BIOLOGY 2F03 Lecture Notes - Lecture 14: Phosphatidylserine, Annexin A5, Apoptosis
19 Jun 2018
School
Department
Course
Professor
Surgery and Anaesthesia Sheena Cheung ssc211
Module 1: Cancer repair and regeneration With additions from ABS’ notes
8
7. Phagocytosis to clear debris
Cell is broken into smaller apoptotic bodies by pinching off that can be up-taken by
phagocytes
Poorly understood mechanisms for changing phagocytic behaviour to engulf apoptotic
bodies
o Phosphatidyl serine is a key molecule for phagocytosis of debris. It flips and
become present on the surface of the surface when apoptotic signal is detected
(normally on the inside therefore cannot be detected).
o Release of lysophosphatidyl choline is chemo attractant for immune cells.
Identification of apoptosis in labs
In labs, characteristic 180 base pairs laddering pattern appears because of apoptotic
mediated snipping (activated CAD snipping between histone cores) therefore
fragmentation of chromatin
PARP cleavage
a. PARP (poly ADP-ribose polymerase) has a caspase recognition site and gets
cleaved by caspase 3
b. Two peptides as a result of the cleavage can be detected by western blotting to see
some shorter cleaved PARP rather than only full length PARP detected to see that
caspases are active
Annexin V
o Most common way for a while, more complicated
o Propidium iodine binds to DNA when it can get into the cell - if cell surface is
intact, it cannot get in therefore it will appear as propidium iodine negative cells
o Annexin V binds to phosphatidyl serine if it is on the outside of the cell or the
membrane integrity is completely gone
o Staining analysis
Healthy cells will have both negative for PI and Annexin V
Apoptotic cells: Staining for annexin V but negative for PI
Necrotic = Membranes ruptured: positive for both therefore freely available
Caspase 3/7 activation
o Recognition sequence for caspase tagged onto Luciferin
o Recognition is chopped off by caspase 3/7 if apoptotic, giving an activated enzyme
o Activated enzyme binds to Luciferase to produce light
o Measure light to see how much apoptosis is going on
Document Summary
With additions from abs" notes: phagocytosis to clear debris. Cell is broken into smaller apoptotic bodies by pinching off that can be up-taken by phagocytes. Poorly understood mechanisms for changing phagocytic behaviour to engulf apoptotic bodies: phosphatidyl serine is a key molecule for phagocytosis of debris. It flips and become present on the surface of the surface when apoptotic signal is detected (normally on the inside therefore cannot be detected): release of lysophosphatidyl choline is chemo attractant for immune cells. In labs, characteristic 180 base pairs laddering pattern appears because of apoptotic mediated snipping (activated cad snipping between histone cores) therefore fragmentation of chromatin. Healthy cells will have both negative for pi and annexin v. Apoptotic cells: staining for annexin v but negative for pi. Necrotic = membranes ruptured: positive for both therefore freely available. Dna damage that cannot be repaired initiates apoptotic change. P53 is a tumour suppressor gene (50% is mutated in all cancers)
