UNIT 16 – Cell Death and Renewal (Q&A) | MZO-001 MSCZOO | IGNOU
SAQ
i) Match the items in column I with column II:
Answer: a) → iv; b) → iii; c) → vii; d) → viii; e) → vi; f) → i; g) → ii; h) → x; i) → v; j) → xi; k) → ix
ii) Fill in the blanks:
a) Autophagy includes the formation of three different vesicles including ....................., which circularizes to form ......................, which then fuses with a lysosome to form the ..................... .
Answer: phagophore, autophagosome, autolysosome
b) Apoptosis can be mediated by two modes or pathways, ....................... or ....................... .
Answer: intrinsic, extrinsic
c) Pharmacological inhibitors such as ....................... block the lysosomal proton transport and thus autophagy.
Answer: Bafilomycin A1
d) ...................... is released from the mitochondria upon MOMP and binds to the adapter protein Apaf1 to form the complex known as ...................... .
Answer: Cytochrome C, Apoptosome
e) ....................... and ....................... are the examples of non-fluorescent stains used to measure cell death.
Answer: methyl green, trypan blue
f) The process of chromatin condensation during apoptosis is also known as ........................ .
Answer: pyknosis
g) The inhibition of RIPK1 by ...................... and its derivatives can inhibit TNFR1-driven necroptosis.
Answer: necrostatin-1 / Nec-1
h) The intrinsic apoptotic pathway is also known as the ........................ pathway.
Answer: mitochondrial apoptotic
i) The ligand that binds to the Fas receptor is ...................... .
Answer: Fas ligand or FasL
j) ........................ is an example of a DRD2 inhibitor used in anticancer treatment.
Answer: ONC201
iii) State whether the following statements are true (T) or false (F):
a) Activation of mTOR in turn leads to the activation of the Atg1/ULK1 complex in the autophagic process.
Answer: False
b) Initiator caspase 3 activates effector caspase 9 to bring about cell death.
Answer: False
c) RIPK3 can only be activated in a RIPK1-dependent method.
Answer: False
d) TP53 can up regulate the expression of BH3-only proteins in response to DNA damage or chromosomal aberrations.
Answer: True
e) Chromatin condensation is an important morphological feature of NETosis.
Answer: False
TERMINAL QUESTIONS
1. What are autophagosomes and autolysosomes? Indicate the major difference between the two.
Inside eukaryotic cells, there is a special self-cleaning process known as autophagy. This process helps the cell to remove its own damaged organelles, misfolded proteins, or other unwanted cytoplasmic materials. It is especially active during stress, starvation and low energy conditions. Autophagy allows the cell to recycle these unwanted components and use them again for energy or rebuilding. During this pathway, the cell forms two important vesicle-like structures at two different stages of the process: autophagosome and autolysosome. These two structures work one after the other to complete the cleaning and recycling task.
Autophagosome
An autophagosome is a double-membrane vesicle that forms during the early stage of autophagy. It begins by surrounding the targeted material, such as damaged mitochondria and protein aggregates. This membrane keeps extending until it completely encloses the selected material, forming a sealed vesicle. At this stage, the autophagosome contains only the material to be degraded. It does not have any digestive enzymes, so no breakdown or digestion happens yet. Its role is mainly to collect and isolate the unwanted material from the rest of the cell. After forming completely, the autophagosome moves towards a lysosome to begin the next step.
Autolysosome
An autolysosome is formed when the autophagosome fuses with a lysosome, which is an organelle that contains digestive (hydrolytic) enzymes such as proteases, lipases and nucleases. After fusion, these enzymes enter the inner compartment and start digesting the enclosed material. This is the final breakdown stage. The broken-down products like amino acids, sugars and fatty acids are released into the cytoplasm and are reused by the cell. So, the autolysosome is the structure where actual digestion and recycling takes place.
Major Difference between Autophagosome and Autolysosome
The main difference between the two lies in their function and enzyme content:
An autophagosome is a double-membraned vesicle that collects and isolates cellular waste. It does not contain any digestive enzymes, so no degradation happens at this stage.
An autolysosome is formed after fusion with a lysosome. It contains active digestive enzymes and performs the actual breakdown and recycling of the material.
In short:
- Autophagosome = Collection stage (no enzymes)
- Autolysosome = Digestion stage (with enzymes)
2. What is mitochondrial outer membrane permeabilization (MOMP)? How is it important in the process of apoptosis?
Mitochondrial outer membrane permeabilization (MOMP) is an essential and highly regulated step in the intrinsic pathway of apoptosis, which refers to the programmed and controlled death of a cell. In this process, the outer membrane of the mitochondria becomes permeable, meaning it develops openings or pores. As a result, certain proteins that are usually stored safely inside the mitochondrial intermembrane space get released into the cytoplasm. These released proteins serve as strong intracellular signals that trigger a cascade of events leading the cell towards apoptosis. Thus, MOMP acts as a critical checkpoint in deciding whether a cell will survive or enter the death pathway.
MOMP does not happen suddenly or accidentally. It is highly regulated by a group of proteins known as the Bcl-2 family. This family includes both pro-apoptotic proteins and anti-apoptotic proteins.
- The pro-apoptotic proteins members such as BAX and BAK promote MOMP by forming pores in the outer mitochondrial membrane.
- In contrast, the anti-apoptotic proteins like Bcl-2 and Bcl-xL try to prevent this pore formation.
When pro-apoptotic signals are stronger, BAX and BAK get activated and insert themselves into the outer membrane. They then oligomerize and form channels that allow proteins inside the mitochondria to escape into the cytoplasm. This escape marks the point where the cell becomes committed to die. Hence, MOMP is often called the "point of no return" in apoptosis.
How MOMP is important in the process of apoptosis?
Once MOMP occurs, cytochrome c is released from the intermembrane space of mitochondria. In the cytoplasm, cytochrome c binds to a protein called Apaf-1 (apoptotic protease activating factor-1). In the presence of ATP or dATP, Apaf-1 and cytochrome c form a large complex called the apoptosome. This apoptosome then activates initiator caspase-9, which further activates executioner caspases such as caspase-3 and caspase-7. These caspases begin to break down cellular components in an organized way, leading to cell death without causing inflammation.
Apart from cytochrome c, other important proteins are also released due to MOMP. SMAC/DIABLO helps by removing inhibitors of apoptosis (IAPs), allowing caspases to work more effectively. AIF (Apoptosis-inducing factor) travels to the nucleus and causes DNA fragmentation in a caspase-independent manner.
Therefore, MOMP is not just a small step in apoptosis. It is a central and irreversible stage. Once it happens, the cell cannot go back. It guarantees that the cell will follow the death pathway, either through caspases or other methods.
3. How do pro-apoptotic proteins differ from pro-survival proteins? Give examples (two each) for both classes of proteins mentioned.
In the intrinsic pathway of apoptosis, a special group of proteins called the Bcl-2 family plays a major role in controlling whether the cell will live or die. This family has two opposite types of proteins. First type is called pro-apoptotic proteins, which promote cell death. Second type is called pro-survival proteins, which protect the cell and stop it from dying. These pro-survival proteins are also called anti-apoptotic proteins, because their function is to stop apoptosis.
These two groups work by controlling a key event called mitochondrial outer membrane permeabilization (MOMP). If pro-apoptotic proteins win, then MOMP happens and apoptosis starts. But if pro-survival proteins are stronger, then MOMP is blocked and the cell survives.
Pro-apoptotic proteins
These proteins promote apoptosis by causing damage to the outer mitochondrial membrane. They help in a critical step called mitochondrial outer membrane permeabilization (MOMP). When MOMP happens, proteins like cytochrome c, SMAC/DIABLO and AIF are released into the cytosol. This activates the caspase cascade and leads to apoptosis.
There are two types of pro-apoptotic proteins:
- BH3-only proteins (like BID, BIM, BAD): These are sensors of stress. They get activated by cellular damage, then go and activate the effector proteins.
- Effector proteins (like BAX and BAK): These directly form pores in the mitochondrial membrane. Once activated, they oligomerize (join together) and create large channels for cytochrome c to exit.
Examples of Pro-apoptotic proteins:
- BAX: Normally present in the cytosol, but moves to mitochondria when activated. It forms pores and promotes cytochrome c release.
- BAK: Already attached to the mitochondrial outer membrane. It undergoes conformational change during apoptosis and forms pores with BAX.
Pro-survival (anti-apoptotic) proteins
These proteins inhibit apoptosis. Their main function is to bind and neutralize the pro-apoptotic proteins. They either block BH3-only proteins or stop BAX and BAK from forming pores. Thus, they prevent MOMP and keep the mitochondria stable.
They are very important in normal cells to avoid unwanted cell death, but their overexpression is also linked to cancer.
Examples of Pro-survival (anti-apoptotic) proteins:
- Bcl-2: It binds to activated BH3-only proteins and prevents BAX activation. This blocks the release of apoptotic factors and protects the cell.
- Bcl-XL: Similar in function to Bcl-2, but has a stronger protective role in neurons. It binds directly to BAX or BAK and stops pore formation.
4. Name the components of an apoptosome and a necrosome. What is the difference in the function of an apoptosome and a necrosome?
In the cell, programmed forms of death are controlled by special protein complexes. Two important complexes among them are the apoptosome, which helps in the process of apoptosis, and the necrosome, which is involved in necroptosis. Though both are connected with cell death, their components are different and they function through separate pathways.
Components of the Apoptosome:
The apoptosome is a large protein complex that plays a crucial role in the intrinsic pathway of apoptosis. It forms when mitochondrial outer membrane permeabilization (MOMP) occurs, leading to the release of various mitochondrial factors into the cytosol. The main components of the apoptosome are:
- Cytochrome c: Released from the mitochondria into the cytosol during MOMP. It plays a key role in activating the apoptosome.
- Apaf-1 (Apoptotic protease-activating factor 1): A cytosolic protein that binds to cytochrome c and undergoes a conformational change to form a complex with other proteins.
- Caspase-9: Activated by the apoptosome, caspase-9 triggers the activation of downstream caspases (such as caspase-3 and caspase-7) that execute the apoptosis process.
- ATP: Necessary for the formation of the apoptosome complex. ATP binds to Apaf-1, enabling its conformational change and the subsequent formation of the apoptosome.
Components of the Necrosome:
A necrosome is a protein complex that plays a role in necrosis, specifically regulated necroptosis, which is a form of programmed cell death distinct from apoptosis. The components of a necrosome include:
- RIPK1 (Receptor-interacting protein kinase 1): A key protein that initiates the necroptosis signaling pathway.
- RIPK3 (Receptor-interacting protein kinase 3): Works with RIPK1 to form a necrosome complex.
- MLKL (Mixed lineage kinase domain-like protein): The final executor protein in necroptosis, which, when activated, leads to cell membrane rupture and necrosis.
Difference in Function between Apoptosome and Necrosome:
The apoptosome and necrosome differ in their roles in programmed cell death:
- Apoptosome: It is involved in apoptosis, a form of programmed cell death that is clean, controlled, and non-inflammatory. The apoptosome triggers caspase activation, leading to the orderly dismantling of the cell and its components.
- Necrosome: It is involved in necroptosis, a form of programmed necrosis. Unlike apoptosis, necroptosis leads to cell rupture and the release of cellular contents, causing inflammation. The necrosome complex activates MLKL, which causes the cell membrane to rupture, leading to the uncontrolled release of cellular material.
5. What are BH domains? What is their importance in apoptosis?
BH domains or Bcl-2 Homology domains, are conserved amino acid sequence regions found in the members of the Bcl-2 family of proteins, which play an essential role in regulating apoptosis, especially in the intrinsic mitochondrial pathway. The term "BH" comes from the first discovered member of this family, Bcl-2 (B-cell lymphoma 2). These domains help in protein-protein interactions that either promote or prevent apoptosis, depending on the type of protein in which they are present.
These BH domains are very important because they determine the pro-apoptotic or anti-apoptotic nature of the Bcl-2 family proteins. Some proteins, like Bcl-2 and Bcl-xL, help the cell survive by blocking apoptosis. Others, like Bax, Bak and Bid, help in killing the cell by starting the apoptotic process. The BH domains control these actions.
Types of BH Domains
There are four types of BH (Bcl-2 homology) domains and each one plays a different role in the process of apoptosis.
1. BH1 Domain
- This domain is mostly found in anti-apoptotic proteins like Bcl-2 and Bcl-xL. BH1 helps in the formation of a hydrophobic groove along with BH2 and BH3 domains. This groove is important because it binds to pro-apoptotic proteins, especially to their BH3 domain and thus prevents them from initiating apoptosis. So, BH1 plays a role in protecting the cell from death.
2. BH2 Domain
- BH2 is also found in anti-apoptotic proteins like Bcl-2 and Bcl-xL. It works together with BH1 to maintain the proper structure of the protein and to form the binding groove. It helps the anti-apoptotic proteins in neutralizing pro-apoptotic proteins. Without BH2, the anti-apoptotic proteins cannot work properly.
3. BH3 Domain
This is the most critical and central domain for pro-apoptotic activity. It is present in all pro-apoptotic proteins, including both:
- BH3-only proteins like Bid, Bad, Bim
- Multi-domain pro-apoptotic proteins like Bax, Bak
The BH3 domain is required for binding to anti-apoptotic proteins like Bcl-2 and Bcl-xL. When the BH3 domain binds to the groove of anti-apoptotic proteins, it blocks their function, allowing apoptosis to proceed. It is like a key that switches on the death signal in the cell.
4. BH4 Domain
This domain is found only in anti-apoptotic proteins such as Bcl-2 and Bcl-xL. It helps in maintaining the stability and function of these proteins. The BH4 domain can also interact with non-Bcl-2 proteins and may have additional roles in cell survival, calcium homeostasis and autophagy. Without BH4, the anti-apoptotic proteins cannot protect the cell efficiently.
Importance of BH Domains in Apoptosis
The BH domains are very important in regulating the balance between cell survival and cell death. Here's how they help:
- Apoptosis is regulated by the interaction between pro-apoptotic and anti-apoptotic proteins and these interactions are controlled by BH domains.
- Proteins with BH1, BH2 and BH4 are generally anti-apoptotic and help the cell survive under stress.
- Proteins with only BH3 domain are called BH3-only proteins and act as sentinels. They sense cell stress or damage and then activate Bax or Bak to start apoptosis.
- The interaction between BH3 domain and the groove made by BH1/BH2 is the key control point for apoptosis.
- If the pro-apoptotic signals (BH3-only proteins) overcome the anti-apoptotic protection, then MOMP (mitochondrial outer membrane permeabilization) happens, which releases cytochrome c and starts apoptosis.
Thus, BH domains are like molecular switches that determine the cell’s fate, whether it will live or die. They are essential for mitochondrial apoptosis and are often studied in cancer biology, where apoptosis regulation is disturbed.
6. What are necrostatins?
In normal conditions, cells die in a controlled way through apoptosis, which is a peaceful and clean process that does not harm nearby tissues. But sometimes, when apoptosis is blocked or does not work properly, the body uses another backup method to kill the damaged or infected cell. This second method is called necroptosis. Necroptosis is a form of programmed cell death, but unlike apoptosis, it causes the cell to swell, burst and release harmful substances, which leads to inflammation and tissue damage.
Necrostatins are a group of small synthetic chemical molecules that are used to block necroptosis, which is a type of programmed cell death. They work by stopping the activity of a key protein called RIPK1 (Receptor Interacting Protein Kinase 1). This protein is very important for starting the necroptosis process. If RIPK1 is blocked, then necroptosis cannot happen.
The first necrostatin that was discovered is called Necrostatin-1 (Nec-1). It was the original molecule that showed scientists that necroptosis can be stopped. However, Nec-1 was not very stable and had some problems, so researchers made a better version called Nec-1s (Nec-1 stable). This new version is more effective and is now commonly used in lab research to study necroptosis in detail.
Necrostatins are not used as regular medicines yet, but they are very important for understanding how necroptosis works and how it can be stopped. Their study helps researchers find new ways to treat diseases where too much cell death and inflammation cause harm.
Importance in Disease and Research
Necrostatins have become important tools in scientific research. They are helping scientists understand the role of necroptosis in many diseases such as:
- Neurodegenerative diseases like Alzheimer's and Parkinson's
- Ischemic injuries like stroke and heart attack
- Inflammatory diseases like rheumatoid arthritis and inflammatory bowel disease
By controlling necroptosis, necrostatins have the potential to reduce inflammation and protect tissues from damage. That's why they are being studied as possible future drugs for many conditions related to cell injury and inflammation.
7. Differentiate between necrosis and apoptosis.
Necrosis and apoptosis are two different types of cell death. Both occur in different situations and have different effects on cells and tissues. Here are the differences between necrosis and apoptosis bssed on different key criteria:
1. Based on Process Type and Regulation
Necrosis is an uncontrolled, passive and pathological type of cell death. It happens when a cell is injured suddenly due to harmful external factors like infection, mechanical trauma, or loss of blood supply. The cell does not participate in its own death.
Apoptosis, on the other hand, is a highly regulated and controlled physiological process. It is also called programmed cell death. Here, the cell itself activates a sequence of steps to die in a clean and safe way, especially during development or when it becomes damaged.
2. Based on Morphological Changes in Cell
In necrosis, the cell swells, organelles also swell and finally the plasma membrane breaks. This leads to the bursting of the cell and release of its contents into the surrounding tissue.
In apoptosis, the cell shrinks, the chromatin condenses in the nucleus and the membrane starts forming small blebs (bulges). Finally, the cell breaks into small sealed fragments called apoptotic bodies which are then removed by other cells like macrophages.
3. Based on Inflammation and Tissue Response
Necrosis causes inflammation because when the membrane ruptures, it releases enzymes and toxic substances that damage nearby healthy tissue.
Apoptosis does not cause inflammation because the cell contents are never leaked outside. Everything is packed into apoptotic bodies which are cleaned up quickly by phagocytosis, maintaining tissue health.
4. Based on Energy Requirement and Enzymatic Involvement
Necrosis is an energy-independent process. It happens passively without needing ATP or special enzymes.
Apoptosis is an energy-dependent process. It requires ATP and involves a well-organised cascade of enzymes, mainly caspases, along with proteins like Bcl-2 family and cytochrome c, to carry out the cell death steps.
5. Biological Role and Outcome
Necrosis is usually harmful for the body and linked to diseases. It often leads to tissue damage, scarring or further infection.
Apoptosis is a beneficial and protective process. It removes unwanted or abnormal cells in a safe way. It plays a key role in embryonic development, immune system regulation, and cancer prevention.
8. Indicate the difference between intrinsic and extrinsic apoptotic pathways.
Apoptosis is a highly regulated process of programmed cell death that removes unwanted or damaged cells without causing inflammation. There are two main apoptotic pathways: the intrinsic (mitochondrial) pathway and the extrinsic (death receptor) pathway. The intrinsic pathway is triggered by internal stress like DNA damage, while the extrinsic pathway is activated by external signals such as death ligands. Even though both pathways lead to programmed cell death, they are quite different in many ways. These differences are based on various criteria such as:
1. Based on Type of Triggering Signal
Intrinsic pathway is activated by internal cellular stress signals such as DNA damage, oxidative stress and ER stress. These arise from within the cell itself, usually due to damage or malfunction that threatens cell survival.
Extrinsic pathway is triggered by external signals like binding of death ligands (e.g., FasL, TNF-α) to death receptors on the plasma membrane, usually sent by immune cells to remove unwanted or infected cells.
2. Based on Initiating Mechanism and Location
Intrinsic pathway begins with mitochondrial outer membrane permeabilization (MOMP), leading to the release of cytochrome c into the cytosol. This process is highly controlled by Bcl-2 family proteins and occurs inside the cell without any ligand-receptor interaction.
Extrinsic pathway is initiated at the cell surface when death ligands bind to specific death receptors like Fas or TNFR1. This forms a death-inducing signaling complex (DISC) that activates downstream caspases.
3. Based on Initiator Caspases and Activation Complex
Intrinsic pathway uses caspase-9 as the initiator, which is activated through the formation of the apoptosome complex (Apaf-1 + cytochrome c + procaspase-9). This complex forms in the cytoplasm only after mitochondrial cytochrome c release.
Extrinsic pathway uses caspase-8 (or caspase-10 in humans) as the initiator, which is directly activated by DISC complex formed at the plasma membrane without requiring mitochondrial involvement.
4. Based on Role of Mitochondria
Intrinsic pathway is mitochondria-dependent and completely relies on mitochondrial signaling. Without mitochondrial membrane permeabilization, this pathway cannot proceed, making mitochondria the central regulator.
Extrinsic pathway is mitochondria-independent in its primary form. However, in some cells (called Type II cells), caspase-8 cleaves Bid protein, which indirectly connects it to mitochondria, forming a crosstalk with the intrinsic pathway.
5. Based on Physiological Role and Cell Type Specificity
Intrinsic pathway mainly functions in response to internal damage, like mutations and metabolic failure. It plays a major role in tumor suppression, organ development and response to stress in most mammalian cells.
Extrinsic pathway is primarily active in immune regulation, where cytotoxic T-cells and NK cells use it to kill virus-infected or cancer cells. It is more common in immune target cells like hepatocytes and lymphocytes.
9. What are the major stages of autophagy?
Autophagy is a highly regulated catabolic process in which cells degrade and recycle their own components like damaged organelles, misfolded proteins or excess cytoplasmic material. The word "autophagy" means "self-eating" and it is important for maintaining cellular health, especially during stress, starvation or damage. The process is controlled by autophagy-related genes (ATG) and it plays a crucial role in cell survival, immunity, aging and disease regulation. There are different types of autophagy, but the most studied and important one is macroautophagy, commonly referred to simply as autophagy.
Major Stages of Autophagy
Autophagy does not occur randomly, but in an orderly step-wise manner. There are five major stages of autophagy and each stage is controlled by specific proteins and molecular signals.
1. Initiation (Induction)
In this first step, autophagy is triggered by signals such as nutrient deprivation, oxidative stress and damage. These signals activate the ULK1 complex (Unc-51-like kinase 1), which starts the autophagy machinery. The ULK1 complex is inhibited by mTOR (mechanistic target of rapamycin) under normal conditions, but during starvation, mTOR becomes inactive, allowing ULK1 to function.
2. Nucleation (Phagophore Formation)
Now, a small cup-shaped double-membrane structure begins to form. This is called the phagophore or isolation membrane. This stage is controlled by the Beclin-1 complex (includes Beclin-1, Vps34, and others). This complex helps produce PI3P (phosphatidylinositol 3-phosphate), which attracts other ATG proteins to the membrane.
3. Expansion and Elongation of Phagophore
The phagophore expands and forms a closed double-membrane vesicle called the autophagosome. This stage requires the action of ATG proteins and two ubiquitin-like conjugation systems. One of them helps in the conversion of LC3-I to LC3-II, which gets inserted into the membrane and is used as a marker for autophagosomes.
4. Fusion with Lysosome
The mature autophagosome moves towards a lysosome and fuses with it to form an autolysosome. This fusion requires proteins like SNAREs, Rab7 and LAMP-2. The contents trapped inside the autophagosome are now ready for degradation.
5. Degradation and Recycling
Inside the autolysosome, lysosomal enzymes break down the captured material into basic components like amino acids, fatty acids and sugars. These materials are then sent back to the cytoplasm to be reused by the cell for energy or biosynthesis.
10. What is the principle of LDH assay for the measurement of cell death?
LDH assay is one of the most commonly used biochemical methods to measure cell membrane damage and cell death, especially during necrosis or late-stage apoptosis. LDH stands for Lactate Dehydrogenase, which is an intracellular enzyme found in the cytoplasm of almost all cells. Under normal conditions, LDH remains inside the cell. But when the cell membrane becomes damaged or ruptured due to stress, toxin, or cell death, LDH leaks out into the surrounding medium. The LDH assay takes advantage of this leakage to estimate the extent of cell death.
Principle of the LDH Assay
The principle of the LDH assay is based on the ability of LDH enzyme to catalyze a reaction that changes lactate to pyruvate. This reaction also results in the conversion of NAD⁺ to NADH. The NADH produced can then react with specific substrates to create a colored product, which can be measured using a spectrophotometer.
The overall reaction looks like this:
Lactate + NAD⁺ → Pyruvate + NADH + H⁺
The NADH produced is responsible for reducing a tetrazolium salt or INT reagent in the assay. This reduction results in a color change and the intensity of the color correlates with the amount of LDH released.
The more LDH in the culture medium, the more NADH is formed, leading to more intense color production. This color is measured at a specific wavelength (usually 490 nm or 540 nm) and the resulting absorbance value indicates the level of cell death.
Importance and Use of LDH Assay
- It is used to measure cytotoxicity in drug testing and cell viability studies.
- It helps to distinguish between necrotic and apoptotic cell death.
- It is non-radioactive, easy to perform and suitable for high-throughput experiments.
- It does not require destruction of the remaining cells, so the same sample can be used for other tests.
11. Name any two each of fluorescent and non-fluorescent stains to measure cell death.
To measure cell death in cells, scientists use special types of chemical dyes called stains. These stains help to identify whether the cells are alive, dead, or undergoing a specific type of death like apoptosis or necrosis. These stains can be broadly divided into two types based on their properties: fluorescent stains and non-fluorescent stains.
1. Fluorescent Stains
Fluorescent stains emit visible light (usually green, red and blue) when exposed to specific wavelengths of light under a fluorescence microscope. These stains are commonly used in cell biology and molecular biology labs to identify apoptotic and necrotic cells by detecting changes in their membranes or internal cell structures.
Examples of fluorescent stains:
1. Propidium Iodide (PI):
- This dye is impermeable to live cells but easily enters dead or damaged cells due to their leaky plasma membrane. Once inside, it binds strongly to DNA and gives a bright red fluorescence. It is especially used for identifying necrotic cells or cells in late-stage apoptosis.
2. Annexin V-FITC (Fluorescein-labeled Annexin V):
- This protein-based dye binds to phosphatidylserine, a molecule that flips from the inner side of the plasma membrane to the outer side during early apoptosis. The FITC gives off green fluorescence, allowing early detection of apoptotic cells even before they lose membrane integrity.
2. Non-Fluorescent Stains
These stains are visible directly under a light microscope. They are generally used in routine cell culture labs for simple viability tests.
Examples of non-fluorescent stains:
1. Trypan Blue:
- This is a commonly used vital dye. It is excluded by healthy cells with intact membranes, but dead cells absorb it and appear blue. This helps in calculating the percentage of live vs dead cells using a hemocytometer.
2. Eosin:
- Eosin is an acidic dye that stains cytoplasmic proteins in dead cells more strongly than in living ones. It is usually used in combination with Hematoxylin in tissue sections, where dead or necrotic areas take deeper pink staining.
12. What are the initiator and effector caspases? Give examples of each.
Caspases are a special family of protease enzymes that play a very important role in apoptosis, which is also known as programmed cell death. These enzymes are present in an inactive form inside the cell and get activated when the cell receives a signal to die. Caspases work like a chain reaction. Some caspases get activated first and then they activate other caspases. Based on their function in the apoptosis process, caspases are mainly divided into two groups: initiator caspases and effector caspases. Both types work together to ensure proper and controlled death of damaged or unnecessary cells in the body.
1. Initiator Caspases:
Initiator caspases are the first enzymes to be activated when the cell receives a death signal. These caspases act like a starting point in the apoptotic pathway. They do not break down the cell directly but instead activate other caspases (effector caspases) by cutting them at specific places. Initiator caspases are usually activated through multi-protein complexes. For example, in the extrinsic pathway, initiator caspases are activated through death receptor complexes like DISC (Death-Inducing Signaling Complex), and in the intrinsic pathway, they are activated by the apoptosome.
Examples of initiator caspases:
- Caspase-8:
- Caspase-8 is activated in the extrinsic pathway when death receptors like Fas or TNF receptor are stimulated by their ligands. Once activated, it cleaves and activates downstream effector caspases like caspase-3, initiating the execution phase of apoptosis.
- Caspase-9:
- Caspase-9 is involved in the intrinsic pathway and becomes active after the release of cytochrome c from mitochondria. It forms a complex with Apaf-1 and ATP to create the apoptosome, which activates effector caspases to begin cell dismantling.
2. Effector Caspases:
Effector caspases are also known as executioner caspases because they are directly responsible for breaking down the important components of the cell. Once they are activated by initiator caspases, they start cleaving various structural and regulatory proteins inside the cell, leading to the classic features of apoptosis like DNA fragmentation, chromatin condensation and cell membrane blebbing.
Examples of effector caspases:
- Caspase-3:
- Caspase-3 is a major executioner caspase that is activated by both intrinsic and extrinsic pathways. It cleaves several important substrates like PARP, leading to DNA fragmentation, nuclear condensation and other typical apoptotic changes in the cell.
- Caspase-7:
- Caspase-7 works alongside caspase-3 and is also activated by initiator caspases. It targets structural proteins in the cytoskeleton and nuclear membrane, playing a key role in dismantling the cell in a controlled and clean manner during apoptosis.
13. What are the different sub-classes of BCI2 proteins? Explain briefly based on structure and function.
The Bcl-2 family of proteins is a very important group of regulatory proteins that play a major role in the intrinsic (mitochondrial) pathway of apoptosis, which is a kind of programmed cell death. These proteins mainly control the permeability of the mitochondrial outer membrane and thus regulate the release of apoptotic factors like cytochrome c. This family includes both pro-apoptotic proteins (which promote cell death) and anti-apoptotic proteins (which protect the cell from dying).
Sub-classes of Bcl-2 Family Proteins
These proteins are classified into different sub-classes based on the number and type of BH (Bcl-2 Homology) domains they contain, and also based on their functional role in apoptosis. There are three major sub-classes of Bcl-2 family proteins.
1. Anti-apoptotic Bcl-2 Proteins (BH1-BH4 containing proteins)
These proteins inhibit apoptosis and protect cells from death. Structurally, they have all four BH domains: BH1, BH2, BH3 and BH4. Functionally, they bind to pro-apoptotic proteins like Bax, Bak, or BH3-only proteins and neutralise their apoptotic action. By doing this, they prevent the permeabilization of the mitochondrial outer membrane, which stops the release of cytochrome c, a key step in activating caspases.
Examples:
- Bcl-2: First identified in B-cell lymphomas. It stabilises mitochondria and prevents MOMP (mitochondrial outer membrane permeabilization).
- Bcl-XL: Expressed in many cell types and inhibits apoptosis by binding to Bax and Bak.
- Other proteins: Mcl-1, Bcl-w, A1/Bfl-1.
2. Pro-apoptotic Effector Proteins (BH1-BH3 containing)
These proteins help in the execution of apoptosis. They have three BH domains: BH1, BH2 and BH3, but lack BH4. Structurally, they can form homodimers or heterodimers and functionally they insert into mitochondrial membranes to form pores. These pores allow the release of cytochrome c, which then activates the downstream caspases like caspase-9.
Examples:
- Bax: Normally present in the cytosol in inactive form. After apoptotic signals, it changes shape and moves to the mitochondria to form pores.
- Bak: Always anchored to the mitochondrial membrane. On activation, it forms oligomers that disturb membrane integrity.
Both Bax and Bak are essential for the proper induction of intrinsic apoptosis.
3. BH3-only Pro-apoptotic Proteins
These proteins act as initiators or sensors of apoptosis. They contain only the BH3 domain and do not form pores themselves. Their main job is to activate Bax and Bak directly or to inhibit the anti-apoptotic Bcl-2 proteins, which frees Bax/Bak to do their job. These proteins are often upregulated or activated in response to cellular stress, DNA damage, or growth factor deprivation.
These BH3-only proteins work like messengers that sense damage and then pass the death signal to the main effectors (Bax/Bak).
Examples:
- Bid: Cleaved by caspase-8 into tBid during extrinsic apoptosis. tBid then activates Bax/Bak.
- Bad: Becomes active when dephosphorylated. It binds to Bcl-2 or Bcl-XL and frees Bax/Bak.
- Bim: Activated under stress like growth factor withdrawal.
- Puma and Noxa: Induced by p53 during DNA damage.
14. Briefly explain how BCI-2 and MCL-1 inhibitors help in cancer treatment. Give examples (two each) of BCI-2 and MCL-1 inhibitors.
Bcl-2 and Mcl-1 are anti-apoptotic proteins that belong to the Bcl-2 family, which controls the process of programmed cell death or apoptosis. In many types of cancers, these proteins are found in high levels. Because of this, the cancer cells avoid apoptosis, even when they are damaged or abnormal. This helps the cancer cells survive for a long time, grow uncontrollably and become resistant to chemotherapy or radiation.
To stop this abnormal survival of cancer cells, scientists have developed special drugs called Bcl-2 inhibitors and Mcl-1 inhibitors. Bcl-2 and Mcl-1 inhibitors are special drugs that block the action of these anti-apoptotic proteins. When these proteins are blocked, the cancer cells lose their ability to avoid apoptosis. As a result, the natural process of cell death restarts. These inhibitors allow the pro-apoptotic proteins (like Bax and Bak) to become active again, which helps in triggering the death of cancer cells.
By doing this, these inhibitors help shrink tumors and reduce the spread of cancer. They also make cancer cells more sensitive to other therapies. That is why these inhibitors are often used in combination with chemotherapy or targeted therapy to improve the overall effect of treatment.
So, the main way Bcl-2 and Mcl-1 inhibitors help in cancer treatment is by removing the protection that cancer cells have against cell death, allowing them to die naturally and stop multiplying. This improves the success of cancer treatment and may also reduce relapse or resistance.
Examples of Bcl-2 Inhibitors
1. Venetoclax (ABT-199):
- It is a highly selective Bcl-2 inhibitor approved for treating chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). It binds directly to the Bcl-2 protein and prevents it from protecting the cancer cell. By doing this, it promotes apoptosis and helps reduce tumour burden, especially in blood cancers.
2. Obatoclax:
- This is a broad-spectrum Bcl-2 family inhibitor that can target multiple anti-apoptotic proteins including Bcl-2, Bcl-xL and Mcl-1. It has been tested in clinical trials for leukemia, lymphoma and some solid tumours. It increases the sensitivity of cancer cells to chemotherapy by promoting apoptosis.
Examples of Mcl-1 Inhibitors
1. S63845:
- It is a selective Mcl-1 inhibitor that binds strongly to Mcl-1 protein and blocks its anti-apoptotic action. This allows Bax and Bak to become active and induce apoptosis. It is effective against cancers that depend mainly on Mcl-1 for survival like multiple myeloma, AML and some solid tumours.
2. AZD5991:
- This is a potent Mcl-1 inhibitor developed for treating hematologic malignancies such as leukemia and myeloma. It promotes apoptosis by targeting Mcl-1 and is currently under clinical trial studies for its safety and effectiveness.
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