The 3D structure of the apical complex and association with the flagellar apparatus revealed by serial TEM tomography in Psammosa pacifica, a distant relative of the Apicomplexa

Abstract

The apical complex is one of the defining features of apicomplexan parasites, including the malaria parasite Plasmodium, where it mediates host penetration and invasion. The apical complex is also known in a few related lineages, including several non-parasitic heterotrophs, where it mediates feeding behaviour. The origin of the apical complex is unclear, and one reason for this is that in apicomplexans it exists in only part of the life cycle, and never simultaneously with other major cytoskeletal structures like flagella and basal bodies. Here, we used conventional TEM and serial TEM tomography to reconstruct the three dimensional structure of the apical complex in Psammosa pacifica, a predatory relative of apicomplexans and dinoflagellates that retains the archetype apical complex and the flagellar apparatus simultaneously. The P. pacifica apical complex is associated with the gullet and consists of the pseudoconoid, micronemes, and electron dense vesicles. The pseudoconoid is a convex sheet consisting of eight short microtubules, plus a band made up of microtubules that originate from the flagellar apparatus. The flagellar apparatus consists of three microtubular roots. One of the microtubular roots attached to the posterior basal body is connected to bypassing microtubular strands, which are themselves connected to the extension of the pseudoconoid. These complex connections where the apical complex is an extension of the flagellar apparatus, reflect the ancestral state of both, dating back to the common ancestor of apicaomplexans and dinoflagellates.

Figure 1. General morphology of Psammosa pacifica.

a. Surface structure of the ventral side of Psammosa pacifica, showing two flagella inserted subapical region of the ventral side. The opening of the gullet (arrowhead) is located at the cell apex. b. A longitudinal section of the cell showing a cluster of micronemes (M), dense vesicles (d) and the nucleus (N). c. The longitudinal section of the apical region shows a cluster of micronemes (M), dense vesicles (D) between the opening of the gullet (arrowhead) and a mitochondrion (mit). Single membrane bound trichocysts (T) are located near the cluster of micronemes. Alveoli vesicles (asterisk) are absent at the gullet and trichocysts. N: nucleus. Scales: 5 µm in a, 1 µm in b, 500 nm in c.

Figure 2. Tomographic reconstruction of the Psammosa pacifica apical complex (Cell 1).

The microtubule component of the P. pacifica apical complex is composed of eight short conoid microtubules (C, red) and a similar number of extended conoid microtubules (ECM, brown) that are longer and extend towards the posterior of the cell. The vesicular components includes two clusters of rhoptries; one of which (M1, turquoise) is on the ventral side of the pseudoconoid, and the other (M2, green) is on the right side of ECM. It also includes the large gullet (G, purple) and several spherical dense granules (D, blue).

Figure 3. Serial TEM of the extended conoid microtubules (ECM) and microtubular strands.

a–g. Excerpts from a series of serial sections proceeding from the ventral to the dorsal. The extended conoid microtubules (ECM) are aligned between to short conoid microtubules (CM) and microtubule strands (MS), and extend to the posterior towards the basal bodies, following the line of the gullet (G) and a large elongated vesicle. Arrowhead: the boundary between CM and ECM. Arrow: the boundary between ECM and MS. Scale bars = 500 nm.

Figure 4. Tomographic reconstruction of mature and nascent apical complexes of P. Pacifica (Cell 2).

In the second cell where the apical complex was reconstructed by serial tomography, two sets of apical complexes were found, one large and mature, and a second smaller. The structures observed are as in Figure 1, but in each case the number is doubled, so there are two pseudoconoids consisting of eight CMs, two ECMs, four clusters of micronemes, and several spherical dense granules. All abbreviations are as in Figure 1.

Figure 5. Overview of the apical complex and flagellar apparatus of P. pacifica.

Dorsal view. b. Ventral view. The pseudoconoid (C) consists of eight conoid microtubules and a band of extended conoid microtubules (ECM) that are positioned on the ventral side of the gullet and extend posteriorly towards the basal body. The ECM meet the microtubular strands (MS) that bypass the ventral side of the basal bodies along the ridge of right anterior part of the cell. The MS is connected to the 6 ventral microtubules of the R1/LMR. R1/LMR is lined with a fibrous sheet on the dorsal side and is connected to the longitudinal basal body (LB). A fibrous connective (dorsal root connective: DRC) material on the left side of LB connect to the posterior side of transverse basal body (TB). The LB and TB are at approximately right angles to one another. TB has a fibrous “collar” (transverse basal body collar: TBC) of partial donut shape.

Figure 6. Longitudinal basal body and associated structures.

a–h. series of nearly transversal serial sections, proceeding from anterior to posterior. Arrowheads: dorsal root connective material. Asterisk: thin connective fibers. L: longitudinal basal body. MS: microtubular strands. R1: microtubular root 1, which is also called the longitudinal microtubular root (LMR). R1’: a part of R1 that connects to MS. R2: microtubular root 2, which is composed of 6 very short microtubules. T: transverse basal body. Arrowhead: the fibrous connective between MS and R1. The inset in panel (a) shows the relationship of the plane of these sections to the cell. Scale bars = 500 nm.

Figure 7. Transversal basal body and associated structures.

a–f. A series of nearly transversal serial sections, proceeding from anterior to posterior. MS: microtubular strands. R4: microtubular root 4. T: transverse basal body. TC: transverse collar. TSR: transverse striated root. Scale bars = 500 nm.

Figure 8. Root 4 and transverse striated root (TSR).

a–c. A series of nearly longitudunal serial sections, priceeding from ventral to dorsal. M: mitochondrion. MS: microtubular strands. N: nucleus. R1: microtubular root 1. R4: microtubular root 4. TF: transverse flagellum. TSR: transverse striated root. Scale bars = 500 nm.

Figure 9. Comparison of the flagellar apparatus and the apical complex in myzozoans.

a. General dinokaryotes. b. Psammosa pacifica (this study). c. Rastorimonas subtilis . d. Colpodella vorax . e. Chromera velia , f. Toxoplasma gondii . R1: root 1; R2: root 2; R3: root 3; R4: root 4. Green: bypassing microtubule strands (solid line) or SF-assemblin containing fibre (broken line); red: pseudoconoid or conoid; orange: basal bodies or centorioles.

Figure 10. Character evolution of the apical complex among myzozoans.

Color of the tree branches indicate trophic strategy of the linages; orange: photysynthetic, blue: predatory; grey: parasitic. Character evolution is indicated with triangles; green: acquisition of a character, yellow: transition of a character, white: loss of a character.