Apoptosis : Mechanism and Morphologic Changes

  Apoptosis, also called "programmed cell death" is the process where the cell regulates its own death through the production of certain enzymes. These enzymes cause degradation of nuclear and cytoplasmic material and the cell breaks into fragments called apoptotic bodies. These apoptotic bodies are then removed by the process of phagocytosis.

  Apoptosis could be triggered by factors such as infections, especially viral infections, misfolding of proteins due to mutations and DNA damage due to mutation, radiation, hypoxia and free radicals.

  Apart from pathologic factors inducing programmed cell death, apoptosis is also a homeostatic mechanism, where cells that are not needed are killed, thereby maintaining a steady state population of cells.

Cell undergo Apoptosis

Morphological changes during Apoptosis

  During apoptosis, the cell undergoes certain morphologic changes that can be seen in light and electron microscopy.
• The cell shrinks and becomes smaller in size.
• The cytoplasm and organelles become tightly packed.
• The nuclear chromatin shrinks and becomes condensed at the center or at the periphery, a process called pyknosis.

Following this, chromatin material undergoes  karyorrhexisi.e it disintegrates and becomes fragmented. Under the microscope, a cell undergoing apoptosis would appear shrunken, with a dense eosinophilic cytoplasm and small clumps of hematoxophilic chromatin material.

Further, the cell starts to form blebs on its surface and starts to break off into small fragments called apoptotic bodies. These apoptotic bodies have portions of cytoplasm, organelles and nuclear fragments of the cell.

Apoptosis does not elicit inflammation

It is important to understand that apoptosis does not elicit inflammation, unlike another form of cell death called necrosis. This is because; apoptotic bodies have an intact plasma membrane and prevent any content from leaking out into the interstitial space. Also, apoptotic bodies are quickly recognised by phagocytes and removed from the environment.

Pathways of inducing cell death

  A cell can undergo apoptosis due to several reasons and depending on the etiologic factors may have 3 different pathways of initiating cell death. Apoptosis could be initiated by signals from
- The intrinsic pathway,
- The extrinsic pathway and
- The perforin /granzyme pathway.

Intrinsic pathway

  In the intrinsic pathway, mitochondria become leaky and ooze out proteins called cytochrome C, which initiate apoptosis.

  Usually the cytoplasm and mitochondrial membrane harbour proteins called Bcl-2 and Bcl-x which are anti-apoptotic and preserve the integrity of the mitochondrial membrane, preventing apoptotic proteins like cytochrome C from leaking into the cytoplasm.

However, in the absence of a growth signal, or insults due to radiation or protein misfolding, stress proteins called “BH3 only” proteins are stimulated. “BH3 only” proteins comprising of Bim, Bid and Bad  proteins block the function of Bcl-2 and Bcl-x.

These proteins further activatetwo pro-apoptotic effectors called Bax and Bak, which create channels in the mitochondrial membrane, allowing intra-mitochondrial proteins like cytochrome C to leak into the cytoplasm.

  Cytochrome C, in the cytoplasm, binds with a protein called Apaf - 1 (Apoptosis activating factor 1) to form a complex called “apoptosome”.

Apoptosome complex binds with Caspase 9 and begins to cleave and activate adjacent caspase-9 molecules.
  Caspase 9 is an initiator caspase and activated Caspase 9 molecules activate executioner Caspases like caspase 3 and caspase 6 leading to apoptosis of the cell.

Causes Apoptosis

Cytotoxic T-lymphocytes or CD8 T cells cause apoptosis of infected cells or tumor cells by Fas Ligand and Fas/death receptor interaction. Many cell types express a receptor called the Fas receptor and cytotoxic T cells express Fas Ligand. These Cytotoxic T cells bind to the Fas receptor on tumor cells or infected cells through their Fas Ligands.

This interaction may produce apoptotic signals through two pathways -
-  Extrinsic pathway
-  Granzyme/Perforin pathway.

Extrinsic pathway

The Extrinsic Pathway involves binding of an adapter protein called Fas-associated death domain (FADD) to the cytoplasmic end of at least 3-4 Fas ligands. This then binds with caspase 8, another initiator caspase, which gets cleaved to become active.

  The active caspase 8 further activates other caspase 8 molecules. These in turn activate executioner Caspases 3 and 6 leading to apoptosis of the cell.

Granzyme/Perforin pathway

At times the Granzyme/Perforin pathway is initiated on FasL/Fas receptor interaction. T cells release perforins, which form trans-membrane pores on the cells, through which granzymes, another protein secreted by T-cells, enter.

Granzymes, could either directly activate executioner caspase molecules or cause DNA cleaving, leading to apoptosis.

Execution of Apoptosis

Executioner caspases 3 and 6 cause degradation of
chromosomal DNA and also degradation of cytoskeletal proteins, which cause the morphological changes such as nuclear fragmentation and cellular shrinkage respectively. However, it is not yet known what causes changes like cellular blebs and apoptotic bodies.

Apoptotic bodies are coated with a phospholipid called  phosphotidylserine, which is recognised by phagocyte receptors. Also, apoptotic bodies may be coated with opsonins like antibody IgG or complement proteins like C3b which are recognised by phagocytes thus facilitating rapid phagocytosis of apoptotic bodies.