Plasmodium falciparum PfMyoF contains a Rab-like tail domain and associates with perinuclear membrane trafficking proteins

Figures

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  Fig. 1. Predicted structural features and dimerisation potential of PfMyoF. A Schematic of the full-length PfMyoF, highlighting the N-terminal motor domain, neck with six IQ motifs, coiled-coil regions, Rab-like domain, and C-terminal WD40 domain. B AlphaFold-3-predicted structure based on the PfMyoF sequence. Ba PfMyoF coloured by pLDDT value. Bb PAE plot of PfMyoF. Bc PfMyoF coloured according to key domains. C AlphaFold-3-predicted structures of the coil domains of PfMyoF, HsMYO5A (positive control), and HsMYO6 (negative control). Structures are coloured by pLDDT with corresponding PAE plots presented below. D AlphaFold-3 prediction of PfMyoF dimers shows coiled-coil interaction with a backfolded architecture. Black arrowheads point to the coiled-coil interaction within the PAE plot. E Sequence alignment showing the six putative IQ motifs within PfMyoF neck domain. Green sequences highlight the canonical IQ motif amino acids, and yellow sequences show degenerate sequences. F AlphaFold-3 prediction of putative light chains PfCaM, PLC3, and PF3D7_1418300 shows that only PfCaM binds to all six IQ motifs in the PfMyoF neck with high ipTM confidence (0.63). G AlphaFold-3 modelling predicts…

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  Fig. 2. Identification and structural characterisation of the PfMyoF Rab-like domain. A AlphaFold-3 predicted structure of PfMyoF Rab-Like domain. B DALI structural homology search identifies significant similarity to Plasmodium and human Rab GTPases. A table detailing the top hits from a DALI server search of the protein data bank (PDB) and AlphaFold servers for proteins with a similar structure to the PfMyoF Rab-Like domain. Z-scores, a measure of significant similarities, are shown. C AlphaFold-3 predicted structure of PfMyoF Rab-Like domain and PfRab6 bound to GTP. The additional helix in PfMyoF appears to disrupt the GTP binding pocket and reposition the key Switch I and Switch II sites essential for GTP hydrolysis. D Sequence alignment of PfMyoF Rab-Like domain, PfRab6 and HsRab24 reveals a conserved GTP-binding site but divergence in Switch I and II regions. Residues are coloured by conservation, and regions of interest are labelled.

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  Fig. 3. Characterisation and centrosomal localisation of the PfMyoF WD40 domain in mammalian cells. A AlphaFold-3 predicted structure of PfMyoF WD40 domain, forming a 7-bladed β-propeller structure with a Plasmodium-specific insertion. B A schematic of PfMyoF WD40 (left) highlighting the 7-blade structure as well as an N-terminal to C-terminal rainbow colour scheme in top view (middle) and side view (right). C A table detailing the top hits from a DALI server search of the protein data bank (PDB) and AlphaFold servers for proteins with a similar structure to the PfMyoF WD40 domain. Z-scores, a measure of significant similarities, are shown. D Confocal microscopy of GFP-tagged PfMyoF WD40 expressed in RPE cells stained with anti-GFP (magenta), anti-γTubulin (yellow) and Hoechst (blue). Scale bars represent 10 µm. E iU-ExM of RPE cells transiently expressing GFP-tagged PfMyoF WD40 domain and stained with anti-GFP (magenta), Hoechst (blue) and either anti-Centrin or anti-Acetyl-αTubulin (yellow). Scale bars represent 10 µm, or 2 µm for zooms.

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  Fig. 4. PfMyoF is a plus-end directed myosin motor. A AlphaFold-3 prediction of the PfMyoF converter-neck region highlights a larger insertion within the converter domain. B Alignment of HsMYO6 with PfMyoJ and K (left panel) and alignment of PfMyoA, PfMyoF, TgMyoF, HsMYO1E, HsMYO5A, HsMYO6+ by their converter domain (middle panel). In the right panel, the overlay of both minus-ended (left panel) and plus-ended subgroups (middle panel) is shown. C PfMyoF aligns along actin filament bundles and accumulates in membrane ruffles in RPE cells transiently transfected with GFP-PfMyoF motor neck; GFP (yellow), Phalloidin (magenta) and Hoechst (blue). Scale bars represent 10 µm. D PfMyoF localises to filopodia tips. RPE cells were transfected with GFP-tagged PfMyoF motor neck and mCherry-tagged MYO6+ and stained with anti-GFP (yellow), Phalloidin (magenta) and Hoechst (blue). Scale bars represent 10 µm. E Quantification of filopodia tip localisation as a measure of their directionality across different human myosin classes and PfMyoF constructs. RPE cells were co-transfected with various GFP-tagged myosins alongside mCherry-tagged MYO6+. Per construct, 100 filopodia were counted in 3 independent…

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  Fig. 5. PfMyoF interactome identified by native co-immunoprecipitations. A Immunoblot validation of α-PfMyoF antibody against recombinant WD40 fragment (left panels) and endogenous PfMyoF from parasite lysates (right panels). Sf9 cell lysate expressing 6His-WD40 domain (left panels) was probed with anti-6His (left lane) or anti-PfMyoF antibodies (right lane). P. falciparum lysate (right panels) was probed with Pre-Immune (left lane) or anti-PfMyoF antibodies (right lane). B Co-immunoprecipitation from trophozoite-stage lysates identifies 25 enriched proteins; 15 were reproducible across three replicates and 9 across two replicates. C Gene Ontology (GO) analysis of the Co-IP hits shows enrichment in intracellular vesicles, secretory pathway and rhoptry-associated proteins. D A map of proteins enriched in the anti-PfMyoF Co-IP. Hits were defined as a 5-fold enrichment in iBAQ scores of anti-PfMyoF relative to the Pre-Immune control. Hits are arranged based on their predicted functions within the parasite. Solid border lines are for proteins identified in all 3 repeats, and dotted lines for proteins only found in one or two of the repeats.

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  Fig. 6. PfMyoF localises to the perinuclear region around the Golgi apparatus in P. falciparum parasites. A U-ExM of trophozoite stage parasites stained with anti-PfMyoF (magenta), the ER marker anti-PvBiP (yellow) and Hoechst (blue) shows enrichment of PfMyoF near the nucleus, partially overlapping with BiP (ER marker). B U-ExM of schizont stage parasites stained with anti-PfMyoF (magenta) and Hoechst (blue). PfMyoF remains concentrated in the perinuclear region. C U-ExM of schizont stage parasites stained with anti-PfERD2 (yellow) and Hoechst (blue) shows that PfMyoF localisation closely resembles that of the cis-Golgi marker PfERD2. All scale bars represent 10 µm. D Immunogold labelling of schizont stage parasites with anti-PfMyoF detected with Protein A conjugated 15 nm gold and anti-PfERD2 detected with Protein A conjugated 10 nm gold beads confirms co-localisation of PFMyoF and ERD2 on Golgi-proximal membranous structures. Red and yellow circles highlight 15 nm and 10 nm gold particles, respectively.

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  Fig. 7. Conditional knockdown of PfMyoF impairs parasite growth. A HA-Trap affinity enrichment of PfMyoF-HA from PfMyoF-HAglmS parasites grown in the absence or presence of 5 mM glucosamine (GlcN). Blot shows enrichment of PfMyoF-HA from untreated parasites but is depleted following GlcN treatment. Anti-HA immunoblot (upper panel); anti-LDH antibodies were used as a loading control (lower panel). MK marker, IN input, FT flow-through, W1 wash 1, CT control bead eluate, E HA bead eluate. B The percentage reduction in growth across four replication cycles was compared between 3D7-wildtype and PfMyoF-HAglmS lines in the presence of 5 mM GlcN. To control for baseline variability, the untreated parasites for each line were used as its own internal control. Data are mean ± SEM for three replicates shown. n = 4. C PfMyoF-HAglmS parasites were stained with anti-HA (magenta), anti-PfERD2 (yellow) and Hoechst (blue). Scale bar: 2 µm.