Visualization of Cellulose Synthase Demonstrates Functional Association with Microtubules
Alex R. Paredez, Christopher R. Somerville, David W. Ehrhardt

Supporting Online Material

This supplement contains:
Materials and Methods
Figs. S1 to S3
Table S1
Movies S1 to S8
References

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Movie S1
Distribution and motility of YFP:CESA6 in the cell membrane. Time lapse movie of the etiolated Arabidopsis hypocotyl cell shown in Fig. 1 A-F. Note the bi-directional movement of particles and their linear trajectories. Red arrow heads track two distinct particles. Movie acquired over 10 min, images acquired at 10 sec intervals and averaged in sequential groups of 3 images.

Movie S2
Isoxaben causes loss of YFP:CESA6 particles at the cell membrane. Time lapse movie showing the effect of the cellulose synthase inhibitor isoxaben. The movie begins 1 minute after exposure to 100 nM isoxaben and ends at 13 minutes after exposure. Note that the label in the plasma membrane is depleted while the label in the Golgi appears unaffected. The Golgi become more pronounced at the end of the movie because they are no longer masked by the fluorescence of the membrane localized particles. The red arrow indicates a particle remaining in the cell membrane at the end of the treatment. It has been marked to demonstrate that the cell membrane remains in the focal plane. Images acquired at 10 sec intervals.

Movie S3
Dynamic co-localization of YFP:CESA6 and microtubules. Time lapse movie showing co-localization of YFP:CESA6 (green) CFP:TUA1 (red) in the cells portrayed in Figure 2 A-F. White arrows are in the same positions as in Figure 2 A-F; they mark prominent regions of co-localization. A treadmilling microtubule has been marked with a yellow arrow. Note that single, rapidly-treadmilling microtubules do not often show association with YFP:CESA6. The blue arrow marks a cytosolic particle labeled with YFP:CESA6, displaying rapid and erratic movement. Movie acquired over 10 min, images acquired every 10 sec and averaged in sequential groups of 3 images.

Movie S4
Concerted re-orientation of microtubules and YFP:CESA6. Time lapse movie showing a transition in MT and YFP:CESA6 organization. YFP:CESA6 is green, CFP:TUA1 is red. This time lapse movie was acquired from an adjacent region of a cell already imaged for 10 min. Note that in the beginning of the movie both the MTs and the CESA6 are in a transverse orientation and by the end of the movie the MTs and the CESA6 are longitudinally arranged. Images acquired every 10 sec, and averaged in sequential groups of 2 images.

Movie S5
Loss and creation of YFP:CESA6 and microtubule co-localization. Time lapse movie of MT catastrophe and bundle creation events as shown in Fig. 3. Carets delimit locations where microtubules depolymerize and abandon arrangements of YFP:CESA6, which continue to translocate in the absence of the microtubule, and show significant dissipation by the end of the image sequence. Yellow arrows delimit a microtubule bundle which is created and subsequently populated by YFP:CESA6. Movie occurs over 10 min; images were acquired every 10 sec and averaged in sequential groups of 2 frames.

Movie S6
Evidence for arrival and immediate motility of a YFP:CESA6 particle at the cell membrane. A new particle arrives within the yellow circle and appears to move as soon as it is detected. The red arrow tracks the new particle. Images acquired every 10 sec, and averaged in sequential groups of 3 images.

Movie S7
Intermediate oryzalin treatment causes partial depolymerization of the microtubule cytoskeleton and disorganization of CESA6 patterns. Cells have been treated for 3.6 hours in 10 µM oryzalin. YFP:CESA6 is green, CFP:TUA1 is red. Most of the oryzalin resistant microtubules have YFP:CESA6 associated with them. Movie acquired over 10 min., images acquired every 10 sec and averaged in sequential groups of 2 images.

Movie S8
Increased oryzalin treatment causes nearly complete depolymerization of microtubules and reveals re-organization of YFP:CESA6. Cells have been treated for 8 hours in 20 µM oryzalin. The distribution of YFP:CESA6 appears more regularly distributed in this cell than in cells with intact microtubules, compare to Movies S3, S4, S5 and S7. Movie acquired over 10 min, images acquired every 10 sec and averaged in sequential groups of 2 images.

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