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