Vesicular structures after PfRbsn5L-inactivation are host cell cytosol uptake intermediates. (A) Electron microscopy images of PfRbsn5L knock-sideways (8 h rap) and control parasites. One representative image of n = 42 (rapalog) and 32 (control) cells. Top right shows the top left image with false coloring. E, erythrocyte (red); P, parasite (blue); FV; food vacuole (yellow); V; vesicular structure (red). White arrows indicate putative intraluminal bodies. Scale bars, 2 μm. (B, C) Live-cell images of PfRbsn5L (B) and PfSand1 (C) knock-sideways (rap+) and controls (ctrl) in parasites treated with E64 (E64+). Left: Live-cell images of represent-tative DHE-stained parasites. Right: Quantif-ication of number of cells with bloated FVs. Fisher’s exact test. Pooled from n = 3 independent experiments with PfRbsn5L: 20, 26, and 22 cells (control) and 22, 20, and 20 cells (rapalog), and PfSand1endo: 22, 21, and 21 cells (control) and 29, 18, and 23 cells. P-values indicated. Rapalog was added in young trophozoites (see Materials and methods). (D) Electron microscopy images of PfRbsn5L-knock-sideways (8h rap) and control parasites treated 8 h with E64. False coloring and labels as in (B). Scale bars, 2 μm. (E) Live-cell images of PfRbsn5L knock-sideways (1 h, 2 h, 3 h, and 6 h rapalog) and control parasites, co-expressing the mScarlet tagged PI3P marker P40 (P40epi). White arrows: PI3P positive structures adjacent to the food vacuole. Yellow arrows: PfRbsn5L signal at circular structure adjacent to the food vacuole. Blue arrows: accumulations of PI3P near the food vacuole. Purple arrows: overlap of PI3P signal with the vesicle-like structures visible in the DIC. Merge, overlay of red and green channels. Areas in dashed boxes are magnified on the right.

Sabitzki R, Roßmann AL, Schmitt M, Flemming S, Guillén-Samander A, Behrens HM, Jonscher E, Höhn K, Fröhlke U, Spielmann T. Role of Rabenosyn-5 and Rab5b in host cell cytosol uptake reveals conservation of endosomal transport in malaria parasites. PLoS Biol. 2024 22(5):e3002639. PMID: 38820535