Cell division in Apicomplexan parasites is organized by a homolog of the striated rootlet fiber of algal flagella

Abstract

Apicomplexa are intracellular parasites that cause important human diseases including malaria and toxoplasmosis. During host cell infection new parasites are formed through a budding process that parcels out nuclei and organelles into multiple daughters. Budding is remarkably flexible in output and can produce two to thousands of progeny cells. How genomes and daughters are counted and coordinated is unknown. Apicomplexa evolved from single celled flagellated algae, but with the exception of the gametes, lack flagella. Here we demonstrate that a structure that in the algal ancestor served as the rootlet of the flagellar basal bodies is required for parasite cell division. Parasite striated fiber assemblins (SFA) polymerize into a dynamic fiber that emerges from the centrosomes immediately after their duplication. The fiber grows in a polarized fashion and daughter cells form at its distal tip. As the daughter cell is further elaborated it remains physically tethered at its apical end, the conoid and polar ring. Genetic experiments in Toxoplasma gondii demonstrate two essential components of the fiber, TgSFA2 and 3. In the absence of either of these proteins cytokinesis is blocked at its earliest point, the initiation of the daughter microtubule organizing center (MTOC). Mitosis remains unimpeded and mutant cells accumulate numerous nuclei but fail to form daughter cells. The SFA fiber provides a robust spatial and temporal organizer of parasite cell division, a process that appears hard-wired to the centrosome by multiple tethers. Our findings have broader evolutionary implications. We propose that Apicomplexa abandoned flagella for most stages yet retained the organizing principle of the flagellar MTOC. Instead of ensuring appropriate numbers of flagella, the system now positions the apical invasion complexes. This suggests that elements of the invasion apparatus may be derived from flagella or flagellum associated structures.

Figure 1. T. gondii tachyzoites express two SFAs that localize to a structure close to the nucleus.

(A) Electron micrograph and schematic of a dividing T. gondii parasite. Two daughters are assembled within a mother cell. Centrosomes are shown in blue, apicoplast in green. (B) Robust multi-array averages of SFA2 and SFA3 transcripts over two consecutive division cycles based on dataset collected by Behnke and colleagues ,. (C) Parasites do not express TgSFA2 at all times. Parasites expressing SFA2-HA were scored by IFA using anti-HA antibody in an asynchronous parasite population (n = 250). (D) Western blot analysis shows a 30-kDa band when probed with anti-HA antibody in SFA2-HA transgenics but not the parental strain. Anti-SFA3 antibodies recognize the recombinant protein used for immunization (rSFA3), as well as the native protein in parental parasites. Lower mobility bands of approximately 60 kDa likely correspond to dimers formed both by the recombinant and native SFA3 proteins. Anti-SFA3 antibodies also recognize YFP tagged SFA3 in the endogenously tagged TgSFA3-YFP strain. The expected masses for SFA3 and SFA3-YFP are 36 kDa and 56 kDa, respectively. Anti-GFP antibodies specifically recognize SFA3-YFP in the endogenously tagged strain, but not in the parental strain. (E) IFA of SFA2-HA parasites stained with anti-SFA3 and anti-HA antibody and DAPI. Note that both SFA proteins localize to two fiber-like structures per parasite.

Figure 2. T. gondii SFAs are expressed only during cell division.

(A–C) IFAs of SFA2-HA. Parasites were labeled with anti-HA (green), in combination with anti-IMC1 (blue) and anti-centrin (red). IMC1 labels the IMC in both the mother and the daughter cell and centrin is a marker of the centrosome. Robust HA staining is only apparent in parasites with duplicated centrosomes. Scale bar = 5 µm. (D and E) SFA2-HA parasites were fixed, cryo-sectioned, and probed with anti-HA antibody, followed by incubation with gold conjugated protein A. (D) The spindle (Sp) is visible in an invagination of the envelope of the nucleus (Nu), note vertical white lines representing spindle microtubules. Gold particles are highlighted by white arrowheads. (E) IMC outlining a daughter bud is highlighted by black arrowheads. Scale bar = 500 nM. Insets show further enlargements for detail.

Figure 3. TgSFA2 and TgSFA3 are required for parasite growth.

(A) Simplified schematic of iΔSFA3 promoter insertion mutant (see supplemental Figure S2 for further detail). Promoter activity is inhibited by addition of ATc to the growth medium. (B) Top, Western blot using anti-SFA3 antibodies to measure SFA3 in the iΔSFA3 strain upon ATc treatment (P, parent; tubulin is shown as a loading control). Bottom, reverse transcriptase PCR analysis of SFA2 transcript in the iΔSFA2 strain upon ATc treatment (RT-PCR of TGGT1_021600 transcript is shown as control). Note that the expression of both targeted genes is susceptible to ATc. (C) Plaque assay measuring growth of mutants and parental strains in the presence (+) or absence of ATc (−). Note that both mutant strains fail to form plaques (clearings) in a monolayer of fibroblast in the presence of ATc in the medium. The parental strains and untreated cultures of all strains are shown for comparison. The asterisk indicates that the parental strains (P and P*) of the mutants are distinct (see supplementary Figure S2).

Figure 4. Parasites lacking TgSFA2 or TgSFA3 show a pronounced cell division defect.

IFAs of cells infected with iΔSFA3 (A) or iΔSFA2 (B) mutant parasites cultured for 48 h in presence or absence of ATc prior to fixation. Note that both mutants accumulate multiple nuclei (blue), and fail to form proper buds (IMC1, green) under knock down conditions. Untreated controls divide normally. (C) Transmission electron micrograph of iΔSFA3 parasites grown in presence of ATc for 48 h. This section through a cell shows three nuclei (N; scale bar = 500 nm). (D) Multi-nucleated (≥2) parasites were quantified for iΔSFA2 parasites (48 h ± ATc) using DAPI and IMC1 staining. 30 randomly chosen fields were counted and the percentage of vacuoles containing parasites with multiple nuclei is graphed. Error bars represent standard deviation (n = 3).

Figure 5. Parasite lacking SFA go through mitosis normally but fail to form daughter cells.

(A) iΔSFA2 parasites grown in the presence or absence of ATc for 24 h were labeled with anti-IMC1 (green) and DAPI (blue) anti-centrin (red, centrosomes, upper panels) or anti-CenH3 (red, centromeres, lower panels). Representative examples are shown for cells in interphase or division. Interphase nuclei associate with a single centrosome, while larger 2N nuclei associate with two. This is unchanged by ATc treatment. Interphase nuclei contain a haploid genome and exhibit one CenH3 dot representing a cluster of the centromeres of all 14 chromosomes bundled in the nucleus in close proximity to the centrosome (see [9]). Dividing nuclei exhibit two CenH3 dots representing duplicated chromosomes. Again, this labeling pattern is not affected in the mutant when judged on a per nucleus basis. Note though that in both cases the ATc-treated cell is already tetraploid with no sign of cytokinesis. (B) The number of CenH3 dots per nucleus was quantified in IFA experiments for untreated or ATc-treated iΔSFA2 parasites. The number of CenH3 dots, representing the number of chromosome sets per nucleus, is graphed, error bars show standard deviation (>50 parasite vacuole per experiment counted, n = 3). (C) Immunofluorescence of iΔSFA2 parasites after 48 h ATc treatment showing IMC1 (green), DAPI (blue), and anti-ISP1 (red). ISP1 labels the apical cap of the IMC of both mother and daughter cells and is an early marker of budding . ISP1 staining for daughter cells is absent in the ATc-treated mutant. Note that the nuclei have completed mitosis in these cells and compare to a similar stage shown for untreated parasite.

Figure 6. The SFA fiber extends in the apex of the forming daughter.

IFAs showing SFA2-HA stained with anti-HA (green) in combination with markers for daughter bud. (A) anti-tubulin (red) labels the sub-pellicular microtubules. SFA2 staining extends beyond the sub-pellicular microtubules and into the conoid. (B) Anti-IMC3 (blue) labels the IMC of emerging daughter cells and is shown as a reference for the position of daughter cells. RNG1-YFP was detected using anti-GFP antibodies (red). RNG1 is a marker of the conoid . The SFA2-HA signal extends into and slightly beyond the ring of RNG1 staining. (C) anti-ISP1 (blue) labels the apical cap of the IMC. Anti-Morn1 (red) labels the basal complex of both the mother's and the daughter's IMC, as well as, a nuclear structure in immediate proximity of the centrosome ,. The SFA2-HA signal spans from the centrosome region to the apex of the daughter bud. Scale bars = 5 µm.

Figure 7. A fiber links the centrosome and the apical complex of the daughter bud.

Transmission electron micrographs of serial sections of dividing wild-type parasites. (A and B) Two consecutive sections through a daughter cell are shown. An electron dense structure is highlighted in (A) (arrowheads, note some striation in this section, in particular toward the conoid end) emanating from close to one centriole (Ce; B) and reaching up to the conoid of the daughter cell (Cn; A and B). The structure ends in close proximity of the central pair of microtubules within the conoid (arrow; A and B). (C–E) Three consecutive sections perpendicular to the conoid and the orientation of the parasite depicted in (A and B) are shown. A bent electron dense structure (arrowheads) runs within the conoid (Cn) towards the apical ring. The end of this structure appears in contact with the conoid-associated microtubule pair (arrow, also see Figure 10 for a schematic outline). (F–I) Four consecutive sections through an early daughter cell. Both parallel centrioles (Ce) of the centrosome can be seen in (F). A fiber is visible reaching into the conoid and touching the apical ring (Cn; F–H). The fiber emerges between the centrioles (arrow; F and G). The last section shows intra-nuclear microtubules as part of the centrocone (CC, a nuclear envelope structure associated with centromere organization in Apicomplexa); these link the kinetochores of the chromosomes to the centrosome. Scale bars = 250 nM.

Figure 8. The SFA fiber grows in a polar fashion away from the centrosome.

(A) Time-lapse imaging of SFA3-YFP parasites expressing Centrin1-RFP. Images were taken every 10 min for 220 min. Note that SFA-YFP forms a centrosome associated spot that extends away from the centrosome (min 60–120). The fiber breaks at its distal end (min 140–160) and then shortens in reverse order. See Video S2 for an animated version. (B–E) Photo bleaching assay of TgSFA3-YFP parasites expressing FNR-RFP (Ferredoxin/NADPH Reductase-RFP). (B) The fiber in the focal plane (arrowhead) was 0.45 µM prior to bleaching. (C) The target fiber was bleached using a 488-nm laser and is no longer visible after the laser pulse. (D) Images of the photo-bleached fiber were taken after 1 h to monitor recovery of the YFP signal. YFP labeling of the fiber can again be appreciated spanning 0.35 µM. (E) Image showing the YFP labeled fiber after bleaching, its sister unbleached fiber, and FNR-RFP for reference. The right most fiber (unbleached) spans 0.85 µM. The bleached fiber appears shorter and that (new) labeling appears proximal to the FNR-RFP signal. (F) IFA showing the close proximity and relative orientation of apicoplast (FNR-RFP, red), centrosome (blue), and SFA fiber (green). The apicoplast (FNR-RFP) associates with the centrosome during division , and was used as a marker for the position of the centrosome. Scale bar = 1 µm.

Figure 9. Daughter microtubules depend on SFA fiber, but the fiber does not depend on microtubules.

(A and B) Immunofluorescence of SFA2-HA parasites treated with oryzalin, a microtubule-disrupting agent that prevents formation of new microtubules in daughter cells, and that has a more moderate effect on existing stable microtubules in the mother ,. Note that after 12 h of treatment parasite cells fail to assemble buds (B) and compare to the matched control (A). SFA2-HA fibers are nonetheless detected. Note that in untreated parasites, daughter cells are normally detectable when similar fibers are observed. (C) SFA3-YFP parasites were treated with oryzalin and scored for the presence of SFA3-YFP after 0, 12, or 24 h of treatment. Note that treated parasites accumulate fibers. (D) Fiber length was measured in control parasites and parasites treated with oryzalin for 24 h. Data points reflect the mean fiber length per field of view scored (n = 18–81 fiber/field, four fields for each of three independent repeats). Fibers of treated parasites are overall shorter and more uniformly distributed in size. iΔSFA2 parasites were grown in the absence (E) or presence of ATc (F) and stained for acetylated tubulin (red), centrin (green), and DAPI (blue). The anti-acetylated tubulin antibody labels daughter buds strongly (as do antibodies to unmodified tubulin) in untreated mutants. ATc-treated iΔSFA2 parasites exhibit acetyl-tubulin staining exclusively in the mother cell cytoskeleton (note that two mutant cells are shown with microtubules encaging each entire cell; the cells are abnormally large due to the block in budding). No daughter microtubular-skeletons are discernible in these cells despite the fact that each cell shown has two nuclei, both of which are entering mitosis as indicated by the duplicated centrosomes. Scale bars = 5 µm.

Figure 10. Is the apical host cell invasion complex derived from the flagellum of the algal ancestor?

(A) Schematic of the flagellar rootlet system of Chlamydomonas (simplified after [64]). The two flagellar basal bodies are coordinated by rootlet fibers (only SFA and distal fibers are shown here) and bundles of rootlet microtubules (two or four microtubules each) (B). Schematic outline of cell division in the hypothetical flagellated algal ancestor of Apicomplexa. Basal bodies of the flagella also serve to organize the mitotic spindle (flagella are resorbed or shed during mitosis in some flagellated algae, note that number and behavior of flagella in the apicomplexan ancestor is hypothetical). Rootlet fibers (white) may have additional roles in division ,,. (C) Apicoplast, green; nucleus, grey; SFA fiber, white; basal body/centrosome, red; conoid, blue, rhoptries (secretory component of the apical invasion apparatus), light grey. Apicomplexans are intracellular parasites and have lost flagella in most stages. SFA rootlet fiber is only expressed during division and coordinates the centrosome with the MTOC of the daughter bud. This suggests that the system that controlled the positioning and assembly of flagella in the ancestor now organizes the assembly of the apical host cell invasion complex. (D) Schematic of the SFA fiber and its relationship to other cellular structures during T. gondii cell division (only a single daughter bud is shown for simplicity).