1. How is apoptotic cell recognized and engulfed?

2. 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

3. 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?

4. Why no additional mutants were found?
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.

5. Maternal Effects m/+ x m/+ m/m wild-type m/+ x m/+ m/m P0 F1 mutant F2
Rare case
m/+ x m/+
m/m P0 F1
Phenotype
mutant F2
m/m
mutant
Phenotype
A maternal effect is the phenomenon where the genotype of a mother is expressed in the phenotype of its offspring, unaltered by paternal genetic influence

6. Are there more engulfment genes that have maternal effects?P0
ced-1/+
ced-2/+ F1
ced-1/ced-1
ced-2/ced-2
Many corpses
wild-type
Phenotype F2
Many corpses
Many corpses
Are there more engulfment genes that have maternal effects?

7. Regular Genetic Screens
tra-2(n1106) P0 F1
rare
tra-2(n1106); m/+ F2
Egl
suppressor
EMS
m/m
+/+
m/+
Look for rare F2 mutants

8. Maternal Effect Screens
EMS P0
tra-2 (
n1106 )
; m/+ F1
Rare
tra-2
n1106 ( ) F2
+/+
m/+ No
suppressor
m/+
m/m F3
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

9. 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

10. Why are ced-2, -5, -10, -12 mutants defective in both corpse engulfment and cell migration?
It is purely coincidental.
These genes regulate a cellular process common to both corpse engulfment and cell migration
Cell migration is a required step for corpse engulfment
Corpse engulfment is a required step for cell migration
None of above

11. Cell corpse engulfment
Cell-cell signaling between the dying cell and the phagocytic cell

12. Cell corpse engulfment
Cell-cell signaling between the dying cell and the phagocytic cell

14. ced-2, 5, 10, 12 regulate membrane extension

15. ced-2 CRK II (SH2, SH3) ced-5 DOCK180 ced-10 Rac I (GTPase)
ced-2, 5, 10, 12 encode proteins regulate cytoskeleton reorganization
ced CRK II (SH2, SH3)
ced DOCK180
ced-10 Rac I (GTPase)
ced-12 regulator of Rac

16. cytoskeleton Apoptotic cell Engulfing cell CED-7 ? CED-2 CED-5 CED-10

17. 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

18. ced-1 and ced-7 might be involved 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

19. ced-7 may be involved in recognizing or exposing an “eat-me” signal
ced-1 may be involved in recognizing an “eat-me” signal
ced-6 phosphotyrosine-binding protein

20. What are the “eat-me” signals?
Apoptotic cell
Engulfing cell
CED-2
CED-10
CED-1 ?
CED-5
CED-12
cytoskeleton
CED-7
CED-6

21. PS is restricted to the inner leaflet of plasma membrane and its externalization can trigger phagocytosis
Phosphatidylserine(PS)
Normal cell
Dying cell
Apoptosis
Phagocyte
Engulfment

22. * * * * * * * * * *
Secreted AnxV::GFP
Using time-lapse microscopy, we show for the first time that in living C. elegans embryos both apoptotic and neighboring phagocytic cells express exPS during apoptosis, providing strong in vivo evidence for exPS expression by phagocytes.
We thus examined whether genes important for removal of apoptotic cellsmight affect exPS expresion. 22

23. How is PS externalized during apoptosis?

24. 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
CED-3
Out In
Out In
CED-3
cleavage
Living cells
Dying cells

25. 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.

26. Clearance of cell corpses is significantly
compromised in the ced-8(-/-) mutant

27. WAH-1 promotes nuclear and cell surface apoptotic events through CPS-6 and SCRM-1
Death stimulus
SCRM-1
WAH-1
CPS-6

28. Multiple cps genes mediate recognition and engulfment of apoptotic cell corpse
cytoskeleton
PS (phosphatidylserine)

29. How is exposed PS recognized by phagocytes?

30. PSR-1 is a PS binding protein
Yang et al., (2015) Nature Communications

31. 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-5
CED-10 PS
CED-2
CED-12
Wang et al., (2003) Science 302:

32. Wang et al. Nature Cell Biology 2010

33. How do living cells maintain PS asymmetry?
What happens if PS asymmetry is disrupted in living cells?

34. 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)

35. tat-1 functions to restrict PS to the inner leaflet of plasma membrane
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

36. Can externalized PS in living cells induce phagocytosis?
Inactivation of tat-1 causes random loss of living cells
Pmec-4GFP
AVM
PLM
PVM
ALM
ADE
PHA/PHB
HSN VC
Pida-1GFP
WT %
tat-1(lf) %
WT %
tat-1(lf) %
tat-3(lf) %
tat-3(lf) %
% animals missing at least one touch cell
% animals missing at least one neurons

37. Two partially redundant pathways promote removal of apoptotic cells in C. elegans
Engulfing cell
CED-2
CED-10
CED-1 ?
CED-5
CED-12
cytoskeleton
CED-7
CED-6
ced-6
Exposed PS
ced-1
ced-7 PS
PSR-1
ced-2
Exposed PS
ced-10
psr-1
ced-5
ced-12

38. Living cells in the tat-1 mutant are removed by a phagocytic mechanism
Pmec-4GFP
Pida-1GFP
AVM
PLM
PVM
ALM
ADE
PHA/PHB
HSN VC
% animals missing at least one touch cell
% animals missing at least one neurons
WT %
WT %
tat-1(lf) %
tat-1(lf) %
ced-1(lf) %
ced-1(lf) %
ced-1(lf); tat-1(lf) %
ced-1(lf); tat-1(lf) %
psr-1(lf) %
psr-1(lf) %
tat-1(lf); psr-1(lf) %
tat-1(lf); psr-1(lf) %