Detailing organelle division and segregation in Plasmodium falciparum

Abstract

The malaria-causing parasite, P. falciparum, replicates through schizogony, a tightly orchestrated process where numerous daughter parasites are formed simultaneously. Proper division and segregation of one-per-cell organelles, like the mitochondrion and apicoplast, are essential, yet remain poorly understood. We developed a new reporter parasite line that allows visualization of the mitochondrion in blood and mosquito stages. Using high-resolution 3D imaging, we found that the mitochondrion orients in a cartwheel structure, prior to stepwise, non-geometric division during last-stage schizogony. Analysis of focused ion beam scanning electron microscopy data confirmed these mitochondrial division stages. Furthermore, these data allowed us to elucidate apicoplast division steps, highlighted its close association with the mitochondrion, and showed putative roles of the centriolar plaques in apicoplast segregation. These observations form the foundation for a new detailed mechanistic model of mitochondrial and apicoplast division and segregation during P. falciparum schizogony and pave the way for future studies into the proteins and protein complexes involved in organelle division and segregation.

Figure 1.

Comparison of MitoTracker and a new mitochondrial marker for fluorescence imaging. (A) Fluorescent imaging of WT parasites stained with MitoTracker Orange CMTMRos (MT orange) or MitoTracker Red CMXRos (MT red). (B) Fluorescence microscopy of MitoRed. The mito-mScarlet signal was observed in all asexual life-cycle stages including rings, trophozoites, and early and late schizonts. No antibody staining was used and the fluorescent signal observed is exclusively the mito-mScarlet signal. (C) Fluorescence microscopy of MitoRed, either unstained (no MT) or stained with MT orange, MT red, or MitoTracker Deep Red FM (MT deep red). Mito signal is the combined MitoTracker and mito-mScarlet signal that is observed in this channel. DAPI (blue) is used to visualize DNA and DIC (differential interference contrast) for general cellular context. All images are maximum intensity projections of Z-stacks (41 slices, 150 nm interval) taken with an Airyscan confocal microscope. Scale bars, 2 μm.

Figure S1.

Generation and verification of MitoRed parasite line. (A) Schematic overview of strategy to generate a parasite line harboring a fluorescent mitochondrial marker. CRISPR-Cas9 is used to create two double-strand breaks at SIL7 (indicated by scissors). A construct containing the promotor and targeting sequence of the mitochondrial protein HSP70-3 (PF3D7_1134000) fused with mScarlet is integrated by double homologous recombination. Once integrated, the mitochondrial-targeted mScarlet is expressed and leads to fluorescent staining of the mitochondrion. (B) Diagnostic PCR of MitoRed parasite line with WT- and integration-specific primer combinations (indicated in panel A) demonstrating successful 5′ and 3′ integration and the absence of WT parasites in the MitoRed line. (C) Growth assay showing similar growth of MitoRed and WT parasites. Three independent cultures were set up from one tightly synchronized parasite culture for both MitoRed and WT. Samples were taken over a 5-day period and parasitemia (corrected for dilution factors) was determined with flow cytometry. Error bars (note they are quite small) indicate standard deviation. Source data are available for this figure: SourceData FS1.

Figure S2.

Comparison of MitoTracker and the mitochondrial marker for live fluorescence imaging. (A) live imaging of WT parasites stained with MitoTracker Orange CMTMRos (MT orange) or MitoTracker Red CMXRos (MT red). (B) Live imaging of MitoRed stained with MT orange, MT red, MitoTracker Deep Red FM (MT deep red), Rhodamin123, or without staining. Mito signal is the combined MitoTracker and mito-mScarlet signal that is observed in this channel. Parasites were stained with Hoechst 33342 to visualize DNA. All images are single slices of Z-stacks taken with Airyscan confocal microscope. Scale bars, 2 μm.

Figure 2.

Mitochondrial dynamics during gametocyte development and activation. (A) Immunofluorescence assay on MitoRed gametocytes stages IIa, IIb, III, IV, and V, stained with anti-β-tubulin (green) and DAPI (DNA, blue). The mito-mScarlet signal is shown in magenta. In stages IV and V, male (M) and female (F) gametocytes are distinguished based on the intensity of the tubulin signal (males high, females low). (B) Immunofluorescence assay on MitoRed parasites during different stages of gametocyte activation (2, 5, 10, and 20 min after activation). (C) Immunofluorescence assay on MitoRed exflagellating male gamete 20 min after activation. (A–C) Images are maximum intensity projections of Z-stacks (41 slices, 150-nm interval) taken with an Airyscan confocal microscope. (D) 3D visualization of male and female MitoRed parasites 2, 5, 10, and 20 min after activation. The mito-mScarlet fluorescent signal is segmented based on manual thresholding. Scale bars, 2 μm.

Figure S3.

Mitochondrial morphology in stage IV and V male and female gametocytes. (A and B) Immunofluorescence assay on male and female MitoRed gametocytes stage IV (A) and stage V (B), stained with anti-β-tubulin (green) and DAPI (blue). Male (M) and female (F) gametocytes are distinguished based on the intensity of the tubulin signal (males high, females low). Images are maximum-intensity projections of Z-stacks taken with an Airyscan confocal microscope. Scale bars, 2 μm.

Figure S4.

Mitochondrial association with axonemes in activated male gametocytes. (A) Exflagellation events in MitoRed and NF54 parasites 20 min after activation in four cultures in two independent experiments. Unpaired t test showed no significant difference. (B) Three examples of activated males where the mitochondria (magenta) localize closely to the axonemal tubulin (green) in MitoRed parasites. Left images are single slices, right images are maximum intensity projections (MIPs). (C) Exflagellating MitoRed male gametocyte with apposition of the mitochondria with the axonemal tubulin. Top images are single slices and crops from the bottom image, which is a MIP. Scale bars, 2 μm.

Figure 3.

Mitochondrial dynamics during ookinete development. (A) Live imaging of MitoRed ookinetes 1 day after mosquito feed. Different stages of ookinete maturation (II–V) were distinguished based on description by Siciliano et al. (2020). Cells were stained with an Alexa Fluor 488-conjugated anti-Pfs25 antibody to visualize the parasite outline (green). Images are maximum intensity projections of Z-stacks (30 slices, 185 nm interval) taken with an Airyscan confocal microscope. Scale bars, 2 μm. (B) 3D visualization of different ookinete maturation stages. The mito-mScarlet fluorescent signal is segmented based on manual thresholding. Two smaller images in the upper right corner of stages II–III and stage III are crops of the mitochondrial fluorescent signal with increased brightness and contrast. Scale bars, 1 μm.

Figure S5.

Mitochondrial dynamics in oocyst development. (A–C) Live imaging of MitoRed oocysts on day 7 (A) day 10 (B) and day 13 (C) after mosquito infection. (A) Oocyst at day 7 after infection with the left image showing a maximum intensity projection of the mito-mScarlet signal. The right image shows a segmentation of the mito-mScarlet fluorescent signal by thresholding in Arivis software. Scale bar, 4 μm. (C) Two oocysts on day 13 after infection. Images on the right are crops of the mito-mScarlet signal of the image on the left, indicated by the dotted-line areas. Yellow arrowheads indicate mitochondrial organization centers (MOCs). (D) Oocysts at day 13 show beginning MOCs (yellow arrowheads). (E) Oocyst at day 13 show globular mitochondrial signal which could be a sign of unhealthy or dying parasites. (B–E) Scale bars, 10 μm; scale bars crops, 2 μm.

Figure S6.

Time-lapse imaging of MitoRed. (A) Live time-lapse imaging of MitoRed schizonts that show changes in parasite morphology. Mitochondria fall apart after ∼75–90 min of imaging. (B) Live time-lapse imaging of MitoRed schizonts that leave the RBC after 45–52.5 min of imaging. Parasites were stained with Hoechst to visualize DNA. All images are maximum-intensity projections of Z-stacks taken with Airyscan confocal microscopy. Timestamps in the upper right corner represent the time points of the time-lapse experiment in minutes. Scale bars, 2 μm.

Figure 4.

Mitochondrial fission in asexual blood-stage parasites. Immunofluorescence assay on MitoRed schizonts stained with anti-GAP45 antibody (green) to visualize IMC and DAPI (DNA, blue). The mito-mScarlet signal is shown in magenta. Four different stages of schizont maturity are distinguished: pre-segmentation (pre seg), schizonts still undergo nuclear division (nuclei are large and irregularly shaped) and there is no or very little IMC staining without clear curvature. Early-segmentation (early seg): schizonts have (almost completely) finished nuclear division (nuclei are small and round) and there is a clear IMC signal that has a curved shape at the apical end of the forming merozoites but is less than halfway formed. Mid-segmentation (mid seg): the IMC of the segmenting merozoites in these schizonts is more than halfway formed, but there is still a clear opening at the basal end of the merozoite. Late-segmentation (late seg): in these schizonts the IMC seems to be completely formed with no clear opening at the basal end of the forming merozoites. Images are single slices of a Z-stack taken with an Airyscan confocal microscope. Images of the mito-mScarlet signal in the seventh column are maximum intensity projections (MIPs) (41 slices, 150 nm interval). Images in the eighth column are crops of the GAP45 signal depicted in the first column, indicated by the dotted-line areas. Scale bars, 2 μm; scale bars crops, 1 μm.

Figure S7.

Mitochondrial division stages in ABS schizogony. Fluorescent imaging of MitoRed parasites in different mitochondrial division stages (described on the left). Images are representatives of the 17 parasites that were analyzed in the second independent experiment. Mito-mScarlet signal is shown in magenta and DAPI (DNA) in blue. Images are maximum-intensity projections of Z-stacks taken with an Airyscan confocal microscope. The fifth column shows the 3D image of the fluorescent mito-mScarlet signal, while the sixth column shows the 3D visualization of the segmented mitochondrial signal. The color of the mitochondrial fragment represents the size of this fragment, as is shown in the color bar at the bottom. Scale bars, 2 μm.

Figure 5.

3D analysis of mitochondrial fission stages during schizogony. (A) 3D visualization of mitochondrial segmentations based on thresholding of the mito-mScarlet signal in Arivis image analysis software. Smaller images in the top row are a single slice of the Z-stack with anti-GAP45 labeling (IMC, green), DAPI (DNA, blue) and mito-mScarlet (magenta), and a maximum intensity projection of the mito-mScarlet signal. The larger bottom picture is a 3D visualization of the segmented mitochondrial signal. The color of the mitochondrial fragment represents the size of this fragment, as is shown in the color bar at the bottom. Two representative parasites are depicted for each of the four segmentation stages defined in Fig. 4. Scale bars, 2 μm. (B) Boxplot indicating the number of nuclei per parasite in the different segmentation stages. Two-sided t test was performed; **, P < 0.01; ***, P < 0.001. (C) Boxplot indicating the number of mitochondrial fragments per parasite in the different segmentation stages. Two-sided t test was performed; ***, P < 0.001. (D) Boxplot indicating the size of the mitochondrial fragments in the different segmentation stages. A total of 40 schizonts were analyzed, pre-segmentation (n = 6), early-segmentation (n = 9), mid-segmentation (n = 15), and late-segmentation (n = 10).

Figure 6.

3D rendering of mitochondrion and apicoplast during different stages of schizogony. The first column contains representative micrograph images from different schizont stages. The numbers between brackets indicate the parasite ID number and detailed information can be found in Tables S2 and S3. The red blood cell (RBC) and food vacuole (FV) are indicated by their abbreviations. Rhoptries are indicated by white arrowheads, parasitophorous vacuole membrane is indicated by red arrowheads, and parasite membrane invaginations are indicated by black arrowheads. Scale bars, 1 μm. The second, third, and fourth columns contain 3D renderings of parasite membrane (gray, 5% transparency), nuclei (teal, 50% transparency), mitochondrion (red), and apicoplast (yellow). Red arrows indicate merozoite entrance bulins.

Figure 7.

Association of apicoplast and not the mitochondrion with centriolar plaques during schizont development. (A) Micrographs of nuclei (teal) with centriolar plaques (CPs, purple). Scale bars, 0.5 μm. (B) 3D rendering of nuclei (teal), apicoplast (yellow), mitochondrion (red), CPs (purple), and parasite membrane (gray, 5% opacity). Parasite ID numbers are indicated on the left side of the micrograph images. Right column shows measured distances between CPs and the closest point to the apicoplast or mitochondrion in each parasite. Two-sided t test was performed; ***, P < 0.001.

Figure S8.

Shape and volume of mitochondrial fragments during final stages of schizogony. (A) 3D rendering of mitochondria in a late and fully segmented schizont. The number between brackets indicates the parasite ID number (Table S3). In the right images, each mitochondrial fragment is depicted in an arbitrary color to distinguish separate and connected structures. (B) Bar graphs indicating the mitochondrial fragment volumes of each parasite in μm³ and bar colors correspond to colors of the mitochondrial fragments in A.

Figure S9.

Interaction between mitochondrion and apicoplast in different stages of schizogony. (A) Micrograph images of contact sites between the apicoplast (yellow) and mitochondrion (red) in early-stage schizont, indicated by orange arrows. Nuclei are marked in teal. Scale bars, 0.5 μm. (B) 3D rendering of the mitochondrion (red, 50% opacity) and apicoplast (yellow), with contact sites indicated by orange arrows. (C) Micrograph images of mid- and fully segmented schizonts showing contact sites where apicoplast and mitochondrion are aligned over the total apicoplast length (black arrows). Red arrow indicates where the basal end of the apicoplast is in contact with the bulbous part of the mitochondrion at the merozoite entrance. Scale bars, 1 μm. (D) 3D rendering of apicoplast and mitochondrion in different stage schizonts. Blue arrows indicate where the basal end of an apicoplast interacts with the end of a mitochondrial branch. The number between brackets indicates schizont ID number (Table S3).

Figure S10.

Bulbous membrane invagination structures (bulins) in the mitochondrion. (A) 3D rendering of the mitochondrion in a mid-segmentation schizont with a thin (blue arrow) and thick (red arrow) part. (B) Fluorescent microscopy of mito-mScarlet showing bulbous mitochondrial parts at the base of a mitochondrial branch. Scale bars, 2 μm. (C) Micrograph image of mid-segmentation schizont showing four bulins. Scale bar 1 μm. (D) Micrograph images of mitochondrial bulins in different schizont stages. (E) micrograph images of apicoplast bulins (yellow arrows) in early stage schizont. (D and E) Scale bars, 0.5 μm. (F) 3D rendering of the mitochondrion (gray, 7% opacity) and membrane invaginations (blue). Red arrows indicate bulins at the base of a mitochondrial branch just outside the forming merozoite entrance. Orange arrows indicate membrane invaginations at a branching point in the mitochondrial network. Green arrows indicate membrane invaginations in the middle of a continuous mitochondrial branch. The number between brackets indicate schizont ID number (Table S3).

Figure S11.

Different interaction between apicoplast and centriolar plaques in an early schizont (3). (A) Two centriolar plaques (CPs, purple) from a single nucleus (teal) associating with one apicoplast branch (yellow). (B) Two CPs from a single nucleus associating with two different apicoplast branches.

Figure 8.

Schematic model for mitochondrial and apicoplast division and segregation in P. falciparum during schizogony. (1) Nuclear division is ongoing, while inner membrane complex (IMC) formation has not started and both the mitochondrion and apicoplast are branched networks. The apicoplast localizes more to the center of the cell, while the mitochondrion is stretched throughout the whole cell. (2) When IMC formation starts, the apicoplast branches associate with the centriolar plaques (CPs) at the periphery of the parasite. (3) The apicoplast divides in a non-2ⁿ progression, while it keeps its interaction with the CPs. (4) When the nuclear division is in its final stages, apicoplast division is completely finished. The apical end of the apicoplast fragments associate with the CPs, while mitochondrial branches associate with the basal end of the apicoplast fragments. (5) The IMC develops further and envelops large parts of the nuclei. The mitochondrion orients itself in a cartwheel structure, while its branches align with the apicoplast fragments. (6) IMC formation is almost finished, and just a small opening connects the merozoites to the residual body. The mitochondrion divides in a non-2ⁿ progression. The apicoplast still associates with the CPs and aligns with mitochondrial branches/fragments. (7) Merozoite segmentation is complete, the apicoplast loses its clear association with the CPs since they become smaller and do not have a clear extranuclear compartment anymore. The mitochondrion is fully divided and still aligns with the apicoplast. Red blood cell (RBC), parasitophorous vacuole (PV).