HepG2 cells were infected with fluorescent P. berghei parasites and detached cells were collected at either 65 or >75 hours of infection. They were then stained with the nuclear dye Hoechst 33342 (blue) and several additional live stains to characterize the nature of the surrounding membrane. The existence of a membrane in general was shown by staining with Vybrant DiO (green) in cells infected with P. berghei-mCherry (red, top panel). The presence of phosphatidylserine in the outer membrane leaflet, an indicator for ongoing cell death, was tested using pSIVA (green) in cells infected with P. berghei-mCherry (red, middle panel). Propidium iodide (red) is a membrane-impermeable nuclear stain and served to check the integrity of the membrane of P. berghei-GFP (green)-infected detached cells (lower panel). Quantification of the phenotypes (mean + SEM of three independent experiments) clearly shows that the membrane of freshly formed detached cells was intact and phosphatidylserine-negative while the membrane of older detached cells slowly lost phosphatidylserine asymmetry and integrity. Total n for 65hpi - Vybrant DiO: 712, pSIVA: 1483, PI: 1291. Total n for 75hpi - Vybrant DiO: 899, pSIVA: 874, PI: 1564. Bars  = 10 µm; WF (wide-field).

HepG2 cells were infected with P. berghei parasites and fixed at different time points after infection. They were then stained with an anti-MSP1 antibody to visualize the PM (red) and anti-Exp1 antibody to label the PVM (green). The parasite cytoplasm was labeled using a transgenically expressed protein (cyan) and the nuclei were stained with DAPI (blue). While the PM surrounded the parasite as a whole during the late schizont stage (A), it began to invaginate around groups of nuclei during cytomere formation (B). It eventually surrounded individual merozoites both before (C) and after (D) PVM breakdown. Representative images are shown. Bars  = 10 µm; CPS (confocal point scanning).

A The coding sequence of the Exp1 protein, which localizes to the PVM, was fused to the coding sequence of mCherry. Expression of the construct in the parasite was driven by a liver stage-specific promoter (LS). B Correct localization of the expressed fusion protein was confirmed by fixation and immunofluorescence staining of infected cells with anti-Exp1 antibody (green). Nuclei were stained with DAPI (blue) and the fusion protein with anti-RFP antibody (red). Bar  = 10 µm, CPS. C HepG2-GFP cells were infected with P. berghei-Exp1-mCherry parasites and imaged starting at 62 hpi. Stills from a representative movie (Video S1) clearly show that the PVM broke down completely. Quantification of this event confirmed that 100% of detached cells had cytosolic distribution of mCherry-Exp1. n = 20 from six independent experiments, bar  = 10 µm, CLS (confocal line scanning). D and E HepG2-GFP cells were infected with P. berghei-mCherry parasites. Representative cells were chosen to generate fluorescence profiles of both channels before and after PVM breakdown. Loss of the PVM barrier could be seen by entry of host cell GFP into the parasitophorous vacuole (D) and the spread of the densely packed merozoites into the host cell cytoplasm (E, see also Video S2). Bar  = 10 µm, CLS.

HepG2 cells were infected with P. berghei-mCherry (red), stained with Vybrant DiO (green) and imaged starting at 62 hpi. Stills from a representative movie (Video S3) are shown and demonstrate that the HCM became the membrane surrounding the detached cell. n = 34 (of which 10 covered development from PVM breakdown to complete detachment) from 11 independent experiments, bar  = 10 µm, CLS.

A HepG2 cells were transfected with pDisplay-mCherry and infected with P. berghei-GFP. From 62 hpi onwards cells were imaged. Stills from a representative movie (Video S4) show increasing loss of Display-mCherry from the HCM, eventually resulting in a spotty distribution throughout the cytoplasm of the detached cell. n = 3, bar  = 10 µm, CLS. B HepG2 cells were transfected with pDisplay-mCherry, infected with P. berghei-GFP and fixed at 24 and 60 hpi. Cells were stained for immunofluorescence analysis with anti-GFP and anti-RFP antibody. Late stages were additionally stained with anti-Exp1 antibody to determine the state of the PVM. Detached cells (65–68 hpi) were not fixed but collected from the culture supernatant and observed live. In all cases, the distribution of Display-mCherry was quantified (means of at least three independent experiments shown). Before PVM breakdown, Display-mCherry distribution was partly vesicular (grey columns) and partly membranous (black columns) while after PVM breakdown Display-mCherry could only be observed in vesicles. Total n for 24 hpi: 201, 60 hpi before PVM breakdown: 252, 60 hpi after PVM breakdown: 53, 65–68 hpi: 574. C HepG2 cells were infected with P. berghei-parasites and treated with 5 µg/ml cycloheximide for the indicated time periods. At 70 hpi detached cells were collected from the supernatant and counted (means of three independent experiments shown). The number of detached cells resulting from an untreated control was set to 100% and the other values were calculated in relation to it. Treatment at earlier stages blocked parasite development while treatment during the late stage had no effect on the number of detached cells in comparison to untreated samples. Viability of the host cells after cycloheximide treatment was confirmed by Live/Dead staining. Total number of detached cells: untreated: 2435, 36–44 hpi: 32, 36–70 hpi: 0, 62–70 hpi: 2714.

A HepG2 cells were infected with P. berghei-Exp1-mCherry and stained with MitoTracker GreenFM at 62 hpi. Stills from a representative movie (Video S6) show that host cell mitochondria drew together and disintegrated. n = 17 from three independent experiments, bar  = 10 µm, CLS. B pDsRed1-N1-Cox8-transfected HepG2 cells were infected with Pb^cGFP_MITO parasites to visualize parasite mitochondria (green). Detached cells were collected at 68 hpi and stained with Hoechst 33342 (blue). The overlay shows that the mitochondria remnants were of host cell origin. Representative images are shown. Bars  = 10 µm, CPS. C pEGFP-N1-Cox8-transfected HepG2 cells were infected with P. berghei parasites. Detached cells were collected at 68 hpi and stained with Hoechst 33342 (blue) and TMRE (red). Representative images show that most of the host cell mitochondria remnants (green) had no TMRE staining, indicating a loss of membrane potential. An enlarged part of the merged image (indicated by a white box) is shown in the inset. Bars  = 10 µm, CPS.

After the PVM has invaginated around individual merozoites the PVM breaks down and releases the merozoites and the parasitophorous vacuole contents into the host cell cytoplasm. At this point host cell mitochondria begin to disintegrate, protein biosynthesis becomes dispensable and the host cell detaches. Merosomes bud from the detached cells, which initially retain both phosphatidylserine asymmetry and membrane integrity.