This study was carried out in strict accordance with the guidelines of the German Tierschutzgesetz (TierSchG; Animal Rights Laws). Mice were obtained from Charles River Laboratories. The protocol was approved by the Department of Veterinary Affairs of the Hamburg state authorities (Permit Number: FI 28/06). Blood feeding was performed under ketavet/rompun anesthesia, and all efforts were made to minimize suffering.
All parasite strains used in the present paper have a P. berghei
ANKA background. The wild type strain does not express any fluorescent proteins. P. berghei-
mCherry and -GFP express mCherry or GFP under the constitutive eef1aa promoter and show cytosolic localization of the respective fluorescent protein 
. In P. berghei
-Exp1-mCherry the PVM protein Exp1 
is fused to mCherry and expression is driven by the late liver stage-specific promoter of the gene PBANKA 100300 (PB103464.00.0) 
. In the Pbc
parasite strain, GFP is targeted to mitochondria by fusion with the predicted mitochondrial targeting sequence of P. berghei
heat shock protein 60 (Hsp60, PBANKA_121400); the sequence was amplified using the oligonucleotides ATATGGATCCATGCTATCTAGATTGTGTGGG
. Expression of the fusion protein in the parasite is controlled by the eef1aa promoter.
Transgenic P. berghei
strains were generated as described previously 
. Briefly, expression plasmids were introduced into blood stage schizonts using electroporation and transfected schizonts were then injected intravenously into mice for replication and selected with pyrimethamine.
Culture of HepG2 cells
HepG2 cells were purchased from the European cell culture collection and kept in Minimum Essential medium with Earle's salts supplemented with 10% FCS, 1% penicillin/streptomycin and 1% L-glutamine (all from PAA Laboratories, Austria). They were cultured at 37°C and 5% CO2 and split using Accutase (PAA Laboratories, Austria).
Transfection of HepG2 cells
HepG2 cells were harvested by Accutase treatment and 2x106
cells were pelleted by centrifugation at 160g. They were resuspended in Nucleofector V solution (Lonza, Cologne, Germany) and transfected with 3–5 µg plasmid DNA using program T-28 of the Nucleofector transfection device according to the manufacturer's instructions. The mCherry-pDisplay-plasmid was generated by inserting the coding sequence of mCherry into the pDisplay vector (Invitrogen, Darmstadt, Germany) and kindly provided by Isabelle Tardieux (Universite Paris Descartes, Paris, France). The pDsRed1-N1-Cox8 and pEGFP-N1-Cox8 plasmids contain the respective fluorescence protein fused to the targeting sequence of the mitochondrial protein Cox8. The hIR-GFP plasmid was purchased from addgene (Addgene plasmid 22286) and originally created by Rowena Ramos 
HepG2 cell infection
HepG2 or HepG2-GFP cells were seeded into either glass bottom dishes (WillCo Wells BV, The Netherlands) or on cover slips in 24-well plates. At 12–24 hours after seeding, P. berghei
sporozoites were isolated from the salivary glands of infected Anopheles stephensi
mosquitoes and added to the HepG2 cells. After an incubation period of 2 hours the sporozoite-containing medium was removed and fresh medium was added. At the indicated time points, infected cells were prepared for imaging. Detached cells and merosomes were collected from the supernatant of infected cell cultures as a mixture. Since merosomes have been shown to bud from detached cells 
, they share the same membrane and were therefore examined as one.
Cycloheximide treatment of infected HepG2 cells
HepG2 cells were seeded at 8x104 cells per well into 24-well plates and infected with P. berghei sporozoites. They were then treated with 5 µg/ml cycloheximide (Sigma-Aldrich, Taufkirche, Germany) for the indicated time periods. The medium was changed twice daily and detached cells were removed and counted at 70 hpi.
was added at 1 µg/mL for 10 min at 37°C and 5% CO2
. MitoTracker Green
(Molecular Probes, Invitrogen, Darmstadt, Germany) was added to a concentration of 300 nM and incubated for 30 min at 37°C and 5% CO2
. Propidium iodide
(Sigma-Aldrich, Taufkirche, Germany) was added at a concentration of 3 µM for 15 min at room temperature. pSIVA
(kindly provided by Yujin Kim, University of Southern California) 
was added at 20 µg/ml and incubated for 1 h at 37°C and 5% CO2
(Molecular Probes, Invitrogen, Darmstadt, Germany) was added to a concentration of 25 nM and incubated for 30 min at 37°C and 5% CO2
. Vybrant DiO
(Molecular Probes, Invitrogen, Darmstadt, Germany) was added at 5 µl/ml for adherent and 10 µl/ml for already detached cells and incubated for 1 h at 37°C and 5% CO2
. For all stainings of adherent cells, the staining medium was removed after the incubation period and fresh medium was added. For detached cells, the staining solution was diluted by adding an equal amount of fresh medium before imaging.
Indirect immunofluorescent staining
8x104 HepG2 cells were seeded on cover slips in 24-well plates. At appropriate time points after infection cells were fixed in 4% paraformaldehyde/PBS for 20 minutes at room temperature. They were washed three times with PBS and subsequently incubated in ice-cold methanol for 10 minutes at −20°C. After washing with PBS, unspecific binding sites were blocked by incubation in 10% FCS/PBS for 1 h at room temperature. Primary antibodies were diluted in 10% FCS/PBS and incubated for 2 hours at room temperature. Cells were then washed three times with PBS and secondary antibodies in 10% FCS/PBS were added for 1 hour at room temperature. Cells were washed again with PBS and mounted on microscope slides with Dako Fluorescent Mounting Medium (Dako, Cambridgeshire, UK). For the staining of detached cells and merosomes, the cell culture supernatant was collected at 68 hpi and centrifuged at 160g for 5 minutes. The cells were then resuspended in 4% PFA/PBS and incubated for 20 minutes at room temperature. Afterwards, they were spun down at 160g for 5 minutes and resuspended in a small amount of 4% PFA/PBS. The solution was subsequently spread onto a microscope slide using a cytospin centrifuge at 800g for 4 minutes. The detached cells and merosomes were incubated with 4% PFA/PBS for 15 minutes and then washed three times with PBS. They were then incubated with ice-cold methanol for 5 minutes at room temperature. After washing with PBS, they were blocked and stained immediately as described above. For anti-MHCI staining, cells were incubated with anti-MHCI antibody in culture medium on ice for 30 minutes. They were then washed three times with culture medium and fixed in 4% paraformaldehyde/PBS for 10 minutes at room temperature. After washing with PBS, they were incubated in 0.25% Triton X/PBS for 10 minutes at room temperature. They were again washed three times with PBS and subsequently blocked and stained as described above. The following antibodies were used: mouse FITC anti-MHCI antibody (BD Biosciences, Heidelberg, Germany), rat anti-RFP antibody (Chromotek, Planegg-Martinsried, Germany), chicken anti-Exp1 antibody (Heussler Lab, BNI, Hamburg, Germany), mouse anti-MSP1 antibody (kindly provided by Anthony Holder), anti-rabbit AlexaFluor594 antibody (Invitrogen, Darmstadt, Germany), anti-mouse AlexaFluor488 antibody (Invitrogen, Darmstadt, Germany), anti-rat AlexaFluor594 antibody (Invitrogen, Darmstadt, Germany), anti-chicken Cy2 antibody (Dianova, Hamburg, Germany) and anti-mouse Cy5 antibody (Dianova, Hamburg, Germany). Nuclei were stained with 1 µg/ml DAPI (Invitrogen, Darmstadt, Germany) during the secondary antibody incubation period. All images of fixed cells were acquired using a confocal laser point scanning microscope (see below for specifications).
Time lapse imaging
8x104 HepG2 or HepG2-GFP cells were seeded into glass bottom dishes and infected with P. berghei sporozoites. At 60–62 hpi they were either imaged directly or stained as indicated. All time lapse imaging was performed using a confocal laser line scanning microscope (see below for specifications). Images were acquired every 10–20 minutes.
HepG2 cells were infected with P. berghei-GFP parasites. They were subsequently FACS-sorted to enrich for infected cells and seeded on Thermanox cover slips. At 48 hpi cells were first washed twice with PBS and then fixed with 2% glutaraldehyde in sodium-cacodylate buffer pH 7.2. This was followed by a postfixation step with 1% osmium tetroxide and dehydration at increasing ethanol concentrations and propylene oxide. Cells were then embedded in an epoxy resin (Epon). Ultrathin sections were made with an Ultra Cut E microtome (Reichert Microscope Services, Depew, USA) and stained for imaging with uranyl acetate and lead citrate. Images were then acquired using a Tecnai Spirit transmission electron microscope (FEI, Eindhoven, The Netherlands) at an acceleration voltage of 80 kV.
Immunofluorescence microscope specifications
For wide-field (WF) imaging, an Axiovert 200 base was used in combination with an X-Cite 120 Fluorescence Illumination System (EXFO, Mississauga, Canada). Excitation and emission light was passed through Chroma filter sets (AHF Analysentechnik AG, Tuebingen, Germany). Images were taken using a FAST1394 QICam camera (QImaging, Surrey, Canada) and the Openlab software. For all images, a Zeiss 20x LD A-Plan objective was used. For confocal point scanning (CPS) microscopy, an Olympus IX81 microscope was used. Images were acquired using an Olympus 100x UPlanSApo 1.4 Oil objective and the Olympus Fluoview software version 1.7b. Fluorescence was excited by a 405-nm diode laser (Olympus, Germany), a 488-nm argon laser (Olympus, Germany), a 559-nm diode laser (NTT Electronics, USA) and a 635-nm diode laser (Olympus, Germany). Emission light passed through a 405/Mar/5437635 filter set before detection via PMTs. Confocal line scanning (CLS) was performed with a Zeiss Observer. Z1 inverted microscope integrated into an LSM5 Live imaging setup. Images were acquired using a Zeiss 63x Plan-Apochromat 1.4 Oil objective and the Zeiss Efficient Navigation 2008 and 2009 software. Fluorescence was excited by a Sapphire 488-nm diode and a Compass 561-nm diode pumped laser (both Coherent, USA) and emission light passed through a 505-nm and a 575-nm long-pass filter before detection via a CCD line detector. During imaging, cells were kept in a CO2 incubator at 37°C.
Image processing and analysis
After acquisition, contrast and brightness levels were optimized using either the Zeiss Efficient Navigation 2009 software or Adobe Photoshop CS 8.0. Images were only enhanced as a whole. Fluorescence profiles were generated using the Zeiss Efficient Navigation 2009 software.
Cytochrome c oxidase subunit VIII (Cox8), Protein/Entrez ID CAG28615; exported protein 1 (Exp1), PlasmoDB ID PBANKA_092670; green fluorescent protein (GFP), Protein/Entrez ID ADN93293; insulin receptor (IR), GenBank/Entrez ID M10051; mCherry, Protein/Entrez ID ACY24904; merozoite surface protein 1 (MSP1), Protein/Entrez ID AAA66185.