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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02458

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Plastid segregation and cell division in the apicomplexan parasite Sarcocystis neurona

¹ Department of Cellular Biology, University of Georgia, Athens, GA, 30602, USA
² Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, 30602, USA
³ Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, 40546, USA
⁴ Department of Cell and Development Biology, University of Pennsylvania, PA 19104, USA
⁵ Institut für Zoologie, Technische Universität Dresden, Dresden, 01062, Germany

View larger version (85K): [in a new window]   Fig. 1. Short intranuclear spindles are maintained throughout the cell cycle. S. neurona-infected cultures were double labeled with anti-tubulin (A,D) and anti-centrin (B,E) antibodies to detect microtubules and centrosomes, respectively. (C,F) The merged images of the red (tubulin), green (centrin) and blue (DAPI) channels. Centrosomes organize a complex network of spindle microtubules during mitosis (A-C). The `dots' of tubulin staining observed during interphase are flanked by two centrosomes (D-F, also see inset in F). (G-I) S. neurona-infected cultures were fixed in situ, embedded in Epon and ultrathin sections were cut for electron microscopy. (G) A section through a young schizont. The nucleus (N) is located at the center of the cell and plastid (P) and Golgi (G) are equally discernible. (H,I) Serial sections of the boxed region of G at higher magnification. A short basket of microtubules is evident within the nucleus (black arrowhead), which emanates from a centrosome on the cytoplasmatic site of the nuclear envelope (white arrowheads with black outline). Interestingly the end of this minute spindle seems to be in contact with condensed nuclear material (white arrows).

View larger version (34K): [in a new window]   Fig. 2. S. neurona forgoes nuclear division and cytokinesis for five cell cycles prior to the budding of 64 daughter cells. Cell cultures were infected with S. neurona merozoites and fixed and processed for immunofluorescence 24-72 hours after infection. Cells were incubated with a monoclonal antibody against -tubulin (red) and DAPI to stain DNA (blue). Developing schizonts present two mutually exclusive staining patterns: multiple spindles throughout the nucleus coinciding with DNA condensation or dots in proximity to the nuclear envelope (see Fig. 1). The number of these dots increased with the size of the schizont in a geometric progression (2-64, A-F). (G) Plot of the intensity of DNA staining of each nucleus (in arbitrary units, as shown for a parasite and host cell nucleus in inset) against the number of tubulin dots per nucleus (100 random fields were analyzed, individual measurements are shown as red dots, black lines and error bars indicate the mean and standard deviation, respectively). Progression from 32 to 64 (E,F) produces 64 daughter nuclei. Note that the dots often appeared as doublets (D).

View larger version (94K): [in a new window]   Fig. 3. The plastid in S. neurona schizonts is a single tubular structure in close association with the nucleus and its spindles. Extracellular S. neurona merozoites (A-D) were fixed and incubated with an affinity-purified rabbit antiserum raised against the T. gondii plastid protein ACP (A) and DAPI (B). A single round organelle was detected close to the nucleus colocalizing with the extranuclear plastid DNA (arrowhead). S. neurona-infected cultures were fixed 48 hours post-infection (E-H), and simultaneously incubated with antibodies against ACP (green, E), -tubulin (red, F) and DAPI (G). The plastid in developing schizonts appeared to be a single tubule wrapped around the nucleus (H). (I-L) Transfection plasmids were constructed that place the genes of two plastid targeted proteins from T. gondii (FNR and ACP, fused to RFP (red) or YFP (green), respectively) under control of a S. neurona promoter element. Plasmids were introduced into S. neurona merozoites by electroporation prior to infection. Transformed cultures were observed by fluorescence microscopy in living cells 36 hours after transfection.

View larger version (73K): [in a new window]   Fig. 4. In vivo laser bleaching experiments show that the S. neurona plastid is a continuous organelle. In vivo laser bleaching experiments were performed to test if a fluorescent marker freely diffuses along the entire length of the tubular plastid. The extent of bleaching predicted for organelles with continuous (A) and discontinuous (B) lumen is depicted schematically. FNR-RFP-expressing S. neurona (C-H) and T. gondii (I-K) parasites were imaged in vivo. Plastids were exposed to a series of short laser pulses at the position indicated by the laser symbol (see Materials and Methods for detail). Cells were imaged before (pre) and after (post) bleaching. (C,F,I,L) Single fluorescence images at the focal plane; (D,G,J,M) merged images of C,F,I and L with the respective DIC image; (E,H,K,N) rendered 3D projection of the entire z-stack. (A quicktime movie of these data is available in supplementary material.) Upon exposing the distal end of the S. neurona plastid to multiple laser pulses the entire organelle is bleached (F-H). (I-K) Experiments to control for the spatial precision of the laser were performed with FNR-RFP expressing T. gondii parasites. (L-N) A single plastid was exposed to the laser resulting in bleaching of only the targeted organelle (white arrowhead) without affecting its nearest neighbors.

View larger version (125K): [in a new window]   Fig. 5. The tubular plastid shows tight association with centrosomes. (A) Infected cultures were double labeled with antibodies to ACP (green) and centrin (red). Centrosomes (arrowheads) can be seen in association with the tubular plastid. Cultures were fixed in situ, embedded in Epon (B-D) and ultrathin sections were cut parallel to the culture surface and analyzed by electron microscopy. (B,C) Two consecutive serial sections through a developing schizont with its host cell (HC). The nucleus (N) is located in the center of the cell and as seen in a higher magnification (C), four spindle poles (large arrowheads indicate the centrioles) are present. Several intranuclear microtubules (small arrowheads) indicate that this cell is in the early stages of mitosis. Wrapped around the upper right corner of the nucleus is the plastid (P) as a continuous tubular organelle, which is surrounded by four membranes and closely apposed to the centrioles of the centrosome (D, white arrowheads indicate the four plastid membranes; black arrowheads, the two membranes of the nuclear envelope). This organelle is devoid of tubular cristae, which are clearly identifiable on the multiple sections through mitochondria (M). Ultrathin sections were also cut from material that was fixed with formaldehyde and embedded in LR-White resin to preserve antigenicity. Sections were reacted with the ACP antibody followed by an anti-immunoglobulin labeled with 10 nm gold particles. Again a tubular organelle in close proximity to the nucleus was observed (E and enlarged in F) which under this conditions showed heavy and specific gold labeling (arrowheads).

View larger version (86K): [in a new window]   Fig. 6. Ablation of spindles perturbs plastid organization and faithful segregation, but does not prevent fission. (C-F) Infected cultures were allowed to develop for 48 hours and then treated with 2.5 µg/ml oryzalin for 24 hours and stained for plastids using an anti-ACP antibody. (A,B) Untreated controls. The plastid loses its organization and attachment to the nuclear envelope (D,F) but remains associated with centrosomes detected by centrin staining (E). (G,H) After 48 hours of drug treatment plastid fission is observed in some schizonts. (I-L) Labeling of identically treated cultures with an antibody to IMC3 shows that daughter cell budding still occurs (K), however when compared to controls (I,J) treated schizonts seem unable to segregate nuclei and produce anucleate daughter cells (L). (H) Contact of nuclei and plastids is equally lost.

View larger version (77K): [in a new window]   Fig. 7. Plastid fission concurs with the last mitosis and cytokinesis. (A-D) In order to precisely time plastid fission the micronemal protein SnMic10 was used as molecular marker. Infected cultures were fixed 72 hours post-infection and labeled with antibodies to SnMIC10 (red), ACP (green) and DAPI (blue). (A) Three S. neurona stages: (1) developing schizont, (2) late schizont initiating budding and (3) final stage schizont with emerging daughter cells. (B) Expression of SnMIC10 coincides with budding and daughter cell formation and is undetectable in developing schizont (see also Hoane et al., 2003). (C,D) In the absence of SnMIC10 expression, plastids are always tubular (D, 1) and plastid fission concurs with the onset of SnMIC10 expression (C). (E-H) Cultures were also triple labeled for plastid, microtubules and DNA. Concurrent with the formation of daughter cell buds (red halos in inset in F), the plastid labeled with anti-ACP antibody starts to fragment (E). Merged images of anti--tubulin and anti-ACP staining (G) revealed that plastid spindle pole association is maintained through this last division similar to plastid segregation in T. gondii (Striepen et al., 2000). Note that the nuclear DNA is still unsegregated. The emerging fully separated merozoites each contain a nucleus and a plastid (D 3).

View larger version (34K): [in a new window]   Fig. 8. A schematic outline of the S. neurona cell cycle. (1) Merozoites infect a host cell and initiate intracellular development. For 2-3 days the nucleus cycles from mitosis (2) to interphase (3) growing in size and ploidy. Intranuclear microtubular structures are always evident either as full mitotic spindles (2) or as peculiar `mini-spindles' in interphase (3, constant kinetochore spindle association as drawn, is an attractive hypothesis but has not been experimentally confirmed yet). Concurrent with the last mitosis, budding (4) and cytokinesis (5) occur, giving rise to 64 merozoites (only 8 shown here for simplicity). Plastid (green) development mirrors nuclear (turquoise) events and both organelles are organized by centrosomes (black dots) and spindles (red). S, S phase; M, mitosis; B, budding.

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