Fig 1.
U-ExM allows reconstruction of P. falciparum 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.
Fig 2.
Mitotic structures and axonemes are highlighted by U-ExM during P. falciparum gametogenesis.
A-C. Representative full projections of activated P. falciparum gametocytes. α/β-tubulin: magenta; amine reactive groups / NHS-ester: shades of grey. Column 4 shows the 3D surface topology reconstruction of α/β-tubulin and NHS-ester. A. A 1–2 min activated macrogametocyte shows degrading of the subpellicular microtubules and NHS-ester dense osmiophilic bodies. B. A 1–2 min activated microgametocyte shows the NHS-ester dense basal bodies (BB) giving rise to axonemes (1 to 8 visible in S1 Movie). The mitotic spindle (yellow asterisk) is highlighted by α/β-tubulin staining and at each extremity the intranuclear bodies (INB) show an NHS-ester dense staining. NHS-ester positive kinetochores are present along the spindle. The white dotted line highlights the nuclear periphery. At this stage, the subpellicular microtubules are completely lost. C. A 10–15 min activated microgametocyte displays a round shaped and full length axonemes. Scale bars = 5 μm.
Fig 3.
The MTOC coordinating axoneme formation and mitosis shows a bipartite structure across the nuclear membrane during P. berghei 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.
Fig 4.
U-ExM and Pan-ExM of P. berghei 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.
Fig 5.
γ-tubulin displays dynamic localisation from the amorphous MTOC to basal bodies and intranuclear bodies during P. berghei microgametogenesis.
A-C. Representative full projections of non-activated and activated P. berghei gametocytes. α/β-tubulin: magenta; amine reactive groups/NHS-ester: shades of grey; γ-tubulin: green. Boxed areas indicate area shown in zoom for each image, highlighting the basal bodies (BB), the amorphous MTOC (black arrow), the intranuclear body (INB) stained with NHS-ester, γ-tubulin and α/β-tubulin. Individual axonemes are numbered 1–4. The yellow asterisks highlight mitotic spindles. Scale bars = 5 μm.
Fig 6.
Molecular organisation of the basal body during P. berghei microgametogenesis.
A-F. Representative full projections of activated P. berghei gametocytes. α/β tubulin: magenta; amine reactive groups/NHS-ester: shades of grey; centrin: blue; yellow: SAS4-HA (A-D) or SAS6-HA (E-H). Boxed areas indicate areas shown in insets. Column 5 represents zoomed-in images highlighting the basal bodies (BB), the amorphous MTOC (black arrow), and intranuclear bodies (INB) stained with NHS-ester and for SAS4-HA (A-D) or SAS6-HA (E-H). Scale bars = 5 μm.
Fig 7.
3D reconstruction of the bipartite MTOC during P. berghei microgametogenesis.
A and B. 3D surface topology of the amorphous MTOC and intranuclear body of non-activated microgametocytes shown in Fig 6. Amine reactive groups/NHS-ester: shades of grey; centrin: blue; yellow: SAS4-HA (A) or SAS6-HA (B); amorphous MTOC: black arrow; intranuclear body: INB. Scale bar = 0.5 μm. C. xy and xz or yz views of individual basal bodies in 10–12 min activated microgametocytes, SAS4-HA (left) and SAS6-HA (right). Scale bar = 2 μm. D. 3D surface topology of exflagellated microgametes of Fig 6, SAS4-HA (top row) and SAS6-HA (bottom row). Scale bar = 2 μm.
Fig 8.
Characterisation of SAS4-KO and SAS6-KO lines highlights different requirements of both proteins for the integrity of the basal body.
A. Deletions of sas4 or sas6 lead to a profound defect in exflagellation (error bars show standard deviation from the mean). B-C. Quantification of the phenotypes shown in D and E in 15 individual cells for each line. BB = basal body; INB = intranuclear body. ND = Not Detected. D-E. Representative full projections of (D) 1–2 min activated and (E) 10–12 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. Boxed areas correspond to close-ups. Yellow star = mitotic spindle; yellow arrow = non-bundled microtubules; green arrow = bundled microtubules; INB = intranuclear body; BB = basal body. Scale bar = 5 μm.
Fig 9.
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.
Fig 10.
Working model showing the molecular organisation and dynamics of the bipartite Plasmodium 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.