How is apoptotic cell recognized and engulfed?
In 1983, E. Hedgecock isolated two cell death mutants
(ced-1 and ced-2) which are pivotal for identification
of the other cell death mutants.
Wild-type
ced-1
Similar screens to look for more
engulfment defective mutants
Wild-type
ced-x?
Did not identify any additional mutant except more ced-1 alleles
Why?
Why no additional engulfment genes were found?
A) Only one gene is needed
B) Phenotypes are too weak and thus mutations are
difficult to isolate
C) The graduate students did not try hard enough
D) Maternal effect
E) Other engulfment mutants are all lethal.
Maternal Effects
Rare case
P0
m/+ x m/+
m/+ x m/+
F1
m/m
m/m
mutant
wild-type
F2
m/m
m/m
Phenotype
mutant
mutant
Phenotype
A maternal effect is the phenomena where the genotype of a
mother is expressed in the phenotype of its offspring, unaltered by
paternal genetic influence
Maternal Effects
P0
ced-1/+
ced-2/+
F1
ced-1/ced-1
ced-2/ced-2
Phenotype
F2
corpses
wild-type
corpses
corpses
Are there more cell death genes that have
maternal effects?
Regular Genetic Screens
EMS
P0
tra-2(n1106)
rare tra-2(n1106); m/+
F1
F2
+/+
m/+
Egl suppressor
m/+
m/m
Look for rare F2 mutants
Maternal Effect Screens
EMS
tra-2(n1106)
P0
Rare
F1
tra-2(n1106); m/+
F2
+/+
m/+
No suppressor
m/+
F3
m/m
suppressor
R. Ellis and R.H. Horvitz performed F3 screen and identified mutations
in four additional genes affecting cell corpse engulfment
ced-5, ced-6, ced-7 and ced-10
Two partially redundant pathways
control cell corpse engulfment in C. elegans
ced-1
ced-6
ced-7
cell corpse engulfment
ced-2
ced-5
ced-10
ced-12
Cell migration
Why are ced-2, -5, -10, -12 mutants defective in
both corpse engulfment and cell migration?
A) It is purely coincidental.
B) These genes regulate a cellular process common to
both corpse engulfment and cell migration
C) Cell migration is a required step for corpse
engulfment
D) Corpse engulfment is a required step for cell
migration
E) None of above
Cell corpse engulfment
Cell-cell signaling between the dying cell and the phagocytic cell
Cell corpse engulfment
Cell-cell signaling between the dying cell and the phagocytic cell
ced-2, 5, 10, 12 regulate membrane extension
ced-2, 5, 10, 12 encode proteins regulate cytoskeleton reorganization
ced-2
ced-5
ced-10
ced-12
CRK II (SH2, SH3)
DOCK180
Rac I (GTPase)
regulator of Rac
CED-7
?
CED-2
CED-5
Apoptotic cell
CED-10
cytoskeleton
Engulfing cell
CED-12
ced-1 and ced-7 might involve in cell corpse recognition
ced-1 EGF-like receptor
ced-7 ABC transportor
In ced-7(lf) mutants, CED-1::GFP no longer encloses apoptotic cells
ced-7 may be involved in recognizing or exposing an “eatme” signal
ced-1 may be involved in recognizing an “eat-me” signal
ced-6 phosphotyrosine-binding protein
What is the “eat-me” signal?
CED-6
cytoskeleton
CED-7
CED-1
CED-7
?
CED-2
CED-5
Apoptotic cell
CED-10
cytoskeleton
Engulfing cell
CED-12
PS is restricted to the inner leaflet of plasma membrane
and its externalization can trigger phagocytosis
Normal cell
Phosphatidylserine(PS)
Apoptosis
Dying cell
Phagocyte
Engulfment
*
*
*
*
*
*
*
*
*
*
Secreted AnxV::GFP
21
How is PS externalized during apoptosis?
CED-8, an unexpected activator of apoptotic
PS externalization
• ced-8 was first identified as a gene involved in regulating the
timing of apoptosis and encodes a homologue of human XK
transporters (Stanfield and Horvitz, Mol Cell 2000)
CED-8
acCED-8
Out
Out
CED-3
In
CED-3
In
cleavage
Living cells
Dying cells
Ectopic expression of acCED-8 is sufficient to induce
PS externalization in all living cells in C. elegans
Surface-exposed PS is labeled by the PS-binding Lactadherin::GFP
Chen et al., Nature Communications 2013.
Clearance of cell corpses is significantly
compromised in the ced-8(-/-) mutant
WAH-1 promotes nuclear and cell surface
apoptotic events through CPS-6 and SCRM-1
PS
Death stimulus
SCRM-1
WAH-1
CPS-6
PS
Multiple cps genes mediate recognition and
engulfment of apoptotic cell corpse
cps-7
cps-9
cytoskeleton
psr-1
cps-12
cps-13
cps-14
PS (phosphatidylserine)
How is exposed PS recognized by
phagocytes?
PSR-1 is a PS binding protein
Yang et al., (2015) Nature Communications
PSR-1 may transduce the “PS eat-me” signal
through CED-5 and CED-12 signaling pathway
cps-7
cps-9
cps-12
cps-13
cps-14
psr-1
CED-10
CED-5
PS
CED-2 CED-12
Wang et al., (2003) Science 302: 1563-1566
Wang et al. Nature Cell Biology 2010
How do living cells maintain PS asymmetry?
What happens if PS asymmetry is disrupted in
living cells?
Aminophospholipid translocases are implicated in
restricting PS to the inner leaflet of plasma membrane
There are 6 aminophospholipid translocases in C. elegans, which are
annotated as tat genes (transbilayer amphipath transporter)
Genetic inactivation of tat-1 but not other tat genes causes
stronger PS exposure on the surface of living cells
DIC
Annexin V
Hoechst
DIC
Annexin V
Hoechst
tat-1(-/-)
tat-4(-/-)
tat-2(-/-)
tat-5(-/-)
tat-3(-/-)
tat-6(-/-)
tat-1 functions to restrict PS to the inner leaflet of plasma
membrane
Darland-Ransom et al. Science 320, 528, 2008
Can externalized PS in living cells
induce phagocytosis?
Pida-1GFP
Pmec-4GFP
AVM
ALM
PLM
PVM
ADE
HSN
PHA/PHB
VC
% animals missing at
least one touch cell
% animals missing at
least one neurons
WT
tat-1(lf)
1%
19%
WT
tat-1(lf)
1%
24%
tat-3(lf)
1%
tat-3(lf)
2%
Inactivation of tat-1 causes random loss of living cells
Two partially redundant pathways promote removal
of apoptotic cells in C. elegans
Exposed
PS
CED-6
ced-1 ced-6
ced-7
cytoskeleton
CED-7
CED-1
CED-7
PSR-1
PS
?
CED-2
CED-5
Apoptotic cell
Exposed
PS
psr-1
ced-2
ced-5
ced-12
CED-10
cytoskeleton
ced-10
Engulfing cell
CED-12
Living cells in the tat-1 mutant are
removed by a phagocytic mechanism
Pmec-4GFP
AVM
ALM
Pida-1GFP
PLM
PVM
ADE
HSN
PHA/PHB
VC
% animals missing at
least one touch cell
% animals missing at
least one neurons
WT
tat-1(lf)
1%
19%
WT
tat-1(lf)
1%
24%
ced-1(lf)
0%
ced-1(lf)
2%
ced-1(lf); tat-1(lf)
0%
ced-1(lf); tat-1(lf)
1%
psr-1(lf)
1%
psr-1(lf)
2%
tat-1(lf); psr-1(lf)
2%
tat-1(lf); psr-1(lf)
2%