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doi: 10.1242/10.1242/jcs.00055

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Meiotic telomere clustering is inhibited by colchicine but does not require cytoplasmic microtubules

¹ Department of Plant and Microbial Biology, University of California — Berkeley, Berkeley, CA 94720-3200, USA
² Department of Molecular and Cell Biology, University of California — Berkeley, Berkeley, CA 94720-3200, USA

View larger version (20K): [in a new window]   Fig. 1. Meiotic prophase stages and timing of the bouquet in Secale cereale cv. `Blanco'. Telomeres (green) were detected by FISH using a probe to the telomere repeat, and chromatin (red) was stained with DAPI. Single optical sections of meiotic nuclei are shown. (a) Premeiotic interphase, (b) leptotene and (c) zygotene. The bouquet is evidenced by the close spacing of the telomere FISH signals. (d) Pachytene. Bar, 10 µm.

View larger version (29K): [in a new window]   Fig. 2. (a) Diagram of rye plant and meiotic development and anther culture methodology. A single rye floret contains three anthers at an equivalent developmental stage. Meiosis occurs synchronously within a single anther and among the three anthers from one floret. The anthers are shown in yellow-green, and meiotic cells are depicted in blue. The meiotic cells cannot be observed through the anther wall; the diagram merely suggests their location. One anther half provides the time 0 control, whereas the halves of a second anther are put into culture. (b) Synchrony of meiosis in rye anthers. A projection of a 3D image of meiotic cells from a single anther. All cells appear to be in metaphase of the first meiotic division, as indicated by the presence of seven bivalents. Bar, 10 µm.

View larger version (82K): [in a new window]   Fig. 3. The effect of colchicine on telomere clustering. To convey the 3D architecture of the cell, the data corresponding to the entire nuclear volume (approximately 100 z-sections) was divided into quarters, and each quarter was projected into a single image (Bass et al., 1997). The resulting images are displayed sequentially. Control and 100 µM colchicine-treated anthers were cultured for 16 hours. Telomeres (green) were detected by FISH using a probe to the telomere repeat, and chromatin (red) was stained with DAPI. Bar, 10 µm.

View larger version (76K): [in a new window]   Fig. 6. The effect of APM, a plant-specific microtubule-depolymerizing agent, on telomere clustering. Representative nuclei are displayed as sequential projections of the three-dimensional nucleus, as in Fig. 3a. Control and 25 µM APM-treated anthers were cultured for 14 hours. Telomeres (green) were detected by FISH using a probe to the telomere repeat, and chromatin (red) was stained with DAPI. Bar, 10 µm.

View larger version (79K): [in a new window]   Fig. 5. The effect of colchicine on bouquet dispersal. Representative nuclei are displayed as sequential projections of the 3D nucleus as in Fig. 3. Control and 100 µM colchicine-treated anthers were cultured for 18 hours. Telomeres (green) were detected by FISH using a probe for the telomere repeat, and chromatin (red) was stained with DAPI. Bar, 10 µm.

View larger version (16K): [in a new window]   Fig. 4. Telomere distributions in colchicine-treated nuclei. Box-whisker plots of the (a) mean telomere distances and (b) nearest neighbor distances are shown. Time 0, n=26; control, n=37; 100 µM colchicine, n=20. Boxes represent the second and third quartiles (50th and 75th percentiles); the bold line through the box is the median, and whiskers extend to the range.

View larger version (70K): [in a new window]   Fig. 7. The effect of colchicine and APM on cytoplasmic microtubule organization. MTs were detected using an antibody against sea urchin -tubulin. The cells shown are of roughly equivalent meiotic stages. (a) Control, (b) 25 µM APM, (c) 5 mM colchicine and (d) 100 µM colchicine. Bar, 10 µm.