Additional data for MyoF.

(A) Extended panel of live cell microscopy images of parasites expressing the MyoF-2xFKBP-GFP-2xFKBP fusion protein from the endogenous locus with episomally expressed mCherry-K13. Nuclei were stained with Hoechst. Scale bar, 5 μm. (B) Localisation of MyoF-2xFKBP-GFP by live-cell microscopy across the intra-erythrocytic development cycle. Nuclei were stained with DAPI. Scale bar, 5 μm. (C) IFA microscopy images of acetone-fixed parasites expressing MyoF-3xHA with episomally expressed mCherry-K13 across the intra-erythrocytic development cycle. Nuclei were stained with DAPI. Scale bar, 5μm. Foci were categorized into ‘overlap’ (black), ‘partial overlap’ (dark grey), close foci (light blue) and ‘non overlap’ (light grey) in n = 31 parasites. Scale bar, 5μm. (D) Live-cell microscopy of knock sideways (+ rapalog) and control (without rapalog) MyoF-2xFKBP-GFP-2xFKBP^endo+1xNLSmislocaliser parasites at 0, 1, 2, 4 and 22 hours after the induction of knock-sideways by addition of rapalog. (E) Individual growth curves of MyoF-2xFKBP-GFP-2xFKBP^endo+1xNLSmislocaliser with (red) or without (blue) addition of rapalog shown in Fig 1F. Summary of individual growth curves shown as percentage of control parasites (without rapalog). Three (3D7, black) and eight (MyoF-2xFKBP-GFP-2xFKBP^endo, red) independent experiments. Error bars, mean ± SD. (F) Relative growth of synchronised MyoF-3xHA^endo compared with 3D7 wild type parasites after two growth cycles. Each dot shows one of six independent growth experiments. P-values determined by one-sample t-test. (G) Number of vesicles per parasite in trophozoites determined by live-cell fluorescence microscopy (DIC) in 3D7 and MyoF-3xHA^endo parasites. Three independent experiments with each time n = 27–38 (mean 31.7) parasites analysed per condition. Representative DIC images shown on the right. (H) Experimental setup of the bloated food vacuole assay shown in Fig 1H. (I) Bloated food vacuole assay with 3D7 parasites 8 hours after rapalog addition compared with controls (- rapalog). Cells were categorized as with ‘bloated FV’ or ‘non-bloated FV’ and percentage of cells with bloated FV is displayed; n = 2 independent experiments with each n = 26–40 (mean 31,3) parasites analysed per condition. Representative DIC and fluorescence microscopy images are shown. Parasite cytoplasm was visualized with DHE. (J) Representative images of bloated food vacuole assays with knock sideways (+ rapalog; bottom row) and control (without rapalog; top row) MyoF-2xFKBP-GFP-2xFKBP^endo+1xNLSmislocaliser parasites shown in Fig 1I and 1J. Scale bar, 5 μm. (K) Experimental setup of the RSA shown in Fig 1I. Schematic representation of the cell lines depicted above the corresponding panel.

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