U-ExM allows reconstruction of <i>P</i>. <i>falciparum</i> gametocytogenesis in 3D.

A-F. Representative full projections of P. falciparum gametocyte stages. α/β-tubulin: magenta; amine reactive groups / NHS-ester: shades of grey. Columns 4 and 5 represent the 3D surface topology reconstruction of α/β-tubulin and NHS-ester staining. A. A stage III lemon shaped gametocyte. The black arrow shows the host erythrocyte. B. A stage III to IV transitioning macrogametocyte, the tips start to get pointed and the cell displays a more elongated shape. Osmiophilic bodies show strong NHS-ester staining as indicated by a white arrow head. C and D. A stage IV microgametocyte characterised by the presence of a cytoplasmic amorphous MTOC (black arrow) and the intra nuclear body (INB). The nuclear contour is distinguished by a light NHS-ester staining and the white asterisk indicates the position of the nucleus. Subpellicular microtubules start to surround the gametocyte. E and F. Stage V micro- and macrogametocytes displaying rounded ends and an increase in width. Subpellicular microtubules start disassembling but IMC plates remain visible by NHS-ester staining (top Z section, blue arrow heads). In macrogametocytes, NHS-ester dense osmiophilic bodies are visible (white arrow heads). At the gametocyte extremities (dotted square area) 3 to 4 ring-like structures are present. Black arrow heads highlight a region with a lower NHS-ester density that may correspond to the mitochondrion. G and H. Close up on the apical ends highlighting NHS-ester dense annuli (orange arrows). Scale bars = 5 μm.</p

3D surface topology reconstruction of the microgametocyte shown in Fig 3B.

α/β tubulin: magenta; amine reactive groups/NHS-ester: grey. (MP4)</p

Oligonucleotides used in this study.

(DOCX)</p

Generation and characterisation of SAS4-HA, SAS6-HA and SAS6-GFP transgenic lines.

A-D. Genetic modification scheme and genotyping of the transgenic lines. E. SAS6-GFP microgametocytes display an abnormally shaped amorphous MTOC densely stained for SAS6-GFP. This does not seem to affect mitosis but axoneme formation and arrangement is compromised. α/β-tubulin: magenta; amine reactive groups/NHS-ester: shades of grey; centrin: blue; SAS6-GFP: yellow. Scale bars = 5 μm. (TIF)</p

Working model showing the molecular organisation and dynamics of the bipartite <i>Plasmodium</i> MTOC coordinating mitosis and axoneme formation during microgametogenesis.

In the non-activated microgametocyte the intranuclear body is connected to the amorphous MTOC across the nuclear membrane. The amorphous MTOC corresponds to a deuterosome-like structure where components of the basal body and associated proteins localise in distinct or overlapping subdomains. The molecular organisation of the amorphous MTOC and its link with the intranuclear body relies on SRPK1. Upon activation of CDPK4, the intranuclear body gives rise to eight intranuclear bodies that coordinate the assembly of mitotic spindle while the amorphous MTOC differentiates into eight basal bodies to form eight axonemes (one representation shown). γ-tubulin shows dynamic localisation from the amorphous MTOC to the basal bodies and the intranuclear bodies to sequentially nucleate microtubules of the axonemes and mitotic spindles.</p

The MTOC coordinating axoneme formation and mitosis shows a bipartite structure across the nuclear membrane during <i>P</i>. <i>berghei</i> microgametogenesis.

A and B. Localisation of the nuclear membrane protein GEX1-HA in P. berghei gametocytes. α/β-tubulin: magenta; amine reactive groups/NHS-ester: shades of grey; centrin: blue; GEX1-HA: cyan. A. Non-activated and activated microgametocytes show a lobulated nuclear architecture, as seen by GEX1-HA signal and tubulin negative areas. Column 3 represents a close up of the boxed areas. The nuclear contour is distinguished by a light NHS-ester staining, which matches GEX1-HA staining. Nucleus: white asterisk; amorphous MTOC: black arrow (centrin-positive); basal bodies: BB (centrin-positive); intranuclear body: INB (centrin-negative). B. Non-activated and activated macrogametocytes. Macrogametocytes display a crescent-shaped nucleus, very low levels of α/β-tubulin, and NHS-ester dense osmiophilic bodies. Scale bars = 5 μm.</p

Generation and characterisation of SAS4-KO, and SAS6-KO transgenic lines.

A. Genetic modification scheme and genotyping of the transgenic lines. B-C. D-E. Representative full projections of (B) non-activated and (C) 5–6 min activated microgametocytes; wild-type (1st row), SAS4-KO (2nd row) and SAS6-KO (3rd row). α/β-tubulin: magenta; amine reactive groups/NHS-ester: shades of grey; centrin: blue; γ-tubulin: green. Boxed areas correspond to close-ups. Yellow star = mitotic spindle; yellow arrow = non-bundled microtubules; green arrow = bundled microtubules; white arrow = intranuclear body; BB = basal body. Scale bar = 2 μm. (TIF)</p

A single MTOC coordinates mitosis and axoneme assembly during <i>Plasmodium</i> microgametogenesis.

Circulating microgametocytes are arrested at a G0-like stage at the haploid level showing an amorphous MTOC (black arrow) lying on the cytoplasmic face of a nuclear pore that is physically linked to another electron dense aggregation called the intranuclear body (INB) in the nuclear face of the same pore. The molecular organisation of both structures and their link is unknown. One minute after activation, the first genome replication is completed and the spindle of mitosis I (spindle: yellow, kinetochores: pink) is observed between the two intranuclear bodies (INB). At the same time, the amorphous MTOC gives rise to 8 basal bodies (BB) which initiate nucleation of eight axonemes (magenta). By 8 minutes three successive rounds of genome replication and endomitosis have happened and at 10 minutes, full length axonemes become motile. As each basal body remains attached its corresponding intranuclear body, they drag a haploid genome that is incorporated into the exflagellating gametes. (TIF)</p

U-ExM and Pan-ExM of <i>P</i>. <i>berghei</i> microgametogenesis allows further insights into the dynamics of mitotic structures and axoneme formation.

A-E. Representative full projections of activated P. berghei gametocytes. α/β-tubulin: magenta; amine reactive groups/NHS-ester: shades of grey; centrin: blue. Boxed areas indicate areas shown in insets. A. A non-activated microgametocyte showing an amorphous MTOC (black arrow, centrin-positive) and an intranuclear body (INB, centrin-negative). B. A 1–2 min activated microgametocyte, showing two tetrads of basal bodies and eight growing axonemes (1 to 8). In the nucleus the mitotic spindle is highlighted by a yellow asterisk. C. A 5–6 min activated microgametocyte is undergoing mitosis II with two mitotic spindles (yellow asterisks). Each intranuclear body (INB) is connected to two cytoplasmic basal bodies (BB). D. A 10–12 min activated microgametocyte has undergone mitosis III. Each intranuclear body (INB) is connected to a single cytoplasmic basal body (BB). The yellow asterisk highlights a remnant mitotic spindle. E. Exflagellated male gametes display a centrin (blue) and NHS-ester dense basal body at their proximal extremity while DNA (Hoechst: green) runs along the axoneme. F. Pan-ExM of a 10–12 min activated and NHS-ester labelled microgametocyte. The central panels represent four different z sections (top left numbers) of the same microgametocyte. Each mitotic spindle is numbered from 1 to 8. Top panels are close ups of four different z sections (top left numbers) of mitotic spindle 7; Arrows indicating the proteinaceous filaments linking the basal body with the intranuclear body. Lower panels show four different z sections (top left numbers) of the boxed area highlighting individual axonemal microtubules. Scale bars = 5 μm.</p

Formation of the amorphous MTOC and its differentiation into functional basal bodies relies on two kinases: CDPK4 and SRPK1.

A-C. Representative full projections of (A) non-activated, (B) 1–2 min activated and (C) 10–12 min activated microgametocytes; wild-type (1st row), CDPK4-KO (2nd row) and SRPK1-KO (3rd row). α/β-tubulin: magenta; amine reactive groups/NHS-ester: shades of grey; centrin: blue. Boxed areas correspond to close-ups shown in panel 5. Amorphous MTOC: black arrow; intranuclear body: INB; basal body: BB; mitotic spindle: yellow asterisk. Scale bars = 5 μm.</p