C. elegans apoptosis pathway EGL-1 CED-9 CED-4 CED-3 ... documents/Franc.pdfC. elegans apoptosis...
Transcript of C. elegans apoptosis pathway EGL-1 CED-9 CED-4 CED-3 ... documents/Franc.pdfC. elegans apoptosis...
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!!!!!!!