Fig 1.
TGGT1_238895 localizes to the daughter IMC body and is recruited at the earliest stages of endodyogeny.
A) Gene model of TGGT1_238895 (IMC43) showing the endogenous 3xHA tag fused to the C-terminus for localization studies. B) The cell-cycle expression profile for TGGT1_238895 closely matches the cyclical pattern of the known daughter IMC protein IMC29. RMA = robust multi-array average [24]. C) IFA showing that TGGT1_238895 is undetectable in mature parasites. Magenta = anti-HA detecting TGGT1_2388953xHA, Green = anti-IMC6. D) IFA showing that TGGT1_238895 localizes to daughter buds and colocalizes with IMC6. Magenta = anti-HA detecting TGGT1_2388953xHA, Green = anti-IMC6. E) IFA of budding parasites showing that TGGT1_238895 is excluded from the apical cap, marked by ISP1. Magenta = anti-HA detecting TGGT1_2388953xHA, Green = anti-ISP1. F) IFA showing that IMC43 recruits to the daughter IMC before IMC6. Magenta = anti-HA detecting IMC433xHA, Green = anti-IMC6. G) IFA showing that IMC43 recruits to the daughter IMC before ISP1. Arrow points to the mother cell apical cap. Asterisk indicates daughter buds. Magenta = anti-HA detecting IMC433xHA, Green = anti-ISP1. H) IFA showing that IMC43 recruits to the daughter IMC at the same time as FBXO1. Magenta = anti-Ty detecting IMC432xStrep3xTy, Green = anti-HA detecting FBXO13xHA. I) IFA showing that IMC43 recruits to the daughter IMC at the same time as AC10. Magenta = anti-HA detecting IMC433xHA, Green = anti-Ty detecting AC102xStrep3xTy. J) IFA showing that IMC43 recruits to the daughter IMC at the same time as IMC32. Magenta = anti-HA detecting IMC433xHA, Green = anti-V5 detecting IMC323xV5. Scale bars = 2 μm.
Fig 2.
IMC43 is essential for parasite replication and survival.
A) Diagram showing the mAID-3xHA degron tag fused to the C-terminus of IMC43 in a TIR1-expressing strain to facilitate proteasomal degradation upon treatment with IAA. B) IFA showing that the IMC43AID protein localizes normally to the daughter IMC and is depleted when the parasites are treated with IAA. Magenta = anti-HA detecting IMC43AID, Green = anti-IMC6. C) IFA showing the broad range of morphological and replication defects observed after treating IMC43AID parasites with IAA for 24 hours. White arrows point to large gaps in the IMC marked by IMC6. Yellow arrows point to daughter buds present outside of the maternal parasite. Asterisks mark parasites producing more than two daughter buds. All three +IAA vacuoles also display desynchronized endodyogeny, where parasites in the same vacuole are at different stages of replication. Green = anti-IMC6. D) Quantification of vacuoles displaying morphological and/or replication defects after 24 hours of IMC43 depletion. Statistical significance was determined using a two-tailed t test (****, P < 0.0001). E) IFA of IMC43AID parasites treated with IAA for 40 hours. White arrows point to large gaps in the IMC marked by IMC6. Asterisks indicate swollen parasites. Green = anti-IMC6 F) Diagram of the full-length smOLLAS-tagged IMC43 complementation construct driven by its endogenous promoter (IMC43WT) and integrated at the UPRT locus in IMC43AID parasites. G) IFA showing that IMC43WT localizes normally to the daughter IMC and rescues the morphological and replication defects observed upon treatment with IAA. Magenta = anti-OLLAS detecting IMC43WT, Green = anti-IMC6. H) Plaque assays for IMC43AID and IMC43AID + IMC43WT parasites grown for seven days -/+ IAA. Depletion of IMC43 results in a severe defect in overall lytic ability, which is fully rescued by complementation with the wild-type protein. Scale bars = 0.5 mm. I) Quantification of plaque number for plaque assays shown in panel H. IMC43-depleted parasites form fewer than 10% as many plaques compared to control. Statistical significance was determined using multiple two-tailed t tests (****, P < 0.0001; ns = not significant). J) Quantification of plaque size for plaque assays shown in panel H. Plaques formed by IMC43-depleted parasites are <1% the usual size. Statistical significance was determined using multiple two-tailed t tests (****, P < 0.0001; ns = not significant). Scale bars for all IFAs = 2 μm.
Fig 3.
Assessment of key structures involved in endodyogeny.
A) IFA showing that the daughter IMC protein IMC29 is unaffected by depletion of IMC43. Magenta = anti-V5 detecting IMC293xV5, Green = anti-IMC6. B) IFA showing that the apical cap marker AC10 is unaffected by depletion of IMC43. Arrows point to the apical cap of daughter buds. Magenta = anti-V5 detecting AC103xV5, Green = anti-IMC6. C) IFA showing that the basal complex marker MORN1 is unaffected by depletion of IMC43. Arrows point to the basal complex of daughter buds. Magenta = anti-V5 detecting MORN13xV5, Green = anti-IMC6. D) IFA showing that the conoid and subpellicular microtubules still assemble on nascent daughter buds when IMC43 is depleted. Arrows point to daughter bud conoids. Magenta = transiently expressed Tubulin1-GFP, Green anti-IMC6. E) IFA showing that centrosome duplication continues when IMC43 is depleted. Centrosomes, marked by Centrin1, appear to duplicate and associate with daughter buds and dividing nuclei. Parasites forming more than two daughter buds also form more than two centrosomes. White arrow points to a single parasite containing two centrosomes (normal). Yellow arrows point to single parasites containing three or more centrosomes (abnormal). Magenta = anti-Centrin1, Green = anti-IMC1, Blue = Hoechst. F) IFA showing that nuclear division and centrosome duplication continue after 40 hours of IAA treatment. Magenta = anti-Centrin1, Green = anti-IMC1, Blue = Hoechst. Scale bars = 2 μm.
Fig 4.
Deletion analyses reveal regions involved in IMC43 localization and function.
A) Diagram of the nine deletion constructs generated for domain analysis of IMC43. B) IFA showing the localization of IMC43Δ402–494 compared to IMC43AID. IMC43Δ402–494 partially mislocalizes to the cytoplasm of parasites (arrow). Magenta = anti-OLLAS detecting IMC43Δ402–494, Green = anti-HA detecting IMC43AID. C) IFA showing that IMC43Δ1441–1653 fails to rescue the morphological and replication defects caused by depletion of endogenous IMC43. Arrow points to a single maternal parasite producing five daughter buds, indicating dysregulation of endodyogeny. Asterisks indicate disruption of the cytoskeleton. Magenta = anti-OLLAS detecting IMC43Δ1441–1653, Green = anti-IMC6. D) Quantification of plaque number for IMC43AID, IMC43AID + IMC43WT, and all nine IMC43 deletion strains shown in panel A after seven days of growth -/+ IAA. Y axis represents the number of plaques formed in +IAA conditions divided by the number of plaques formed in -IAA conditions for each strain. Statistical significance was determined by one-way ANOVA (****, P < 0.0001; ns = not significant). E) IFA showing that IMC43Δ1441–1561 fails to rescue the morphological and replication defects caused by depletion of endogenous IMC43. Arrow points to a single maternal parasite producing three daughter buds, indicating dysregulation of endodyogeny. Asterisks indicate disruption of the cytoskeleton. Magenta = anti-OLLAS detecting IMC43Δ1441–1561, Green = anti-IMC6. F) IFA showing that IMC43Δ1562–1653 rescues the morphological and replication defects caused by depletion of endogenous IMC43. Magenta = anti-OLLAS detecting IMC43Δ1562–1653, Green = anti-IMC6. G) Quantification of plaque number for IMC43AID, IMC43AID + IMC43WT, IMC43AID + IMC43Δ1441–1561, and IMC43AID + IMC43Δ1562–1653 parasites after seven days of growth -/+ IAA. Y axis represents the number of plaques formed in +IAA conditions divided by the number of plaques formed in -IAA conditions for each strain. Statistical significance was determined by one-way ANOVA (****, P < 0.0001; ns = not significant). Scale bars = 2 μm.
Fig 5.
TurboID and Y2H screens yield 30 candidate IMC43 binding partners.
A) Diagram of IMC43TurboID. B) IFA showing that IMC43TurboID localizes normally to the daughter IMC and biotinylates proximal proteins in a biotin-dependent manner. Arrows point to endogenously biotinylated apicoplasts. Magenta = anti-HA detecting IMC43TurboID, Green = streptavidin. Scale bars = 2 μm. C) Venn diagram comparing genes identified in the Y2H screen and TurboID experiments. All Y2H hits that were ranked as “D” (moderate confidence) or higher were included. TurboID results were filtered to include only genes that were at least two-fold enriched with a difference of >5 spectral counts when comparing IMC43TurboID to control. 30 genes were identified in both experiments after filtering results as described. D) Table showing the 30 genes identified in both the TurboID and Y2H screens. The two candidate binding partners analyzed in this study are highlighted in yellow. “Y2H” column indicates the confidence score assigned in the Y2H screen (B = high confidence, C = good confidence, D = moderate confidence). “TurboID Ctrl / Exp” column indicates the average spectral count for each gene in the control and IMC43TurboID mass spectrometry results. “GWCS” refers to the phenotype score assigned to each gene in a genome-wide CRISPR/Cas9 screen [35]. “Localization” column reports the known localization of each protein [13,15,16,27,29,48–50,54–56,76]. Localizations followed by “(LOPIT)” indicate predicted localizations based on hyperplexed localization of organelle proteins by isotope tagging (hyperLOPIT) [42].
Fig 6.
IMC44 is a novel IMC protein which depends on IMC43 binding for regulation of its dynamic localization.
A) Gene model of TGGT1_297120 (IMC44) showing the region determined to interact with IMC43 by the Y2H screen. B) The cell-cycle expression profile for TGGT1_297120 closely mirrors the cyclical pattern of IMC43. RMA = robust multi-array average [24]. C) IFAs showing the localization of TGGT1_297120 at different stages of endodyogeny. Arrows indicate the point at which TGGT1_297120 can be first seen localizing to the basal complex of daughter buds. Magenta = anti-Myc detecting TGGT1_2971203xMyc, Green = anti-IMC6. D) IFAs comparing the localization of TGGT1_297120 with the basal complex marker MORN1. Arrows point to the basal complex of daughter buds. Magenta = anti-Myc detecting TGGT1_2971203xMyc, Green = anti-V5 detecting MORN13xV5. E) IFA showing the localization of IMC44 in IMC43AID parasites -/+ IAA. Depletion of IMC43 causes IMC44 to fail to localize to the body of the IMC. Arrows point to the body of the IMC in daughter buds. Magenta = anti-Myc detecting IMC443xMyc, Green = anti-IMC6. F) IFA showing the localization of IMC44 in IMC43AID + IMC43WT parasites -/+ IAA. IMC43WT rescues the mislocalization of IMC44 observed in IMC43-depleted parasites. Arrows point to the body of the IMC in daughter buds. Magenta = anti-Myc detecting IMC443xMyc, Green = anti-IMC6. G) IFA showing the localization of IMC44 in IMC43AID + IMC43Δ1441–1561 parasites -/+ IAA. IMC43Δ1441–1561 fails to rescue the mislocalization of IMC44 observed in IMC43-depleted parasites. Arrows point to the body of the IMC in daughter buds. Magenta = anti-Myc detecting IMC443xMyc, Green = anti-IMC6. Scale bars = 2 μm. H) Diagrams showing the fragments of IMC43 and IMC44 that were fused to LexA/GAL4AD for pairwise Y2H assays. I) Pairwise Y2H assays assessing the interaction of IMC43 and IMC44. Growth on restrictive (-L/W/H) media indicates binding between the indicated fragments of each protein.
Fig 7.
IMC43 binding is required for maintenance of IMC32 during later stages of endodyogeny.
A) Gene model of IMC32 showing its predicted N-terminal palmitoylation site, Ig-PH domain, 5 predicted CC domains, and IMC43-interacting region identified by the Y2H screen. B) IFA comparing the localization of IMC43 and IMC32 at the mid/late budding stages of endodyogeny. Top: Daughter buds viewed from the side. Bottom: Daughter buds viewed from below. Magenta = anti-V5 detecting IMC323xV5, Green = anti-HA detecting IMC43AID. C) IFA showing the localization of IMC32 during bud initiation in IMC43AID parasites after 18 hours of growth -/+ IAA. Depletion of IMC43 has no effect on IMC32 localization during bud initiation. Magenta = anti-V5 detecting IMC323xV5, Green = anti-IMC6. D) IFA showing the localization of IMC32 during the late-budding stage in IMC43AID parasites after 18 hours of growth -/+ IAA. Depletion of IMC43 causes IMC32 to lose its characteristic striped localization pattern at this stage of endodyogeny. Magenta = anti-V5 detecting IMC323xV5, Green = anti-IMC6. E) IFA showing the localization of IMC32 in IMC43AID parasites after 30 hours of growth -/+ IAA. Depletion of IMC43 over a longer time course results in an accumulation of mislocalized IMC32 in the cytoplasm. White arrows point to IMC32 enriching at early daughter buds. Yellow arrows point to daughter buds at the mid/late-budding stage with diffuse and disorganized IMC32 staining. Magenta = anti-V5 detecting IMC323xV5, Green = anti-IMC6. F) IFA showing the localization of IMC32 in IMC43AID + IMC43WT parasites after 18 hours of growth -/+ IAA. IMC43WT rescues the mislocalization of IMC32 caused by depletion of IMC43. Magenta = anti-V5 detecting IMC323xV5, Green = anti-IMC6. G) Quantification of vacuoles with mislocalized IMC32 at different stages of endodyogeny after 18 hours of growth -/+ IAA. Approximately 76% of vacuoles at the mid and late budding stages exhibited mislocalization of IMC32 when IMC43 was depleted. This phenotype was fully rescued by complementation with IMC43WT. Statistical significance was determined using multiple two-tailed t tests (****, P < 0.0001; ns = not significant). H) IFA showing the localization of IMC32 in IMC43AID + IMC43Δ1441–1561 parasites after 24 hours of growth -/+ IAA. IMC43Δ1441–1561 fails to rescue the mislocalization of IMC32 caused by depletion of IMC43. Magenta = anti-V5 detecting IMC323xV5, Green = anti-IMC6. Scale bars = 2 μm. I) Diagrams showing the fragments of IMC43 and IMC32 that were fused to LexA/GAL4AD for pairwise Y2H assays. J) Pairwise Y2H assays assessing the interaction of IMC43 and IMC32. Growth on restrictive (-L/W/H) media indicates binding between the indicated fragments of each protein.
Fig 8.
A) Diagram summarizing the effects of IMC43 depletion. Depletion of IMC43 causes gross morphological defects such as swelling, cytoskeletal instability, asynchronous division, excess budding & nuclear division, and loss of overall lytic ability. Assembly of the basal complex and apical cap remain unaffected. B) Diagram summarizing the relationship between IMC43 and its binding partners IMC32 and IMC44. The newly identified Localization domain (LOC) and Essential Interaction Domain (EID) of IMC43 are shown. Grey boxes indicate interactions between the Essential Interaction Domain of IMC43 and IMC32/IMC44. Boxed panels include a diagram showing how depletion of IMC43 affects the localization of IMC32 and IMC44 throughout bud initiation (1), early/mid-budding (2), and late budding (3).