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First published online 9 May 2006
doi: 10.1242/jcs.02949

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A MORN-repeat protein is a dynamic component of the Toxoplasma gondii cell division apparatus

Marc-Jan Gubbels^1,*, Shipra Vaishnava², Nico Boot^1,, Jean-François Dubremetz³ and Boris Striepen^1,2,

¹ Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, Athens, Georgia 30602, USA
² Department of Cellular Biology, University of Georgia, Paul D. Coverdell Center, Athens, Georgia 30602, USA
³ UMR CNRS 5539, Université de Montpellier 2, Montpellier, 34095, France

View larger version (78K): [in a new window]   Fig. 1. MORN1 is highly conserved among apicomplexans. The MORN1 protein consists of 14 MORN repeats. (Top) The PFam (Protein families database) consensus sequence is aligned with one of the repeats and identical residues are highlighted in black. (Bottom) T. gondii (Tgon) MORN1 was aligned with homologous proteins identified in P. falciparum (Pfal; PF10_0306; www.plasmodb.org), C. parvum (Cpar: EAK89899; www.cryptodb.org) and E. tenella (Eten; Contig3337, nt 6439-7527; www.sanger.ac.uk). Residues identical in at least three out of four species are highlighted in black, those that are similar are in grey. Nucleotide sequence data are available in the GenBankTM, EMBL and DDBJ databases under the accession numbers DQ181547-48.

View larger version (53K): [in a new window]   Fig. 2. Antibodies to MORN1 label a structure at the anterior and posterior end of the inner membrane complex (IMC). (A) Anti-MORN1 antiserum reacted specifically with purified HIS-tagged MORN1 (HM1, 42.8 kDa, second band probably represents a dimer) but not MORN2 protein (HM2, 51.5 kDa) in western blot analysis. A band of slightly smaller molecular mass consistent with native MORN1 was detected in parasite lysates (RH, 40.9 kDa); no reactivity was observed with the preimmune serum (PI). (B-D) In immunofluorescence assays this antiserum (green) produces staining at the apical and posterior end of the IMC (red) of the parasite as well as a dot mid-cell (anterior label indicated by arrowheads). (E-G) In dividing cells daughter IMCs are equally labeled. An additional structure in the IMC, most probably the micropore, is also detected (arrow).

View larger version (73K): [in a new window]   Fig. 3. MORN1 is associated with the nucleus and undergoes cell cycle dependent changes. (A-C) Combined MORN1 antibody (green) and DNA staining using DAPI (blue) reveals a structure (arrowhead) associated with the nucleus that doubles (B, note higher DNA content) and associates with the daughters during division (C). (D) DNA content of nuclei was measured by image analysis of DAPI-stained preparations and plotted against the number of nuclear MORN1 dots (n=105, P<0.0001). (E-G) Co-staining of MORN1 with anti--tubulin antibody resulted in colocalization of nuclear MORN1 (arrowhead) and tubulin (F). (H-K) In vivo labeling using a MORN1-YFP transgene (see 3D projection of this dataset as Movies 1 and 2 in the supplementary material). These parasites were transfected with (I) GRASP55-RFP (Golgi), (J) FNR-RFP (apicoplast) and (K) centrin-RFP (centrosome). (L-Q) Immunoelectron microscope analysis of MORN1 structures. (L) Gold particles mark the posterior end of a tachyzoite (arrowheads), higher magnification of the same area (M) shows the labeling at the inside of the IMC. In parasite cross sections the MORN1 antibody labels a ring structure (N). O shows a tachyzoite early in division. White arrowheads highlight the newly forming daughter IMC apical of the nucleus (N). The centrocone (black arrow) appears electron dense in the lumen and below the membrane that separates it from the nucleoplasm (see Fig. 7E for a schematic view of this structure). (P,Q) Higher magnifications show gold particles located with this dense material. The edge of the daughter IMC (white arrowheads) is equally labeled.

View larger version (71K): [in a new window]   Fig. 4. Time-lapse analysis of the dynamic MORN1 localization during T. gondii cell division. (A) MORN1-YFP (green) and H2B-mRFP (red, nucleus) transgenic parasites were imaged every 10 minutes. The entire dataset is available as Movie 3 in the supplementary material. Distances between the daughter and mother cell MORN1 structures (B) are plotted over time (C, average of both daughters given). (D-K) Selected time points (as indicated in C) are shown at higher magnification for one cell (boxed in A). Arrow in (D) highlights centrocone, double arrows the first sign of daughter buds. Centrally forming apical ring is marked by arrow before (G) and after extrusion (H). An additional weakly labeled MORN1 is apparent in I, arrow. Note ring constriction from J to K.

View larger version (33K): [in a new window]   Fig. 5. MORN1 interacts with the cytoskeleton. Parasites were extracted using different detergents and separated into pellet (P) and soluble (S) fraction. Controls consist of intact parasites (PBS, all protein in pellet) and parasites solubilized by boiling in 1% SDS (all proteins in supernatant). (A) Extracts were subjected to SDS-PAGE, blotted and probed with antibodies. Cytoplasmic YFP-YFP (Gubbels et al., 2003) served as fully soluble control. (B) MORN1 signals were quantified and expressed as percentages of the pellet plus supernatant sum. (C-F) Parasites expressing Myc-tagged myosin C were labeled with antibodies to MORN1 (red, C) and Myc epitope (green, D). A vacuole containing four dividing parasites is shown; note posterior division rings (indicated by arrows and in inset) labeled with both antibodies.

View larger version (98K): [in a new window]   Fig. 6. Overexpression of MORN1-RFP abolishes parasite cell division. (A-D) Expression of MORN1-RFP driven by the strong -tubulin promoter results in ring-shaped accumulations of RFP signal (24 hours after transfection) and morphological defects of the parasite. Note large size of cell (B) and nucleus (C) along with changes in shape when compared to untransfected controls (D). (E) Nuclear DNA content in transfected (+) and untransfected (-) parasites was quantified using image analysis 48 hours after transfection (arbitrary units of DAPI fluorescence; n=120, P<0.0001, error bar show standard deviation). (F-M) Parasites expressing IMC3-YFP (F-I, green) or YFP-tubulin (J-M, green) were transiently transfected with tubMORN1-RFP (red). Untransfected controls are shown in I and M. Some IMC3-YFP colocalized with MORN1 (arrows) but no discernable buds were formed. Multiple daughter tubulin baskets have been initiated and extended in the mutant, however, microtubules are not constricted at the posterior end as observed in the wild type (dotted lines in L and M). (N-P) RH-infected cultures were treated with 0.05 µM oryzalin and stained with anti-MORN1 (N) and anti-IMC1 (O) antibody. Note multiple overlapping MORN1 and IMC1 structures (arrowheads) and a single centrocone in the nucleus (arrow, P).

View larger version (55K): [in a new window]   Fig. 7. MORN1 in apicomplexan cell division. MORN1 is shown in green, tubulin and centrosome in red, IMC in dark blue and the nucleus in light blue. (A) The centrocone persists through interphase. (B) The centrosome duplicates and sets up the mitotic spindle, splitting the centrocone into two. (C) Mitosis occurs and apical and posterior MORN1 rings form for each daughter. (D) During budding, subpelicular microtubules push the MORN1 rings down while the spindle pushes the centrocones up. Microtubule driven movements are indicated by red arrows, constriction is shown in green (C,D). (E) Schematic representation of the centrocone: C, centrosome; CC, centrocone; MT, microtubules; NE, nuclear envelope; K, kinetochores. (F) A model for the structure and function of the posterior ring in daughter buds.