L4: Apoptosis I Flashcards
purpose of cell death and apoptosis specficially?
cell death allows us to fight infections, get rid of malfunctioning agents, necessary for physiological processes e.g: menstruation, allows immune system to develop, essential for development.
apoptosis helps sculpt tissues, regulate number, and is required for immune system function.
apoptosis in development: finger development of mouse embryo- a protein called PU1 is important for macrophage function, cells death cases gaps between dingers. macrophages fill the area of dying cells to clear apoptotic fragments ‘cleaning’ the tissue,
apoptosis vs necrosis?
necrosis: severe or sudden injury e.g: ischaemia, heat
plama membrane is the main site of damage, leakage of intracellular contents, inflammatory, necroptosis refers to programmed necrosis.
apoptosis: programmed cell death. no leakage, not inflammatory, rapid clearance of apoptotic cells
apoptosis vs necrosis morphological features?
necrosis: a cell undergoes chromatin clumping, has swollen organelles and flacculent mitochondria.
it undergoes disintegration causing the release of intracellular contents that lead to inflammation.
phagocytosis: milkd convulation, chromatin compaction and seggregation, condensation of cytoplasm. then nuclear fragmentation and blebbing occurs and phagocytosis of apoptotic bodies,
regulation of apoptosis?
cells require tophic factors to stay alive. without apoptosis some cells are triggered to undergo apoptosis by specific signals. Immune cells could also bind to a cells plasma membrane or a bacteria/toxin binds to cell to breakdown plasma membrane inducing apoptosis.
cellular changes in apoptosis?
Phosphatidylserine (PS) externalisation:
In healthy cells, PS is usually located on the inner leaflet of the plasma membrane.
During apoptosis, it is translocated to the outer leaflet, serving as an “eat me” signal for phagocytes.
Charge and protein recruitment:
PS is a negatively charged phospholipid, so its externalisation attracts positively charged, PS-binding proteins, facilitating recognition by immune cells.
Phospholipid scramblase:
A Ca²⁺-activated transmembrane protein that disrupts the asymmetric distribution of phospholipids, allowing PS to flip to the outer leaflet.
High intracellular Ca²⁺ activates scramblase, promoting PS externalisation.
Flipase:
An ATP-dependent enzyme that normally returns PS to the inner leaflet to maintain membrane asymmetry.
During apoptosis, caspases inhibit flipase activity, allowing PS to accumulate on the outer membrane surface.
Membrane blebbing:
Caused by cytoskeletal breakdown and osmotic imbalances.
Despite blebbing, the osmotic gradient is preserved to prevent cell lysis, and the membrane remains sealed.
Membrane sealing and ESCRT:
There’s evidence that the ESCRT (Endosomal Sorting Complex Required for Transport) machinery helps in membrane remodeling during apoptosis to maintain integrity.
The role of transglutaminase is debated—while it’s involved in some cell death processes (e.g., crosslinking cytoskeletal proteins), it’s not essential for membrane sealing. Worth researching the ESCRT connection more.
Nuclear Changes in Apoptosis:
Chromatin condensation and nuclear shrinkage (pyknosis):
One of the hallmarks of apoptosis, making the nucleus appear darker and smaller under a microscope.
DNA fragmentation:
Activation of endonucleases (e.g., CAD—caspase-activated DNase) leads to internucleosomal cleavage of DNA, resulting in the characteristic DNA ladder seen during apoptosis.
add transglutimase from the slides
assays?
- annexin v binding to phosphotidylserine on outer leaflet
- cell morphology: blebbing
- nuclear condensation and fragmentation
- dna fragmentation
annexin v assay?
annexin v normally acts in the cytosol to repair damaged pm. recombinant annexin v made in vitro is labelled with a fluorescent to detect external phosphotidyl serine upon binding.
blebbing assay?
microscopic detection of blebbing e.g by a scanning electron microscope. normal cells are smoother? cytochrome c release from the mitochondria, causes activates caspases to cleave rock1 so continuously active by removing its regulatory domain. this causes mlc phosphorylation and causes actin contraction, constant phosphate inhibition of ml phosphatase so hyperphosphorylation of mlc so increases actin contractility so so actin cytoskeleton contracts inwards and is released by pm?
nuclear condensation and fragmentation?
in apoptotic cells nucleus is no longer visible as nuclear envelope disintegrates as nuclear lamina are destroyed. see large black bubbles of highly condensed DNA. DNA binding dyes can show normal vs apoptotic cells. apoptotic= smaller, brighter more condensed fragments by dyes such as merge or acradine orange (binds DNA and rna).
through confocal microscopy you can see intermediate fragment networks under nuclear envelope
through wildfield screening microscopy see round nuclear stain of nuclear lamina.
no longer see protective meshwork around apoptotic cells
dna changes i.e cad
chatgpt: DNA is Wound Around Nucleosomes
DNA is wrapped around histone proteins, forming nucleosomes (like beads on a string).
The DNA between nucleosomes is called linker DNA.
- Linker DNA is More Exposed to Nucleases
The nucleosome-bound DNA is protected, but the linker DNA is more accessible to enzymes.
Under normal conditions, inactive nucleases exist in the cell but do not degrade DNA.
- Caspase-Activated DNase (CAD) Triggers DNA Fragmentation in Apoptosis
In apoptosis, caspase-3 activates Caspase-Activated DNase (CAD) by cleaving its inhibitor (ICAD).
CAD then cuts linker DNA between nucleosomes, creating fragments in multiples of ~180-200 base pairs (bp).
- DNA Laddering Pattern
The regular spacing of nucleosomes results in even-sized DNA fragments.
When apoptotic DNA is run on an agarose gel, it produces a ladder-like pattern.
This is different from necrosis, where DNA degradation is more random and smeared.
