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First published online 25 November 2008
doi: 10.1242/jcs.039065

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MOR1, the Arabidopsis thaliana homologue of Xenopus MAP215, promotes rapid growth and shrinkage, and suppresses the pausing of microtubules in vivo

Department of Botany, University of British Columbia, Vancouver, BC, Canada V6T 1Z4

View larger version (84K): [in this window] [in a new window]   Fig. 1. Comparison of microtubule dynamics in the mor1-1 mutant and the wild type. (A) Wild type at 21°C (corresponds to Movie 1 in supplementary material); (B) wild type at 31°C (corresponds with Movie 2 in the supplementary material); (C) mor1-1 at 21°C (corresponds to Movie 3 in supplementary material); (D) mor1-1 at 31°C (corresponds with Movie 4 in supplementary material). Representative time-lapse series of microtubules labelled by expression of the 35S driven GFP-TUB reporter are shown, as collected from epidermal cells on the abaxial surface of first leaves from 11- to 12-day-old seedlings. Microtubule plus ends are indicated with yellow dots and minus ends are labelled with blue triangles. Time between frames was 8 seconds (see supplementary material Movies 1-4) but 16 second intervals are shown here. Images were collected with a spinning disc confocal microscope equipped with a temperature-controlled stage to keep specimens at 21°C or 31°C. Bars, 5 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 2. Kymograph analysis of microtubule dynamics in wild type and mor1-1. Kymographs were created with ImageJ Multiple Kymograph using representative microtubules labelled with GFP-TUB in wild type and mor1-1 at 21°C and 31°C, as described in Fig. 1. Two different kymographs are shown for mor1-1 at the restrictive temperature (31°C).

View larger version (42K): [in this window] [in a new window]   Fig. 3. Quantification of growth and shrinkage rates in wild type and mor1-1 at 21°C and 31°C. Growth and shrinkage rates calculated from time-lapse images of microtubules expressing GFP-TUB as described in Fig. 1 are presented as frequency distribution histograms. Mean values are shown in the insets. (A) Growth rates at 21°C. (B) Growth rates at 31°C. (C) Shrinkage rates at 21°C. (D) Shrinkage rates at 31°C. Shrinkage rates greater than –22 µm/minute are shown as one data group (). Data were collected from 115 microtubules from six cells from four different plants for wild type at 21°C, 28 microtubules from seven cells from four different plants for wild type at 31°C, 98 microtubules from five cells from three different plants for mor1-1 at 21°C, and 26 microtubules from 12 cells from nine different plants for mor1-1 at 31°C.

View larger version (69K): [in this window] [in a new window]   Fig. 4. EB1-GFP comet tracking of microtubule plus end dynamics in wild type and mor1-1. (A) Representative time-lapse images are displayed so that the relative velocities of EB1 comets are easily compared at 21°C and 31°C for wild type and mor1-1. Images were collected from the abaxial surface of first leaves of 11- to 12-day-old plants. The images of the wild type were obtained from single cells at both temperatures using identical confocal settings and digital processing. Contrast adjustments for the mor1-1 images shown here varied, so the fluorescent intensities cannot be compared. (B) Quantification of ProEB1::EB1-GFP comet velocities in wild type and mor1-1 at 21°C and 31°C. Data were generated from time-lapse images taken every 5 seconds, as shown in A. The velocities were measured from 296 comets from 18 cells from five different plants for wild type at 21°C, 126 comets from 12 cells from three different plants for wild type at 31°C, 157 comets from 10 cells from three different plants for mor1-1 at 21°C and 142 comets from 12 cells from five different plants for mor1-1 at 31°C. Bars indicate the standard deviation.

View larger version (114K): [in this window] [in a new window]   Fig. 5. EB1 comet intensity fluctuates in mor1-1 mutants at 21°C and is greatly reduced at 31°C. (A,B) Transgenic ProEB1::EB1-GFP line (in wild-type background) at 21°C (A) and 31°C (B). (C,D) mor1-1 expressing ProEB1::EB1-GFP at 21°C (C) and 31°C (D). Time-lapse images of ProEB1::EB1-GFP were collected as described in Fig. 4 but at 8 second intervals. To compare fluorescence between treatments, images were collected from the same cell with identical confocal settings before and after the temperature increase. The same contrast adjustments were applied to the images obtained from wild type and mor1-1. Arrowheads follow one comet between time frames in each series. Comets in mor1-1 at 31°C (D) were less abundant and rarely persisted for several time frames (upper arrowheads) or were detected at only one time point (lower arrowhead) within a series. A-D correspond to Movies 5-8 in supplementary material. Bars, 5 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 6. EB1-GFP associates with microtubule side walls in mor1-1 at 31°C. Changes in GFP-EB1 distribution patterns are shown for the same cells before and after the temperature increase using identical confocal settings and contrast adjustments. (A,B) Transgenic ProEB1::EB1-GFP in wild type at 21°C (A) and 31°C (B). Side wall labelling was not detected in wild type at 31°C. (C,D) mor1-1 expressing ProEB1::EB1-GFP at 21°C (C) and 31°C (D). At 31°C, side wall labelling (arrowheads) became obvious in mor1-1. Bars, 5 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 7. Quantification of time spent in growth, shrinkage and pause, and transition frequencies between phases at 21°C and 31°C. (A,B) Relative duration of growth, shrinkage and pause events at 21°C (A) and 31°C (B). The sum of time every microtubule end spent in each phase was divided by the sum of time for which all microtubule plus ends were measured. (C,D) Transition frequencies between growth (G), shrinkage (S) and pause (P) at 21°C (C) and 31°C (D) were calculated by dividing the total number of transitions from one state to another by the total time spent in the initial state. Data were collected from 115 microtubules from six cells from four different plants for wild type at 21°C, 28 microtubules from seven cells from four different plants for wild type at 31°C, 98 microtubules from 5 cells from three different plants for mor1-1 at 21°C, and 26 microtubules from 12 cells from nine different plants for mor1-1 at 31°C. Epidermal cells from the abaxial surface of the first leaves from 11- to 12-day-old seedlings were used. Data were obtained from time-lapse images of microtubules expressing GFP-TUB in both wild type and mor1-1 (as described in Fig. 1).

View larger version (27K): [in this window] [in a new window]   Fig. 8. MOR1 promotes the constant growth of microtubules. Distribution frequencies of the durations for one constant growth event were calculated from time-lapse images of GFP-TUB-labelled microtubules in wild type (WT) and mor1-1 at 21°C and 31°C. Growth events were categorized as lasting at least 8 seconds (8 sec), at least 16 seconds (16 sec) or for more than 24 seconds. Constant growth was inhibited in mor1-1 at 31°C. Data were collected from 115 microtubules from six cells from four different plants for wild type at 21°C, 28 microtubules from seven cells from four different plants for wild type at 31°C, 98 microtubules from five cells from three different plants for mor1-1 at 21°C, and 26 microtubules from 12 cells from nine different plants for mor1-1 31°C.

View larger version (36K): [in this window] [in a new window]   Fig. 9. A model for the interaction of MOR1 with microtubules and free tubulin. The predicted domain structure of MOR1 includes five N-terminal TOG domains and conserved C-terminal domains (red bars). The predicted 60 nm length of MOR1 (Cassimeris et al., 2001) and the 5.4-6.0 nm of each TOG domain (Al-Bassam et al., 2007; Slep and Vale, 2007) suggest that each TOG domain may be spaced one tubulin dimer apart along the microtubule. For simplicity, only one tubulin protofilament is shown. In vitro studies suggest that the C-terminal region (indicated with bracket) confers microtubule polymer binding (Twell et al., 2002). The location of the mor1-1 point mutation, which substitutes a single amino acid in the first TOG domain, is indicated (yellow triangle). According to the data presented in our current study, this mutation site is critical for maintaining microtubule dynamics. Moreover, suppressor mutagenesis data from yeast suggests that this region interacts directly with β-tubulin (Wang and Huffaker, 1997). The model shown here suggests that MOR1 facilitates the docking of a free tubulin dimer via its first TOG domain (TOG1A), and then stabilizes the polymerization event by moving processively. The high affinity of TOG1A for free tubulin may stimulate a partial dissociation that is propagated along all the TOG domains in an inch worm-like manner to enable movement toward the microtubule plus end.

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