JCS030221 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Table S2 -

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• Supplemental Figure S1 -

  Fig. S1. Dynamics of GFP-EB1- and GFP-TUB6-labelled microtubules in guard cells. Cotyledon guard cells in 4-day-old seedlings expressing GFP-EB1b (A) or GFP-TUB6 (B) were analyzed by confocal scanning. GFP-EB1 labeled the plus end of growing microtubules, whereas GFP-TUB6 decorates the microtubule lattice with no apparent preference to the plus end. Kimographs on the right were drawn along the yellow lines in the left images. Time is given in seconds.

• Supplemental Figure S2 -

  Fig. S2. Microtubule distribution in seedling petioles. (A) Cortical microtubules were visualized by GFP-TUB6 in the petiole epidermal cells in 7-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 and **P< 0.01).

• Supplemental Figure S3 -

  Fig. S3. Recombinant SP2L binds with taxol-stabilized microtubules. SP2L fused to Trigger Factor (TF; Takara) was incubated with (+MT) or without (−MT) taxol-stabilized microtubules, centrifuged at 100,000g, and then separated to supernatants (S) and pellets (P). Proteins were separated on SDS-PAGE gels, and stained with Coomassie Brilliant Blue. Tubulin is indicated by an asterisk, TF-SP2L by arrowheads.

• Supplemental Figure S4 -

  Fig. S4. Parameters of microtubule dynamics in vitro. Dynamic behavior at the minus end of in vitro assembling microtubules was measured at 27°C in presence of TF, TF-SPR2 or TF-SP2L at the concentrations of 0.2 µM and 0.4 µM, by using a dark field microscopy. (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. (D) Microtubule dynamicity. Data shown are the mean ± s.d. Statistical significance (*P<0.05 and **P<0.01) is shown for TF-SPR2 or TF-SP2L vs. TF control at the same concentration.

• Supplemental Figure S5 -

  Fig. S5. GST-SPR2 bundles microtubules in vitro. Recombinant SPR2 fused GST and a poly-His tag was produced in E. coli and purified. Tubulin at the final concentration of 15 µM was incubated with (A) or without (B) 0.7 µM GST-SPR2 for 10 minutes at 37°C, and then was observed by using a dark field microscopy.

• Supplemental Figure S6 -

  Fig. S6. Full-length SPR2 is required to localize at microtubules in vivo. Full-length SPR2 (SPR2^1-864), N-terminal fragment (SPR2^1-331), middle fragment (SPR2^327-499), and C-terminal fragment (SPR2^498-864) were fused to the N-terminus of GFP and expressed stably under the control of the CaMV 35S promoter in tobacco BY-2 cells. Transgenic tobacco cells were observed with laser scanning confocal microscopy. Only full-length SPR2 efficiently labeled cortical microtubules.

• Movie 1 -

  Movie 1. SPR2-GFP associated with a shrinking microtubule end (arrowhead) in a hypocotyl epidermal cell. Time points are every four seconds for 28 seconds.

• Movie 2 -

  Movie 2. SPR2-GFP particles that merge on a microtubule (arrowheads). Time points are every four seconds for 108 seconds.

• Movie 3 -

  Movie 3. SPR2-GFP associated with a growing microtubule end (arrowhead). This movie precedes Movie 1 for the same microtubule. Time points are every four seconds for 64 seconds.

• Movie 4 -

  Movie 4. SPR2-GFP moving along a growing microtubule end in a guard cell (arrowhead). Time points are every 4 seconds for 92 seconds.

• Movie 5 -

  Movie 5. Movement of GFP-EB1b in a hypocotyl epidermal cell of spr2-2. An arrowhead shows the same GFP-EB1b comet that is shown by an orange line in Fig. 6B. Time points are every 4 seconds for 176 seconds.