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doi: 10.1242/10.1242/jcs.00054

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Reorganization and polarization of the meiotic bouquet-stage cell can be uncoupled from telomere clustering

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 (31K): [in a new window]   Fig. 1. A diagram of a bouquet-stage meiotic rye cell indicating the cellular components used for assessing polarity. Terms are defined in Materials and Methods. Details of individual measurements are given in Figs 4 and 5 and Fig. 8C.

View larger version (18K): [in a new window]   Fig. 4. (A) The position of the nucleus in early meiotic prophase. The cell center-nucleus distance is the distance between the center of the cell and the center of the nucleus. A diagram of the distance () used to quantify nuclear displacement is shown. A box-whisker plot of the cell center-nucleus distance in pre-bouquet (n=12), bouquet (n=14) and post-bouquet (n=18) cells is shown. Distances were standardized to the nuclear radius. The cell center-nucleus distance units are based on the nuclear radius, such that the radius equals 1 unit. (B) Bouquet-stage polarization. The telomere-cell cortex angle is the angle created between the center of the telomeric heterochromatin, center of the nucleus and the center of the cell cortex. The cell cortex is the subset of the short side cell cortex bounded by tangents perpendicular to the nucleus. The telomere-NP angle is the angle created between the center of the telomeric heterochromatin, the center of the nucleus and the center of the NP-containing region. Diagrams of the telomere-cell cortex and telomere-NP angles used to determine bouquet-stage polarization are shown. Angles are indicated by . A box-whisker plot of telomere-cell cortex (n=22) and telomere-NP (n=30) angles in bouquet-stage cells is shown. Bouquet-stage angles were compared with the distribution of random angles between two points in a sphere through the center of the sphere (n=50). In both (A) and (B), the box represents the second and third quartiles, the horizontal line through the box is the median and whiskers extend to the range (see also Materials and Methods).

View larger version (42K): [in a new window]   Fig. 2. Rye heterochromatin as an indicator of telomere distribution. Single z-sections of early meiotic prophase nuclei are shown. Telomeres (green) were detected by FISH. Chromatin (red) was stained with DAPI. Heterochromatin is visible as intensely staining DAPI regions (indicated with *). (A) Dispersed telomeres prior to the bouquet. Five of the eight FISH signals in this section are associated with heterochromatin. (B) Clustered telomeres during the bouquet. A single heterochromatic region is evident. Bar, 10 µm.

View larger version (94K): [in a new window]   Fig. 3. Cytoplasmic MT distribution during early meiotic prophase. To convey the 3D architecture of the cell, the data corresponding to the entire cellular volume (approximately 100 z-sections) were divided into quarters, and each quarter was projected into a single image (Bass et al., 1997). The resulting images are displayed sequentially. MTs (green) were detected with an antibody against -tubulin. Chromatin (red) was stained with DAPI, and telomere positions were inferred from the telomeric heterochromatin (a subset indicated with *) in (A) and (B). (A) Pre-bouquet, (B) bouquet and (C) post-bouquet. Bars, 10 µm.

View larger version (19K): [in a new window]   Fig. 5. Telomere position relative to the cell in Rabl or bouquet organizations. The telomere-cell angle is the angle created between the center of the telomeric heterochromatin, the center of the nucleus and the cell center. A diagram of the telomere-cell angle used to calculate telomere position within the cell is shown. The angle is indicated by . A box-whisker plot (as in Fig. 4) of the telomere-cell angles for pre-bouquet (n=11) and bouquet (n=14) stage cells, compared with the distribution of random angles in a sphere (n=50), is shown.

View larger version (67K): [in a new window]   Fig. 6. Nuclear pore reorganization during early meiotic prophase. Sequential z-axis projections of meiotic cells, as described in Fig. 3. NPs (green) were immunolocalized using an antibody against nuclear pore proteins (mAb414). Chromatin (red) was stained with DAPI, and telomere positions were inferred by the telomeric heterochromatin (a subset indicated with *) in (A) and (B). (A) Pre-bouquet, (B) bouquet and (C) post-bouquet. Bars, 10 µm.

View larger version (55K): [in a new window]   Fig. 7. Nuclear displacement and telomere positioning in colchicine-treated bouquet-stage cells. (A) Sequential z-axis projections of meiotic cells, as described in Fig. 3. Control and 250 µM colchicine-treated anthers were cultured for 14 hours. MTs (green) were detected with an antibody against -tubulin. Chromatin (red) was stained with DAPI, and telomere positions were inferred from the telomeric heterochromatin (a subset indicated with *). Bar, 10 µm. (B) Nuclear positioning in colchicine-treated cells. A box-whisker plot (as in Fig. 4) of telomere-cell center distance, as calculated in Fig. 4, for control (n=14) and 250 µM colchicine-treated (n=10) cells is shown. The cell center-nucleus distance units are standardized to the nuclear radius, such that the radius equals 1 unit. (C) Telomere polarization relative to the cell cortex in colchicine-treated cells. A box-whisker plot of telomere-cell cortex angle, as described in Fig. 6, for control (n=22) and 100 µM and 250 µM colchicine-treated (grouped together; n=22) cells, compared with the distribution of random angles in a sphere (n=50).

View larger version (53K): [in a new window]   Fig. 8. Nuclear pore redistribution and polarization in colchicine-treated bouquet-stage cells. (A) Sequential z-axis projections of meiotic nuclei, as described in Fig. 3. Control and 100 µM colchicine-treated anthers were cultured for 12 hours. NPs (green) were immunolocalized using an antibody against nuclear pore complex proteins (mAb414). Chromatin (red) was stained with DAPI. Positions of telomeres were inferred from heterochromatin (a subset indicated with *). Bar, 10 µm. (B) NP clustering in colchicine-treated cells. A box-whisker plot of the percentage of the nuclear surface occupied by NPs in control (n=22) and 100 µM colchicine-treated (n=22) nuclei. (C) Polarization of NPs in colchicine-treated cells. A box-whisker plot (as in Fig. 4) of telomere-NP angles (as in Fig. 6) in control (n=30) and 100 µM colchicine-treated (n=34) nuclei, compared with the distribution of random angles in a sphere (n=50). The NP-cell cortex angle is the angle created between the center of the NP-containing region, the center of the nucleus and the center of the cell cortex (Fig. 4B). A diagram of NP-cell cortex angle is shown; the angle is indicated by . A box-whisker plot of NP-cell cortex angle in control (n=8) and 100 µM colchicine-treated (n=12) cells is shown. Also shown is the distribution of random angles in a sphere (n=50).