TgDrpC, an atypical dynamin-related protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division

Abstract

Dynamin-related proteins (Drps) are involved in diverse processes such as organelle division and vesicle trafficking. The intracellular parasite Toxoplasma gondii possesses three distinct Drps. TgDrpC, whose function remains unresolved, is unusual in that it lacks a conserved GTPase Effector Domain, which is typically required for function. Here, we show that TgDrpC localizes to cytoplasmic puncta; however, in dividing parasites, TgDrpC redistributes to the growing edge of the daughter cells. By conditional knockdown, we determined that loss of TgDrpC stalls division and leads to rapid deterioration of multiple organelles and the IMC. We also show that TgDrpC interacts with proteins that exhibit homology to those involved in vesicle transport, including members of the adaptor complex 2. Two of these proteins, a homolog of the adaptor protein 2 (AP-2) complex subunit alpha-1 and a homolog of the ezrin-radixin-moesin (ERM) family proteins, localize to puncta and associate with the daughter cells. Consistent with the association with vesicle transport proteins, re-distribution of TgDrpC to the IMC during division is dependent on post-Golgi trafficking. Together, these results support that TgDrpC contributes to vesicle trafficking and is critical for stability of parasite organelles and division.

Figure 1.. TgDrpC localization.

A) Diagram of scheme used to introduce a triple hemagglutinin (3xHA) epitope tag into the endogenous TgDrpC gene. DHFR represents modified allele that confers resistance to pyrimethamine. B) Protein extracts from the parental or TgDrpC-HA expressing strain were analyzed by Western blot using anti-HA antibody. SAG1 was used as loading control. C-E) Intracellular TgDrpC-HA expressing parasites were stained with antibodies against HA. To detect dividing parasites, cultures were co-stained with either DAPI (blue in D) or IMC3 antibodies (green in E). The outlines in C delineates two individual parasites within the vacuole and arrows point at examples of bright puncta. Arrow in E point at the growing edge of daughter parasites. Scale bars = 3 μm.

Figure 2.. Redistribution of TgDrpC during parasite division.

Intracellular TgDrpC-HA expressing parasites were stained with anti-HA antibodies to detect TgDrpC (red) and with anti-MORN1 antibodies (green) to monitor parasites at different stages of division. Scale bar = 3 μm.

Figure 3.. TgDrpC association with parasite organelles.

A) Schematic diagram comparing the domain architecture of the three dynamin related proteins from Toxoplasma (TgDrpA, TgDrpB, and TgDrpC) with mammalian dynamin-1 and dynamin-1-like proteins. Domains indicated are GTPase, Central domain, Pleckstrin homology domain (PH), GTPase Effector Domain (GED), and WW-domain ligand protein domain (WWbp). B) Intracellular TgDrpC-HA expressing parasites were stained with anti-F1B ATPase to visualize the mitochondrion (red) and anti-HA antibodies to detect TgDrpC (green). Arrowheads point at the brightest dots that co-localize to the mitochondrion (top row) and arrows point at TgDrpC puncta enriched at constricted points along mitochondrion (bottom row). Scale bars = 2 μm. C) Parasites were stained for TgDrpC with anti-HA antibodies (red) and for the Golgi with anti-Sortilin (green, top row), the IMC with anti-IMC3 (green, middle row) or the apicoplast with anti-Atrx1 (green, bottom row). Circled areas show bright TgDrpC puncta that associate to the organelles analyzed. Scale bars = 6 μm.

Figure 4.. Establishment and growth of parasite strain with conditional expression of TgDrpC.

A) Western blots of extracts from parasites in which the endogenous TgDrpC was tagged with either HA or HA-DD grown for 24 or 48 hours in presence (+) or absence (-) of Shld1. Blots were probed with antibodies against HA to detect TgDrpC or against the surface antigen SAG1 as a loading control. B) Parasites expressing TgDrpC-HA-DD were allowed to grow for 5 and 8 days in the presence and absence of Shld1. Cultures were stained with Crystal Violet, which allows visualization of plaques (white areas) arising from parasites lysing their host cells (dark areas). C) Doubling assays quantifying the number of TgDrpC-HA-DD parasites per vacuole following 24 and 48 hours of growth in the presence or absence of Shld1 (n=3, ±SD). D) The total number of vacuoles of TgDrpC-HA-DD parasites at 24 and 48 hours with and without Shld1 was determined for 10 randomly selected fields of view. (n=3, ±SD) (***p<0.001). E) Phase image of TgDrpC-HA-DD parasites grown without Shld1 for 48 hours reveals aberrant vacuole structures. F) The distribution of vacuoles that appear normal, swollen, or vacuolated when parasites are grown with or without Shld1 for 48 hours (n=3, ±SD).

Figure 5.. Organellar morphology in parasites lacking TgDrpC.

A-C) Intracellular TgDrpC-HA-DD parasites were grown for 48 hours without Shld1 and stained with anti-F₁B-ATPase to detect mitochondria (A), with anti-ATrX1 to detect apicoplast (B), or with antibodies against the Sortilin-like receptor (SORTLR) to detect Golgi (D). In A and B both normal (left) and abnormal right) vacuoles within the same culture are shown. In B, DNA is visualized with DAPI and circled area shows parasites that do not have an apicoplast based on lack of DAPI staining and diffuse ATrX1 staining. In A, B, and D, the left panels show vacuoles with normal morphology and the right panels show vacuoles that appear swollen, both within the same culture. C) Enlarged images of the areas circled in B. In parasites highlighted by arrows, there is no signal for DAPI staining and a diffused pattern of ATrX1. E) Graph shows the percentage of vacuoles within each category (i.e. normal, swollen, and vacuolated vacuoles) that have altered mitochondria (m), apicoplast (a), or Golgi (g), (n=3, ±SD). Scale bars = 6 μm.

Figure 6.. TgDrpC and organellar morphology.

A-C) Intracellular TgDrpC-HA-DD parasites grown without Shld1 for 42 hours were stained with HA to detect TgDrpC and with anti-F1B-ATPase to detect mitochondria (A), with antibodies against the Sortilin-like receptor (SORTLR) to detect Golgi (B) or with anti-ATrX1 to detect apicoplast (C). In A, circles highlight areas within a single vacuole in which parasites with and without TgDrpC are detected, in B circles highlight areas with TDrpC and normal Golgi, and in D circles highlight areas without TgDrpC. Arrows indicate normal mitochondria. Scale bar = 6 μm.

Figure 7.. Inner membrane complex formation and parasite division in absence of TgDrpC.

A) Immunofluorescence analysis of DrpC-HA-DD parasites grown for 42 hours in absence of Shld1. Anti-IMC3 antibody was used to detect the inner membrane complex. Images of parasites with either normal or altered IMC are shown. Arrows indicate IMC alterations. Scale bar = 3 μm. B) Percentage of vacuoles with altered IMC structure at 42 hours with or without Shld1. (n=3, ±SD) (*p<0.001). C) Intracellular DrpC-HA-DD expressing parasites grown for 42 hours without Shld1 were stained with antibodies against HA to detect TgDrpC (red). To detect dividing parasites, samples were co-stained with either DAPI (blue) and IMC3 antibodies (green). Circled area indicates dividing parasites. Scale bar = 6 μm. D) Percentage of vacuoles with or without DrpC signal in which parasites were dividing at 42 hours without Shld1. (n=3, ±SD) (*p<0.001).

Figure 8.. Probing interaction between TgDrpC and homolog of ERM family proteins.

A myc epitope tag was added to the endogenous gene TGGT1_262150 in the parasite strain expressing TgDrpC-HA. A) Dually tagged strain stained with anti-myc (green) and IMC3 (red) antibodies to detect TGGT1_262150 and TgDrpC. Bottom row shows dividing parasites. Boxed area was enlarged to show co-localization in the overlay on the left. Arrows indicate co-localization of TGGT1_262150 and IMC3. Scale bar = 3 μm (top row), 6 μm (bottom row). B) Dually tagged strain stained with anti-myc (green) and anti-HA (red) antibodies to detect TGGT1_262150 and TgDrpC. Bottom row shows dividing parasites. Arrows indicate co-localization of TGGT1_262150 and TgDrpC. Circled area shows localization to the TgDrpC-containing ring structure and protein recruitment to the daughter parasites (arrow point to the TgDrpC ring structure). Scale bar = 3 μm. C) Immunoblot showing reciprocal co-immunoprecipitation of TgDrpC-HA and TGGT1_262150-MYC using HA magnetic beads and IgG as a negative control.

Figure 9.. Localization of TgDrpC in Brefeldin A treated parasites.

A) Disruption of the Golgi apparatus in intracellular tachyzoites after 5μg/ml Brefeldin A (BFA) treatment for 30 minutes was confirmed by staining with antibodies against sortilin-like receptor (SORTLR). Control tachyzoites were incubated with ethanol (EtOH), which was used as vehicle. Scale bar = 3 μm B) Intracellular parasites were treated with EtOH (control) or 5μg/ml BFA for 30 min and stained IMC3 (green) to detect dividing parasites and with HA to detect TgDrpC (red). Both dividing and non-dividing parasites are shown. Scale bar = 3 μm C) Graph shows the percentage of vacuoles with TgDrpC ring structures among those that are dividing in cultures treated with BFA or ethanol as control. (n=3, ±SD) (*p < 0.0001).