All tags Apoptosis Introduction to apoptosis

Introduction to apoptosis

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​​​​​A first glance at apoptosis, a regulated form of cell death that plays an essential role in multicellular organisms.


Apoptosis is characterized by several biochemical features including cell shrinkage, membrane blebbing, chromosome condensation (pyknosis), nuclear fragmentation (karyohexis), DNA laddering and the eventual engulfment of the cell by phagosomes. In contrast to necrosis, the apoptotic cell does not provoke an inflammatory response, and only individual cells are affected by apoptosis in vivo.

Apoptosis and necrosis represent two extremes of cell death, with a full range of variations in between. There are other forms such as necroptosis or autophagy, which share some of the characteristics of either form. As cell death can occur by several different paths, both morphologically and biochemically, researchers need to examine multiple biochemical markers at carefully selected time points to determine the mechanism of cell death in their particular experimental system.

Apoptotic mechanism

During apoptosis, a family of cysteine-aspartate proteases known as caspases accelerates cell death through restricted proteolysis of over 400 proteins. The two main pathways through which this cell death process is initiated are the intrinsic and extrinsic cell death pathways, both converging on caspase activation.

​​​​​​​​The intrinsic cell death pathway is governed by the Bcl-2 family of proteins, which regulate commitment to cell death through the mitochondria. A myriad of intracellular death signals are communicated through the intrinsic cell death pathway, such as DNA damage, oncogene activation, growth factor deprivation, ER stress and microtubule disruption. The key step in the intrinsic cell death pathway is the permeabilization of the mitochondrial outer membrane, which has been identified as a ‘point of no return’ after which cells are committed to cell death. Following permeabilization, release of various proteins from the mitochondrial intermembrane space promotes caspase activation and apoptosis. Cytochrome C binds APAF-1 (apoptosis protease-activating factor 1), inducing its oligomerization and thereby forming a structure called the apoptosome that recruits and activates an initiator caspase, caspase 9. Caspase 9 cleaves and activates the executioner caspases, caspase 3 and 7, leading to apoptosis. 

Activation of the extrinsic cell death pathway occurs following the binding on the cell surface of “death receptors” to their corresponding ligands such as Fas, TNFR1 or TRAIL. These death receptors have two distinct signaling motifs, death domains (DD) and death effector domains (DED), that allow them to interact and recruit other adaptor molecules such as Fas-associated death domain protein (FADD) and caspase 8, which can then directly cleave and activate the executioner caspases, caspase 3 and caspase 7, leading to apoptosis.

Activation of the extrinsic cell death pathway occurs following the binding on the cell surface of “death
receptors” to their corresponding ligands such as Fas, TNFR1 or TRAIL. These death receptors have
two distinct signaling motifs, death domains (DD) and death effector domains (DED), that allow them to
interact and recruit other adaptor molecules such as FAS-associated death domain protein (FADD), and
caspase 8, which can then directly cleave and activate caspase 3 and caspase 7, leading to apoptosis.

Crosstalk between the extrinsic and intrinsic pathways occurs through caspase 8 cleavage and activation of the BH3-only protein BID (BH3-interacting domain death agonist), the product of which (truncated BID, known as tBID) is required in some cell types for death receptor-induced apoptosis.

​​​​Parameters of apoptosis

Apoptosis occurs via a complex signaling cascade that is tightly regulated at multiple points, providing many opportunities to evaluate the proteins involved. The image below shows the main parameters of apoptosis and the approximate relative time when they are likely to be detected.

Cell death parameters

These parameters do not happen in a sequential order, and many of them will overlap and occur at the same time. Loss of membrane asymmetry or initiation of caspase cascade are biochemical features of apoptosis which do not necessarily lead to cell death. However, other downstream features such as decrease of the mitochondrial membrane potential (ΔΨm) and concomitant release of cytochrome C into the cytosol are generally considered points of no return, after which it is very unlikely the cell will survive.

Some of these biochemical and morphological features associated with apoptosis can also be observed in other types of cell death such as necrosis or necroptosis. Therefore, it is recommended to analyze more than one parameter to identify apoptosis as a cause of death in the studied population.

The table below shows the main parameters associated with apoptosis and the most common methods to study them. 

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Apoptotic parametersDetection methodsSample type

Loss of membrane asymmetry/ PS exposure

Flow cytometry analysis of annexin V bindingLive cells
Cleavage of anti-apoptotic Bcl-2 family proteinsWestern blot assessment of protein cleavage

Cell extracts

Tissue extracts







Caspase activation

Colorimetric / fluorometric substrate- based assays in microtiter plates

Cell extracts

Tissue extracts

Detection of cleavage of fluorometric substrate in flow cytometry / microscopy or by microtiter plates analysisLive cells
Western blot analysis of pro- and active caspase

Cell extracts          

Tissue extracts

Flow  cytometry / microscopy analysis with antibodies specifically recognizing the active form of caspases

Live cells (Flow Cyt)                  

Fixed cells (Microscopy)

Microplate spectrophotometry analysis with antibodies specifically recognizing the active form of caspases

Cell extracts

Tissue extracts



Caspase substrate (PARP) cleavage

Microplate spectrophotometry analysis with antibodies specific for cleaved PARP

Cell extracts

Tissue extracts

Live cells (In cell ELISA)

Western blot analysis of cleaved PARP

Cell extracts

Tissue extracts

Non-caspase proteases (cathepsins and calpain) activationColorimetric / fluorometric substrate-based assays in microtiter plates

Cell extracts

Tissue extracts


Mitochondrial transmembrane potential (Δ​ψm) decrease

Flow cytometry/ microscopy / microplate spectrophotometry analysis with Δ​ψm sensitive probesLive cells
Oxygen consumption studiesLive cells



Cytochrome C release

Western blot analysis of presence of cytochrome C in the cytosolFixed cells
Antibody-based microscopy analysis of presence of cytochrome C in the cytosolFixed cells
Increase of sub G1 populationFlow cytometry analysis of sub G1 peakFixed cells


Nuclear condensation

Flow cytometry analysis of chromatin condensationLive cells
Microscopy analysis of chromatin condensationLive cells


DNA fragmentation

Analysis of DNA ladder in agarose gelDNA
Analysis of DNA fragmentation by TUNELLive cells


Membrane blebbing

Light microscopy analysis of membrane blebbingLive cells
Western blot analysis of cleaved substrate (gelsolin, ROCK1)

Cell extracts

Tissue extracts





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