Fig 1.
P. falciparum microgametogenesis and our live cell imaging approach.
(A) Details of the cell biological transformations occurring during microgametogenesis from activation at t = 0 minutes to exflagellation at t = 15 minutes. At t = 0 minutes, microgametocytes start with a falciform morphology and an in-tact 4-layer membrane, comprised of the red blood cell (RBC) membrane, parasitophorous vacuole membrane (PVM), plasma membrane (PPM) and inner membrane complex (IMC). Following the first round of DNA replication (1n-2n) at t = 1 minute, the microtubule organising centre (MTOC) transforms to two tetrads of basal bodies joined by a mitotic spindle. Further replication of DNA (2n-4n, 4n-8n) occurs after ~ t = 3–4 minutes, simultaneously to the separation of basal bodies and egress. During egress, PVM rupture precedes erythrocyte egress (inside-out-mechanism) and parasites egress from an erythrocyte pore. Axonemes nucleate from basal bodies following the first DNA replication at t = 1 min and elongate from t = 1–15 min, coiling around the parasite cell body. At 15 minutes post-activation, axonemes emerge attached to a haploid genome as microgametes, in the process of exflagellation. (B) The workflow of live gametocyte labelling and fluorescence microscopy of microgametogenesis. P. falciparum NF54 gametocytes are labelled with SiR-Tubulin, WGA-488 and Vybrant DyeCycle Violet at 37°C, at which point inhibitors of microgametogenesis may be added. Labelled gametocytes are subsequently activated with ookinete medium in pre-positioned imaging slides at room temperature (RT). SiR-tubulin labelled mitotic spindles are used to identify microgametogenesis events, which are continually imaged through T and Z, alternating between brightfield and fluorescence to minimise phototoxicity. When parasites are deemed unviable based on photobleaching or morphology, exflagellation events are captured as single Z-slice timelapses through T. Panel B was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com.
Fig 2.
Tubulin dynamics, egress and DNA replication during P. falciparum microgametogenesis.
Individual time-frames acquired from fluorescent time-lapses of microgametocytes labelled with (A) SiR-Tubulin (magenta) only and (B-D) a combination of SiR-Tubulin (magenta), WGA-488 (green) and Vybrant DyeCycle Violet (blue) in the early developmental stages of microgametogenesis. See S1, 2 and 3 Videos for corresponding timelapses of A-C. (A) Microtubule labelling (magenta) portrays MTOC transformation into basal body tetrads and axoneme nucleation, depicted as maximum intensity projections (MIP) of 3D data. (B-D) DNA replication (blue), microtubule dynamics (magenta), host erythrocyte egress and morphological transformations (host erythrocyte membrane, green). (B) Microtubule labelling showing the formation of mitotic spindles, basal bodies and axonemes. DNA (blue) segregation is visible. (C) Erythrocyte membrane possible echinocytosis (white dashed box), pore formation (white dashed box) and egress are shown. (D) A change in DNA content between early and late stages is visible. (E) SiR-Tubulin volume of individual cells from distinct developmental stages; spindle (n = 17), newly nucleated axonemes (n = 10) and developed axonemes (n = 14). (F) A graph plotted to show cell circularity and SiR-Tubulin intensity (arbitrary units), with each plot representing individual cells of a given developmental stage; falciform (n = 12), spindle (n = 46), newly nucleated axonemes (n = 48) and developed axonemes (n = 82). Representative images of developmental stages are above the plots. (G) Vybrant DyeCycle Violet intensity (arbitrary units) quantified in distinct developmental stages; spindle (n = 11), newly nucleated axonemes (n = 11) and developed axonemes (n = 17). (H) DNA segregation angles measured between basal body tetrads and segregated DNA are shown (n = 7). (I) Egress phenotypes were quantified (n = 5). Significant differences in label intensities between developmental stages were calculated using one-way ANOVA tests with Tukey multiple comparisons (**** p < .0001). A-D Time is depicted as minutes and seconds (mm:ss). 2D maximum intensity projection (MIP) of 3D data, scale bars = 10 μm. B-D 3D projections of volume rendered data are shown. Individual channels of B and C can be found in S1A and S1B Fig, respectively. A-D All images here depict the observations made from >10 biological replicates.
Fig 3.
Exflagellation of P. falciparum microgametes.
2D individual time-frames acquired from timelapses of exflagellation imaged by (A) brightfield and (B-C) fluorescence microscopy. See S8 and S15 Videos for corresponding timelapses of A-B. (B-C) SiR-Tubulin-labelled axonemes (magenta) emerge from the parasite cell as microgametes. (B) Emerging microgametes carry a 1n genome from the newly replicated 8n genome (blue) and adhere to neighbouring erythrocytes (green). (C) 3D projections of volume rendered data of exflagellating microgametes, see S17 Video for a 3D rotated view. (D) 3D intensity plot of SiR-Tubulin labelling intensity to reveal dense regions (white dashed line) of axoneme overlap. A-C Time is depicted as minutes, seconds and milliseconds (mm:ss:ms). Scale bars = 10 μm. Individual channels of C can be found in S1E Fig. All data depicted reflect observations from >10 biological replicates.
Fig 4.
Cellular phenotypes of PKG, CDPK4 and proteasome-inhibited parasites.
Cellular phenotypes upon inhibition of P. falciparum (A) CDPK4, (B) PKG and (C) proteasome by 1294, ML10 and bortezomib, respectively, during microgametogenesis. Perturbations to microtubule rearrangement (SiR-Tubulin, magenta), the host erythrocyte (WGA, green) and DNA replication (Vybrant DyeCycle Violet, blue) are shown as 2D maximum intensity projections of 3D data and alongside 3D sectioned views. Individual channels can be found in S3 Fig. (A) Failed DNA replication, cytoskeletal rearrangement and MTOC (white dashed line) transformation under 1294-treatment are shown. Stress induced egress prior to activation is also depicted. See S19 Videofor the corresponding time-lapse. (B) The failed DNA replication and cytoskeletal rearrangement due to PKG inhibition by ML10 is shown. Mixed egress phenotypes were observed, including (i) incomplete and (ii) failed egress. See corresponding timelapses in S23 and S24 Videos. (C) Perturbations to (i) MTOC transformation (white dashed line) and (ii-iii) formation of two-three truncated axonemes resulting from proteasome inhibition are shown. See S25, S27 and S28 Videos for the corresponding timelapses. (D) A continuum of SiR-tubulin labelling intensity (arbitrary units) in untreated (n = 4), 1294 (n = 3), ML10 (n = 3) and bortezomib (n = 6) treated parasites. (E) SiR-tubulin labelling (arbitrary units) at 10 minutes post-activation under different treatments. Untreated (n = 14), 1294 (n = 16), ML10 (n = 16), bortezomib (n = 9). Significance was calculated using one-way ANOVA tests with Tukey multiple comparisons (**** p < .0001). (F) Vybrant DyeCycle Violet labelling (arbitrary units) was significantly reduced (one-way ANOVA tests with Tukey multiple comparisons (**** p < .0001)) at 10 minutes post-activation under different treatments. Untreated (n = 17), 1294 (n = 15), ML10 (n = 9), bortezomib (n = 16). (G) A graph depicting the cell circularity and SiR-tubulin labelling intensity of individual cells across the entirety of microgametogenesis under varying treatments. Untreated (n = 188), 1294 (n = 58), ML10 (n = 106), bortezomib (n = 105). (H) Percentage egress at 10 minutes post-activation under different treatments was quantified, with distinct egress phenotypes depicted beside the stacked bar graph. Untreated (n = 58), 1294 (n = 20), ML10 (n = 25), bortezomib (n = 24). All imaging data depicted reflect observations from >3 biological replicates.
Fig 5.
Novel insights into microgametogenesis with and without drug inhibition.
Schematic diagrams showing the transformations observed by our live-cell microscopy of microgametogenesis. The observations depicted are of (A) untreated, (B) CDPK4-inhibited, (C) PKG-inhibited and proteasome-inhibited microgametocytes, treated with DMSO, 1294, ML10 and bortezomib, respectively. (A) Pore-formation and possible echinocytosis of the host cell membrane was found to occur during egress of untreated microgametocytes, which ejected from a single spindle pole of the parasite. Nuclear segregation was found to occur perpendicularly to the mitotic spindle. (B) CPDK4 inhibition prevented MTOC transformation and full rounding-up with three distinct egress phenotypes: 1) stress-induced egress prior to activation, 2) no egress and 3) incomplete egress. (C) Inhibition of PKG prevented rounding-up and any microtubule polymerisation, with 3 distinct egress phenotypes: 1) no egress, 2) full egress and 3) incomplete egress. (D) Proteasome inhibition resulted in abhorrent MTOC division and some nuclear segregation, with few truncated axonemes nucleating from one pole of the transformed parasite. Rounding-up of proteasome-inhibited parasites was observed.