Figure 1.
Clathrin interactome and localisation in T. gondii.
(A) Putative clathrin interactome with green symbols conserved in T. gondii, orange symbols conserved in most eukaryotes and red symbols indicating fungi specific interactors. For details see also Table S1. (B) Schematics of the chc1 endogenous ha tagging construct and its integration into the genomic chc1 locus. UTR, untranslated region. (C) Analytical PCR on genomic DNA using oligos indicated as orange arrows in (B). The obtained 1484 bp confirms endogenous tagging of chc1. (D) Immunoblot of indicated parasite lines probed with anti-HA, anti-Catalase, and anti-Aldolase antibodies. Catalase and aldolase were used as loading controls. (E and F) Immunofluorescence analysis of endogenously chc1 tagged parasites with indicated antibodies. CHC1-HA shows no colocalisation with IMC1 (E) and predominantly accumulates apical to the nucleus (E and F). It colocalises with DrpB and resides like DrpB adjacent to the trans-Golgi weakly overlapping with GRASP-RFP. (F). R, Pearson correlation coefficient ± SD averaged for the indicated number (n) of parasites. Scale bars represent 1 μm and 10 μm in (E) and 10 μm in (F). Inlets show twofold enlargements of the indicated area.
Figure 2.
Ultrastructural analysis of CHC-HA expressing T. gondii parasites.
Routine (A) and immuno (B and C) electron micrographs. (A) Section through the anterior of a tachyzoite showing nucleus (N), apicoplast (A), Golgi (G), rhoptries (R), micronemes (M), and mitochondrion (Mi). Scale bar is 100 nm. Insert: Enlargement of the enclosed area showing a coated vesicle. Bar is 50 nm. (B) Similar area to that in (A) of a parasite processed for immuno electron microscopy showing gold particles located predominately at the periphery of the Golgi (G). N, nucleus; A, apicoplast; DG, dense granules. Bar is 100 nm. Insert: Detail of the enclosed area showing a number of gold particles around a possible vesicle. Bar is 50 nm. (C) Section through a parasite undergoing endodyogeny showing the two partially formed daughters (D1, D2) with gold particles (arrowheads) associated with the periphery of the daughter Golgi bodies (G). N, nucleus. Bar is 500 nm. Insert: Enlargement of the enclosed area showing gold particles at the periphery of the Golgi body. Bar is 100 nm.
Figure 3.
Rapid ablation of CHC1 function in T. gondii
(A) Schematic of the dominant negative DD-Hub construct and of the chlathrin triskelion structure. The Hub fragment is highlighted in pink. dd, destabilization domain; myc, myc epitope tag; p, promoter; UTR, untranslated region. (B) Schematic (left) and immunofluorescence analysis (right) of Shield-1 dependent regulation of DD-Hub. Immunofluoresence has been performed with indicated antibodies 24 hours after incubation of parasites in presence or absence of 1 μM Shield-1. Hub fragments localise to the cytoplasm and their expression lead to deformed IMCs (C) Immunoblot of parasite lysates probed with indicated antibodies. Catalase and aldolase were used as loading controls. Intra- or extracellular parasites were treated with 0 μM, 0.1 μM and 1 μM Shield-1 for the indicated time. (D) In contrast to the controls DD-Hub expressing parasites show no plaque formation after 7 days incubation in presence of 1 μM Shield-1. Scale bar represents 500 μm. (E) For analysis of the invasion rate intra- or extracellular parasites were treated for 6 hr with or without 1 μM Shield-1 prior to the experiment. Data represent mean values of three independent experiments ±SD. (F) For analysis of the replication rate indicated parasite lines were cultured in absence or presence of 1 μM Shield-1 and fixed 24 hr post invasion. Data represent mean values of three independent experiments ±SD. (G) Immunofluorescence analysis with indicated antibodies of DD-Hub parasites treated with indicated Shield-1 concentrations for 24 hours. Scale bar represents 10 μm. Immunoflourescence images are representative of at least three independent experiments and depicted abnormalities have been observed in 100% of 200 random examined vacuoles compaired to controls.
Figure 4.
CHC1 is essential for Golgi function and segregation.
(A) Immunofluorescence analysis of DD-Hub expressing parasites stable transfected with the cis-medial Golgi marker GRASP-RFP cultured for 24 hr in absence or presence of 0.1 and 1 μM Shield-1. Only under high Shield-1 concentrations daughter parasites show abnormal Golgi morphology (white arrow heads) or no Golgi at all (yellow arrow heads, N highlighted in yellow). N, nucleus. (B-D) Immunofluorescence analysis of DD-Hub expressing parasites stable transfected with SAG1∆GPI-dsRed. After treatment for 24 hr with 0.1 and 1 μM Shield-1 DD-Hub expression causes a block in constitutive secretion of SAG1∆GPI-dsRed. (C and D) Overnight treatment with 5 μg/ml Brefeldin A (BFA) or 24 hr treatment with 1 μM Shield-1 respectively show an identical block in transport for SAG1∆GPI-dsRed and the micronemal protein MIC3 (arrow heads). Immunoflourescence images are representative of at least three independent experiments and depicted abnormalities have been observed in 100% of 200 random examined vacuoles compaired to controls.
Figure 5.
Impact of DD-Hub expression on mitochondrial and apicoplast segregation.
Immunofluorescence analysis of DD-Hub expressing parasites stable transfected with either HSP60-RFP (A) or FNR-RFP (B). Parasites were cultured for 24 hr in absence or presence of 0.1 and 1 μM Shield-1. In presence of high Shield-1 concentrations mutants show mitochondrial and apicoplast segregation defects. Scale bars represent 10 μm. Immunoflourescence images are representative of at least three independent experiments and depicted abnormalities have been observed in 100% of 200 random examined vacuoles compaired to controls.
Figure 6.
Ultrastructural analysis of stably dominant negative DD-Hub expressing parasites.
Parasites were cultured in absence (A) or presence (B-E) of Shield-1 for 24 hrs. (A) Section through a parasitophorous vacuole containing two daughters each showing normal morphology with nucleus (N), Golgi body (G) and apical organelles. C, conoid; R, rhoptries; M, micronemes; DG, dense granules. Note the small residual body (RB) is free of organelles. Bar is 1 µm. (B) Section of a Shield-1 treated sample showing two daughters enclosed by the double layer pellicle with limited anterior organelles, such as micronemes (M) and rhoptries (R). Note the large residual body (RB), limited by the plasmalemma, containing remnants of parasite organelles consisting of mitochondrion (Mi) and dense granules (DG). N, nucleus; G, Golgi body. Bar is 1 µm. (C) Irregularly shaped parasite showing abnormally located daughter inner membrane complexes (IMC), multiple nuclei (N), abnormal pre-rhoptries (R) and convoluted mitochondrion (Mi). Bar is 1 µm. (D) Apical end of a daughter exhibiting the nucleus (N), dilated Golgi body (G), rare micronemes (M) and abnormal rhopties (R) which lack ducts. Bar is 100 nm. (E) Detail of the perinuclear region showing an expanded Golgi body with a few vesicles with electron dense contents around the periphery (arrowheads). Mi, mitochondrion. Bar is 100 nm.
Figure 7.
Model of CHC1 function in post-Golgi trafficking and organellar biogenesis in T. gondii.
Generation of various transport vesicles (V) at the trans-Golgi is reliant on clathrin. In concert with DrpB and SORTLR clathrin is involved in vesicle formation from which secretory organelles such as micronemes (Mic) and rhoptries (Rop) derive. Whereas post-endosome-like compartments (ELC) sorting of secretory vesicles destined for the rhoptries occurs only in a Rab5A/C dependent fashion, targeting to the micronemes occurs cargo specific either dependent or independent of Rab5A/C. In addition, clathrin functions independently of DrpB/SORTLER in constitutive secretion and generation of transport vesicles at the Golgi involved in pellicle biogenesis. Whereby Rab11B mediates inner membrane complex (IMC) formation, Rab11A mediates plasma membrane (PM) formation and is important for IMC maturation. Whereas apicoplast (Api) segregation is regulated by DrpA, fission of mitochondria (Mito) might be regulated by DrpC. A direct function of clathrin in apicoplast and mitochondrial replication remains to be confirmed. CCV, clathrin-coated vesicle; C, conoid; DG, dense granule.