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 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.

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

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?

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

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

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? 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

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

cytoskeleton Apoptotic cell Engulfing cell CED-7 ? CED-2 CED-5 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

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

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

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

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

* * * * * * * * * * 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

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 CED-3 Out In Out In CED-3 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 Death stimulus SCRM-1 WAH-1 CPS-6

Multiple cps genes mediate recognition and engulfment of apoptotic cell corpse cytoskeleton 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-5 CED-10 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)

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

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 1% tat-1(lf) 19% WT 1% tat-1(lf) 24% tat-3(lf) 1% tat-3(lf) 2% % animals missing at least one touch cell % animals missing at least one neurons

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

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 1% WT 1% tat-1(lf) 19% tat-1(lf) 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%