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First published online June 4, 2008
doi: 10.1242/10.1242/jcs.026427

Journal of Cell Science 121, 2046-2053 (2008)
Published by The Company of Biologists 2008
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Differential association of Orc1 and Sir2 proteins to telomeric domains in Plasmodium falciparum

Liliana Mancio-Silva1, Ana Paola Rojas-Meza1,2, Miguel Vargas2, Artur Scherf1,* and Rosaura Hernandez-Rivas2,*

1 Unité de Biologie des Interactions Hôte-Parasite, CNRS URA 2581, Institut Pasteur, 25, Rue du Dr Roux, 75724 Paris, France
2 Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (IPN), Apartado postal 14-740, 07360 México, D. F., México


Figure 1
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  		Fig. 1. Orc1 localizes to telomeric foci and the nucleolus. (A) Double IF using anti-rabbit Orc1 (red) and anti-rat Sir2 (green). Orc1 is preferentially localized at the periphery of the parasite nucleus and colocalizes with Sir2 signals. (B) Dual-colour IF by using anti-rabbit Orc1 (red) and anti-rat Nop1 (green). A fraction of Orc1 signals colocalize with Nop1, suggesting that Orc1 also localizes in the nucleolus of the parasite. (A,B) Parasites are in ring stage and nuclear DNA was stained with DAPI (blue). Scale bars: 1 µm.

 

Figure 2
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  		Fig. 2. Orc1 and Sir2 specifically recognize elements on the telomeric and subtelomeric repeats. (A) Schematic representation of telomere and subtelomeric regions of P. falciparum chromosome ends. The probes used in the EMSA and ChIP assays are indicated. (B) Binding of nuclear proteins to telomeric and subtelomeric regions. 32P-labelled telomere (left panel), TARE3 (middle panel) and TARE6 (right panel) probes were incubated with nuclear extracts. Competition shift assays were done using either 50-fold molar excess of unlabelled homologous (third lane) or heterologous (KAHRP and Sp1, fourth and fifth lanes) competitor. A free probe was run in the first lane in all cases. The telomere probe formed three protein-DNA complexes, whereas TARE3 and TARE6 formed only a single retarded complex. Arrowheads indicate DNA-protein complexes. (C) Binding of Orc1 and Sir2 to telomeric and subtelomeric repeats. A total of 30 µg of antiserum against Sir2 and Orc1, and respective non-immune sera, were pre-incubated for 15 minutes in the binding reaction, followed by the addition of the labelled telomere, TARE3 and TARE6 probes. Telomere and TARE3 probes produced supershifted complexes with both anti-Sir2 and -Orc1 antibodies (left and middle panels), whereas the TARE6 probe only formed a supershifted complex in the presence of Sir2 (right panel). NE, nuclear extracts; C, complex; SC, supershifted complex; KAHRP, upstream region of kahrp gene; Sp1, consensus Sp1-binding-site factor; PI, pre-immune serum.

 

Figure 3
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  		Fig. 3. Orc1 and Sir2 distribution at telomeric and subtelomeric chromatin. (A,B) ChIP analysis of ring-stage parasites using antibodies against Orc1 (A) and Sir2 (B). Immunoprecipitated DNA was analyzed by dot-blots hybridized with probes specific to telomeric sequences, subtelomeric sequences (TARE1, TARE2, TARE2-3, TARE3 and TARE6) and HRP. A representative dot-blot is shown for each antibody (left panels). The right panels show a quantitative presentation of the data shown in the left panels. In all cases, ChIP values represent a percentage of the total input DNA after subtraction of the background signal value (i.e. the material immunoprecipitated by the pre-immune sera or IgG). Association of Orc1 with telomeric and subtelomeric repeats is similar to that of Sir2.

 

Figure 4
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  		Fig. 4. Sir2 and Orc1 relocalize during the developmental cycle of P. falciparum. (A) DNA synthesis during the P. falciparum cell cycle. During the 48-hour blood-stage cycle, the parasite differentiates through ring [between 0- and 18-hours post-invasion (p.i)], trophozoite (18- to 36-hours p.i.) and schizont (36- to 48-hours p.i.) stages. DNA replication in P. falciparum takes place in the trophozoite stage; it peaks at 30 hours and can be blocked by adding aphidicolin to the culture medium. Nuclear division occurs by schizogony, leading to the production of 16-32 merozoites; these are released in the bloodstream and can initiate a new cycle by invasion of a new red blood cell. (B-D) IF analysis of Sir2 (green) and Orc1 (red) during the blood-stage cycle. (B) Ring stages display a punctate pattern at the nuclear periphery. (C) In the trophozoite stages, anti-Sir2 and -Orc1 antibodies reveal an apparent increase of both protein levels, and an additional punctate and diffuse pattern inside and outside of the nucleus. (D) The schizont stage, Sir2 and Orc1 seem to relocalize at the nuclear periphery. (E-G) Double-labelling IF using parasites cultivated with aphidicolin for ~24 hours. (E) Aphidicolin-treated parasites demonstrate that the redistribution of Sir2 (green) and Orc1 (red) occurs prior to initiation of DNA replication. (F) Labelling with an anti-mouse Hsp70 (red) revealed that Sir2 (green) is displaced into the cytoplasm of the parasite before S-phase. (G) Histone H3 (dimethyl K4, 2mK4H3; red) remained associated with the nucleus. Nuclei were detected by DAPI staining (blue) in all the figures. Scale bars: 1 µm.

 

Figure 5
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  		Fig. 5. Visualization of the telomeric clusters during the blood-stage cycle. (A-D) FISH analysis of nuclei stained with DAPI (blue) and hybridized with TARE6 (green) probe to visualize the chromosome ends on rings (A), trophozoites (B), schizonts (C) and aphidicolin-treated parasites (D). Scale bars: 1 µm. (E) Quantification of TARE6 FISH signals in rings, trophozoites and aphidicolin-treated parasites. n refers to the chromosome number per nucleus; in trophozoite stage, n>1, depending on the number of nuclear divisions that occurred on each individual parasite. In the case of aphidicolin-treated parasites, all signals (weak and strong) were counted. Error bars are 95% confidence intervals (±1.96 s.e.m.). The standard deviations for rings, aphidicolin-treated parasites and trophozoites are 1.20, 3.93 and 4.71, respectively.

 

Figure 6
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  		Fig. 6. Model for dynamic telomeric heterochromatin assembly and relocation during blood-stage development. (A) Hypothetical model of the differential spreading of P. falciparum telomeric proteins. Orc1 (green ovals) might bind directly to telomere and to TARE1-3 repeats via its N-terminal DNA-binding domain. The interaction of Orc1 with other unknown TARE6-binding molecules (adaptor proteins) might lead to telomere bending. Sir2 (red circles) might be recruited via interaction with an unknown telomere-associated protein (black curve) or alternatively with Orc1. Sir2 might then deacetylate telomeric histone tails (Merrick and Duraisingh, 2007Go), promoting heterochromatin formation and spreading towards the coding region. (B) Model for the dynamics of telomere chromatin factors during the P. falciparum blood-stage cycle. During ring stage (left), parasite telomeres form clusters at the nuclear periphery and associate with Sir2 (red circles) and Orc1 (green circles) (i). This period (G phase) is followed by multiple rounds of DNA synthesis and nuclear mitosis (trophozoite stage, middle), which produce a multinucleate schizont (right). Our data show that Orc1, Sir2 and telomeric clusters disassemble prior to DNA replication (ii). Telomeric components assemble in the newly formed nuclei (iii) and will be maintained for the next cycle. We speculate that the relocation events are driven by specific post-translational modifications. Alternatively, cytoplasmic Orc1 and Sir2 might correspond to newly synthesized proteins necessary to accommodate the demands of the rapid nuclear divisions occurring during S-M phase.

 

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© The Company of Biologists Ltd 2008