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![The Distribution of Two Basal Complex Components, TgMORN1 and TgCentrin2, in Mature Parasites (A) The major structural reference points of the T. gondii cytoskeleton referred to throughout this paper are illustrated in the cartoon drawing of an interphase adult parasite. The cytoskeleton of T. gondii includes the apical and the basal complexes, the spindle pole, the centriole, and the cortical cytoskeleton, which includes all of the cytoskeletal elements aligning the parasite body (i.e., cortical microtubules, the IMC, and the filamentous network underlying the IMC), except for the apical and basal complexes. (B) In interphase parasites, TgMORN1 (green, EGFP-TgMORN1) forms a cap at the extreme basal end of the parasite (green arrow), filling the gap at the basal end of the IMC (blue, labeled by anti-IMC1 monoclonal antibody plus Alexa350-anti-mouse IgG). TgMORN1 also weakly concentrates in the apical complex (dotted square indicated by the white arrow. Stronger contrast enhancement of TgMORN1 labeling is applied to this region to highlight the apical complex labeling of TgMORN1). TgMORN1 is also localized to the spindle pole (green arrowhead), which is juxtaposed to the centriole (red, mCherryFP-TgCentrin1, red arrowhead) during interphase. (C) In interphase parasites, the basal labeling of TgMORN1 (green arrows) is clearly separated from the cortical microtubules (red, mCherryFP-TgTubA1, small gray arrows). The inset shows a magnified view of the centriole/spindle pole assembly, showing red TgTubA1 in the centriole and green TgMORN1 in the spindle pole. At this point in the cell cycle, tubulin labeling of the spindle pole is weak, but will become stronger in early cell division (cf. Figure 5). White arrow, conoid labeling by mCherryFP-TgTubA1. (D) TgMORN1 and TgCentrin2 occupy different subcompartments within the basal complex, with the TgCentrin2 compartment (red arrow at the basal) located posterior to the TgMORN1 basal labeling (green arrow). These parasites are at the very end of interphase, as indicated by the recent duplication of the centriole in the parasite on the left, but not in the parasite on the right. Green, mCherryFP-TgMORN1 (pseudo-color, for consistency in the color scheme); red, EGFP-TgCentrin1 (pseudo-color); blue, anti-IMC1 antibody detected by Alexa350-anti-mouse IgG. Red arrowheads, TgCentrin2 labeling of the apical polar ring (cf. [A], [16]). Red arrows near the apical portion of the parasite apical, TgCentrin2 peripheral annuli (cf. [A], [16]). Insets are at 2 3 magnification. The insets in (B) and (D) do not include the anti-IMC1 labeling in order to emphasize the differences in localization betweenTgMORN1/ TgCentrin1 (B) and TgMORN1/TgCentrin2 (D) labeling. (E) An EM image of an EGFP-TgMORN1 transgenic parasite extracted with 0.5% TritonX-100, immunogold labeled with anti-GFP antibody and negatively stained with phosphotungstic acid. Numerous gold particles are located in the basal complex (bottom inset, green arrows). There is also a small concentration of gold particles in the apical polar ring (top inset, green arrowhead). The cluster of gold particles aligned together (black arrow) at the bottom half of the parasite body is likely to be the labeling of an EGFP-TgMORN1 fiber sometimes seen to form in extracellular parasites [16]. (F) An immuno-EM image of an EGFP-TgCentrin2 transgenic parasite, extracted, labeled, and stained as described in (E). As previously reported [16], there are clear concentrations of EGFP-TgCentrin2 labeling in the apical polar ring (top inset, red arrowheads) and several peripheral annuli (red arrows). Consistent with the light microscopy data, the basal complex labeling of EGFP-TgCentrin2 is considerably lighter than that of EGFP-TgMORN1 (bottom inset, red arrows). A concentration of gold particles within a ; 250 nm patch at the extreme basal end of the parasite is often seen, which likely corresponds to the concentration of TgCentrin2 basal labeling at the light microscopy level (cf. [D]). Both TgMORN1 and TgCentrin2 display certain levels of localization along the parasite body, which is likely to be from the proteins in the cytoplasmic pool. Scale bars 1⁄4 500 nm; Insets are at 1.5 3 magnification. doi:10.1371/journal.ppat.0040010.g001](publication/5644041/figure/fig1/AS:280323976908802@1443845852121/The-Distribution-of-Two-Basal-Complex-Components-TgMORN1-and-TgCentrin2-in-Mature.png)
The Distribution of Two Basal Complex Components, TgMORN1 and TgCentrin2, in Mature Parasites (A) The major structural reference points of the T. gondii cytoskeleton referred to throughout this paper are illustrated in the cartoon drawing of an interphase adult parasite. The cytoskeleton of T. gondii includes the apical and the basal complexes, the spindle pole, the centriole, and the cortical cytoskeleton, which includes all of the cytoskeletal elements aligning the parasite body (i.e., cortical microtubules, the IMC, and the filamentous network underlying the IMC), except for the apical and basal complexes. (B) In interphase parasites, TgMORN1 (green, EGFP-TgMORN1) forms a cap at the extreme basal end of the parasite (green arrow), filling the gap at the basal end of the IMC (blue, labeled by anti-IMC1 monoclonal antibody plus Alexa350-anti-mouse IgG). TgMORN1 also weakly concentrates in the apical complex (dotted square indicated by the white arrow. Stronger contrast enhancement of TgMORN1 labeling is applied to this region to highlight the apical complex labeling of TgMORN1). TgMORN1 is also localized to the spindle pole (green arrowhead), which is juxtaposed to the centriole (red, mCherryFP-TgCentrin1, red arrowhead) during interphase. (C) In interphase parasites, the basal labeling of TgMORN1 (green arrows) is clearly separated from the cortical microtubules (red, mCherryFP-TgTubA1, small gray arrows). The inset shows a magnified view of the centriole/spindle pole assembly, showing red TgTubA1 in the centriole and green TgMORN1 in the spindle pole. At this point in the cell cycle, tubulin labeling of the spindle pole is weak, but will become stronger in early cell division (cf. Figure 5). White arrow, conoid labeling by mCherryFP-TgTubA1. (D) TgMORN1 and TgCentrin2 occupy different subcompartments within the basal complex, with the TgCentrin2 compartment (red arrow at the basal) located posterior to the TgMORN1 basal labeling (green arrow). These parasites are at the very end of interphase, as indicated by the recent duplication of the centriole in the parasite on the left, but not in the parasite on the right. Green, mCherryFP-TgMORN1 (pseudo-color, for consistency in the color scheme); red, EGFP-TgCentrin1 (pseudo-color); blue, anti-IMC1 antibody detected by Alexa350-anti-mouse IgG. Red arrowheads, TgCentrin2 labeling of the apical polar ring (cf. [A], [16]). Red arrows near the apical portion of the parasite apical, TgCentrin2 peripheral annuli (cf. [A], [16]). Insets are at 2 3 magnification. The insets in (B) and (D) do not include the anti-IMC1 labeling in order to emphasize the differences in localization betweenTgMORN1/ TgCentrin1 (B) and TgMORN1/TgCentrin2 (D) labeling. (E) An EM image of an EGFP-TgMORN1 transgenic parasite extracted with 0.5% TritonX-100, immunogold labeled with anti-GFP antibody and negatively stained with phosphotungstic acid. Numerous gold particles are located in the basal complex (bottom inset, green arrows). There is also a small concentration of gold particles in the apical polar ring (top inset, green arrowhead). The cluster of gold particles aligned together (black arrow) at the bottom half of the parasite body is likely to be the labeling of an EGFP-TgMORN1 fiber sometimes seen to form in extracellular parasites [16]. (F) An immuno-EM image of an EGFP-TgCentrin2 transgenic parasite, extracted, labeled, and stained as described in (E). As previously reported [16], there are clear concentrations of EGFP-TgCentrin2 labeling in the apical polar ring (top inset, red arrowheads) and several peripheral annuli (red arrows). Consistent with the light microscopy data, the basal complex labeling of EGFP-TgCentrin2 is considerably lighter than that of EGFP-TgMORN1 (bottom inset, red arrows). A concentration of gold particles within a ; 250 nm patch at the extreme basal end of the parasite is often seen, which likely corresponds to the concentration of TgCentrin2 basal labeling at the light microscopy level (cf. [D]). Both TgMORN1 and TgCentrin2 display certain levels of localization along the parasite body, which is likely to be from the proteins in the cytoplasmic pool. Scale bars 1⁄4 500 nm; Insets are at 1.5 3 magnification. doi:10.1371/journal.ppat.0040010.g001
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The apicomplexans are a large group of parasitic protozoa, many of which are important human and animal pathogens, including Plasmodium falciparum and Toxoplasma gondii. These parasites cause disease only when they replicate, and their replication is critically dependent on the proper assembly of the parasite cytoskeletons during cell division. In...
Contexts in source publication
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... adult parasites, the basal complex occupies the basal gap of the IMC, which, together with the underlying filamentous network, encloses the entire parasite body except for the extreme apical and basal ends ( Figure 1A and 1B) (the gap at the apical end of IMC is occupied by the cytoskeletal apical complex, an intricate assembly that includes the conoid, a tubulin-based molecular machine that does not utilize conventional microtubules; three polar rings; and two intra- conoid microtubules [ Figure 1A and 1C] [6,7]). The basal complex is separated by more than 1.5 lm from another set of major cytoskeletal elements, the cortical microtubules, which emanate from the most posterior of the three polar rings and extend ;2/3 of the length of the parasite body ( Figure 1A and 1C). ...
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... adult parasites, the basal complex occupies the basal gap of the IMC, which, together with the underlying filamentous network, encloses the entire parasite body except for the extreme apical and basal ends ( Figure 1A and 1B) (the gap at the apical end of IMC is occupied by the cytoskeletal apical complex, an intricate assembly that includes the conoid, a tubulin-based molecular machine that does not utilize conventional microtubules; three polar rings; and two intra- conoid microtubules [ Figure 1A and 1C] [6,7]). The basal complex is separated by more than 1.5 lm from another set of major cytoskeletal elements, the cortical microtubules, which emanate from the most posterior of the three polar rings and extend ;2/3 of the length of the parasite body ( Figure 1A and 1C). ...
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... adult parasites, the basal complex occupies the basal gap of the IMC, which, together with the underlying filamentous network, encloses the entire parasite body except for the extreme apical and basal ends ( Figure 1A and 1B) (the gap at the apical end of IMC is occupied by the cytoskeletal apical complex, an intricate assembly that includes the conoid, a tubulin-based molecular machine that does not utilize conventional microtubules; three polar rings; and two intra- conoid microtubules [ Figure 1A and 1C] [6,7]). The basal complex is separated by more than 1.5 lm from another set of major cytoskeletal elements, the cortical microtubules, which emanate from the most posterior of the three polar rings and extend ;2/3 of the length of the parasite body ( Figure 1A and 1C). ...
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... basal complex contains several distinguishable regions organized along its anterior-posterior axis, and is composed of substructures defined by different protein markers, including TgMORN1, TgCentrin2 and TgDLC, a dynein light chain of T. gondii [16]. Interestingly, these basal complex proteins are also components of the apical complex and the centriole/spindle pole assembly ( Figure 1A-1D). The similar- ity in protein composition among these structurally and spatially distinct cytoskeletal assemblies is particularly in- triguing given the de novo nature of the construction of the apical and the basal complex, because the centriole/spindle pole assembly are the only structures inherited by the daughter parasites during cell division, and the centrioles are the only cytoskeletal structure in T. gondii that can self- replicate, thus capable of propagating the structural infor- mation for building a new cytoskeleton to the daughters. ...
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... out this paper, ''the cortical cytoskeleton'' is used to refer to the entire framework of cytoskeleton elements that aligns the parasite body [i.e. cortical microtubules, the IMC and the filamentous network underlying the IMC] except for the apical and the basal complexes [ Figure 1A]). ...
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... previously identified basal complex components in T. gondii, TgMORN1 and TgCentrin2 [16], occupy two distinct compartments in the basal complex of mature parasites. The TgMORN1 compartment is shaped into a cone that forms the main body of the basal complex ( Figure 1A-1E), whereas TgCentrin2 is concentrated at the posterior tip of the basal complex ( Figure 1D and 1F). Both TgCentrin2 and TgMORN1 are also components of the apical complex. ...
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... previously identified basal complex components in T. gondii, TgMORN1 and TgCentrin2 [16], occupy two distinct compartments in the basal complex of mature parasites. The TgMORN1 compartment is shaped into a cone that forms the main body of the basal complex ( Figure 1A-1E), whereas TgCentrin2 is concentrated at the posterior tip of the basal complex ( Figure 1D and 1F). Both TgCentrin2 and TgMORN1 are also components of the apical complex. ...
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... addition, they are localized to the spindle pole and the centrioles, respectively. The spindle pole and the centrioles are juxtaposed to each other during interphase ( Figure 1B-1D), but well separated after the daughter cortical cytoskeletons have formed in the mother [7,16,18,19] (The slight displace- ...
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... ment between the spindle pole and the centrioles in each interphase cell is a true spatial displacement, not an artifact of mis-registration induced by lens chromatic aberration or optical misalignment between the GFP and mCherryFP filters, because the shift between the red and green fluorescence of a multi-color 0.2 lm bead is clearly much smaller and below the resolution limit of the microscope [ Figure S1]). ...
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... mature basal complex in adult parasite, however, is a conical structure, closed at one end, compartmentalized, and stratified along its anterior-posterior axis (cf. Figure 1). When is the polarity and compartmentation of the basal complex established? ...
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... TgCentrin2 resides in only the most constricted region of the basal complex in the adult parasite (cf. Figure 1D), I investigated the relationships among the recruitment of TgCentrin2, the constriction of the basal complex and the establishment of the polarity of the basal complex, by examining TgCentrin2 localization at several different stages of cell division. ...
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... the TgMORN1 compartment, the TgCentrin2 basal compartment also undergoes significant constriction, from a ;1.0 lm ring to a diffraction limited spot (Figures 8 and 9). Consistent with the involvement of centrin homologs in calcium sensitive contractile apparatus in other systems [22][23][24], the constriction of the TgCentrin2 basal compartment can be artificially induced in daughter parasites at a late stage of cell division when the intracellular calcium concentration is elevated by treatment with calcium ionophore, A23187 (Figure 10; Video S3). ...
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... basal complex is a fascinating cytoskeletal organelle whose biogenesis is an integral part of parasite division during both endodyogeny and polyendodyogeny in apicom- plexan parasites [16,17], including cases where triplets are formed after centriole triplication (Data not shown). In this paper, I investigated the full course of development of the basal complex of T. gondii with respect to the centriole duplication and the construction of the daughter cortical cytoskeleton (Figure 11). This paper yields new insights into the polarity establishment during cell division of T. gondii; however, it also brings to light many puzzles yet to be solved. ...
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... induction of the basal complex constriction however occurred much faster at 378C, which made it difficult to capture the intermediate images. The result from a room temper- ature experiment was thus used for this paper ( Figure 10). After taking a set of pretreatment images, 0.85 ll 5mM A23187 (dissolved in DMSO) diluted in 220 ll CO2 independent medium with 10% FBS was added to the dish containing 1.5 ml medium on the microscope stage, which gave a final concentration of A23187 at ;2.5lM. ...
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... result was confirmed by titrating 1 ml 3.5mM CaCl 2 with 0.1M K 2 EGTA using the same procedure. Figure S1. The Shift between the Red and Green Imaging Channels Induced by the Microscope Setup Is Clearly below the Resolution Limit of the Microscope Found at doi:10.1371/journal.ppat.0040010.sg001 ...
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... ring structures originate? Will they really become the basal complex in the daughters? To address these questions, I followed TgMORN1 distribution together with the daughter cortical cytoskeleton construction and centriole duplication in live parasites expressing EGFP-TgMORN1 and mCherryFP-Tg a 1-tubulin (TgTubA1) (Figure 5; Video S1). The TgMORN1 ring first appears as small extra masses outside the spindle pole, and is located close to the recently duplicated centrioles, which lie on each side of the spindle poles (t 1⁄4 10 min). 20–30 min later (t 1⁄4 30–40 min), the fluorescence of mCherryFP-TgTubA1 in the centriole spot increases, likely correlated with the initial assembly of the conoid in the apical complex, of which TgTubA1 is a major component. At this point, the ring-like nature of the TgMORN1 containing structure becomes apparent, and it is centered around the centriole/conoid mass (t 1⁄4 40 min). At t 60 min, the centriole/conoid assemblies move apically above the plane of the TgMORN1 ring with the extension of the cortical microtubules, and the recruitment of TgMORN1 to the apical complex becomes clear. At this point the future apical and basal complexes are separated far enough to make it clear that the TgMORN1 rings formed at the beginning of the cell division are indeed precursors of the daughter basal complexes. The TgMORN1 rings remain at the basal ends of the daughter cortical cytoskeletons as the daughters grow (t 70–110 min). How is the basal complex able to remain at the constantly growing ends of the daughter cortical cytoskeletons? Studies in mammalian cells have shown that microtubule plus-end (MT-plus-end) binding proteins probably maintain their position at the growing ends of microtubules by rapid association and dissociation [20]. Fluorescence Recovery After Photobleaching (FRAP) analysis of daughter basal complexes in T. gondii reveals constant protein exchange between the daughter basal complex and the cytoplasm (Figure 6). Although it is difficult to calculate an exact t1/2 for the fluorescence recovery because of the noise introduced by constant fluctuation of the basal complex position due to daughter cell movement, the recovery is clearly underway by ; 90 sec after photobleaching. This result indicates that the basal complex is intrinsically a dynamic ‘‘ cap ’’ , thus suggesting a mechanism similar to microtubule association of MT- plus-end binding protein is possibly involved in retaining the basal complex at the growing ends of the daughter cortical cytoskeletons. However, the growth of the daughter cortical cytoskeleton is likely not to be the pre-requisite for the protein exchange in the basal complex, as the fluorescence in the mature basal complex also partially recovers after photobleaching (Figure S2). The daughter cortical cytoskeleton and the basal complex appear to grow in concert during cell division. Is the construction and growth of the basal complex, a seemingly ‘‘ downstream ’’ structure, dependent on the structural integrity of the daughter cortical cytoskeleton? To address this question, the construction of the basal complex was tracked in living parasites whose cortical microtubule extension and the daughter cortical cytoskeleton formation were severely disrupted by treating with oryzalin, a plant herbicide that binds to T. gondii a -tubulin and inhibits the construction of the spindle and the cortical microtubules, but not the centriole replication, during daughter formation (Figure 7; Video S2) [9,10,21]. As expected, the mother’s conoid, cortical microtubules, and basal complex are not affected by the oryzalin treatment, and the overall morphology of the mother cell remains normal until the distorted daughters attempt to bud. Daughter cortical microtubules, however, completely fail to appear. Despite the inhibition of the formation of functional daughter cortical cytoskeleton, the initiation of TgMORN1 ring formation proceeds normally (Video S2, 33 and 48 min). Furthermore, complete TgMORN1 rings are formed ; 30 min after the initiation (Video S2; Figure 7, t 60 min), enlarge to ; 1.2 l m (similar to the diameter of the basal complex in untreated daughters with extending cortical cytoskeletons [cf. Figure 5, t 1⁄4 70 min]), and retain their ring morphology till ‘‘ budding ’’ , at which point the organization of the basal complex becomes unclear due to the distorted parasite morphology. The initiation, construction and maintenance of the daughter basal ring complex are therefore independent of the structural integrity of the daughter cortical cytoskeleton. The organization of a growing daughter ring complex is quite different from the basal complex of an adult parasite. The basal complex of growing daughters is an annulus without any clear polarity (e.g. Figure 5, t 1⁄4 70–110 min). The mature basal complex in adult parasite, however, is a conical structure, closed at one end, compartmentalized, and stratified along its anterior-posterior axis (cf. Figure 1). When is the polarity and compartmentation of the basal complex established? The basal complex starts to constrict before cytokinesis and the constriction continues after cytokinesis when the daughters take over the mother’s plasma membrane, thus closing the basal cap in the mature parasite (cf. Video S1) [16,17]. Because TgCentrin2 resides in only the most constricted region of the basal complex in the adult parasite (cf. Figure 1D), I investigated the relationships among the recruitment of TgCentrin2, the constriction of the basal complex and the establishment of the polarity of the basal complex, by examining TgCentrin2 localization at several different stages of cell division. Interestingly, although TgCentrin2 is hardly detectable in the basal ring complex earlier during cell division (cf. Figure 3B and Figure S3), it is clearly localized to the daughter basal complex as a ring at a late stage when the daughter basal complex appears to be constricted (Figure 8A). Its ring-like localization in the basal complex is also pronounced during cytokinesis when the daughters start to take over mother’s plasma membrane (Figure 8B), and after cytokinesis when the basal complex is still open at both ends (Figure 8C). In all cases, the TgCentrin2 basal ring is located to the posterior of the TgMORN1 ring (Figures 8 and 9). The compartmentation and polarization of the basal complex are thus revealed upon the recruitment of TgCentrin2 prior to the closure of the basal complex. Like the TgMORN1 compartment, the TgCentrin2 basal compartment also undergoes significant constriction, from a ; 1.0 l m ring to a diffraction limited spot (Figures 8 and 9). Consistent with the involvement of centrin homologs in calcium sensitive contractile apparatus in other systems [22– 24], the constriction of the TgCentrin2 basal compartment can be artificially induced in daughter parasites at a late stage of cell division when the intracellular calcium concentration is elevated by treatment with calcium ionophore, A23187 (Figure 10; Video S3). The basal complex is a fascinating cytoskeletal organelle whose biogenesis is an integral part of parasite division during both endodyogeny and polyendodyogeny in apicomplexan parasites [16,17], including cases where triplets are formed after centriole triplication (Data not shown). In this paper, I investigated the full course of development of the basal complex of T. gondii with respect to the centriole duplication and the construction of the daughter cortical cytoskeleton (Figure 11). This paper yields new insights into the polarity establishment during cell division of T. gondii ; however, it also brings to light many puzzles yet to be solved. The results in this study clearly show that although the basal ring complex later becomes the distal end of the daughter cortical cytoskeletons, it is one of the first cytoskeletal structures assembled rather than the last. In addition, the daughter cortical cytoskeleton is unlikely to provide a structural base or template for the initiation of the basal complex, as the initial construction of the basal complex is largely unaffected when the cortical microtubule construction and the structural integrity of daughter IMC complex is abolished by oryzalin treatment. How is a macromolecular assembly like the basal complex built from scratch? Although untemplated de novo assembly of huge macromolecular assemblies certainly can occur (e.g., T4 phage or other large viral particles), templated construction proceeding from an inherited ‘‘ seed ’’ seems to be the rule for most large structures in eukaryotes. Interestingly, the initiation of the basal complex spatially and temporally coincides with the replication of the self-duplicating cytoskeletal organelle- the centrioles, which makes the centrioles a particularly attractive candidate for providing the structural information to initiate the de novo assembly of the basal complex. However, it is also clear that the centriole itself is unlikely to be continuously responsible for the maintenance of the basal ring complex, as the diameters of the rings grow up to 1 l m, much larger than the size of the centrioles while they still surround the centrioles at an early stage of cell division. Thus if the centrioles play a role in the initiation and construction of the basal ring complex, other structures associated with it probably serve as intermediary. Future high resolution EM experiments will be essential to elucidate structural connections between the centrioles and the basal ring complex. TgMORN1 sometimes is seen to form long fibers in the cytoplasm, suggesting that this protein might have the propensity of interacting with itself and possibly form polymeric structures [16]. It is thus conceivable that the basal ring structure could be a product from a TgMORN1 polymer constrained into a ring form through its interaction with other proteins in the basal complex and/or the IMC. This, of course, is an ...
Citations
... While recent work from our lab has determined that the basal complex in P. falciparum exhibits temporal complexity, and that proteins are actively recruited and removed from the basal complex, the smaller size of this organism has hindered the discovery of spatial complexity in the P. falciparum basal complex, despite this being a known aspect of the T. gondii basal complex for over a decade [18,26,27]. In this paper, we report the discovery of two separate subcompartments in the P. falciparum basal complex, which we label the posterior cup and the main ring. ...
... Introduction basal complex, along with a multiplicity of other cellular compartments [25,26,28]. We then generate an inducible knockdown of PfCen2, a protein whose endogenous locus has evaded modification for years, even in the P. berghei system [29][30][31]. ...
... Next, we determine that, among many defects, PfCen2-deficient parasites cannot fully contract their basal complexes, indicating that PfCen2, like TgCen2, is required for the final stage of basal complex constriction [25]. Finally, we demonstrate that treatment with a calcium chelator prevents the latter half of basal complex contraction, suggesting the utilization of calcium-responsive mechanisms for basal complex contraction, although failure to contract in response to calcium ionophore treatment indicates a degree of regulation preventing premature contraction beyond what is present in Toxoplasma [26]. Altogether, we demonstrate that some mechanisms of division used to construct and constrict the basal complex differ significantly between T. gondii and P. falciparum while others seem to be broadly conserved, if differently modulated [25,26]. ...
Asexual replication of Plasmodium falciparum occurs via schizogony, wherein 16–36 daughter cells are produced within the parasite during one semi-synchronized cytokinetic event. Schizogony requires a divergent contractile ring structure known as the basal complex. Our lab has previously identified PfMyoJ (PF3D7_1229800) and PfSLACR (PF3D7_0214700) as basal complex proteins recruited midway through segmentation. Using ultrastructure expansion microscopy, we localized both proteins to a novel basal complex subcompartment. While both colocalize with the basal complex protein PfCINCH upon recruitment, they form a separate, more basal subcompartment termed the posterior cup during contraction. We also show that PfSLACR is recruited to the basal complex prior to PfMyoJ, and that both proteins are removed unevenly as segmentation concludes. Using live-cell microscopy, we show that actin dynamics are dispensable for basal complex formation, expansion, and contraction. We then show that EF-hand containing P. falciparum Centrin 2 partially localizes to this posterior cup of the basal complex and that it is essential for growth and replication, with variable defects in basal complex contraction and synchrony. Finally, we demonstrate that free intracellular calcium is necessary but not sufficient for basal complex contraction in P. falciparum. Thus, we demonstrate dynamic spatial compartmentalization of the Plasmodium falciparum basal complex, identify an additional basal complex protein, and begin to elucidate the unique mechanism of contraction utilized by P. falciparum, opening the door for further exploration of Apicomplexan cellular division.
... At the basal end, T. gondii features a basal complex, devoid of tubulin, which is responsible for completing cytokinesis and thereby facilitating parasite replication [74][75][76]. ...
The success of the intracellular parasite Toxoplasma gondii in invading host cells relies on the apical complex, a specialized microtubule cytoskeleton structure associated with secretory organelles. The T. gondii genome encodes three isoforms of both α- and β-tubulin, which undergo specific post-translational modifications (PTMs), altering the biochemical and biophysical proprieties of microtubules and modulating their interaction with associated proteins. Tubulin PTMs represent a powerful and evolutionarily conserved mechanism for generating tubulin diversity, forming a biochemical ‘tubulin code’ interpretable by microtubule-interacting factors. T. gondii exhibits various tubulin PTMs, including α-tubulin acetylation, α-tubulin detyrosination, Δ5α-tubulin, Δ2α-tubulin, α- and β-tubulin polyglutamylation, and α- and β-tubulin methylation. Tubulin glutamylation emerges as a key player in microtubule remodeling in Toxoplasma, regulating stability, dynamics, interaction with motor proteins, and severing enzymes. The balance of tubulin glutamylation is maintained through the coordinated action of polyglutamylases and deglutamylating enzymes. This work reviews and discusses current knowledge on T. gondii tubulin glutamylation. Through in silico identification of protein orthologs, we update the recognition of putative proteins related to glutamylation, contributing to a deeper understanding of its role in T. gondii biology.
... At the basal end, T. gondii presents a basal complex, without tubulin, responsible for completing cytokinesis and consequently parasite replication [64][65][66]. ...
The success of Toxoplasma gondii (intracellular parasite) host cell invasion relies on the apical complex, a specialized microtubule cytoskeleton structure associated with secretory organelles. The genome encodes three isoforms of both α- and β-tubulin which are altered by specific post-translational modifications (PTMs), changing the biochemical/biophysical proprieties of microtubules, and modulating their interaction with associated proteins. Tubulin PTMs are a powerful and evolutionarily conserved mechanism to generate tubulin diversity, forming a biochemical ‘tubulin code’ that can be ‘read’ by microtubule-interacting factors. The T. gondii tubulin PTMs are: α-tubulin acetylation, α-tubulin detyrosination, Δ5α-tubulin, Δ2α-tubulin, α- and β-tubulin polyglutamylation, and α- and α-tubulin methylation. Tubulin glutamylation is a key candidate to assist microtubule remodeling in Toxoplasma, being involved in the regulation of microtubule stability, dynamics, interaction with motor proteins, and severing enzymes. The correct balance of tubulin glutamylation is achieved by the coordinated action of polyglutamylases and deglutamylating enzymes. In this work we will review and discuss the current knowledge on T. gondii tubulin glutamylation. By in silico identification of mammalian protein orthologs we explored and updated the identification of putative proteins related to glutamylation, contributing to a better understanding of the role of tubulin glutamylation in T. gondii.
... Little is known about SPMT biogenesis during the asexual blood stage of P. falciparum, but it is currently hypothesized that they are nucleated by the APRs (Hanssen et al., 2013;Morrissette and Sibley, 2002), as is the case in Toxoplasma (Morrissette and Sibley, 2002;Tran et al., 2010). Curiously, TgCentrin 2 localizes to the APR of Toxoplasma tachyzoites (Hu, 2008), but no Centrin 2 has been observed to localize to the APRs of P. falciparum. Furthermore, it was recently shown that the SPMTs of P. falciparum gametocytes, which lack an APR, are formed at the outer CP, in the space between the nuclear envelope and PPM . ...
... There is no primary antibody against CINCH at this point, and so it is not possible to determine whether the lack of overlap with NHS ester is due to distance between the smV5 tag and the main protein density of CINCH (CINCH is 230 kDa). It is also possible that this difference in localization reflects basal complex architecture similar to that previously observed in Toxoplasma gondii, where the basal complex consists of multiple concentric rings (Anderson-White et al., 2012;Engelberg et al., 2022;Hu, 2008;Roumégous et al., 2022). ...
Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.
... Curiously, TgCentrin 2 localizes to the apical polar ring of Toxoplasma tachyzoites (K. Hu, 2008 ), but no Centrin 2 has been observed to localize to the apical polar rings of P. falciparum. Furthermore, it was recently shown that the SPMTs of P. falciparum gametocytes, which lack an APR, are formed at the outer CP, in the space between the nuclear envelope and PPM (Li et al., 2022 ). ...
... main protein density of CINCH (CINCH is 230kDa). It is also possible that this difference in localization reflects basal complex architecture similar to that previously observed in Toxoplasma gondii, where the basal complex consists of multiple concentric rings (Anderson-White et al., 2012 ;Engelberg, Bechtel, Michaud, Weerapana, & Gubbels, 2022 ;K. Hu, 2008 ;Roumégous et al., 2022 ). ...
Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample ∼4.5x. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.
Using ultrastructure-expansion microscopy we explore the fundamental cell biology of malaria parasites, providing new insights into processes including establishment of cell polarity and organelle fission.
... In Toxoplasma gondii, a prototypical apicomplexan, the cortical MTs are stabilized by a suite of novel associated proteins [11][12][13][14]. Chemical inhibition of the elongation of the cortical MTs during cell division results in a distorted cortex and non-viable progeny [15][16][17][18][19]. In addition, unlike most polarized microtubule-arrays examined so far, which are typically originated from a centrosome or spindle pole, the cortical MTs of Toxoplasma and other apicomplexans are anchored in the apical polar ring, an annular structure at the parasite apex [4,5,13,20,21]. ...
... During cell division, while the mitotic spindle is assembled in the nucleus, the cortical microtubules of the developing daughter polymerize in the cytosol [12]. Simultaneously the conoid also develops in close proximity to the apical ends of the cortical MTs [17,18]. ...
... Prior to the appearance of the daughter tubulin cytoskeleton, Toxoplasma centrioles duplicate into two pairs located adjacent to the two poles of the nascent spindle, consistent with previous findings [12,17,18,33,[42][43][44]. The two centrioles within each pair are clearly resolved in the ExM images. ...
The tubulin-containing cytoskeleton of the human parasite Toxoplasma gondii includes several distinct structures: the conoid, formed of 14 ribbon-like tubulin polymers, and the array of 22 cortical microtubules (MTs) rooted in the apical polar ring. Here we analyze the structure of developing daughter parasites using both 3D-SIM and expansion microscopy. Cortical MTs and the conoid start to develop almost simultaneously, but from distinct precursors near the centrioles. Cortical MTs are initiated in a fixed sequence, starting around the periphery of a short arc that extends to become a complete circle. The conoid also develops from an open arc into a full circle, with a fixed spatial relationship to the centrioles. The patterning of the MT array starts from a "blueprint" with ~ 5-fold symmetry, switching to 22-fold rotational symmetry in the final product, revealing a major structural rearrangement during daughter growth. The number of MT is essentially invariant in the wild-type array, but is perturbed by the loss of some structural components of the apical polar ring. This study provides structural insights into the development of tubulin-containing structures that diverge from conventional models, insights that are critical for understanding the evolutionary paths leading to construction and divergence of cytoskeletal frameworks.
... In contrast to apical complex, the basal complex (BC) is much less prominent, and the studies on its functions and organization are scant. BC is composed of juxtaposed rings located beneath the basal end of the parasite's internal membrane complex that extends beneath the plasma membrane along the cell body (Hu, 2008). The identification of the molecular framework that composes this complex is extremely important for understanding its role in the parasite's biology and as future targets in chemotherapy. ...
Parasitic diseases are commonly found in various locations across the globe. Some of them do not encounter geographic barriers, having among their infected individuals living in developed and developing countries. The causative agents of these diseases with the greatest medical and social impact are protists, unicellular organisms that are well known for their peculiar structural organization, a consequence of an early divergence from the main lineages of eukaryotic evolution. Many of these unusual features are adaptative mechanisms for a parasitic lifestyle and serve as potential pharmacological targets. In this editorial, we briefly introduce a small collection of articles that bring contributions to different aspects of the Cell Biology of protozoa: (1) raising the state of the art in endoplasmic reticulum biology in trypanosomatids; (2) presenting new molecular components in Toxoplasma gondii and two new potential anti-T. gondii drugs; and (3) revealing new insights into the ultrastructure of the sexual stage of Plasmodium falciparum.
... To further explore this phenomenon, wild-type parasites were transiently transfected with plasmids containing FNR-RFP (a marker for the apicoplast [27])) and Centrin1-GFP (a marker for the centrosome [52]) expression cassettes and visualized using live-cell microscopy. In parasites with highly dynamic, elongated apicoplasts the centrosomes were not always associated with the tips of the apicoplast but were frequently associ ated with the sides of the elongated apicoplast (Fig. 6A, insets 1 and 3) (Video S6). ...
Toxoplasma gondii contains an essential plastid organelle called the apicoplast that is necessary for fatty acid, isoprenoid, and heme synthesis. Perturbations affecting apicoplast function or inheritance lead to parasite death. The apicoplast is a single copy organelle and, therefore, must be divided so that each daughter parasite inherits an apicoplast during cell division. In this study, we identify new roles for F-actin and an unconventional myosin motor, TgMyoF, in this process. First, loss of TgMyoF and actin lead to an accumulation of apicoplast vesicles in the cytosol indicating a role for this actomyosin system in apicoplast protein trafficking or morphological integrity of the organelle. Second, live cell imaging reveals that during division the apicoplast is highly dynamic, exhibiting branched, U-shaped and linear morphologies that are dependent on TgMyoF and actin. In parasites where movement was inhibited by the depletion of TgMyoF, the apicoplast fails to associate with the parasite centrosomes. Thus, this study provides crucial new insight into mechanisms controlling apicoplast-centrosome association, a vital step in the apicoplast division cycle, which ensures that each daughter inherits a single apicoplast.
IMPORTANCE
Toxoplasma gondii and most other parasites in the phylum Apicomplexa contain an apicoplast, a non-photosynthetic plastid organelle required for fatty acid, isoprenoid, iron-sulfur cluster, and heme synthesis. Perturbation of apicoplast function results in parasite death. Thus, parasite survival critically depends on two cellular processes: apicoplast division to ensure every daughter parasite inherits a single apicoplast, and trafficking of nuclear encoded proteins to the apicoplast. Despite the importance of these processes, there are significant knowledge gaps in regards to the molecular mechanisms which control these processes; this is particularly true for trafficking of nuclear-encoded apicoplast proteins. This study provides crucial new insight into the timing of apicoplast protein synthesis and trafficking to the apicoplast. In addition, this study demonstrates how apicoplast-centrosome association, a key step in the apicoplast division cycle, is controlled by the actomyosin cytoskeleton.
... We examined the average number of rounds of replication at 12, 24, and 36 h after infection and found that the rate of replication of the TKO parasite was very similar to that of the wild-type parasite (Fig. 1D). At first glance, it is surprising that a mutant that has destabilized cortical microtubules does not have a replication defect, because microtubule polymerization is coupled with cortex formation, and thus is required for daughter growth (Hu, 2008;Hu et al., 2006;Morrissette and Sibley, 2002b;Stokkermans et al., 1996). However, as we observed previously , in the same cell in which the cortical microtubules of the mother TKO parasite are depolymerized, microtubules do polymerize in growing daughters, which develop normally. ...
Motility is essential for apicomplexan parasites to infect their hosts. In a three-dimensional (3-D) environment, the apicomplexan parasite Toxoplasma gondii moves along a helical path. The cortical microtubules, which are ultra-stable and spirally arranged, have been considered to be a structure that guides the long-distance movement of the parasite. Here we address the role of the cortical microtubules in parasite motility, invasion, and egress by utilizing a previously generated mutant (dubbed "TKO") in which these microtubules are destabilized in mature parasites. We found that the cortical microtubules in∼80% of the non-dividing (i.e. daughter-free) TKO parasites are much shorter than normal. The extent of depolymerization is further exacerbated upon commencement of daughter formation or cold treatment, but parasite replication is not affected. In a 3-D Matrigel matrix, the TKO mutant moves directionally over long distances, but along trajectories significantly more linear (i.e. less helical) than those of wild-type parasites. Interestingly, this change in trajectory does not impact either movement speed in the matrix or the speed and behavior of the parasite's entry into and egress from the host cell.
... The BC is a contractile ring positioned at the IMC's leading edge during segmentation 15,22 . It contracts as segmentation concludes and is hypothesized to act as a docking site for cytoskeletal proteins joining the IMC and mediate abscission of daughter parasites after segmentation 15,[23][24][25][26] . The only known P. falciparum BC proteins are PfCINCH, PfBLEB, PfBCP1, PfBTP1, PfBTP2, PfHAD2, and PfMORN1 15,22,[27][28][29][30] . ...
... In the related Apicomplexan parasite, Toxoplasma gondii, the BC has been extensively studied and multiple proteins have been characterized [23][24][25][26]31,32 . In T. gondii, the class VI-like 33 myosin TgMyoJ and the centrin TgCen2 are hypothesized to mediate BC contraction 25,34 . ...
... In the related Apicomplexan parasite, Toxoplasma gondii, the BC has been extensively studied and multiple proteins have been characterized [23][24][25][26]31,32 . In T. gondii, the class VI-like 33 myosin TgMyoJ and the centrin TgCen2 are hypothesized to mediate BC contraction 25,34 . Moreover, the T. gondii BC is dynamic, 25,35 with a well-constructed timeline of assembly and recruitment 31 . ...
During its asexual blood stage, P. falciparum replicates via schizogony, wherein dozens of daughter cells are formed within a single parent. The basal complex, a contractile ring that separates daughter cells, is critical for schizogony. In this study, we identify a Plasmodium basal complex protein essential for basal complex maintenance. Using multiple microscopy techniques, we demonstrate that PfPPP8 is required for uniform basal complex expansion and maintenance of its integrity. We characterize PfPPP8 as the founding member of a novel family of pseudophosphatases with homologs in other Apicomplexan parasites. By co-immunoprecipitation, we identify two additional new basal complex proteins. We characterize the unique temporal localizations of these new basal complex proteins (late-arriving) and of PfPPP8 (early-departing). In this work, we identify a novel basal complex protein, determine its specific role in segmentation, identify a new pseudophosphatase family, and establish that the P. falciparum basal complex is a dynamic structure.
