Figure 5. A host cell membrane protein is rapidly lost upon PVM breakdown.

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.