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First published online 16 August 2005
doi: 10.1242/jcs.02512

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Quantitative analysis of changes in spatial distribution and plus-end geometry of microtubules involved in plant-cell cytokinesis

Department of Molecular, Cellular and Developmental Biology, University of Colorado, UCB 347, Boulder, CO 80309-0347, USA

View larger version (74K): [in a new window]   Fig. 1. Large-volume tomographic reconstructions of different stages of mitosis and cytokinesis in Arabidopsis meristem cells. All of the tomographic models are cells from root meristems, except the ring phragmoplast stage, which is from an apical-meristem cell. (A) Late-anaphase (5.9x5.9x0.8 µm), which shows two opposing sets of spindle microtubules (mt; green and pink) above and between the migrating sister chomatids (ch) and vesicles (v). (B) Phragmoplast initials (5.9x5.9x0.8 µm) in which the phragmoplast microtubule array is beginning to form between the decondensing chromatin, and cell-plate-forming vesicles and cell-plate assembly matrix (not shown) accumulate at the equatorial plane. (C) Solid-phragmoplast stage (4.3x4.3x1.2 µm), with two dense sets of opposing microtubules between the forming daughter nuclei (n). The growing cell plate (cp) is surrounded by many vesicles and is sandwiched between the two sets of microtubules. (D) Transitional-phragmoplast stage (2.8x2.8x0.8 µm), which lacks a cell-plate assembly matrix and displays many short microtubules on both sides of the maturing cell plate. (E) Ring-phragmoplast stage (5.9x2.8x0.9 µm). Two dense sets of opposing microtubules flank the growing edge of cell plate, which is surrounded by many vesicles, and a cell-plate assembly matrix (not shown). The more mature central cell-plate region has fewer interacting microtubules. Scale bars, 1 µm.

View larger version (72K): [in a new window]   Fig. 2. (A) Serial 2.2 nm tomographic slice images (every third slice is shown) through the plus ends of a horned microtubule in which the protofilaments (arrowheads) are curling, characteristic of a disassembling microtubule. (B-M) Tomographic images and 3D models of reconstructed microtubule plus ends collected from all stages of phragmoplast development. (B-D') Tomographic slices and corresponding models of blunt-ended microtubules. The blunt end architecture is somewhat variable, with one side often being slightly longer. (E-G') Tomographic slices and corresponding models of microtubules with extended ends. (H-I') Tomographic slices and corresponding models of horned microtubules. (J,J') Tomographic slice and corresponding model of a microtubule with a hybrid (horned/extended) end. (K-M') Tomographic slices and corresponding models of microtubules with flared ends. Because the thickness of a tomographic slice is 2.2 nm and the thickness of a protofilament 4 nm, we were unable to model each protofilament individually. Therefore, the architecture is `relatively mapped' and not all of the microtubules modeled exhibit 13 protofilaments. Color coding of the different microtubule plus-end types: blunt-ended, yellow; extended, green; horned, red; flared, blue; hybrid extended/horned, orange. Scale bars, 0.05 µm.

View larger version (63K): [in a new window]   Fig. 3. (A) Projection of five consecutive 2.2 nm tomographic images of cell-plate-associated microtubule plus ends in a root-meristem cell (solid-phragmoplast stage). The black and white arrowheads point to morphologically different plus ends of microtubules. The dashed red line on the right-hand half of the image indicates the interface between the ribosome-rich cytosol and the ribosome-depleted cell-plate assembly matrix region. None of the microtubule plus ends pass across the cell-plate midline. (B-D) Mapping of microtubule plus end types, cell-plate assembly matrix (outlined with small red points) and microtubules (light blue). Color coding of the different microtubule plus-end types: blunt-ended, yellow; extended, green; horned, red; flared, blue. (B) A tomographic model of a solid phragmoplast from a shoot-meristem cell, showing all of the microtubules and the cell-plate assembly matrix outline; only microtubules whose plus ends are found within the cell-plate assembly matrix are color coded. (C) The same solid-phragmoplast tomographic models as shown in B, except that only microtubules whose plus ends are located outside the cell-plate assembly matrix are color coded. (D) A transitional phragmoplast from a root meristem. This tomographic model shows all of the microtubules but notice that this stage of phragmoplast development does not have a cell-plate assembly matrix. Color coding of microtubules' plus ends (see also Fig. 2): blunt, yellow; extended, green; horned, red; flared, blue. Notice the high proportion of blunt-ended microtubules that terminate within the cell-plate assembly matrix and the high proportion of horned microtubules that terminate outside a cell-plate assembly matrix. A quantitative analysis of the microtubule end types is presented in Fig. 4. Scale bars, 1 µm.

View larger version (20K): [in a new window]   Fig. 4. Quantitative analysis of the plus-end types of cell-plate-associated microtubules. At each stage, three different cells were analysed, except for the solid-phragmoplast stage, for which four cells were analysed. (A) Percentages of microtubule plus-end types seen at each stage of cytokinesis. (B) Percentage of microtubule plus-end types that end within a cell-plate assembly matrix. (C) Percentages of microtubule plus ends that do not terminate within a cell-plate assembly matrix. n, number of microtubules counted in each stage.

View larger version (27K): [in a new window]   Fig. 5. Closest-approach analysis of horned (A) and blunt (B) microtubule plus ends to a cell-plate membrane. Solid-phragmoplast-stage cells from shoot and root meristems were examined for this analysis. The bars compose histograms of the actual densities of plus ends of microtubules at different distances from the cell-plate membrane. The line is the density distribution of plus ends of randomly distributed microtubules from the cell-plate membrane. (B) There is a significant peak in the distribution of blunt ends at 30 nm. This population is much greater than the calculated random distribution (thin line) than in A, which lacks such a peak. This suggests that about 30% of blunt-ended microtubules end at 30 nm from the cell-plate membrane.

View larger version (53K): [in a new window]   Fig. 6. Tomographic models showing a solid phragmoplast from a root-meristem cell. (A) Tomographic model with a section depth of 1.2 µm and with the microtubules (green) shown with their actual diameter of 25 nm. (B) Calculated fluorescence image based on the same tomographic model as in A. To produce this image, we increased the diameter of the microtubules to 150 nm (the limit of resolution of fluorescence-microscopy images) and made the microtubules look slightly transparent.

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