Questions about apoptosis

Questions about apoptosis

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  1. Is apoptosis a part of cell division?
  2. Can apoptosis occur if DNA is damaged beyond repair?

I think this question could basically be summarized as "What is apoptosis?" I will attempt to give a brief answer to that question, answering the two aspects of the question along the way.

Apoptosis is at its root programmed cell death (PCD). It is basically when a cell (healthy or not) dies so that it can be replaced. It is happening all the time in multicellular organisms such as me, you, and the lion in the zoo. Basically, it allows all the cells to be replaced as the tissues of the body grow.

For instance, as your skin grows, cells in the dermis are dividing to produce a new epidermal layer. At the same time, cells on the outside are dying (at the stratum corneum) so that the skin does not grow infinitely and we aren't covered with 20 inches thick of skin by the ends of our lives.

The same thing is happening in, for instance, the liver. As the liver cells split, old ones are also dying. This allows us to rebuild the liver to its normal size after a surgery in which we remove a section of it. We simply have to stimulate cell growth so that it counterbalances PCD (apoptosis) and the liver grows to its normal size.

Cell death is initiated, not by the DNA, but rather by caspase activity. All cells have inactive procaspases which will destroy the cell as soon as they become active. When what we call an initiator procaspase bonds with a procaspase within the cell, the procaspase is activated, and the signal is spread across the cell. This initiator procaspase is usually triggered by a death receptor outside the cell.

More reading here

Cells that are damaged by injury, such as by mechanical damage or exposure to toxic chemicals undergo a characteristic series of changes. They (and their organelles like mitochondria) swell (because the ability of the plasma membrane to control the passage of ions and water is disrupted). The cell contents leak out, leading to inflammation of surrounding tissues.

Cells that are induced to commit suicide:

  • shrink
  • develop bubble-like blebs on their surface
  • have the chromatin (DNA and protein) in their nucleus degraded
  • have their mitochondria break down with the release of cytochrome c
  • break into small, membrane-wrapped, fragments
  • release (at least in mammalian cells) ATP and UTP
  • These nucleotides bind to receptors on wandering phagocytic cells like macrophages and dendritic cells and attract them to the dying cells (a "find-me" signal")
  • The phospholipid phosphatidylserine, which is normally hidden in the inner layer of the plasma membrane, is exposed on the surface
  • This "eat me" signal is bound by other receptors on the phagocytes which then engulf the cell fragments
  • The phagocytic cells secrete cytokines that inhibit inflammation (e.g., IL-10 and TGF-&beta)

The pattern of events in death by suicide is so orderly that the process is often called programmed cell death or PCD. The cellular machinery of programmed cell death turns out to be as intrinsic to the cell as, say, mitosis. Programmed cell death is also called apoptosis. (There is no consensus yet on how to pronounce it some say APE oh TOE sis some say uh POP tuh sis.)

1. This is an extracellular messenger of apoptosis
(a) tumor necrosis factor
(b) serine
(c) translation inhibitor
(d) ribozyme

2. This is concerned with the intrinsic pathway of apoptosis
(a) cytochrome d
(b) cytochrome c
(c) cytochrome b
(d) cytochrome a

3. Apoptotic bodies can be recognized with the presence of these on the surface
(a) phosphatidyl tyrosine
(b) phosphatidylinositol
(c) phosphatidylcholine
(d) phosphatidylserine

4. Apoptotic cells detach due to the inactivation of this
(a) PKC
(b) PKB
(c) RAF1
(d) FAK

5. Shrinking of nucleus is caused when this inactivates
(a) gelsolin
(b) tubulin
(c) actin
(d) lamin

6. This cell organelle participates actively in animal apoptosis
(a) nucleus
(b) vacuoles
(c) mitochondria
(d) chloroplast

7. This can stimulate cytochrome release from mitochondria
(a) Akt
(b) Bid
(c) Bad
(d) Smac

8. This cannot be killed by apoptosis
(a) immune cells
(b) cells with DNA damage
(c) cancer cells
(d) cell infected with viruses

9. This is an anti apoptotic protein
(a) Bim
(b) Bcl-Xs
(c) Bfl 1
(d) NOXA

10. This is an active cell death process
(a) necrosis
(b) lysis
(c) apoptosis
(d) senescence

Apoptosis: The Molecular Mechanism of Programmed Cell Death (Short Notes)

What is Apoptosis? Why apoptosis is known as the ‘Programmed Cell Death’?

The total number of cells in an organ or organism is fundamentally fixed to a specific range in all multicellular organisms. In every multi-cellular organism, the cell number is effectively controlled by two strategies- (a) by regulating cell Division and (b) by regulating cell Death. If cells are no longer needed, they commit suicide (self-destruction) by activating an intracellular death signaling programme. Thus, this death process is known as ‘Programmed Cell Death’. This programmed cell death pathway is called Apoptosis.

The term apoptosis in Greek literally mean ‘falling off’. Just like the old leaves ‘falloff’ from the trees without affecting the life of the plant, the apoptotic cell death will not interfere with the functioning of the organ and organism. The most striking feature of apoptosis is that if a cell undergoes the programmed cell death, the neighboring cells are not at all damaged. Apoptotic death of a cell and its subsequent phagocytosis by a neighboring cell or by a macrophage allow the organic components of the death cell to be effectively recycled.

The apoptosis is better known as the ‘Programmed Cell Death’. It is a natural well-orchestrated, well sequenced and timely executed chain of events leads to the death of a cell.

What are the characteristics of Apoptotic Cell Death?

An apoptotic cell death is characterized by:

Ø Shrinkage of the nucleus

Ø Loss of adhesion to the neighboring cells

Ø Formation of membrane blebs (externalization of inner leaflet of membrane)

Ø Condensation and fragmentation of the chromatin (DNA)

Ø Formation of small fragmented chromatin in membrane bounded structures called apoptotic bodies

Ø Rapid engulfment of the apoptotic cell debris by the process of phagocytosis

image source: cc wikipedia

What are ‘apoptotic bodies’?

During apoptotic cell death, the nucleus gets fragmented into many discrete chromatin bodies due to degradation of nuclear DNA. Each such nuclear fragment is surrounded by blebbed plasma membrane and these units were bud-off from the apoptotic cell. Thus, by the completion of apoptosis, the cell content is converted into many small vesicles called ‘apoptotic bodies’. Apoptotic bodies are immediately phagocytosed by the macrophages or surrounding healthy cell.

(image source: cc wikipedia)

Does apoptosis be Natural or Pathogenic?

The apoptosis is a natural process. About 10 10 to 10 11 cells in the human body dies every day by the process of apoptosis. Apoptosis is essential for the proper embryonic development in higher organisms. For example, the separation of fingers and toes in a developing human embryo occurs because cells between the digits undergo apoptosis during the embryonic development. Apoptosis also helps to prevent the perpetuation of lethal genetic damages in the body. The apoptotic cell death can occur in a cell when its genetic material is severely damaged and it cannot be rectified by the inbuilt DNA repair mechanism. Sometimes, the apoptosis can be pathogenic such as the death of healthy neurons which leads to the Alzheimer’s disease.

Webbed toes formation due to the lack of apoptotic cell death during embryonic development (cc wikipedia)

How was apoptosis discovered?

The term apoptosis was coined by John Kerr, Andrew Wyllie and A.R. Currie in 1972. The molecular basis of apoptosis was elucidated for the first time by the studies in a nematode Caenorhabditis elegans. The worm C. elegans constantly maintain their cell number in its embryonic and adult stages. During the embryonic development, the worm produces exactly 1090 cells. Among these 1090 cells, 131 cells are precisely destined to die by apoptosis during the development. Further studies in the worm identified a specific gene involved in controlling the apoptosis process and it is named as CED-3. A worm with inactive CED-3 gene by mutation fails to induce the apoptotic cell death in the embryonic development stage. This shows that CED-3 plays a crucial role in executing the process of programmed cell death. Later, scientists identified genes homologous to the CED-3 of C. elegans in other organisms including humans and subsequently named as Caspases.

Caenorhabditis elegans (image source: cc Wikipedia)

What are caspases? What is the importance of caspases in apoptosis?

Caspases are a family of proteins present in human and other animals which are homologous to the CED-3 gene product of C. elegans. Caspases are cysteine proteases involved in the execution of apoptotic cell death. Cysteine proteases are a category of protease enzymes with a cysteine residue at its active site. The caspases are produced as inactive zymogens called pro-caspases. Pro-caspases are activated to caspases during the early stages of apoptosis. Activation of pro-caspase to caspases is achieved by the catalytic removal of a part of the peptide chain. Activated caspases are responsible for most of the molecular events in the apoptosis signaling pathway.

How caspases execute apoptosis? What are the targets of caspases during apoptosis?

Caspases execute the apoptosis by selectively targeting and cleaving a large array of key molecules in the cells. Most important target molecules of caspases during apoptosis are given below:

(1). Protein Kinases: Protein kinases such as Focal Adhesion Kinase (FAK), Protein Kinase B (PKB), Protein Kinase C (PKC) and Raf1. Inactivation of the FAK cause detachment of the apoptotic cells from its neighboring cells due to the inhibition of cell adhesion.

(2). Lamin: Lamin form the inner lining of the nuclear membrane and thus the cleavage of lamins lead to the disintegration of nuclear lamina (nuclear membrane) and breakage of the nucleus.

(3). Cytoskeleton proteins: The cleavage of cytoskeleton proteins such as actin, tubulin and intermediate filaments lead to the shrinkage of the cells.

(4). CAD (Caspase Activated DNases): CAD is an endonuclease. In a normal cell, the CAD endonuclease exists in an inactive stage. The cleavage of CAD by caspase activates the CAD enzyme. Activated CAD then translocated into the nucleus and it cleaves and degrades the DNA.

What are apoptotic signals?

Any stimuli that can induce and initiate the programmed cell death pathway are called apoptotic signals. The source of apoptotic signals can be of two different types such as those from the external sources and those signals originated in the cell itself. Based on the source of signals, there are essentially two types of apoptotic signaling pathways. They are:

(1). Intrinsic Apoptotic Pathway: Here the apoptotic stimuli are originated internally in the target cell itself. The most important internal signal that induces intrinsic signaling is severe DNA damage that cannot be rectified by the DNA repair mechanism.
(2). Extrinsic Apoptotic Pathway: Here the stimuli are from the external source (not from the cell itself). The most important external apoptotic signals are cytokines such as Tumour Necrosis Factor (TNF). (The exact mechanism of intrinsic and extrinsic pathways of apoptosis will be discussed later).

Even though the signaling cascades of extrinsic and intrinsic pathways are separate, there is always cross-talk between these two pathways. The extrinsic pathway can induce the activation of the intrinsic pathway of apoptosis.

What are the significances of apoptosis?

Apoptosis is a beneficial event. Moreover, failure to regulate apoptosis can result in the damage of organs or organisms. The main significances of apoptosis are given below:

Ø Apoptosis help to maintain the homeostasis in multicellular organisms.

Ø Apoptosis also helps to maintain the proper body size.

Ø Apoptosis maintains the constancy of cell number in an organ or organism.

Ø Apoptotic cell death is a pre-request for the proper embryonic development.

Ø By the process of apoptosis, the body can eliminate unwanted cells such as:

$ A cell with severely damaged DNA

$ A cell with fatal mutation

$ A pathogen (virus) infected cell

$ Unwanted cells formed during embryonic development

$ Cells that to be killed during proper neuronal architecture development

Ø Apoptosis also helps to kill T lymphocytes with receptors for the proteins present on the normal cell. These T cells are produced during the embryonic development. These dangerous T lymphocytes are eliminated by apoptotic cell death.

Ø Apoptotic cell death can be pathogenic in some cases.

Ø Apoptosis is involved in some neurodegenerative diseases such as Alzheimer’s, Parkinson’s disease, and Huntington’s disease by the elimination of essential neurons.

Ø Failure to induce apoptosis is the main reason for most of the cancers.

How the macrophages specifically recognize the apoptotic cells for phagocytosis?

Both the intrinsic and extrinsic pathway of apoptosis converges by activating the same executioner caspases, i.e., caspase-3. As the apoptotic signaling proceeds, the cell loses its contact with the neighboring cell and starts to shrink. The cell ultimately shrinks into one or more condensed membrane-enclosed structures called the apoptotic body. The apoptotic bodies are characterized by the presence of phosphatidyl serine on their outer surface. Phosphatidyl serine is a membrane lipid present only in the inner leaflet of plasma membrane. During apoptotic cell death, the plasma membrane flipping occurs which results in the externalization of phosphatidyl serine residues. These externalized phosphatidyl serine molecules are the ‘eat me signals’ for the macrophages. The macrophages recognize these ‘eat me’ signal and they completely phagocytosis the apoptotic bodies. Thus, the apoptotic cell death is completed without spilling the cellular content into the extracellular environment. This is very significant because the release of cell debris can trigger inflammatory responses which ultimately cause severe tissue damage.

What is the relationship between Apoptosis and Cancer?

Cancer is a pathological process of uncontrolled division of cells leading to tumor development. Some cancerous cells also have the potential to invade healthy tissues by a process called metastasis. The cancer is essentially the uncontrolled division of an abnormal cell with mutations of genetic damage. If the apoptotic signaling is properly working, these unwanted cells can be eliminated from the body by programmed cell death pathway. Thus the main reason for cancer is the failure to induce apoptosis in an unwanted cell and as a result of this, the unwanted cell perpetuates without any control.

Review Questions…

(1). Define Apoptosis.
(2). Why is apoptosis known as the ‘Programmed Cell Death’?
(3). What are the characteristics of Apoptotic Cell Death?
(4). What are apoptotic bodies?
(5). Does apoptosis be Natural or Pathogenic?
(6). What is meant by membrane blebbing?
(7). How was apoptosis discovered?
(8). What are caspases? What is the importance of caspases in apoptosis?
(9). How caspases execute apoptosis? What are the targets of caspases during apoptosis?
(10). What are apoptotic signals?
(11). What are the significances of apoptosis?
(12). What is the relationship between Apoptosis and Cancer?
(13). What is the difference between apoptosis and necrosis?
(14). What is the importance of apoptosis in embryonic development?
(15). What is the role of C. elegans in the discovery of apoptosis?
(16). Who discovered apoptosis?
(17). What is CED? What is its importance in apoptosis?
(18). What are the main targets of caspases in human during apoptosis?
(19). What is meant by intrinsic pathway of apoptosis?
(20). What is meant by extrinsic pathway of apoptosis?
(21). Give some examples of intrinsic apoptotic signals.
(22). Give some examples of extrinsic apoptotic signals.
(23). Differentiate pro-caspases and caspases.

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Intrinsic Pathway of Apoptosis (Apoptosis Molecular Mechanism Part 1)

Two Types of Apoptosis

In the previous post, we have discussed the characteristic features and significance of programmed cell death or apoptosis. As we discussed, the stimuli for the execution of programmed cell death can be of internal or external to the apoptotic cell.

Based on the source of stimuli, there are two types of apoptosis signaling pathways operate in the cells. They are (1) Intrinsic pathway (stimuli are internal) and (2) Extrinsic pathway (stimuli are external) of apoptosis. Even though both the intrinsic and extrinsic pathways considerably different, there is always cross-talk between these two pathways. In the present post, we will discuss the details of INTRINSIC PATHWAY of apoptosis signalling.

What is meant by Intrinsic Pathway of Apoptosis?

In the intrinsic pathway of apoptosis, the death-inducing stimuli are originated inside the target cell itself. Mitochondria, the powerhouse of the cell, have a significant role in executing the intrinsic pathway of apoptosis. Thus, the intrinsic pathway of apoptosis is also known as the Mitochondria-mediated death pathway.

What are the stimuli for the intrinsic pathway of apoptosis?

Most commonly observed internal stimuli for the initiation of the intrinsic pathway of apoptosis are:

Ø Very high concentration of cytosolic Ca 2+ ions

Ø Presence of some viral proteins

Ø Severe oxidative stress due to the production of free radicals

What are Bcl-2 (B-cell lymphoma-2) family proteins?

The intrinsic pathway of apoptosis is facilitated by the members of Bcl-2 family proteins. The members of the Bcl-2 family proteins are characterized by the presence of one or more BH domains (Bcl-2 Homology Domain). The first identified member of Bcl-2 family proteins is Bcl-2 itself. The Bcl-2 was first identified as a cancer-causing oncogene in some human lymphomas. The gene which codes for the Bcl-2 protein was over-expressed in these cancer cells due to translocation. However, later studies have shown that Bcl-2 is not directly acting as an oncogene. They act as the oncogene by promoting the survival of the cancerous cells that would otherwise die by apoptosis.

The Bcl-2 family proteins were classified into THREE subcategories:

(1). Pro-apoptotic Bcl-2 proteins

(2). Anti-apoptotic Bcl-2 proteins

(3). BH3-only proteins

(1). Pro-apoptotic Bcl-2 members

Ø Pro-apoptotic Bcl-2 proteins promote apoptosis.

Ø In a normal cell, the pro-apoptotic Bcl-2 members are in inactive stage.

(2). Anti-apoptotic Bcl-2 members

Ø Anti-apoptotic Bcl-2 proteins inhibit apoptosis and ensure cell survival.

Ø Example: Bcl-2, Bcl-xL and Bcl-w

(3). BH3-only proteins

Ø BH3-only proteins only have a small BH3 domain.

Ø They can promote or inhibit apoptosis through an indict mechanism.

Ø Examples: Bid, Bad and Bim

The BH3-only proteins can be pro-apoptotic and they can promote apoptosis in two different ways. In some cases, they promote apoptosis by inhibiting the anti-apoptotic Bcl-2 members. In other cases, they stimulate apoptosis by activating pro-apoptotic Bcl-2 members. In both the cases, the BH3-only proteins are the key determinant in the cell survival or apoptotic cell death.

In a normal and healthy cell, the level of BH3-only proteins will be maintained in a very low concentration. Besides, in a healthy cell, the anti-apoptotic Bcl-2 proteins are able to restrain the pro-apoptotic members. If an apoptosis inducing stimulus is evoked in the cell, the levels of BH-3 only proteins are dramatically increased. This increase in the level BH3-only protein causes an imbalance in the level of pro-apoptotic and anti-apoptotic factors and beside that they shift the balance towards apoptotic cell death. When this balance is lost, the inhibitory effects of the anti-apoptotic Bcl-2 proteins are also compromised. In this critical situation, a mitochondria-mediate signaling cascade is initiated inside the cell and that will eventually result in the execution of programmed cell. The detailed mechanism of the intrinsic pathway of apoptosis is summarized below:

Ø When the cell lost the balance between pro-apoptotic and anti-apoptotic factors, a pro-apoptotic factor called Bax is translocated from the cytosol to the outer mitochondrial membrane.

Ø Bax protein undergoes a conformational change and gets inserted into the outer mitochondrial membrane.

Ø The assembly of Bax protein to the outer mitochondrial membrane is in such a way that it creates protein-lined channels or pores on the outer membrane.

Ø Due to the formation these pores, the permeability of the outer mitochondrial membrane dramatically increased.

Ø Through these pores, cytochrome c is released out of the mitochondria to the cytosol.

image source cc wikipedia

Ø The increase in permeability of the membrane also causes a dramatic loss in the electrical potential of the mitochondria.

Ø The loss of mitochondrial membrane permeability is accelerated by the increased level of Ca2+ ions in the cytoplasm which are released by the endoplasmic reticulum.

Ø The membrane potential compromised mitochondria also release SMACs (second mitochondria-derived activator of caspases) into the cytosol.

Ø SMACs bind to and inactivate all anti-apoptotic proteins in the cytosol.

Ø The complete release of cytochrome c to the cytoplasm is a ‘point of no return’. This means that the cell cannot be reverted back to its normal stage and moreover the cell should commit the apoptotic cell death.

Ø In the cytoplasm, the cytochrome c molecules combine together with Apaf-1 and Pro-caspase-9 in an ATP-dependent manner to form a multi-subunit complex.

Ø This multi-subunit complex of cytochrome c, Apaf-1 and pro-caspase-9 is called Apoptosome

image source cc wikipedia

Ø A single apoptosome may contain several molecules of pro-caspase-9.

Ø The binding of Apaf-1 induce a conformational change in the pro-caspase-9 and it is activated to its fully proteolytic form (caspase-9).

Ø Caspase 9 is the initiator caspases in the intrinsic pathway of apoptosis.

Ø Caspase 9 activates the executioner caspases such as caspases -3 and Caspase-7.

Ø Activated caspase-3 and caspase-7 cleaves its target molecules in the cell and thus the apoptotic cell death is executed.

Cell Biology 11: Apoptosis & Necrosis

This lecture will cover two different ways cells can die: apoptosis (programmed cell death) and necrosis (unplanned cell death). It is easy to tell these two apart morphologically under the microscope, as shown in this Wikimedia Commons image:

Necrosis is when cells die accidentally due to, say, trauma (ex. a poisonous spider bite), or lack of nutrients (ex. lack of blood supply). Necrosis begins with cell swelling, the chromatin gets digested, the plasma and organelle membranes are disrupted, the ER vacuolizes, the organelles break down completely and finally the cell lyses, spewing its intracellular content and eliciting an immune response (inflammation).

Apoptosis can constitute cell suicide or cell murder. Cells will commit suicide when they lack any incoming survival signal in the form of trophic factors, or when they detect extensive DNA damage in their own nucleus. Cells will murder other cells to clear out unneeded cells or to eliminate potentially self-attacking immune cells.

Either of these processes constitutes programmed cell death. During embryonic development, people have webbed hands and feet and tails the cells that constitute those parts later apoptize. Apoptosis also goes on constantly in many tissues including the intestines.

Here’s a stunning Wikimedia Commons image of apoptosis (read left to right, top to bottom) thanks to Egelberg:

  • Cell shrinks
  • Cell fragments
  • Cytoskeleton collapses
  • Nuclear envelope disassembles
  • Cells release apoptotic bodies

Notably absent from this list is ‘send out a signal.’ Apoptotic cells do not send out any signal, with one exception: they release apoptotic bodies and ‘engulfment proteins’ to induce other cells (‘phagocytic’ cells) to engulf the apoptotic bodies and and break them down in their lysosomes, but this is not much of an immune response.

Proteins important in apoptosis:

  • ‘killer proteins’: the caspases (discussed in detail below).
  • ‘destruction proteins’ that digest DNA, fragment the cell and break down the cytoskeleton
  • ‘engulfment proteins’ that elicit and promote phagocytosis by other cells

C. elegans has been the major model organism for understanding apoptosis, both by forward and reverse genetics. Forward genetics is observing a phenotype and then determining which gene gives rise to it reverse genetics is introducing a mutation into a known gene in order to see what phenotype results.

The key pathway in C. elegans apoptosis is shown in this Google Drawing I created:

Here’s an explanation of how each of these proteins does its job, from bottom up:

  • CED-3 pulls the trigger, activating apoptotic proteins that destroy the cell. (In the mammalian equivalent, CED-3 is Caspase 9, which cleaves-thereby-activating Caspase 3, which in turn destroys the cell.)
  • CED-4 activates CED-3.
  • CED-9 binds to CED-4, preventing its activation
  • EGL-1 is transcriptionally activated in response to death signals and catalyzes the release of CED-4 from CED-9.

Note that there is no robustness in this system – it is single points of failure all the way through. If CED-3 is knocked out, no apoptosis can occur. If CED-4 is knocked out, no apoptosis can occur. If CED-9 is knocked out, every cell in the worm will apoptose. If EGL-1 is knocked out, no apoptosis can occur. Note that the order the arrows point in the above diagram reflects the flow of information in the system. For instance, if EGL-1 and CED-9 are both knocked out, it’s the same as if CED-9 alone was knocked out: every cell will apoptose.

In mammals, apoptosis is governed chiefly by caspases (cysteine-aspartic proteases). The entire caspase pathway is post-translationally regulated: the caspases are always present in inactive form (called procaspases, containing a prodomain, which contains a caspase recruitment domain (CARD)) and can be activated by cleavage. This allows a very quick response if cell suicide is needed. In order for apoptosis to occur, the initiator caspases must be cleaved and dimerize. Thus activated, they must then cleave the effector caspases (aka pro-caspases), triggering a ‘caspase cascade’. This amplifies the number of activated caspases in the cell. The effector caspase have many targets including the nuclear lamina and cytoskeleton.

There are both pro-survival and pro-apoptotic caspases, and they share many common domains. Pro-survival caspases have BH1, 2, 3 and 4 pro-apoptosis caspases have either BH1, 2 and 3 or just BH3.

Inhibitor of apoptosis proteins (IAPs) restrain both the initiator and effector caspases. They each have a zinc binding domain that binds directly to caspases, inhibiting their activity.

However, there are also mitochondrial proteins called SMAC and DIABLO which inhibit the inhibitors. Upon mitochondrial injury they are released and will bind IAPs, freeing the caspases to go cause apoptosis. Another collection of mitochondrial proteins called Htra2/Omi, apoptosis-inducing factor (AIF) and endonuclease G can also be released and will cleave IAPs. AIF also causes chromosome condensation and DNA fragmentation independent of caspases.

Indeed, the mitochondria are central regulators of apoptosis. Outer mitochondrial membrane proteins Bcl-2, the BH3-only proteins and Bax are involved: Bax can form a pore in the membrane to allow cytochrome c, normally located in the intermembrane space, out into the cytosol. Bax monomers move from the cytoplasm to the outer mitochondrial membrane, where they oligomerize and permit the influx of ions through the membrane. This has also been shown in in vitro experiments where you can show that vesicles made of outer mitochondrial memrbanes are permeabilized in the presence of Bax. It is not currently known why this influx of ions leads to cytochrome c release.

Bcl-2 prevents release of cytochrome c, thus blocking apoptosis. Bcl-2 was the first mammalian apoptosis gene to be cloned. In some lymphomas, it gets translocated to a position under a stronger promoter, causing overexpression that prevents the cancer cell from apoptosing. See also bad & bid.

Once cytochrome c is released, it binds to Apaf-1 (apoptotic protease activating factor), causing the latter to hydrolyze the ATP to which it is usually bound, thus causing a conformational change that activates Apaf-1 and triggers the caspase cascade. Apaf-1 forms a disc-shaped heptamer called the ‘wheel of death’ or apoptosome which activates caspases (Wikimedia Commons image by Org1012):

When a trophic factor is present, the receptor activates PI3K, which activates PKB/Akt, which phosphorylates Bad. p-Bad is then retained in the cytosol by 14-3-3, preventing p-Bad from inhibiting Bcl-2. Thus apoptosis is prevented.

Trophic factors are an example of a cell extrinsic signal that promotes survival. There are also extrinsic signals that promote death (this is cell murder). Tumor necrosis factor (TNF-alpha) is released by macrophages to trigger cell death by binding to ‘death receptors’. Death receptors have a single transmembrane domain. They must trimerize in order to activate FADD (Fas-associated death domain). These serve as adapters for caspase-8 and -10 and form a death-inducing signaling complex (DISC) which can initiate the caspase cascade. Though this whole process originates independent of mitochondria, it can also activate (?) t-Bid, leading to a mitochondrial apoptosis signal as well.

Cells can become murder-resistant by expressing decoy receptors which have only the ‘death ligand’ binding domain and no active cytosolic domain. This occurs sometimes normally in animal cells but is also a trick that some viruses use – they encode decoy receptor proteins to keep their host cells safe from immune attack.

TNF-alpha usually promotes death, but can also promote survival in certain cell types by activating NF-κB. Sometimes cells use decoy receptors to promote an inflammatory response instead of death.

p53 is a key regulator of DNA damage response and can promote DNA repair, apoptosis or cell cycle arrest. It does this by binding to promoters of target genes. It is still not clear what determines when p53 will induce cell cycle arrest versus apoptosis.

experimental methods

Apoptotic cells exhibit a particular chemical signature. One of these is that an endonuclease cleaves DNA into fragments in the linker regions between nucleosomes and the resulting fragments form a ladder when run on a gel. Another is TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labeling) staining. This involves adding a Tdt enzyme and a BrdU which Tdt will add to the ends of cleaved DNA. After giving it a chance to do this you wash away excess BrdU and then use an antibody against BrdU. Yet another method is that phosphatidylserine (PS) is normally located in the cytosolic leaflet of the plasma membrane during apoptosis, it flips to the exoplasmic leaflet, where it serves as a signal to request other cells to phagocytose the dying cell. A fluorescently labeled annexin V protein can label PS on the outside of apoptotic cells.

Double-stranded DNA cannot get through the plasma membrane of intact cells – and that means healthy cells and apoptotic cells. If it does get out, that is a sign of necrosis. So you can stain with annexin V for exoplasmic PS and with 7-AAD for dsDNA apoptotic cells are those which are positive for annexin V but negative for 7-AAD.

concluding video

In sum, here is a disturbing video about apoptosis:

relevance to PrP

In general, in nature, cells either die by apoptosis, necrosis or by autophagy (meaning, in this case, getting engulfed whole by other cells). There aren’t really any other ways to go. One of the many mysterious things about prion diseases is how neurons die in the prion-infected brain – they do not obviously appear to follow any of these paths. Here’s a quote from an excellent recent paper on toxic mechanisms of prion disease [Moreno 2012]:

Caspase 12 cleavage occurred at 10wpi, following rising CHOP expression… coincident with onset of neuronal loss… however the exact effector mechanism of neuronal death is unclear: we found neither apoptosis, nor autophagy, nor necrosis on examination of hippocampal slices… and neither Bax deletion, nor Bcl-2 overexpression, nor caspase 12 deficiency are neuroprotective in prion disease.

In addition to her own evidence, Moreno cites the study of prion infection in mouse models with Bax (a pro-apoptotic protein) deleted or Bcl-2 (an anti-apoptotic protein) overexpressed – two different ways of blocking apoptosis. Neither of these mouse models had any delay or amelioration of prion disease [Steele 2007a]. Another apoptotic protein, Caspase-12, undergoes proteolytic processing during prion infection, but deletion of Caspase-12 also did not change the course of prion disease [Steele 2007b].

About Eric Vallabh Minikel

Eric Vallabh Minikel is on a lifelong quest to prevent prion disease. He is a scientist based at the Broad Institute of MIT and Harvard.

AP Biology Practice Questions 2

The figure below shows a model generated from a study of interactions in a Serengeti ecosystem. This figure describes hypothesized relationships between a parasite that causes disease in wildebeest, rinderpest, and other important components of the ecosystem. Thick arrows represent dominant effects, and “grass” is in a dotted circle because that variable was not directly measured in the study.

Which of the following can most reasonably be predicted based on this figure?
A: Increasing the amount of rinderpest would decrease the amount of grass.
B: Increased fire would lead to higher tree and elephant populations.
C: Increasing the amount of rinderpest could indirectly lower tree numbers.
D: Increased human populations have a net positive effect on the elephant population.

C: Increased rinderpest prevalence would lower wildebeest populations, increasing the amount of grass (because wildebeests feed on the grass) and providing more mate-rial to start and spread fires, thereby decreasing the number of trees. Choice (C) is thus correct. Choice A is incorrect because more rinderpest means fewer wildebeests, which in turn leads to more grass. Choice B is incorrect because fire reduces the tree population. Choice D is incorrect because the figure suggests that humans have only an adverse effect on the elephant population.

Human red blood cells lack mitochondria. Which of the following correctly explains the primary pathway that red blood cells use to produce energy?
A: Red blood cells generate ATP and NADH via aerobic respiration.
B: Red blood cells generate ATP and NADH via anaerobic fermentation.
C: Red blood cells metabolize glucose via glycolysis followed by carbon dioxide and ethanol production to produce ATP.
D: Red blood cells produce ATP via glycolysis followed by lactic acid production.

D: Lacking mitochondria, red blood cells predominantly produce energy by anaerobic respiration. Thus, red blood cells produce ATP via glycolysis followed by lactic acid fermentation. The correct answer is (D). Choice A is incorrect because aerobic cellular respiration occurs in mitochondria, which red blood cells lack. Choice B is incorrect because the products of anaerobic fermentation are ATP and NAD+ (glycolysis produces ATP and NADH, and fermentation regenerates NAD+). Choice C is incorrect because human red blood cells undergo lactic acid fermentation, not alcohol fermentation.

Before a cell can undergo cellular division, it must progress through an interphase stage in which the cell matures and produces proteins needed for division. Which of the following best describes the interphase stage?
A: The cell grows, replicates its DNA, and prepares to divide during interphase. If a genetic mutation is introduced during interphase and not repaired, the daughter cells will inherit the mutation.
B: The cell grows, replicates its DNA, and prepares to divide during interphase. None of the genetic mutations introduced during interphase are inherited by the daughter cells.
C: The cell replicates its DNA and prepares to divide during interphase, but does not grow. None of the genetic mutations introduced during interphase are inherited by the daughter cells.
D: The cell replicates its DNA and prepares to divide during interphase, but does not grow. If a genetic mutation is introduced during interphase and not repaired, the daughter cells will inherit the mutation.

A: During the 3 stages of interphase (G1, S, and G2), there is pronounced growth, DNA replication, and crucial preparation for cellular division. DNA replication errors during this process, if not detected and fixed, would result in mutations being passing on to daughter cells. (A) is thus correct. (B) is incorrect because unrepaired mutations would be passed on. (C) and (D) are incorrect because cells do in fact grow during interphase.

Saturated fats have a high melting point and remain solid at room temperature. Unsaturated fats are liquid at room temperature and solidify at much lower temperatures than saturated fats. In order for the membrane to function properly, membrane fluidity must stay within a certain physiological range. Chemical analysis of membrane lipids in bacteria adapted to different temperature ranges is shown in the table below.
Which of the following conclusions can be reasonably drawn from the data provided in the table?

A: The composition of phospholipids in the membrane is not correlated to ambient temperature.
B: Bacteria that thrive in cold environments adapt by increasing membrane rigidity.
C: The membranes of G. stearothermophilus are adapted to increase fluidity at high temperatures.
D: Fatty acids in B. psychrophilus allow the membrane to maintain its fluidity at low temperatures.

D: Based on the information provided, cell membranes require a significant proportion of unsaturated fatty acids in order to maintain fluidity at low temperatures. Thus, B. psychrophilus needs to contain a larger proportion of unsaturated fatty acids because it tends to be found at lower temperatures, making (D) correct. (A) is incorrect because there is a correlation found in the table: temperature is inversely correlated with unsaturated fatty acid content. (B) is incorrect because bacteria in colder environments require additional fluidity, not additional rigidity. (C) is incorrect because the high temperatures that G. stearothermophilus is exposed to require greater rigidity, not greater fluidity, which is why the species contains such a small proportion of unsaturated fatty acids.

Which of the following conclusions can most reasonably be drawn if a host cell’s function has been impaired following a viral infection?
A: The host cell will lyse or undergo apoptosis regardless of the severity of damage.
B: The host cell will lyse or undergo apoptosis if the severity of damage exceeds the cell’s ability to repair itself.
C: The host cell will only lyse.
D: The host cell will only undergo apoptosis.

B: If a cell loses its ability to function and cannot be repaired, then it could lyse (burst open) or undergo apoptosis (programmed cellular death). Whether it does either depends on the severity of the damage to the cell. Choice (B) is thus correct. Choice A is incorrect because the cell will remain intact if it is capable of repairing the damage. Choices C and D are incorrect because they only present one of the two viable options for a damaged cell.

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Extrinsic Pathway of Apoptosis (Apoptosis Molecular Mechanism Part 2)

In the extrinsic pathway of apoptosis, the death-inducing signal for the programmed cell death is triggered by an external stimulus. For receiving such an external death-inducing signal, the cell possesses plasma membrane receptors specific to each stimulus and thus the extrinsic signalling of apoptosis is also known as the Receptor Mediated programmed cell death pathway.

The external stimuli for the apoptosis in most of the cases will be a cytokine. The most studied cytokine to induce extrinsic pathway of apoptosis is an extracellular messenger protein called Tumor Necrosis Factor (TNF). TNF is so named because it was first discovered as a protein factor which induces cell death in cancerous cells. The TNF cytokine is produced by the cells of the immune system in response towards the adverse conditions. The adverse conditions that can provoke the immune cells to produce TNF are:

Ø Introduction of viral toxins

Ø Exposure to elevated temperature

Ø Exposure to other toxic substances

The detailed signaling mechanism of TNF-mediated extrinsic pathway of apoptosis is summarized below:

Ø TNF first binds to its receptor called TNFR1 (Tumor Necrosis Factor Receptor-1) present on the plasma membrane.

Ø TNFR1 is a member of death receptor family proteins that turn on the apoptotic cell death process in eukaryotic cells.

Ø TNFR1 is a trans-membrane receptor with an external ligand binding domain and a cytosolic domain.

Ø The TNRF1 in the plasma membrane is presented as a pre-assembled trimer.

Ø The cytosolic domain of each TNFR1 subunit contains a segment of about 70 amino acids called ‘death domain’.

Ø Binding of TNF to the TNFR1 receptor cause a conformational change in the death domain.

Ø This conformational change in the ‘death domain’ cause the recruitment of many apoptosis-related adaptor protein factors.

Ø To the activated death domain, two cytosolic adaptor proteins (TRADD and FADD) and Pro-caspase-8 residues are binds to form a multi-protein complex.

Ø The cytosolic death domain of TNFR1, TRADD and FADD interact with one another by homologous regions present on each protein.

Ø Pro-caspase-8 and FADD possess a homologous region called ‘death effector domain’.

Ø The death effector domains of both pro-caspase-8 and FADD interacts each other.

Ø Due to these interactions, the two Pro-caspase-8 molecules cleave each other to generate an active caspase-8.

Ø A single active caspase-8 contains four polypeptide segments derived from two pro-caspases.

Ø Activated caspase-8 is an initiator caspase in the extrinsic pathway of apoptosis, they activate the downstream caspases

Ø Downstream caspases are called executioner caspases (caspase-3) that carry out the self-destruction process (apoptosis) of the cell.

Another commonly observed extrinsic pathway of apoptosis in human is by the killer lymphocytes through Fas ligand and Fas protein by the mechanism given below:

Ø Killer lymphocytes can induce apoptosis by producing a protein called Fas ligand.

Ø The Fas ligand binds to its receptor called Fas on the plasma membrane of the target cell.

Ø Similar to the death domain of TNFR1, the Fas protein can recruit intracellular adapter proteins that can aggregate Pro-caspase-8 molecules.

Ø The pro-caspase-8 molecules are then activated to caspase 8 and that in turn can activate the downstream executioner caspase (caspase-3) to induce apoptosis.


Apoptosis is regarded as a carefully regulated energy-dependent process, characterized by specific morphological and biochemical features in which caspase activation plays a central role. Although many of the key apoptotic proteins that are activated or inactivated in the apoptotic pathways have been identified, the molecular mechanisms of action or activation of these proteins are not fully understood and are the focus of continued research. The importance of understanding the mechanistic machinery of apoptosis is vital because programmed cell death is a component of both health and disease, being initiated by various physiologic and pathologic stimuli. Moreover, the widespread involvement of apoptosis in the pathophysiology of disease lends itself to therapeutic intervention at many different checkpoints. Understanding the mechanisms of apoptosis, and other variants of programmed cell death, at the molecular level provides deeper insight into various disease processes and may thus influence therapeutic strategy.