Introduction to Apoptosis

Introduction to apoptosis

Introduction

Apoptosis or programmed cell death is defined as a mechanism of cellular suicide which occurs after sufficient cellular damage. Apoptosis is first characterized by a change in the refractive index of the cell followed by cytoplasmic shrinkage and nuclear condensation. The cell membrane begins to show blebs and eventually these blebs separate from the dying cell and form "apoptotic bodies". Apoptotic cells also cease to maintain phospholipid asymmetry in the cell membrane, and phosphotidylserine appears on the outer leaflet. The mitochondrial outer membrane also undergoes changes that include loss of its electrochemical gradient, and substances like cytochrome c leak into the cytoplasm. Finally, adjacent cells or macrophages phagocytose apoptotic bodies and the dying cell. The apoptotic cell does not provoke an inflammatory response, and only individual cells are affected by apoptosis in vivo .

The mechanisms of apoptosis

Apoptosis can be induced in response to many external stimuli (extrinsic pathway) including activation of cell surface receptors such as Fas, TNFR1 (tumor necrosis factor receptor 1), TRAIL-R1 (TNF-related apoptosis- inducing ligand receptor 1), TRAIL-R2, p75-NGFR (p75-nerve growth factor receptor) and others. These "death receptors" have two distinct signaling motifs: death domains (DD) and death effector domains (DED) that allow them to interact with other proteins involved in the apoptosis cascade. Typically the extrinsic pathway involves activating the initiator caspase, caspase-8, which in turn either activates caspase-3 or cleaves the Bcl-2 family member, Bid, leading to the formation of the apoptosome and activation of caspase-9.

The alternative mitochondrial pathway (intrinsic pathway) can be activated by events such as DNA damage. The mitochondrial pathway involves members of the Bcl-2 family that regulate cytochrome c release from the mitochondria. Expression of phagocytic recognition molecules at the surface of apoptotic cells may lead at any stage to their removal from the tissue. One of the earliest changes in apoptosis is the movement of negatively charged phospholipids molecules (phosphotidylserine) from the inside of the cell membrane to the outside. Annexin-V labelling of phosphotidylserine exposure is an established procedure for identifying cells at an earlier stage in apoptosis.

Please see our apoptosis pathway diagram on the following link for further details: http://www.abcam.com/ps/pdf/neuroscience/14-3-3_apoptosis.pdf

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Detection of Apoptosis at various stages:

Early detection:

The early stages of apoptosis are characterized by changes to mitochondrial membrane potential and cell membrane asymmetry (but not increased cell permeability). In early stages if apoptosis, the loss of cell membrane asymmetry lease to exposure of phosphatidylserine (PS) residues (normally hidden within the plasma membrane) on the surface of the cell. This can used to detect and measure apoptosis. During apoptosis, PS is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+-dependent affinity for PS. Abcam supplies many antibodies to Annexin V. We also sell several Annexin V kits.

Caspase detection:

Another early event is the activation of the caspase enzyme cascade. Antibodies to the Caspase proteins can be used to detect this cascade at various stages. There are also caspase kits available .

Late detection:

Changes in cellular morphology including cell shrinkage, nuclear condensation, and pyknosis, along with other events leading to loss of mitochondrial membrane potential, loss of plasma membrane asymmetry, and DNA cleavage between nucleosomes characterize later stages in apoptosis. DNA damage can be detected at this stage using Tunnel methods. Several dyes, such as 7-amino actinomycin D (7-AAD) can be used to detect the change in cell membrane potential and appearance of larger pores in the cell membrane. 7-AAD is used in conjunction with Propidium Iodide (PI). 7-AAD is more permeable in apoptosing cells than PI. Over a given time period, more cells will stain with 7-AAD than PI. Apoptosing cells will stain with 7-AAD earlier than with PI. Flow cytometry is a useful tool for measurement and quantification of this process.