toxoplasma gondii tachyzoites phenotypic analysis of mutants · from j-f.dubremetz toxoplasma...
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FromJ-F.Dubremetz
Toxoplasmagondiitachyzoites
Phenotypicanalysisofmutants
MeBOP2016-ModuleII–KarineFrénal,DamienJacot,PacoPino
Toxoplasmagondiitachyzoiteculture
Incubator - 37°C - 5% CO2
Medium - DMEM (Dulbecco's Modified Eagle Medium), high glucose - FCS: fetal calf serum - L-Glutamine - Gentamycin
P2 lab !!
Host cell - HFF Human foreskin fibroblasts - VERO cells
SoldaCandMeissner,CurrentOpinioninCellBiology,2004
InvasionGliding
Egress
intracellulargrowth
A<achment
Ly>ccycleofToxoplasmagondiitachyzoites
Lysisplaques
fixedaNer7daysstainedwithcrystalviolet
48hforonelyCccycle
FromM-A.Hakimi
nucleus
Toxoplasmagondiitachyzoites
Intracellular Extracellular
dense granule
IMC
mitochondrion
ER
nucleus
Golgi
apicoplast
PMrhoptry
microneme
conoidpolar ring
FromJ-F.DubremetzandT.Dowse
Conoid Polar ring
Micronemes
Rhoptries Plasma membrane
Apicoplast Golgi apparatus
Nucleus Endoplasmic reticulum
Mitochondrium
Inner membrane complex Dense granules
2 µm
UltrastructureofToxoplasmagondiitachyzoite:organelles
conoid
Integrityandposi>oningoforganellesbyimmunofluorescence
Cpn60 actin
ROP2-4 MIC2
GRASP
HSP70
IMC1
UltrastructureofToxoplasmagondiitachyzoite:cytoskeleton
2µm
MorrisseYeetSibley,2002
Alveolinnetwork
Pellicle
Apical pole
Posterior pole
plasma membrane
inner membranecomplex (IMC)
microtubules
pellicle
DivisionofToxoplasmagondiitachyzoite
C
PR
Golgi
MyoF
DC
N SM
Ct
ARhMc
Ap
Residualbody
B Replication by endodyogeny and organization in rosettes
Asynchronous culture but synchronized replication within each parasitophorous vacuole
IMC1
MoviefromG.WardhYp://sbb.uvm.edu/~gward/Movies.html
FromJ-F.Dubremetz
Mo>lityandinvasion
Direction of movement
Host cell
Theglidingmo>lityreliesontheglideosome
MoviefromSoldaCetal.,TrendsinParasitology,2004
Glidingassayonpoly-L-lysinecoatedcoverslip
α-SAG1 α-SAG1
Apicalsecretoryorganellesandinvasion
Apical pole
Posterior pole
micronemerhoptry
2 μm
Excreted/SecretedAnCgen(WB)
MovingjuncConformaCon(IFA)
RhoptrysecreConassay
ore-vacuoleassay(IFA)
Invasionassay(red/green)
ASP3-iKDandcontrol+/-ATc
Invasion30minutesat37°C
FixaConandIFA
coverslip
ImmunofluorescenceassayorIFAStep1:Nopermeabiliza>on!!Stainingoftheextracellularparasites(primaryAb:surfacemarker,ex:anC-SAG1)Step2:aSerpermeabiliza>on!!Stainingoftheintracellularandextracellularparasites(primaryAb:insidemarker,ex:anC-GAP45thensecondaryAb)
Invasionassay(red/greenIFA)
ASP3-iKDandcontrol+/-ATc
Invasion30minutesat37°C
FixaConandIFA
coverslip
SAG1-red GAP45-green
ImmunofluorescenceassayorIFAStep1:Nopermeabiliza>on!!Stainingoftheextracellularparasites(primaryAb:surfacemarker,ex:anC-SAG1)Step2:aSerpermeabiliza>on!!Stainingoftheintracellularandextracellularparasites(primaryAb:insidemarker,ex:anC-GAP45thensecondaryAb)
Intracellulargrowthassay(IFA)
ASP3-iKDandcontrol+/-ATc
Incuba>on24-30hoursat37°C
FixaConandIFA
coverslip
IFAWithpermeabiliza>on(primaryAb:anC-GAP45,secondaryAb:anC-rabbit-Alexa594)
Ionophore-inducedegressassay(IFA)
GRA3SAG1
+DMSO
ASP3-iKDandcontrol+/-ATc
Incuba>on30hoursat37°C
SCmulaConwithA23187orDMSOfor7minutes37°C
coverslip
IFAWithpermeabiliza>on(primaryAb:anC-GAP45,secondaryAb:anC-rabbit-Alexa594)(primaryAb:anC-GRA3,secondaryAb:anC-mouse-Alexa488)
+A23187
GRA3SAG1