LEC55: Apoptosis Flashcards
what is necrosis? what happens to the cell?
form of cell death in which cells and organelles **swell and rupture **
this leakage induces inflammatory response from the body
what is apoptosis? what happens to the cell?
cells shrink and condense
organelles and membranes retain their integrity
remnants are then phagocytosed by neighboring cells or macrophages
degredation is rapid; few dead cells are seen
no inflammatory reponse occurs
what assays measure cellular apoptosis?
1) visualize “blebs” of apoptotic cells with DNA dye
2) DNA ladder assay
3) flow cytometry and DNA content
4) TUNEL assay
5) Annexin V assay
what does DNA dye assay show of apoptotic cells?
visualizes DNA in apoptotic cells’ nuclei; shows their distinctive morphology
what does a DNA ladder assay show about apoptotic cells?
reflects the DNA degredation occurring
steps of 180-200 bp in ladder visualize
represents the size of the cuts a nuclease makes in genomic DNA
what does flow cytometry analysis show about apoptotic cells?
apoptotic cells have a hypodiploid DNA content that = <2N
see this on flow cytometer: hypodiploid peak is less than 2N (right graph) as compared to the (short) G2 peak on the L graph)
what makes the flow cytometer to be a rigorous assay for apoptosis?
flow cytometry only works if the membrane of the cell is intact
apoptotic cells’ membranes are intact
what does the TUNEL assay take advantage of re: apoptosis? how does it work?
endonucleases cutting apoptotic cells’ DNA cut the ends unusually
can label those ends with a flourescent deoxynucleotide label
biotinylated dUTP is used in to rxn, can be detected using a streptavidin that has a flourescent tag
visualize flourescence of the apoptotic cells under a microscope
TUNEL: “Terminal Deoxy Uridine Nucleotide End Labeling”
what is signal for macrophages to engulf an apoptotic cell?
phosphatidyl serine flipped localization to the outside of the plasma membrane from the cytoplasmic side where it usually is
how does annexin V and phosphatidyl serine flap assay work for apoptotic cells?
normally, phosphatidyl serine is on cytoplasmic side of plasma membrane
during phagocytosis, it undergoes a flip, is exposed on the extracellular surface of the membrane
this flip is the signal for a macrophage to engulf the cell, incur phagocytosis
can detect the phosphatidyl serine w/ an Annexin V dye, flourescently labeled dye
for an Annexin V assay, do you do anything to the membrane?
you should not permealize the membrane because then it would interact w/ the phosphatidyl serine on the inside of the membrane, which isn’t desired
if you do this, can visualize the nucleus, though
generally, is apoptosis triggered the same way in all cells?
no
how is apoptosis activated in T cells?
by glucocorticoids and antibody binding to the cell surface
what are things that could occur in the cell that would trigger apoptosis? aka what are causes for a cell to undergo apoptosis?
1) specialized cell surface receptors bind ligands
2) DNA damage: ionizing radiation, UV light, genotoxic drugs
3) oncogene activation
4) growth factor withdrawal
what is a caspase
a protease that mediates apoptosis
what is the molecular action of caspases?
hint: think about their name
caspases cleave after specific aspartic acids, NOT GLUTAMIC ACID
they have a cysteine in their active site
they are synthesized as an inactive precursor or zymogen
cysteine + aspases = caspases
what is the structure of caspases?
heterodimer of a large and small subunit; both come from the same original polypeptide
how are caspases activated?
each heterodimer has 2 cleavage sites that must be cleaved in to the large and small subunits in order to free the active caspase from its prodomains and activate it
what different kinds of caspases are there?
1) initiator: caspase-8 and caspase-9
2) executioner: -2, -3, -6, -7
what is the function of each kind of caspase? what initiates each kind of caspase?
initiators: cleave and activate the executioner caspases. are activated by an extracellular signal or DNA damage
executioners: cleave cellular substrates. are activated by the initiator caspases.
what breaks down lamins? result?
executioner caspases break down lamins
result: loss of nuclear membrane
what are executioner caspase’s substrates?
1) DNA degredation proteins: CAD/ICAD
2) DNA repair proteins: PARK:poly ADP ribose polymerase
3) cytoskeletal proteins: gelsolin
4) nuclear structures: lamins
what cleaves DNA?
CAD, an endonuclease
describe how DNA cleavage occurs
CAD is the endonuclease that cleaves DNA. it is bound to and inhibited by iCAD.
executioner caspases cleave ICAD, freeing CAD
CAD then can actively degrade DNA
what are the signaling pathways that initiate apoptosis, activate the initiator caspases?
1) extrinsic pathway: extracellular signal, usually binding of a ligand to cell surface receptor
2) intrinsic pathway: something happens internally in cell; commonly for DNA damage; mitochondria-related pathway
what are the steps of the extrinsic and intrinsic signaling pathways to apoptosis?
what is the caspase of each signaling pathway?
extrinsic: caspase 8
intrinsic: caspase 9
what are the steps of the extrinsic pathway involving cell surface receptors for apoptosis?
1) ligand binds to ligand receptor
induces receptor trimerization
2) intracellular receptor-associated proteins are recruited by the clustering of the cytoplasmic ends of the receptors
3) clustering of many procaspase-8 molecules with low levels of proteolytic activity triggers proteolytic cleavage of procaspase-8, initiates execution of the caspases
apoptosis occurs
in the extrinsic caspase pathway model, what are examples of the ligand? the receptor?
ligand: Fas ligand, Tumor Necrosis Factor, or TRAIL
receptor: Fas **death receptor **or another death receptor
what is the “holoenzyme model”?
cytochrome C is released from mitochondria’s intermembrane space
cytochrome C interacts with cellular protein Apaf1 and dATP
this forms a holoenzyme that cleaves procaspase-9
causes intracellular pathway of apoptosis
what is the apoptosome?
combination of cytochrome C from the mitochondria + Apaf-1 from cytoplasm
together recruit and activate procaspase-9
what is Bcl-2?
anti-apoptotic family of proteins with 4 homology domains (BH1-4)
what are the BH123 proteins?
pro-apoptotic
Bax, Bak
are related to the pores created in the mitochondrial membrane from which cytochrome c is released
it is not clear if the BH123 cause the pores to be formed, but is clear that an apoptosis signal leads to BH123 proteins’ insertion in mitochondrial membrane
they aggregate at the pores’ site
what is the relationship between Bcl-2 and Bax/Bak?
Bcl-2 binds to Bax/Bak and inhibits them from forming pores
this inhibition of pore formation prevents apoptosis, keeps cell alive
this inhibition through heterooligimerization
would cancer cells tend to have high or low Bcl-2 levels?
high
cancer cells w/ high Bcl-2 are resistant to cell death response
what are BH3-only proteins?
apoptosis activating proteins that come in 2 forms:
1) Bid
2) PUMA and NOXA
what is Bid proteins’ action?
when Bid is upregulated, it interacts with Bax and Bak, activates them to do pore formation
cytochrome C is release
apoptosis occurs
what is PUMA and NOXA proteins’ actions?
BH3-onlys that bind to Bcl-2, inhibit it from inhibiting Bax and Bak, and so pores can form and apoptosis occurs
how does p53 interact with BH3-onlys?
it transcriptionally upregulates PUMA and Noxa, which in turn bind to and inhibit Bcl-2, and BAX and BAK can form the pores
what is the phenotype of mice lacking Bax and Bak?
this double knockout is lethal
lacking either Bak or Bax is viable
if mice lack caspase-8, -9, cytochrome C, or APAF-1, what is result?
lethal
shows apoptosis is important in development
what roles does apoptosis play during embryonic development?
1) sculpts structures
2) does digit formation
3) hollows out solid structures to form lumina
4) deletes unneeded structures - vestigial or 1 sex only
what controls cell numbers
apoptosis
neurons, oligodendrocytes are over-produced in vertebrates then eliminated to match cells they innervate
how is apoptotic response important in cancer?
tumor cells suppress apoptosis - cell survival is central to how tumorigenic process occurs
Bax and Bak have been shown to be lost in some tumors
