase image
CCD
objective objective
source
sample
reference
sample
hologram quantitative phase image (QPI)
[pg/µm2]
7.8
5.8
3.9
1.9
0
QPI
Cell death detection using QPI
As a dead cell can be considered:
Cell whose membrane has lost its barrier function.
Cell which has disintegrated into separate bodies, often referred to
as apoptotic bodies.
Cell which was engulfed by professional phagocytes or surrounding
cells.
All these processes are associated with changes in cell mass!
Detekce buněčné smrti pomocí QPI
Detekce buněčné smrti pomocí QPI
Caspase-dependent
(apoptosis)
Caspase-independent
(necrosis/necroptosis)
Rozlišení mezi apoptózou a nekrózou
Apoptotic cells
Necrotic cells
Apoptotic cells Necrotic cells
Rozlišení mezi apoptózou a nekrózou
Based on morphological and dynamic parameters, we are able to automatically distinguish two distinct populations
of cells. Without the use of dyes, only on the basis of a light microscopic method.
Rozlišení mezi apoptózou a nekrózou
Rozlišení mezi apoptózou a nekrózou
Rozlišení mezi apoptózou a nekrózou
Rozlišení mezi apoptózou a nekrózou
Rozlišení mezi apoptózou a nekrózou
Dr. Martina Raudenská
Doc. Michal Masařík
Thanks for your attention.