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First published online August 29, 2005
doi: 10.1242/10.1242/jcs.02497

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Exploiting nuclear duality of ciliates to analyse topological requirements for DNA replication and transcription

¹ Institute of Cell Biology, University of Witten/Herdecke, Stockumer Str. 10, 58453 Witten, Germany
² Department Biology II, Anthropology and Human Genetics, Ludwig Maximilians University Munich, Großhaderner Str. 2, 82152 Martinsried, Germany
³ Department Biology II, Cell and Developmental Biology, Ludwig Maximilians University Munich, Großhaderner Str. 2, 82152 Martinsried, Germany

View larger version (74K): [in a new window]   Fig. 1. Micronucleus and macronucleus morphology. View of four micronuclei and one macronucleus. To display the spherical organization of nuclei, a series of equidistant images (optical sections) were collected using laster-scanning-microscopy. (a) Average intensity planar projections (green) from a series of 42 optical sections were displayed onto planes between the XY-, XZ-, YZ-coordinate axes. 3D reconstruction (red) of the nuclear surface was rendered using To-Pro-3 counterstaining. The surface was partly cut to look inside, above the level of the yellow cutting line marked in planes XZ and YZ. (b) One optical section was selected from the middle of a series of equidistant images to display structural details of a macronucleus (M) and four micronuclei (m) stained with To-Pro-3. The nucleoplasmic connection between the ovoid distal parts of the macronucleus is not visible because it makes a turn, out of the single optical section. (Inset b1) Image taken from another mid optical section; magnification 2.5x. Red arrowhead indicates a central delimitated zone of stained chromatin within an unstained spherical putative nucleolus. (c) Stylonychia lemnae DNA on an agarose gel. Left, DNA size marker. Middle, macronuclear gene-sized DNA molecules of 0.4-20 Kbp. Arrow marks ribosomal DNA. Right, micronuclear DNA.

View larger version (35K): [in a new window]   Fig. 2. Spatiotemporal replication patterns in micronuclei after pulse-chase-pulse labelling with IdU and CldU (pulse, 30 minutes; chase, 2 minutes; pulse, 30 minutes). Mid optical sections of three successive micronucleus replication stages. To-Pro-3 was used for counterstaining. Columns 1-4 (from left) show fluorochrome channels that had been scanned sequentially, generating 8-bit greyscale images. Columns 5 and 6, false colours were assigned to each channel (Alexa Fluor 488TM, green; Cy3, red; To-Pro-3, blue) before being overlaid. Replication patterns resemble type 1 [I, CldU (red); II, IdU (green)] and type 2 and/or type 3 [II, CldU (red); III, IdU (green)].

View larger version (47K): [in a new window]   Fig. 3. The replication band of the macronucleus (rb, replication band; fz, forward zone; rz, rear zone). (a-c) Mid optical section of a macronucleus. a and b, Fluorochrome channels were scanned sequentially generating 8-bit greyscale images; a, To-Pro-3; b, BrdU pulse-labelling of replication foci (pulse 45 minutes) c, Overlay, with false colours assigned to each channel (To-Pro-3, blue; BrdU, red). (d) Average intensity projections (XY plane, YZ plane) of 63 serial optical sections. 3D reconstruction of the nuclear surface (blue) and the disc-shaped replication band (red). The surface was partly cut to look inside. (e) Sequential labelling of replication sites with IdU and CldU (pulse 30 minutes each, chase 5 minutes). Fluorochrome channels were scanned sequentially generating 8-bit greyscale images. Overlay with false colours assigned to each channel (IdU, green; CldU, red). Nuclear surface was rendered using To-Pro-3 counterstaining and marked by a white line in the overlay image. Sites of simultaneous IdU and CldU incorporation appear yellow. Replication occurs in hundreds of replication foci with a size of approximately 0.5 µm each. Initially, replication foci are more concentrated in the periphery (red). Later initiation of replication foci extends to the interior (yellow and green).

View larger version (31K): [in a new window]   Fig. 4. Spatiotemporal replication patterns over the entire macronucleus S phase (approximately 6-8 hours). Mid optical sections of four macronuclei after pulse-chase-pulse labelling with IdU and CldU (pulse, 30 minutes; chase, 2 hours; pulse, 30 minutes) (I-IV) and of one macronucleus after pulse labelling with CldU followed by a 24-hour chase (V). Focal organisation of IdU-labelled post-replicative DNA is preserved even after a total chase of 2.5 hours (chase for 2 hours + second pulse for 30 minutes) or of 4.5 hours (chase for 4 hours + second pulse for 30 minutes; data not shown). These foci still appear as a dispersed band suggesting that mobility of post-replicative foci is strictly limited (II-IV). Focal organisation remains preserved even after amitosis (V), but foci are scattered throughout the entire volume of the macronucleus.

View larger version (46K): [in a new window]   Fig. 5. Proteins associated to replication in Stylonychia lemnae macronuclei. Immunolocalisation of the (a-d) catalytic subunit of telomerase and of (e-f) PCNA. Accumulation of telomerase and PCNA in the replication band. Mid optical sections. (a-d) Immunolocalisation of the catalytic subunit of telomerase by FISH with probes against sequences adjacent to the template region of Stylonychia lemnae telomerase RNA. (a-c) Fluorochrome channels were scanned sequentially generating 8-bit greyscale images. (a) Telomerase RNA (red), (b) To-Pro-3 (blue). (c) Overlay, with false colours assigned to each channel. (d) Overlay; detailed view of a replication band from a-c. (magnification x4). (e-l) Immunolocalisation of PCNA in (e-i) macronuclei and (j-l) one micronucleus. Fluorochrome channels were scanned sequentially generating 8-bit greyscale images. (g,h,i,l) Overlay, with false colours assigned to each channel, PCNA, red (e,j); To-Pro-3, blue (f,k). Detailed views of replication bands (h,i).

View larger version (46K): [in a new window]   Fig. 6. (a-d) Immunolocalisation of nascent RNA after pulse labelling with FU and (e) total RNA staining with SYTO RNASelect in unfixed macronuclei. Transcription occurs in distinct foci colocalised with nucleoli and dispersed chromatin. Mid optical section of one macronucleus (a-c). Fluorochrome channels were scanned sequentially generating 8-bit greyscale images. (a) FU incorporated into newly synthesised RNA. (b) To-Pro-3. (c) Overlay, with false colours assigned to each channel (FU, red; To-Pro-3, blue). (d) Average intensity projection of 62 serial optical sections to a plane between XZ-coordinate axes and 3D reconstruction of the nuclear surface (blue) with one optical section. FU incorporated into newly synthesised RNA (green). Putative nucleoli are marked by a red line, domains of dispersed chromatin are marked by a yellow line. The surface was partly cut to look inside. (e) The rear zone of the replication band remains unstained when SYTO RNASelect is used as a RNA selective dye (green).