Apoptosis is a highly regulated process of programmed cell death that occurs in multicellular organisms. This process is characterised by distinct changes in cell morphology before cell death and it occurs as a homeostatic mechanism to control cell populations in tissues throughout the development of an organism. Apoptosis also acts as a defence mechanism as part of the immune response and in response to cell damage. During the early stages of apoptosis, cell shrinkage and pyknosis occur whereby the cells become smaller and chromatin is irreversibly condensed. This results in the ‘blebbing’ of the plasma membrane and fragmentation of the cell. These fragments are then separated into apoptotic bodies consisting of cytoplasm and tightly packed organelles which are phagocytosed by macrophages, and degraded within phagolysosomes to remove them from the body. The mechanisms of apoptosis are highly complex and sophisticated, involving an energy-dependent cascade of molecular events. There are three main molecular pathways that initiate apoptosis: the intrinsic pathway, the extrinsic pathway and the granzyme B pathway. These pathways all result in the activation of caspase-3 and the execution pathway which results in the degradation of apoptotic cell fragments.
The extrinsic signalling pathways involve transmembrane receptor-mediated interactions. These involve tumour necrosis factor (TNF) receptors which, when bound by TNF ligand (e.g. TNF-α), produce a ‘death’ signal which is transmitted from the cell surface to intracellular signalling pathways. This signal also results in the recruitment of cytoplasmic adapted proteins which associate with and activate caspase-8 via the formation of a death-inducing signalling complex (DISC). This results in the activation of the execution pathway.
The intrinsic pathway is initiated in mitochondria by intracellular signals produced in response to cellular stress. These signals include free radicals, toxins, radiation and the absence of certain growth factors needed for cell survival. All of these stimuli lead to changes in the inner mitochondrial membrane that result in the loss of the mitochondrial transmembrane potential and release of pro-apoptotic proteins from the intermembrane space into the cytoplasm. These proteins activate a caspase-dependent mitochondrial pathway and which results in the activation of caspase-9 and the execution pathway.
The granzyme B pathway is a variant of the type IV hypersensitivity immune response where cytotoxic T cells kill antigen-presenting cells. These cells are able to kill target cells via the extrinsic pathway and are also able to exert their cytotoxic effects on tumour cells and virus-infected cells via a novel pathway that involves secretion of perforin which results in the formation of transmembrane pores and the release of granzyme-containing cytoplasmic granules into the target cell. Following this, granzyme B activates caspase-10 and subsequently the execution pathway.
The execution pathway is the final pathway of apoptosis and follows on from the extrinsic, intrinsic and granzyme B pathways. Caspase-3 is activated by caspase-8/9/10 which results in the activation of the CAD endonuclease for the condensation of chromatin and fragmentation of the cell cytoskeleton. This results in the disruption of cellular processes including cell division and signal transduction. Caspase-3 is also involved in the ‘flipping’ of phosphatidylserine onto the surface of the phospholipid membrane of apoptotic cells. Macrophages then detect the externalized phosphatidylserine, and engulf and destroy the apoptotic cell fragments.
There are numerous assays that can be used to evaluate apoptosis as this process occurs via a complex signalling cascade that is tightly regulated at multiple points, including the detection and sorting of apoptotic cells from live cells. One approach for detecting and differentiating apoptotic cells from live cells utilizes the membrane alterations to apoptotic cells resulting from the apoptosis pathway. In this instance, Annexin V (Annexin A5) binds to the externalized phosphatidylserine molecules present on the outer plasma membrane of apoptotic cells to sort these from live cells.
The Annexin V recombinant protein interacts strongly and specifically with phosphatidylserine and is known to compete for phosphatidylserine binding sites with the blood clot-forming protein prothrombin, thereby acting as an indirect inhibitor of the blood coagulation cascade. The Annexin V protein (blue) has a predominantly helical structure and is also known to bind calcium and sulfate ions (grey). In flow cytometry, Annexin V is conjugated to a fluorophore such as FITC (Fluorescein Isothiocyanate) or PE (R-Phycoerythrin) to allow for the fluorescent detection and sorting of apoptotic cells and dead cells from live cells. Thus, Annexin V-conjugates are capable of detecting individual cells.
In this application, Annexin V is frequently used in conjugation with propidium iodide (PI) or 7-Aminoactinomycin D (7-AAD), fluorescent intercalating agents that can sort apoptotic cells from dead cells. PI and 7-AAD are DNA stains which cannot cross the plasma membrane of live cells, but can enter through pores/holes/gaps in the plasma membrane in late apoptotic/dead cells and bind to nuclear DNA. This allows for the differentiation of early apoptotic cells and late apoptotic/dead cells. The advantage of 7-AAD over PI is the minimal spectral overlap between the emissions of 7-AAD and the Annexin V-conjugate.
|Propidium Iodide (PI)||7-Aminoactinomycin D (7-AAD)|
|Apoptosis Detection kits||Annexin V-FITC, Annexin V-APC||Annexin V-FITC, Annexin V-PE|
|Excitation Laser Line||488, 532, 561 (blue laser)||488, 532, 561 (blue laser)|
|Maximum Excitation Peak||535||546|
|Maximum Emission Peak||617||647|