Identification of PfMICOS candidates based on predictive structural similarities, complexome profiling and the characteristic expression patterns expected for cristae modulators.

A) AlphaFold predictions of ScMIC60 (AFDB ID: AF-A6ZZY0-F1-v4) and PfMIC60 (AFDB ID: AF-Q8IJW5-F1-v4)(32, 33). TED consensus domain predicted mitofilin domain (red), lipid binding site (green) and coiled coil domain (blue, predicted length annotated) are highlighted (34). N- and C-terminus are annotated. (local pLDDT confidence information in Figure S1). B) AlphaFold predictions of ScMIC19 (AFDB ID: AF-P43594-F1-v4) and PfMIC19 (AFDB ID: AF-Q8IIR5-F1-v4)(32, 33). TED predicted CC-helix-CC-helix (CHCH) domain is highlighted (34). N- and C-terminus are annotated. (local pLDDT confidence information in Figure S1). C) Schematic overview of the TED predicted domain architecture of ScMIC60 and PfMIC60. The additional domain annotated for PfMIC60 consists of ten antiparallel β-sheets of unknown function. D) Schematic overview of the TED predicted domain architecture of ScMIC19 and PfMIC19. E) Migration of PfMIC19/PfMIC60 across fractions of a 3-16% BN-PAGE gel. Corresponding apparent molecular mass of gel fractions is indicated on the x-axis. Relative abundance across fractions is indicated through a heatmap. Co-occurrence in gel fractions is indicative of a shared protein complex. F) Sequence logo derived form a MAFFT alignment of non-redundant protein sequences from MIC19 annotated entries listed on NCBI (MMseqs2 filtered at threshold 0.9, aligned sequences listed in Supplementary Information 2). Letter height indicates information content. Amino acid frequencies are shown by relative letter size. The characteristic set of two CX9C motifs found in MIC19 is conserved in PfMIC19 (sequence shown below). G) Western blot analysis of ABS and mature gametocytes of MIC19-HA and MIC60-HA. Tagged proteins were detected with rat anti-HA (PfMIC19 and PfMIC60, top) and rabbit anti-HSP70 was used as a loading control (bottom).

Consequences of deletion of PfMIC60 and PfMIC19 on differentiation and survival of P. falciparum.

A) Growth curve of NF54, mic19−, mic60−, and mic19/60− ABS over time (in days). (n = 3 for NF54, mic19−, mic60− and n = 2 for mic19/60−). B) Exflagellation assay of NF54, mic19−, mic60−, and mic19/60−. (n >= 6). Statistical significance calculated with one-way ANOVA with Sidàk correction for full comparison between all cell lines. C) Infection of A. stephensi mosquitoes with NF54, mic19−, mic60−, and mic19/60− in 6 independent experiments indicated in different colours. Means (black line): NF54 = 43, mic19− = 7.0, mic60− = 15.0 and mic19/60− = 17.1. Statistical significance was assessed with Wald statistics and indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Oocyst count of 0 was plotted at the x-axis for visualisation purposes.

Fluorescence microscopy reveals aberrant mitochondrial morphology in mature P. falciparum mic19−, mic60−, and mic19/60− gametocytes.

A) Representative fluorescent microscopy images of mature NF54 gametocytes with tubular, intermediate, or rounded-up mitochondria. Samples were stained with MitoTracker. Images are maximum intensity projections of Z-stack confocal Airyscan images. Scale bars: 2 µm. B) Analysis of mitochondrial shape based on mean branch length per cell. Rounded-up = branch length < 2 μm, intermediate = branch length between 2 μm and 10 μm, tubular = branch length > 10 μm. Statistical analysis using Chi-squared test with Bonferroni correction for full comparison between all cell lines; significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C) sphericity of mitochondria, D) mitochondrial volume per cell. In all cases n° of replicates > 3 with total number of cells ≥ 75. Statistical significance calculated with one-way ANOVA with Sidàk correction for full comparison between all cell lines; significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Transmission electron microscopy reveals aberrant crista structures in mature P. falciparum mic19−, mic60−, and mic19/60− gametocytes.

A) Representative micrographs of NF54 (WT), mic19−, mic60−, and mic19/60− gametocytes. Mitochondria within a rectangular selection are highlighted in pink. Zoom-ins on these selections are shown on the right side of each micrograph. For each cell line, a male and a female parasite are shown. Scale bars: 1 µm / 200 nm. B - D) Quantification of the length (log) (B), width (log) (C), and area (log) (D) of cross-sections through individual cristae (n ≥ 100, from ≥ 30 mitochondria). E) Quantification of cristae coverage based on the total crista area of a mitochondrial cross section relative to the area of the respective mitochondrial cross section (n ≥ 30). F) Examples of severely aberrant mitochondria as observed in TEM of mic19/60− gametocytes. Scale bars: 200 nm. Statistical analysis of B – E: one-way ANOVA with Sidàk correction for full comparison between all cell lines; significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Transmission electron tomography reveals a drop in crista junction prevalence in mature P. falciparum mic19−, mic60−, and mic19/60− gametocytes.

A) Representative snapshots through TEM tomograms of mature NF54 (WT), mic19−, mic60−, and mic19/mic60− gametocytes. Scale bars: 500 nm. B) Zoom-ins of individual mitochondria from the tomograms shown in A. Crista junctions highlighted with red arrow heads. Scale bars: 200 nm. C) 3D model of 250 nm mitochondrial sections of the mitochondria shown in B. Each model is shown from two angles 180° to each other. Scale bars: 500 nm (full tomograms and mitochondrial segmentations in Movies 1-4). For red coloured cristae, a CJ was identified within the inspected 250 nm section of the mitochondrion. For yellow-coloured cristae we could not identify connection to the boundary membrane within the tomogram. D) Quantification of the relative proportions of cristae for which a crista junction (CJ) was observed within the 250 nm section (n ≥ 56). Statistical analysis using Chi-squared test with Bonferroni correction for full comparison between all cell lines; significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. E) Quantification of the diameter of crista junctions at their narrowest point (n ≥ 12, from several mitochondria per sample). Statistical analysis using one-way ANOVA with Sidàk correction for full comparison between all cell lines; significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. F) Three representative crista junctions from mature NF54, mic19−, mic60−, and mic19/mic60− gametocytes. Scale bars: 100 nm. 2D segmentation of the outer mitochondrial membrane (blue) and the inner mitochondrial membrane (violet) shown in the bottom row.