PCD Flashcards
(17 cards)
- Genetically regulated process leading to controlled elimination of cells
*A.K.A apoptosis
* An orderly disposal of cells
- Sustained too many injury
- Infected with a virus
- Old cells
- Needs to be removed for body parts
Programmed cell death (PCD)
Physiological Roles of Programmed Cell death
In the human body, about 100,000 cells are produced every second by mitosis and a similar number die by apoptosis
Development & Morphogenesis:
- 131 of the 1,090 somatic cells die during C. elegans development
- during limb formation, separate digits evolve by death of interdigital mesenchymal tissue
- ablation of cells no longer needed such as the amphibian tadpole tail during metamorphosis
- demise of cells allows sculpturing of hollow structures
- formation of reproductive organs (Mullerian duct -> uterus, deleted inmales; Wolffian duct -> male organs, deleted in females)
- massive cell death occurs during early development of the nervous system (>50% of all neurons die)
Homeostasis:
a paradigm for the involvement of apoptosis in homeostasis is the immune system: several millions of B and T cells are generated every day and the majority (>95%) of those die during maturation (death by neglect, negative selection) or by AICD or peripheral immune cells)
Deletion of damaged and dangerous cells:
- cells with severely damaged DNA that cannot be repaired appropriately usually are removed by apoptosis
- inappropriate mitogenic signaling that is in conflict with the environmental or cellular status of the cell usually results in cell cycle arrest or apoptosis
- autoreactive cells of the immune system are deleted by apoptosis
- elimination of infected cells
The Morphology of Apoptosis
- Cell shrinks (pyknosis)
- Chromosomes condense and fragment
- Nuclear membrane breaks down
- Apoptotic body formation
- Engulfment of the cell corpse
Apoptosis vs. Necrosis
Normal cell -> Small blebs formation ->
-> Nucleus begins to break apart, and the DNA breaks into small pieces. The organelles are also located in the blebs -> Cell breaks into several apoptotic bodies; organelles are still functional -> Apoptosis
-> Blebs fuse and become larger; no organelles are located in the blebs -> Cell membrane ruptures and releases the cell’s content; the organelles are not functional -> Necrosis
- Physiological programmed cell death
- dying cells shrink, are engulfed by other cells, leave no trace, and don’t result in harmful outcomes
Apoptosis
- Pathological cell death
- dying cells swell and lyse; toxic contents leak out and result in inflammatory response. Cell death after an injury.
Necrosis
- Cysteine aspartyl-specific protease (degrading enzyme) involved in apoptosis
- Effector proteins, cleave key proteins including _______ and _______ leading to cell death
Provide examples also:
Caspases; cytosolic protein; nuclear lamin
E.g. caspase 3, 9,8
Bcl-2 family of proteins, which are crucial regulators of apoptosis (programmed cell death). These proteins contain conserved regions called BH (Bcl-2 Homology) domains, and they are classified into three functional groups based on their domain structure and role in apoptosis:
(A) Anti-apoptotic Bcl-2 proteins
Example: Bcl-2, Bcl-XL
= Cell is still alive
(B) Pro-apoptotic BH123 proteins
Example: Bax, Bak
= Cell is about to die
(C) Pro-apoptotic BH3-only proteins
Example: Bad, Bim, Bid, Puma, Noxa
Molecular Mechanisms of Apoptosis
Intrinsic pathway
- intracellular stress
- Begins at the mitochondria
- Triggered by DNA damage, stress, growth factor withdrawal
- Mitochondria normally display Bcl-2 (inhibits apoptosis) on their surface
Extrinsic pathway
- lethal ligands
- These cells are chosen for death by other cells, usually cytotoxic T cells (immune cells)
- All cells have receptors for Fas and TNF (pro-apoptotic ligands)
Apoptosome -> Caspase 9 -> Caspase cascade -> Apoptosis
More on intrinsic pathway
* Damage to cell alerts p53 which causes protein ___ (pro-apoptotic protein) to migrate to the mitochondrial membrane, inhibiting Bcl-2 and allowing the membrane channel (____) to open
* Opening of —- causes cytochrome C (electron carriers) to leak out (mitochondrial outer membrane permeabilization)
* CytochromeC binds to a protein called _______ (adaptor protein) forming apoptosomes
* These apoptosomes bind and activate caspase-9
* Caspase-9 cleaves and activates other caspases, which lead to proteolysis of everything in the cell
Bad; MAC; Apaf-1
Extrinsic Pathway
* When TNF or FasL binds the cell at these receptors, the cell activates ______, which initiates a cascade of caspases
* This lead to phagocytosis of the cell and its contents
caspase 8
Has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis.
* Detecting DNA fragmentation by labelling the terminal end of nucleic acids
* Relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase (TdT) that will catalyze the addition of dUTPs that are secondary labeled with a marker.
TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay
Bright, green-fluorescent dye label the terminal end of nucleic acid
Alexa-fluor
Cell viability assay
Cell viability
Neutral red assay
MTT
WST
resazurin or alamar blue assay
Cell viability assay
Membrane integrity
Colony formation assay trypan blue
calcein acetoxymethyl (calceinAM)
lactate dehydrogenase (LDH)
hemolytic assays
Cell viability assay
Apoptosis
Propidium iodide
LDH assay
DNA laddering
acridine orange/EtBr
TUNEL assay
caspase-3 activity
a technique used to detect and measure the physical and chemical characteristics of a population of cells or particles.
Flow cytometry
