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First published online 24 July 2007
doi: 10.1242/jcs.009506

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A conserved role for kinesin-5 in plant mitosis

¹ Biology Department, University of Massachusetts, Amherst, MA 01003 USA
² Max Planck Institute for Molecular Plant Physiology, Science Park Golm, 14476 Potsdam, Germany
³ Cold Spring Harbor Laboratory, 1 Bungtown Rd, Cold Spring Harbor, NY 11724, USA
⁴ Carnegie Institution, Department of Plant Biology, Stanford, CA 94305, USA

View larger version (61K): [in this window] [in a new window]   Fig. 1. Identification of RSW7 by recombinational mapping, candidate gene sequencing and complementation. (A) Representation of a part of arabidopsis chromosome 2 showing the positions of SSLP markers used in mapping. White boxes represent AGI-BAC clones. Numbers are recombination events for each marker in a total of 1920 examined chromosomes. (B) Enlargement of the region between SSLP-markers ciw34 and ciw41, showing the names of BAC clones, as well as the position and the number of recombinants. The candidate gene, At2g28620, is depicted as a white box. (C) Exon-intron structure of the kinesin-like gene At2g28620 and the single nucleotide polymorphism in rsw7 (G in wild-type to A in rsw7) found in the fourth exon. Base and amino acid numbers indicate position in the gene. (D) Identification of rsw7 plants by PCR. The polymorphism destroys a BslI restriction site (CCN7GG) in rsw7, therefore representing a CAPS marker (primers shown as underlined sequences). Base numbers on right indicate position in amplified sequence. The gel shows a DNA standard (left), BslI digests of rsw7 (middle) and wild-type (right) PCR products. The predicted sizes of the digested fragments are shown. (E) Complementation of the rsw7 root phenotype with the wild-type At2g28620 gene. Shown is the segregation in the T2 progeny of an rsw7 mutant transformed with an 11.8 kb genomic fragment containing the AtKRP125c gene. Arrows indicate T2 plants with rsw7 phenotype.

View larger version (171K): [in this window] [in a new window]   Fig. 2. AtKRP125c localization and expression. (A) Complementation of the rsw7 phenotype by transformation with AtKRP125c-GFP: 1-week-old wild-type (left), rsw7 (middle) and complemented rsw7 (right) seedlings grown at 19°C. Bar, 5 mm. (B-F) Confocal micrographs showing AtKRP125c-GFP in living cells of complemented rsw7 plants. AtKRP125c-GFP decorates cortical microtubules in the root (B) and hypocotyl (C), and division figures in the root, including preprophase band (D, arrow) and prophase spindle (D, arrowhead), mitotic spindle (E), and phragmoplast (F). Bars, 5 µm. (G) RT-PCR on RNA from whole 2-week-old seedlings (35 cycles). Left-hand gel: EF1- (loading control); right-hand gel: AtKRP125c. The lane order is the same in both. Lane 1: Col genomic DNA. Lane 2: AtKRP125c-GFP genomic DNA. Lane 3: Col cDNA. Lane 4: rsw7 cDNA. Lane 5: AtKRP125c-GFP cDNA. Size standards shown in the middle.

View larger version (66K): [in this window] [in a new window]   Fig. 3. Interphase microtubules in rsw7 root tip cells. (A-D) Confocal immunofluorescence micrographs of microtubules in fixed and immunolabeled 7-day-old root epidermal cells. (A,C) Wild type, (B,D) rsw7; top panels (A,B) grown at the permissive temperature (19°C); lower panels (C,D) grown at 19°C for 6 days and then exposed to the restrictive temperature (30°C) for 12 hours. Images representative of 6-10 roots, per treatment, examined in four different experiments. Bar, 5 µm. (E) Dose-response curve for root diameter as a function of concentration of two different microtubule inhibitors, oryzalin (circles) and RH-4032 (squares). Data presented as mean ± s.e.m. of three replicate plates. The x axis (concentration) is logarithmic. In the absence of inhibitor, the root diamater of rsw7 plants is significantly greater than that of the wild type because the rsw7 phenotype is partially expressed at 19°C (Wiedemeier et al., 2002).

View larger version (127K): [in this window] [in a new window]   Fig. 4. Confocal micrographs of fixed cells with immunolabeled spindles. (A-F) Spindles double labeled for microtubules (green) and DNA (red). (A) Wild-type cells. Typical bipolar spindles in (clockwise from the upper left) anaphase, metaphase and telophase. (B) rsw7 cells grown at 19°C. Most spindles resembled those of the wild type, but a few were aberrant, such as the multi-polar spindle at the top of the panel. (C-F) rsw7 cells exposed to 30°C for 16-24 hours illustrating the range of morphologies, including radial (C,E,F) and linear (D). Radial spindles varied from compact (F) to diffuse (C) and chromosomes were seen either at the centre or periphery of the radial spindle. (G-L) Double labeling for -tubulin (green) and -tubulin (red). In the wild type (G-I), -tubulin is concentrated at the poles at prophase (G) and anaphase (I), but dispersed though the spindle at metaphase (H). In rsw7 (J-L), -tubulin is focused at the poles at prophase (J), spread throughout the diffuse spindles (K), and at the centre of compact, radial spindles (L). Bars, 5 µm.

View larger version (113K): [in this window] [in a new window]   Fig. 5. Pre-prophase bands and phragmoplasts in rsw7 cells. Confocal micrographs of preprophase bands and phragmoplasts in cells of rsw7 plants exposed to the restrictive temperature for 24 hours (A-D) and 6 hours (E,F) prior to fixation. (A,B) Preprophase bands. (C) Cell with an enlarged nucleus and incomplete cross wall. (D) Cell with curved and asymmetrically placed, but structurally normal, phragmoplast. The cell margin is marked with a dashed line. (E) Cell with an aborted cell wall (arrowhead) and unusually deployed phragmoplast fragments. (F) Cell with central DNA mass and radially deployed phragmoplast fragments, possibly reflecting a stage following a spindle as shown in Fig. 4L. Such residual microtubule structures sometimes appeared to be associated with fragments of cell plate (arrowhead). Bars, A,B 5 µm; C-F 10 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 6. Mitosis in live rsw7 cells. Single frames from an image sequence of GFP-tubulin in the rsw7 background. For the complete sequence see Movie 2 in supplementary material. The seedling had been exposed to 30°C for approximately 7 hours by time zero (min:sec, in upper left). This spindle was more or less bipolar to start with, although the poles were less focused than normal. The spindle appeared to be in a prometaphase-like state (0 to 3:00) before the poles rapidly collapsed towards each other (6:00 to 10:30). After a pause in the monopolar configuration, the microtubules migrated away from the poles towards the edges of the cell (12:00 to 13:30), leaving the chromosomes at the centre (visible as a dark mass). Bar, 5 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 7. Eg5 and AtKRP125c in fixed animal epithelial cells. (A,B) Interphase. AtKRP125c-myc localized to microtubules in LLC-Pk1 cells at interphase, whereas Eg5 did not. (C,D) Metaphase. Both AtKRP125c-myc and Eg5 were strongly localized to the spindle in mitotic cells. (E) Treatment of LLC-Pk1 cells with monastrol caused the formation of monopolar spindles and cell cycle arrest, which was not affected by the presence of AtKRP125c-myc. (F,G) Mitotic LLC-Pk1 cells transfected with a hairpin construct against Eg5 were clearly visible by the presence of monopolar spindles and diminished Eg5 labeling (F, arrowheads), whereas cells that were not knocked down had bipolar spindles that labeled strongly for Eg5 (F, arrows). In cells co-transfected with the Eg5 hairpin construct and the AtKRP125c-Myc construct (G), the great majority of spindles were monopolar, despite AtKRP125c binding to spindle microtubules. Bar, 10 µm.