C. elegans apoptosis pathway

EGL-1BH3

CED-9BCL2

CED-3CASPASE-9

CED-4APAF-1

Effector caspases

Subtrates cleavageCELL DEATH

Phagocytosis

Ced-1 Ced-2 Ced-5 Ced-6 Ced-7 Ced-10 Ced-12 PSRLrp CrkII Dock180 Gulp ABC-1 Rac Elmo PSR

Phagocytosis of apoptotic cells in C. elegans

Neighboring cell

Apoptotic corpse

CED-1(SR-EC/LRP)

CED-7(ABC-1)

CED-6(GULP)

Apoptotic ligands ???

CED-5(Dock180)

CED-2(CrkII)

CED-10 (Rac)GTP

CED-12 (Elmo)

CED-7(ABC-1)

PSR1

Model systems for studying Development

-Plants ( A. thaliana)

-Worms (C. elegans)

-Fruit flies ( D.melanogaster )

- Zebrafish (D. rerio )

Drosophila as a Model System

1. Short generation time ~10 days

Embryo

FirstSecond

Third Instar Larvae

Pupae

Adults

fertilization

1 day

3x1 day

3.5-4.5 days

mating

embryogenesis

pupariation2.5-3 days

Drosophila as a Model System

2. Complex body plan

Drosophila as a Model System

3. Only 4 chromosomes

_The first physical and genetic mapCentimorgan : 1% recombination(Sturtevant and Morgan)TH Morgan Nobel lecture 1933

_Polytene chromosomesIn situ hybridization

_Positional cloning, cDNA andchromosomal libraries ( Hogness )

_Genome project completed

Drosophila as a Model System

4. Genetic approach:• Loss of function genetics:

Chemically generated mutants (EMS, ENU) Chromosomal deficiencies (~ 85% of the genome, X-rays, γ-rays, P-elements)Transposable elementsGene knockout by homologous recombination Mosaic analysis: making clones of mutant cells in a normal tissue (FLP/FRT)

• Gain of function genetics: Dominant mutantsChromosome duplicationsTissue specific expression systems: UAS/Gal-4

• BiochemicalHPLC purification from whole organism extracts

•A physical map that corresponds to the genetic map: Fully sequenced, geneticallymarked genome.•Excellent cytogenetics : Genome is arrayed on overlapping Bacterial ArtificialChromosome (BAC) clones, SNPs mapping,•ESTs (Expressed Sequence Tags) characterized for all stages of the life cycle,•Genome-wide microarrays ,•Antibodies and transposable elements to mark gene products, cell morphology andsubcellular structure,•Stable single copy transformation,•Targeted, tissue specific expression systems (UAS/Gal-4),•Genome-wide RNAi library allow double stranded RNA bathing/ transfection of culturedcells to address partial or complete LOF phenotypes,•First genome-wide two hybrid screen (protein-protein interaction)•And the fly community will not stop there!

Drosophila as a Model System

5. Molecular approach :

Normal Cell

Cell shrinkage awayfrom neighbouring cells

Plasma membrane blebbingCytoplasmic and nuclear condensation

Margination of condensedchromatin

Nuclear and cellularfragmentation

Apoptotic BodiesPhagocytosis

Baehrecke Nat Rev Mol Cell Biol. 2002 Oct;3(10):779-87 .

are needed to see this picture.

The importance of PCD in development and homeostasis

Consequences of deregulation of PCD

Excessive cell death :Degenerative neurological disordersStroke, cardiac ischemia,Immune suppression associated with AIDS

Suppression of cell death:Autoimmune diseasesCancer

Programmed Cell Death (PCD)

1. Cell Death Signal

2. Execution of DeathFumiyo KuboGalen Galen

3. Engulfment of remains

Picture by M Janes & P Henson Nature. 2000 Oct 12;407(6805):784-8

http://www.cell death-apoptosis.org/ apop _pic .htm

Programmed cell death during Drosophila embryogenesis

John M. Abrams1, Kristin White1, Liselotte I. Fessler2 and Hermann Steller1Development 117, 29-43 (1993)1Howard Hughes Medical Institute, Department of Brain and Cognitive Sciences and Department ofBiology, Massachusetts. Institute of Technology, Cambridge, Massachusetts 02139, USA2Molecular Biology Institute and Biology Department, University of California, Los Angeles, CA 90024,USA

Allowed for the initiation of a deficiency screen for PCD mutantsAcridine orange staining

Genetic definition of adeficiency:contains at least twocomplementationgroups,

Overlappingdeficiencies provide stillfiner resolution,

Isogenic deletions withprecise breakpoints(drosdel project)

A deficiency kit that covers over 85% of the genome!!!

_allows for the rapid screening of the entire genomefor genes involved in any embryonic phenotype of interest

Homozygous Df(3L)H99 embryos lack all PCD

WT

H99

Acridine orange

The H99 deletion allowed for the identification ofthree regulators of PCD, rpr, hid, and grim

RPR

GRIM

HID

No homology of sequences except in their first 14 aa: RHG/IBM motif65 aa

138 aa

410 aa

239 aa

Rpr

Grim

Hid

Smac

Reaper 2 AVAFYIPDQATLL 14Grim 2 AIAYFIPDQAQLL 14Hid 2 AVPFYLPEGGADD 14Sickle 2 AIPFYEEEHAPKS 14Jafrac2 - AKPE -Smac /Diablo 54 AVPI 66

Jafrac 2 ERS peroxidase domain 242 aa

AKP

Trp block

Sickle 108 aa

Nuclei cleave in a common cytoplasm ( syncytium)At cycle 10, nuclei migrate to the periphery ( blastoderm )

Early embryogenesis in Drosophila

Germ line transgenic making in the fly

Used in:_Rescue experiments (replacement strategy)_Over-expression experiments_Homologous recombination

Genetic screens for enhancers and suppressors of PCD in the eye

QuickTime™ and aGraphics decompressor

are needed to see this picture.

Inhibitors/suppressors of apoptosis

dIAP1 blocks both reaper and hid-induced cell deathdBRUCE blocks reaper-induced cell death but not hiddIAP2 not a very good inhibitor of PCD, cell migration roleDETERIN is most homologous to survivin as it has a single BIR

BIR1 BIR2

BIR

RINGdIAP 1/thread

dBRUCE UBC

438 aa

4876 aa

dIAP 2

DETERIN

BIR1 BIR2 RING 498 aaBIR3

BIR 153 aa

DIAP-1: a central player

thread LOF mutation shows increased PCD in the embryo,and is embryonic lethal.

thread is epistatic to rpr, hid and grim.

GOF of Diap1prevents rpr, hid and grim-induced death.

GMR transgene alone has no effect, but it can inhibitDronc and Debcl -induced death, thus it geneticallyinteracts with Dronc, a caspase gene , and Debcl , a Deathenhancer with Bcl-2 homology (pro-apoptotic Bcl-2homologue).

There are 7 Drosophila CASPASES

Apical DRONC

DECAY

DRICE

Apical DREDD/DCP2

DAMM

DCP-1

STRICA/DREAM

CARD p20

p20

p14

p12

large small

p20 p10

p20 small

p20 p13

p20 smallSer/Thr Rich

Cysteine/ASPartate proteASES related to Ced-3

DED DED

CARD: Caspase Activation Recruitment Domain and DED: Death Effector Domain are protein-protein interactions domains

Caspases phenotypes and interactionsDronc RNAi prevents PCD in the embryo, Dronc deficiency andDronc dominant negative transgene genetically interact with rpr,hid and grim; Dronc over-expression induces cell death.

Dredd LOF have a defect in the immune response.

Damm dominant negative transgene genetically interacts withhid, and over-expression of Damm induces death.

DCP-1 LOF is lethal at third instar larval stage with no imaginaldiscs or gonads and melanotic tumors.

Drice , Decay, Strica : no LOF mutation. Drice over- expression hasno effect.

Drice and Dcp-1 bind to BIR1 of DIAP-1, and act downstream ofDronc

The Drosophila Gas and Brake model

RPRHIDGRIMSICKLE ?JAFRAC2 ?

DIAP-1 DRONC CELLDEATH

Gas Brake

BIR-2

_RPR, HID and GRIM (gas) compete with DRONC forbinding to BIR2 domain of DIAP1 (brake), whichinhibits DRONC._Upon cell death signals, RPR, HID or GRIM (gas) bindto DIAP-1 (BIR-2), competing for it with DRONC. Thusthe brake (DIAP1) is released from DRONC, the caspaseis activated and cell death occurs.

Death initiators and effectors

APAF-1

DARK

WD40 repeatsCARD

CARD

NBD

NBD WD40 repeats

Cytochrome c interaction ?

Apaf -1 related DARK

IAP antagonists of the Bcl-2 family: Buffy and Debcl

DEBCL/DROB-1/DBORG-1

BUFFYMembrane anchorBH3 BH1 BH2α1 α2

Membrane anchorBH3 BH1 BH2

300 aa

The apoptosome in mammals

Closedmonomer

Open

Cyt C

Heptamer

WD40

Inactivemonomer

Activedimer

procaspase ?

Inactiveactive

Drosophila apoptosome: how does it function ?

A

C

B

Cytochrome c release involvement still a question ?

Pro-apoptotic Debcl

Anti-apoptotic Buffy displaced by Debcl ?

RNA interference in Drosophila S2 cells

Introduction ofTRIGGER dsRNA

DICER: processes21-23nt siRNA

RISC: destruction ofTARGET mRNA

mRNA

Loss-of-function phenotype

dsRNA

RNAi Mechanism RNAiIn S2 cells

• dsRNA to serum-free medium (Clemens, et al. 2000 PNAS 97: 6499)

• Partial to complete loss of function • Uniform penetrance

approx. 500nt

438 essential genes !

MicroRNAs in Drosophila apoptosisDevelopmental and environmental death signals

GRIMREAPERSICKLEHID

DIAP1

DRONC

DRICE

CELL DEATH FAT LEVELS

DARK

?

Cell Growth and proliferation

Bantam

mir-2,mir-13 family ?

mir-14

Transcriptional control: RNAi/transcription complex, chromatin silencing

Drosophila intrinsic and extrinsic PCD pathways

Intrinsic death signals

Diap-1

Rpr, Grim. Hid

Dmp53Debcl

Cytochrome c

Buffy

Dark InitiatorCaspasesDroncDredd

EffectorCaspasesDcp-1Drice

BH3 only

CELLDEATH

?

?

?

?

?

Extrinsic death signals Innate immune response

Death receptor TNFR family

dFADD Dredd?

?

UV Sickle?

Phagocytosis

Programmed Cell Death (PCD)

1. Cell Death Signal

2. Execution of DeathFumiyo KuboGalen Galen

3. Engulfment of remains

Picture by M Janes & P Henson Nature. 2000 Oct 12;407(6805):784-8

http://www.cell death-apoptosis.org/ apop _pic .htm

C. elegans apoptosis pathway

EGL-1BH3

CED-9BCL2

CED-3CASPASE-9

CED-4APAF-1

Effector caspases

Subtrates cleavageCELL DEATH

Phagocytosis

Ced-1 Ced-2 Ced-5 Ced-6 Ced-7 Ced-10 Ced-12 PSRLrp CrkII Dock180 hCed-6 ABC-1 Rac Elmo PSR?

A role for Draper in phagocytosis of apoptotic cellsby l(2)mbn and SL2 cells and embryonic macrophages

DRAPER, a CED-1 homologue is expressed at themembrane of glial cells and of embryonic macrophages

Draper RNAi in l(2)mbn and SL2 cells prevents engulfmentof apoptotic cells

Crq RNAi does not prevent engulfment of apoptotic cellsAlthough the mRNA is targeted, the endogenous protein isstill present

Draper RNAi by injection of dsRNA in embryos reduces theengulfment of apoptotic cells in vivo .

Nakanishi and colleagues, 2004.

AND THERE IS SO MUCH MORE TO LEARN!

EGFR/RasEiger /TNFMAPK/Junk pathwayHippo/salvador/warts regulators of cell growthPCD and cell competitionDIAP2 in innate immunityDIAP2 in cell migrationAUTOPHAGYEtc…

John AbramsJulie AgapiteNicholas BakerEric BaehreckeAndreas BergmannSteve CohenMegan GretherErnst HafenIshwar HariharanBruce HaySally KornbluthSharad KumarKim McCallPascal MeierJohn NambuYoshinobu NakanishiGerry RubinHermann StellerNicolas TaponCraig ThompsonKristin White…and colleagues!!!!!!!