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First published online 24 June 2008
doi: 10.1242/jcs.030221

Journal of Cell Science 121, 2372-2381 (2008)
Published by The Company of Biologists 2008
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Arabidopsis SPIRAL2 promotes uninterrupted microtubule growth by suppressing the pause state of microtubule dynamics

Maki Yao1, Yoshinori Wakamatsu1, Tomohiko J. Itoh2, Tsubasa Shoji1 and Takashi Hashimoto1,*

1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan
2 Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan


Figure 1
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  		Fig. 1. Expression analysis of SPR2 and SP2L genes. (A) Structure of Arabidopsis SPR2 and SP2L genes. SPR2 and SP2L consist of four and six exons (shown in thick bars), respectively. The protein-coding regions are in black, whereas the 5'- and 3'-untranslated regions are in white. Tandem copies of GFP were inserted just before the stop codons. PCR amplification results in products with different sizes that arise from cDNAs or genomic regions of SPR2 and SP2L. Mutations in spr2 and sp2l alleles are indicated. (B) Organ-specific expression patterns of SPR2 and SP2L. Various organs of wild-type plants (Col ecotype) were analyzed for expression of SPR2 and SP2L by RT-PCR, using a common primer set. Actin8 was used as a control. The positions of cDNA fragments and genomic fragments are indicated on the left and right sides, respectively. (C) Tissue-specific expression patterns of SPR2 and SP2L. GFP was expressed in the genomic context of SPR2 and SP2L, as indicated in (A). Four-day-old transgenic seedlings were observed by laser scanning confocal microscopy in the same low-power fields and at the equivalent gain levels. Arrows indicate hydathodes and root hairs.

 

Figure 2
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  		Fig. 2. Twisted growth of spr2 is enhanced in spr2sp2l. (A) RT-PCR analysis of SPR2 and SP2L expression in spr2 and spr2sp2l mutant alleles. See Fig. 1A,B for experimental conditions. (B) Twisted petioles in 9-day-old seedlings of spr2 and spr2sp2l. (C) Rotated angles of primary leaves in 2-week-old seedlings. The number of leaves analyzed is shown in the upper right corner. (D) Inflorescence stem of spr2-2sp2l-1 climbs up a support, forming a right-handed helix. Three complete turns are shown by red arrows. (E) Root growth of 7-day-old seedlings grown on vertically placed hard agar plates. (F) Quantitative analysis of the root slanting angles. Roots that grew toward the right side of the plates were assigned the plus sign, whereas roots that grew to the opposite direction had the minus sign in the root-slanting angles. Error bars represent standard deviations of the means. (G) Overexpression of SP2L under the control of the CaMV35S promoter rescued the twisting phenotype of spr2-1. (H) SPR2-GFP expressed in the genomic context rescued the twisting phenotype of spr2-2. Nine-day-old seedlings are shown in G and H.

 

Figure 3
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  		Fig. 3. Intercellular localization of SPR2-GFP and SP2L-GFP proteins. (A) SPR2-GFP and SP2L-GFP are expressed under the control of native regulatory elements in spr2-2 and sp2l-1, respectively. Seven-day-old seedlings were analyzed with laser scanning confocal microscopy. (B) SPR2-GFP particles are found at intersections (arrows) of crossing microtubules (marked by dotted lines) in hypocotyl epidermal cells. (C) SPR2-GFP associated with a shrinking microtubule end (arrowheads). (D) SPR2-GFP particles (arrowheads) moved along the microtubule lattice, collided and then merged. (E) SPR2-GFP is abundant at the polymerizing plus end (arrowheads). (F) SPR2-GFP labels cortical microtubules in cotyledon guard cells of 4-day-old seedlings. Three successive micrographs every 16 seconds are shown. The accumulation of SPR2-GFP at the growing microtubule tip is indicated by arrows. (G) Kymograph of SPR2-GFP movement on the yellow line shown in (F).

 

Figure 4
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  		Fig. 4. Microtubule distribution in seedling hypocotyls. Cortical microtubules were visualized by GFP-TUB6 in the upper and lower regions of epidermal cells in 4-day-old and 7-day-old seedlings, respectively. (A) Images from 4-day-old seedlings. (B) Distribution of microtubule orientations. Transverse microtubule orientation is set at 90 degree, whereas orientations in left-handed helical arrays have values less than 90 degrees. The average microtubule angles are indicated with arrows above histograms. Asterisks show statistically significant differences from control (t-test; *P<0.05, **P<0.01).

 

Figure 5
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  		Fig. 5. Parameters of microtubule dynamics in planta. Microtubule dynamic behavior of the plus end was measured in the GFP-TUB6 background. (A) Growth and shrinkage velocities. (B) Transition frequencies among growth (G), shrinkage (S) and pause (P). (C) Total time spent in growth, shrinkage, and pause. Data shown are means ± s.d. Statistical significance (t-test; **P<0.005) is obtained between spr2 and the SPR2-overexpressing line. (D) Microtubule dynamicity – a measure of the total tubulin exchange at microtubule plus ends. (E) Time spent in the pause state was classified, depending on the previous history of the microtubule ends. When the microtubules were in the pause state at the start of observation, they were categorized as `undefined'. Sample values were: control, n=23 microtubules and t=38.6 minutes; spr2-2, n=32 microtubules and t=58.8 minutes; spr2-2sp2l-1, n=22 microtubules and t=50.7 minutes; SPR2ox;spr2-2, n=22 microtubules and t=36.8 minutes.

 

Figure 6
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  		Fig. 6. Microtubule plus-end tracking with GFP-EB1. (A,B) Tracks of several GFP-EB1b comets were followed for 5 minutes in hypocotyl epidermal cells of control (A) and spr2-2 (B) plants, and are indicated by colored lines. Circles at one end of GFP-EB1 trajectories show the start points of measurements, whereas arrows indicate abrupt changes in the trajectory. Kymographs are shown for two representative trajectories, in which transient stops of GFP-EB1 are indicated by asterisks. (C) Histograms of GFP-EB1 velocity distribution in hypocotyls and petioles of control and spr2-2 plants. Stationary GFP-EB1 comets with a velocity less than 1 µm/minute are shown in black bars. The stationary comets were excluded when the mean values (± s.d.) of the velocity were calculated. Velocity distributions of spr2 cells and control cells were significantly different (t-test; **P<0.01).

 

Figure 7
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  		Fig. 7. Parameters of microtubule dynamics in vitro. Dynamic behavior at the plus end of in vitro assembling microtubules was measured at 27°C in the presence of Trigger Factor (TF), TF-SPR2, or TF-SP2L at concentrations of 0.2 µM and 0.4 µM, by using dark field microscopy. (A) Dynamic instability profiles of microtubules at the plus end in the presence of 0.2 µM SPR2 or 0.2 µM SP2L. The pause state is indicated by thick lines. (B) Growth and shrinkage velocities. (C) Transition frequencies among growth (G), shrinkage (S) and pause (P). (D) Total time spent in growth, shrinkage, and pause. (E) Microtubule dynamicity. Data shown are means ± s.d. Statistical significance (t-test; *P<0.05 and **P<0.01) is shown for TF-SPR2 or TF-SP2L vs TF control at the same concentration. (F) TF-SPR2 increases microtubule polymerization. Turbidity of the 17.5 µM tubulin solution in the presence of 0.5 µM TF (white squares) or 0.5 µM TF-SPR2 (black circles) was monitored at 350 nm and at 37°C. The values are means (± s.d.) from four independent experiments. Sample values; 0.2 µMTF, n=16 microtubules and t=136.9 minutes; 0.4 µMTF, n=12 microtubules and t=130.9 minutes; 0.2 µM TF-SPR2, n=28 microtubules and t=333.1 minutes; 0.4 µM TF-SPR2, n=17 microtubules and t=204.5 minutes; 0.2 µM TF-SP2L, n=20 microtubules and t=245.4 minutes; 0.4 µM TF-SP2L, n=20 microtubules and t=249.8 minutes.

 

Figure 8
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  		Fig. 8. Identification of microtubule binding regions in SPR2. (A) Diagram of SPR2 fragments used in the microtubule co-sedimentation assay. The N-terminal Ser/Thr-rich region is indicated by hatched boxes, while the regions conserved among SPR2, SP2L, and their orthologs in other plant species are shown by gray boxes. Predictable HEAT repeats are numbered from the N-terminus of SPR2. Recombinant proteins are fused to a thioredoxin (TRX)/poly-His tag or to GST, expressed in E. coli, and purified. When fusion proteins were recovered mostly in the pellet fraction after co-sedimentation with taxol-stabilized microtubules, the symbol ++ was given, while + indicates a roughly equal recovery in both supernatant and pellet fractions. The minus symbol shows no microtubule binding. (B) SDS-PAGE analysis of SPR2 fragments after co-sedimentation with taxol-stabilized microtubules. Microtubules and SPR2 fragments were mixed and centrifuged at 100,000 g, and then supernatants (S) and pellets (P) were separated on SDS-PAGE gels. Staining with the Coomassie Brilliant Blue dye (CBB; upper images) detected tubulin (*), BSA and SPR2 fragments (arrowheads). In several samples, TRX-fused SPR2 fragments were also detected by immunobloting (IB) with an anti-polyHis antibody (lower images). (C) Sedimentation of the SPR2 fragments used in (B), in the absence of microtubules, and analysis as in (B). All the tested proteins were recovered in the soluble fraction in the absence of microtubules.

 

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© The Company of Biologists Ltd 2008