programmed cell death Flashcards
characteristics of apoptosis vs necrosis
apoptosis = planned event
cell shrinkage and DNA fragmentation
- Membrane blebbing - protrusion of the cell membrane, actin filament breakdown
- formation of apoptotic bodies
- cell fragmentation
- NO INFLAMMATORY RESPONSE
Necrosis -consequence of trauma/lack of blood supply
- cell swelling
- membrane BREAKDOWN
- cell disintegration
- INFLAMMATORY RESOPONSE
what are the three phases of apoptosis?
- Specification phase (cell instructed to undergo apoptosis)
- Killing phase (apoptotic programme activated)
- Execution phase (cell dismantled and engulfed)
how was gel electrophoresis used to assay for apoptosis? what was seen?
used to look for DNA fragmentation
grew some cardiomyocytes and some cardiac fibroblasts, grown in a culture medium
moved the cells to a serum free medium, and looked at them both periodically
saw -
Fibroblasts - DNA integrity maintained, just one/few high molecular weight bands
Myocytes - DNA is being broken down between nucleosomes, so we see laddering (spreading out as different sized fragments)
what is a TUNEL assay?
when DNA is cut like in apoptosis, the 3’ hydroxyl is exposed
this can be used to fluorescently label broken DNA
what are caspases?
not all caspases are apoptotic proteins
- Proteases
- Cysteine in active site, cleave proteins after aspartic acid residues (so they can target a large number of proteins)
what do caspases target in apoptosis?
Nuclear lamins (breakdown nuclear membrane)
Cell adhesion proteins (break cells apart)
Components of the cytoskeleton
A certain endonuclease
importance of programmed cell death in development?
Metamorphosis - tadpole loses a tail
Immune system
Removal of excess cells, e.g. brain development (more neurons develop than often required)
what are the two kinds of caspases? what is their structure like?
Two kinds, initiator caspases and executioner caspases
All synthesised as inactive procaspases
Structure -
All have at least a large subunit and a small subunit, with a linker in between
Initiator caspases have prodomains at the N terminus (to interact with other components)
The monomers dimerise, but are still inactive…
Until the linkers between large and small subunits are cleaved (and between prodomain and large subunit if an initiator)
Now you have an active tetramer (four subunits)
how is caspase activation a cascade?
initiator caspases must be activated first (dimerise, cleavage of linker domains)
these can then go and cleave the linkers on the executioners to activate them
how come apoptosis doesnt occur all the time?
apoptotic components are present in the cell 24/7, but remain inactive until - for initiator caspases - they dimerise and linkers are cleaved, and then they can go and do the same for executioner caspases
what is CAD -
it’s structure/how it is kept inactive most of the time?
its function?
Caspase-activated DNAse (CAD) -
Enzyme responsible for DNA fragmentation
Needs to be a dimer to function
Folding of monomer requires chaperone ICAD (inhibitor of CAD) so normal conditions cell has monomers of CAD bound to ICAD
Executioner caspases cleave ICAD, allowing CAD to dimerise and activate
what enzyme is responsible for DNA fragmentation?
CAD -
Caspase-Activated DNAse
explain the extrinsic pathway of apoptosis activation
- Requires an extracellular ligand - Fas-L, TNF or TRAIL usually
2.Ligand requires receptor - each one has its own ‘death receptor’
- When ligand binds, receptor partially internalises so it can bind adaptor protein (e.g. FADD)
- Adaptor protein binds inactive initiator caspases into a ‘DISC’ (death-inducing signalling complex). This grouping of I-caspases allows them to activate one another in autoproteolysis
explain the intrinsic pathway of apoptosis activation
- Apoptotic signal triggers mitochondrial leakage, inc.
- cytochrome C
Cyt C activates Apaf1 (apoptotic protease-activating factor-1) - Apaf1 oligomerises into an apoptosome
- The apoptosome exposes binding sites for procaspases, bringing them together so they can activate one another in autoproteolysis
- Now active initiator caspases go on and activate executioner caspases
how does the BCL-2 family tightly control mitochondrial leakage (as this triggers the intrinsic apoptotic pathway)?
BAX/BAK sit on outer mitochondrial membrane
Normally they are bound to BCL-2, preventing the two from forming a pore complex
however, apoptotic signals cause BH3 proteins to activate, displace BCL-2 and allow BAX/BAK to form a pore
allowing cyt-c into the cytoplasm - intrinsic pathway etc…
John E Sulston?
mapped lineage of all cells in adult C. elegans from the embryo
Notice some cells would always undergo apoptosis
E.g. 20% of neural cells undergo apoptosis, inc. some (not all) of the hermaphrodite-specific neurons (HSNs)…
Identified egl-1 gene in a screen for egg-laying defects
Dominant egl-1 mutants (egl-1 always ON) were defective in egg laying…
Why - Some hermaphrodite-specific neurons must be present for egg laying
In the egl-1 mutant, ALL HSNs underwent apoptosis…
So egl-1 must be pro-apoptotic
how were CD4 and CD3 mutants identified, and what was discovered about their role?
Ced-1, an already identified mutant involved in the final phase of apoptosis, results in dying cells persisting for longer
Horvitz group mutagenised ced-1 mutant worms, because dying cells that persist are easier to study
Identified ced-3 and ced-4 mutants - these mutants had NO dying cells so must be involved in the killing stage
how was it disocvered if CED-3 and CED-4 come before or after egl-1 in the apoptotic pathway?
Crossing ced-3 and ced-4 mutants with egl-1 mutants -
The offspring could lay eggs - the HSN neurons did not all die despite the dominant egl-1 mutation
This indicates the ced-3 and ced-4 genes act after egl-1 in the apoptotic pathway
ced-9D is a dominant mutation, i.e. the gene is always ON.
what phenotype does the mutant have?
how was its position in the apoptotic pathway, with respect to the other genes, determined?
same phenotype as ced-3 or 4 mutants, in that cells did not die
BUT - dominant mutant meaning its overactive, ced-9 must be involved in inhibiting apoptosis
Crossing Egl-1 and Ced-9 mutants -
Again HSN cells did not die (could lay eggs) so Ced-9 must be downstream of Egl-1, and likely Egl-1 inhibits Ced-9 normally
Loss of function Ced-9 -
Horvitz lab made LOF Ced-9 mutant = lethal; all cells died
Crossing with Ced3/4 mutants - rescued the cell, so Ced-9 must be before ced-3 and 4 in the pathway
with the experiments in mind, what might the egl-1, ced-3 and 4, and ced-9D code for/be homologous to?
Egl-1 = BH3 (so always being on = always replacing Bcl2 and allowing pore to form)
Ced-9 = Bcl2 (so always on = always inhibiting apoptosis
Ced-4 = APAF-1 (so without it apoptosis pathway is stopped despite Ced-9 or Egl-1 always being on)
Ced-3 = an initiator caspase (caspase 9)
note - the ‘interactions’ are genetic, i.e. does not imply the proteins inhibit each other in every case or something
TUNEL assay?
a super easy way to look for where apoptosis is occurring - tags the 3’ hydroxyls from DNA fragmentation
to look at apoptosis in development, it’s also important to view…?
antiapoptotic proteins to see where it is being inhibited
e.g. screen for BCL-2, (expressed where digits are but not where the webbing is in mice digit development)
why is studying apoptotic GENES a struggle, and where has there been a success?
Classic genetics struggles here - caspases have a lot of redundancy so knockouts are hard. Other apoptotic components are essential in multiple stages of development etc…
One success - BAX/BAK (form the pore in the outer mitochondrial membrane) double mutant showed inhibition of interdigit apoptosis
what are BMPs? what is one thing they might be involved in?
bone morphogenetic proteins
a family of signalling molecules (they are extracellular ligands, so also have receptors)
Circumstantial evidence to associate with apoptosis -
In situ hybridisation show BMP expression in interdigit region (webbing, so prior to apoptosis)
what is a struggle geneticists have when investigating BMP, and what is one experiment that got around this?
Issues - they are involved in multiple processes so can’t just mutate them
BMP Experiment 1 -
Injected chicks developing limb to get localised expression of a dominant negative BMP receptor -
this completely blocks activation of BMP pathway, but only where you want it to be
Result = maintained webbing, so it suggests BMP normally promotes apoptosis
what’s another experiment (beads) used to investigate BMP’s role in apoptosis?
Coat the factor you are interested in onto beads (BMP in this case). Put bead in the region you are interested in seeing its effects
Placed the coated bead on the webbing and…
Result = apoptosis was initiated where the bead was placed (earlier than it normally does)
how was a knockout successful when investigating BMP in apoptosis, and what was seen?
did a tissue specific knockout of BMP-7
Scientists did this somehow idk
Looked at phenotype - some webbing was retained so apoptosis was effected
Using things like RNA sequencing, other genes can be identified that are potentially involved
In this case, when BMP-7 was knocked out there was a reduction is expression of ‘MSX2’ , likely a downstream target of BMP-7
how did tissue specific knockout of BMP-7 show how BMPs and FGFs interact?
FGF = fibroblast growth factors
it is normally expressed at the fingertips ectoderm, not at the interdigit region
knocking out BMP-7 at the interdigit region had no impact on the interdigit region
knockout of BMP-7 in the ectoderm at the fingertips causes expansion of the FGF8 expression domain
SUGGESTING that BMP(7) normally inhibits FGF(8)
what was the experiment that confirmed FGFs inhibit apoptosis?
used fgf inhibitor on a pre-apoptosis mouse limb.
TUNEL assay for apoptosis showed the fgf inhibitor led to much more apoptosis compared to a control (so fgf must be an apoptosis inhibitor)
placed fgf coated bead on an older mouse limb in the IDR (apoptosis has begun).
Saw in TUNEL assay that fgf8 inhibits cell death
summary of FGF and BMPs?
Fgf inhibits BMP preventing apoptosis
BMP promotes apoptosis
They inhibit each other
Expression differs in location and over time
in webbing, how is apoptosis blocked? - is it less BMP, more FGF, or another player?
(include an experiment - hint: bead and ducks)
Analysis of expression levels (ISH) in ducks (webbed) vs chicks (sculpted) for both BMPs and Fgfs showed no difference
So - there must be another player involved…
Addition of BMP coated bead to an IDR of a duck foot, showed there was apoptosis - in this scenario, when BMP is in super high concentration…
So - this new player preventing apoptosis e.g. in ducks webbing, must be an inhibitor of BMPs
what was seen when looking at gremlin expression in a developing chick?
Panel A - pre-apoptosis, gremlin is seen in IDR (interdigit region)
Panel B - when apoptosis has started, gremlin is not expressed in IDR
Panel C - TUNEL assay of limb - when no gremlin is expressed - showing apoptosis beginning coincides with this (the stopping of gremlin expression)
what is gremlin?
an extracellular protein that can bind to BMP ligands - an inhibitor of BMP
gremlin beads experiment confirmed its role?
placing a bead coated in gremlin in a chick’s IDR showed an inhibition of apoptosis (that IDR maintained its webbing)
summarise expression in separated/sculpted digits, vs those that maintain their webbing
separated - gremlin expression declines in IDRs, BMPs are not inhibited, and apoptosis occurs
webbed - gremlin expression persists in IDRs, BMPs are inhibited and cannot trigger apoptosis
