A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii

Abstract

The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member subfamily of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatiotemporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.

Fig. 1

The IMC proteins and their expression during tachyzoite development. (A) Full length and validated open reading frames of each IMC protein in order by IMC number and their corresponding ToxoDB gene name. The alveolin repeat regions are represented in yellow and the N- and C-termini in green. Cysteines are indicated in red and predicted palmytoylation sites marked with blue asterisks. (B) Affymetrix array expression pattern of the IMC mRNAs through two cycles of tachyzoite development. RH strain parasites expressing the herpes simplex thymidine kinase (TK) were synchronized by a thymidine block. Cell cycle stages and timing of budding are indicated at the top. Expression levels are normalized to internal controls on the Affymetrix array. (C) Maximum and minimum expression levels of the IMC genes in the second cycle represented in (B) (hours 6–12). Expression level is shown as the raw fluorescence hybridization data.

Fig. 2

Relative localization of the cortical IMC proteins throughout tachyzoite development. (A–B) Mature (A) and budding (B) parasites co-stained with antibodies against IMC1 (green) and IMC3 (red). (C) Budding parasites expressing ptub driven YFP-IMC6. (D) Budding parasites expressing ptub driven YFP-IMC10. (E) Intensity profile across the budding daughters indicated in (B) panel 3 marked by arrow “E”. Arrowheads indicate specific localization of IMC1 in the mature mother parasites not detected for IMC3. (F) Mature mother intensity profile along the arrow indicated in (C) panel 3 marked with “F”. (G–H) Intensity profiles as indicated by arrows “G” and “H” in (C) panel 3. Relative distance is shown along the length and direction of the arrows in (B,C) on the x-axis and relative intensity is shown on the y-axis.

Fig. 3

IMC7, 12, and 14 associate exclusively with mature cytoskeletons. (A) Selected frames from time-lapse images of YFP-IMC12 (Movie S2). (B–E) Three stages of tachyzoite development are shown for YFP-IMC7 (green) co-stained with IMC3 antibody (red): (B) mature, (C) mid budding, and (D) emerging daughters. (E) Intensity profile as described in Figure 2 across the arrow shown in panel 3 of (C). (F) YFP-IMC7 (green) expressing parasites co-stained with GAP45 antibody (red). (G,H) YFP-IMC7, 12, and 14 associate with the cortical cytoskeleton in the G1-phase of the cell cycle. YFP-IMC7, 12, and 14 expressing parasites were co-stained with centrin antibody (red) and DAPI (blue). YFP-IMC7 is provided as an example in (G). The following three stages of development were defined: cytoplasmic YFP with 1 centrosome (cyto, 1 cent), cortical YFP with 1 centrosome (cort, 1 cent), and cortical YFP with duplicated centrosomes (cort, 2 cent). All stages were blindly counted and G1 distributions plotted in (H) as percentage of parasites in G1. Results from three independent experiments counting 60–160 parasites per experiment are shown; error bars denote standard deviation.

Fig. 4

IMC15 associates with the duplicated centrosomes and transitions to the budding cytoskeleton. (A) live ptub driven YFP-IMC15 expressing parasites showing a total projection (left) and an optical section (middle) of mature parasites and a parasite with two intensely stained budding daughters (right). Arrows indicate the very basal end of the cytoskeleton, arrowheads the very apical end, and the double arrowheads the cap region. (B) YFP-IMC15 (green) expressing parasites co-stained with IMC3 antibody (red) in mid budding parasites. (C) YFP-IMC15 (green) expressing parasites co-expressing myc₂-centrin2 and stained with myc antibody (red) in early budding parasites. Arrowheads indicate the very apical end of the parasite, double arrowheads the apical cap, single arrow the very basal end, double-headed arrows the early bud and double arrowheads the 6 centrin2 foci on the apical cap. Inset is of boxed area. (D) YFP-IMC15 (green) expressing parasites co-stained with centrin antibody (red) in mature parasites. Arrows mark IMC15 localization to the duplicated centrosomes. Inset is of boxed area. (E) YFP-IMC15 (green) expressing parasites co-transfected with CherryRFP-MORN1 (red) and co-stained with anti-human centrin antibody (blue) in very early budding parasites (pre-mitotic as judged from the single, centrally located MORN1 highlighting the spindle pole). The parasite is outlined with a dotted line in the first panel. Insets are of the central region around MORN1. (F) S-phase wild-type parasites stained with anti-IMC15 serum (red) and co-stained with centrin antibody (green) and DAPI (blue). All insets are 2X enlargments.

Fig. 5

IMC5, 8, 9 and 13 localize to the cortical IMC in early buds and then transition to and partially co-localize at the basal complex upon constriction. (A) Live ptub promoter driven YFP-IMC8 expressing parasites at different stages of tachyzoite development (independent parasites are shown in each panel). Mother and daughter parasites are traced by dotted lines in the lower series. (B) Wild type parasites co-stained with anti-IMC5 serum (red), IMC1 antibodies (green), and DAPI (blue). (C) Pair-wise comparisons of the members of the basal complex using co-transfected YFP and CherryRFP constructs. The numbers represent the tagged IMC protein, M1 is MORN1, C2 is centrin2, and the colors correspond with the fluorescent protein fusion. The asterisks in the MORN1 + IM9 panel mark the spindle pole localization of MORN1 (D–G) Electron micrographs identifying an inner collar at the basal end of the cytoskeleton. (D) Cross section through the apical complex demonstrating the absence of a comparable complex at the apical end. (E) Longitudinal section through the posterior end of a parasite displaying the basal inner collar (BIC) and the fold over the alveoli marked with arrowheads. The arrows mark the end of the alveolar vesicles. A unit membrane (UM) of unknown origin with an electron dense coating that is limited to the basal cup region is visible as a clear vesicle sitting within the very basal opening. (F) Longitudinal section through the basal complex. Arrowheads mark the BIC, which bends over the end of the alveolar membrane and connects with the plasma membrane as indicated by the arrow. The area marked by the blue box is enlarged. (G) Cross section through the basal complex displaying the continuity of the BIC and basal inner ring (BIR), which are visible in the enlarged area marked by the blue box. In addition, the two closely apposed UMs can be discerned. P is plasma membrane.

Fig. 6

The alveolin domain contains the localization signal differentiating cortical from basal IMCs. IMC3 (A) and IMC8 (B) are dissected to determine whether the N-terminus (“N”), alveolin domain (“A”), or C-terminus (“C”) is/are responsible for their localization patterns. The domains indicated above or below each panel are fused to an N-terminal YFP and all constructs were driven by the ptub promoter.

Fig. 7

Summarizing schematics of the IMC protein dynamics throughout tachyzoite development and the structure of the basal cytoskeleton. (A) Groups of IMC proteins with a similar behavior are shown in the same color and the groups are introduced at the stage of their defining role; among the yellow, cortical IMC proteins, the ones with a preference for the immature buds are outlined (IMC3, 6, and 10). IMC11 is not included. (B) The tentative structure of the basal complex in mature parasites is composed of three layers. The top layer (green) is composed of MORN1, IMC9, IMC13, and IMC15, the middle layer of IMC5 and 8 and the very basal tip contains centrin2, which overlaps with the middle layer. Data in Figure 5C do not include clear candidates for the bend of the inner collar toward the plasma membrane seen by EM (Fig. 5D–H). Interpretation of the posterior cup is based on data presented in (Mann et al., 2001).