Supplementary Materials for

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1 Supplementary Materials for A Mechanism for Reorientation of Cortical Microtubule Arrays Driven by Microtubule Severing Jelmer J. Lindeboom, Masayoshi Nakamura, Anneke Hibbel, Kostya Shundyak, Ryan Gutierrez, Tijs Ketelaar, Anne Mie C. Emons, Bela M. Mulder, Viktor Kirik, David W. Ehrhardt Corresponding author. ehrhardt@stanford.edu This PDF file includes: Figs. S1 to S8 Captions for Movies S1 to S13 Published 7 November 2013 on Science Express DOI: /science Other Supplementary Material for this manuscript includes the following: (available at Movies S1 to S13

2 Supplementary Figures Fig. S1 Crossover created Microtubule Depolymerized microtubule Severing event 53% 47% Depolymerization Severing at crossover 80% 20% New plus end shrinking New plus end growing Illustration of microtubule crossover outcomes. When a crossover is made, it can disappear either by depolymerization of one of the microtubules forming the crossover or by a microtubule severing event at the crossover. When a microtubule gets severed at a crossover, the new microtubule plus end can either start off shrinking or growing from the crossover. The percentages given for each outcome are calculated from wild type seedlings expressing YFP-TUA5 (N = 1115). The percentages refer to events at both partner microtubules. To simplify the diagram, events involving only one partner are depicted. 2

3 Fig. S2. A WT ktn1-2 GFP-KTN1 / ktn1-2 B C WT 4.0 dark-grown Root Length (cm) GFP-KTN1 ktn1-2 WT (N = 7) ktn1-2 (N = 11) GFP-KTN1 / ktn1-2 (N = 11) 5.0 light-grown ktn Time (Days) 7 Complementation of a ktn1-2 mutant by GFP-KTN1. (A) Light-grown seedling morphologies of wild-type (WT), ktn1-2 and the complemented ktn1-2 plants grown under a 16-h-light/8-h-dark cycle for 7 days. (B) Graph of mean root length over this growth period. Bars are standard errors. (C) Dark-grown seedling morphologies of WT, ktn1-2 and GFP-KTN1 / ktn1-2 plants grown in the total darkness for 6 days. Scale bars, 5 mm. 3

4 Fig. S3. A GFP-KTN1 mcherry-tua5 GFP-KTN1 mcherry-tua5 Kymograph MT shrinking B 10s 35s 60s 85 s 110 s 140 s GFP-KTN1 mcherry-tua5 GFP-KTN1 growing end mcherry-tua5 150s 160s 170s 180s 190s GFP-KTN1 mcherry-tua5 GFP-KTN1 mcherry-tua5 375s 0s Microtubule severing associated with katanin at crossovers. (A) Localization of GFP- KTN1 (yellow arrowheads) to a crossover where severing resulted in a resolved depolymerizing plus end (open blue arrowhead. Dashed blue line location of kymograph in lower panels. Yellow arrowheads in kymograph position of shrinkage of the newly formed plus end. See Movie S5. (B) Microtubule crossover where severing events resulted in growing new plus ends (blue arrowheads). The yellow arrowheads indicate the position of the GFP-KTN1 particle and microtubule severing. Three new 4

5 growing microtubules originated from a single crossover location within ~ 135 s, where in each case GFP-KTN1 localized to the crossover in the moments leading up to the initiation. The first growing end is shown at 65-85s, the second at 110s and the third at 140s. See Movie S6. Scale bars, 5 µm. 5

6 Fig. S4. A B 120 With Severing Without Severing N = N = Counts Crossover angle [degrees] Counts Crossover angles [degrees] Distribution of crossover angles during reorientation. Histograms of the angles of 891 crossovers observed over the course of reorientation in confocal time series acquired from 5 cells. (A) Angles of crossovers where evidence for severing was observed. (B) Angles of crossovers where no evidence for severing was observed. The two observed distributions are not significantly different by a χ 2 test for independence (χ 2 =7.04, 7 degrees of freedom, p<0.425). Thus, the likelihood of severing does not appear to depend strongly on the crossover angle. 6

7 Fig. S5. 0 min 10 min 20 min 30 min 40 min 50 min 60 min phot1 phot2 WT Microtubule reorientation in blue light receptor mutants. Microtubule reorientation in WT and phot1 phot2 mutant etiolated hypocotyl cells. Scale bar, 5 µm. See also Movie S10. 7

8 Fraction of newly generated MTs Fig. S6. A WT 25 phot1 phot Count 15 Count Time [min] Time [min] B Count D WT Angle [degrees] Count phot1 phot Angle [degrees] C Fraction of parallel MT initiations WT phot1 phot2 0.7 WT phot1 phot E blue light PHOT1 PHOT2 -tubulin complex nucleation KTN1 severing at crossovers 0.2 other targets? Microtubule initiations in wild type and phot1 phot2. (A) Microtubule initiations - not at crossovers - over time during reorientation in WT (N = 104) and the phot1 phot2 mutant (N = 219). (B) Histogram of angles between the mother microtubule and the new microtubule not originating from microtubule crossovers in WT and the phot1 phot2 double mutant. (C) Fraction of parallel microtubule initiations - of microtubules not created at crossovers - in WT and the phot1 phot2 mutant. An asterisk indicates that the fraction of parallel microtubule initiations is significantly higher in the phot1 phot2 (N = 219) mutant than in the WT (N = 104) based on Fisher s exact test (p <0.01). (D) 8

9 Proportions of microtubules generated at microtubule crossovers and microtubules not generated at crossovers in WT (N = 243) and phot1 phot2 (N = 313) cells differ significantly (p < , Fisher s exact test, two tailed), indicated by asterisk. For each genotype we analyzed 5 cells for 30 minutes mm 2 and 2158 µm 2 were assayed in WT and phot1 phot2 respectively. (E) Schematic representation of PHOT1 PHOT2 signaling acting on the cortical microtubule cytoskeleton during blue light induced array reorientation. 9

10 Fig. S7. growing end mchy-tua5 GCP2-GFP merge 0s 50s 100s B 0s 20s C 0s 250s 500s mcherry-tua5 growing end mcherry-tua5 history of amplification YFP-TUA5 Generation and amplification of longitudinal microtubules. (A) Two consecutive branching nucleations generate a longitudinal microtubule from a transverse microtubule array. Yellow arrowheads indicate the GCP2-3xGFP nucleation markers. Blue arrowheads indicate growing microtubule ends. See also Movie S11. (B) Example of microtubule bending at microtubule crossover. See also Movie S12. (C) Microtubule severing-dependent amplification of longitudinal microtubules. The blue overlay shows the paths of all microtubules formed by microtubule severing originating from one single microtubule. See also Movie S13. Scale bars, 5 µm. 10

11 Fig. S8. A length time (min) B Hypocotyl elongation speed [ m / h] 250 Before blue light After blue light Col0 WT cry1 cry2 phot1 phot2 Ws WT ktn1-1 C parameter order Longitudinal Longitudinal order after 1 hr in blue diode light Col0 WT phot1 phot2 Suppression of dark-grown hypocotyl elongation by blue light. (A) Kymographs of etiolated hypocotyl elongation for 4-day old seedlings germinated in the dark, imaged for 1 hour using a red safelight, then stimulated with blue light for 1 hr (see methods). The blue arrows mark the moment the blue light was turned on. Each image is representative of the genotypes as quantified immediately below in (B). The seedling image in the rightmost panel indicates the approximate position of the kymograph lines (red line). (B) Mean growth speeds of etiolated hypocotyls of Col0 WT (22 plants), Col0 cry1 cry2 (18 plants), Col0 phot1 phot2 (22 plants), Ws WT (16 plants) and Ws ktn1-1 (22 plants). The asterisks mark significant reductions of etiolated hypocotyl elongation speed after blue light induction as determined by the Mann-Whitney U test (p<0.001). The reduction in phot1 phot2 was not statistically significant in this sample (p>0.05). (C) Longitudinal order parameter of the cortical microtubules in the epidermis cells of Col0 WT (19 plants) and Col0 phot1 phot2 (8 plants) plants used in the growth inhibition experiments 11

12 described in A and B. After 1hr of the blue diode light treatment, the longitudinal order was significantly lower in the phot1 phot2 mutant (p < 0.01, Mann-Whitney U test). 12

13 Supplementary Movie Captions Movie S1. Cortical microtubule array reorientation from transverse to longitudinal in a darkgrown hypocotyl cell expressing mcherry-tua5. Image processing using a time-phased walking subtraction was used to detect growing and shrinking ends, displayed here as green and red comets, respectively (see materials and methods). Highlighting growth and shrinkage in this way is a useful aid for detecting and measuring microtubule initiation events without expression of end-labeling proteins. Movie S2. A new microtubule is generated from a GCP2-3 GFP particle. The blue and yellow arrowheads indicate the new growing microtubule end and the GCP2 signal, respectively (see Fig. 2A). Movie S3. Generation of a new microtubule at a microtubule crossover without a GCP2-3 GFP particle. The solid blue and open yellow arrowheads indicate the new growing end and the microtubule crossover without the GCP2 particle, respectively (see Fig. 2B). Movie S4. Microtubule severing at a crossover marked by a GFP-KTN1 signal, resulting in a new growing end. The blue and yellow arrowheads indicate the growing new end and the GFP-KTN1 particle, respectively (see Fig. 2F). Movie S5. Microtubule severing at a GFP-KTN1 signal, resulting in a depolymerizing new microtubule plus end. The open blue and solid yellow arrowheads indicate the depolymerizing new microtubule plus end and the GFP-KTN1 particle, respectively (see fig. S3A). Movie S6. Three new growing microtubules (blue arrowheads) originate from a single crossover location. In each case, GFP-KTN1 (yellow arrowheads) localizes to the crossover in the moments leading up to the initiation (see fig. S3B). Movie S7. Amplification of the number of longitudinal microtubules by microtubule severing. The blue overlay shows the paths of all microtubules that formed by microtubule severing from one single original microtubule (see Fig. 3I). Movie S8. Time-lapsed imaging of cortical microtubule arrays in WT and ktn1-1 mutant seedlings expressing GFP-TUB6. Array reorientation in the ktn1-1 mutant is severely inhibited (see quantitation in Fig. 4A). Movie S9. Automated analysis of microtubule reorientation. The left panel shows the reorientation movie of a WT cell. The right panels show the output of the ImageJ plugin LOCO, which uses a configurable rotating filter to measure the local orientation of linear structures in the neighborhood of each pixel (see Materials and Methods). Different colors indicate the local preferential orientations for pixels above a threshold level. The contour plot on the upper right shows the evolution of the fraction pixels of each angle over time, as calculated from LOCO output. The graph on the lower left shows the evolution of the transverse and longitudinal order parameters calculated from these data.

14 Movie S10. Cortical microtubule array reorientation from transverse to longitudinal in darkgrown Arabidopsis hypocotyl cells expressing YFP-TUA5. Reorientation in the phot1 phot2 mutant is delayed relative to in the wild type (see quantitation in fig. S5). Movie S11. Two subsequent microtubule nucleations marked by a GCP2-3 GFP labeled complex generating a longitudinal microtubule from a transversely oriented microtubule (see fig. S7A). Movie S12. Example of a diagonally oriented microtubule bending toward longitudinal orientation after making multiple crossovers (see fig. S7B). Movie S13. Amplification of the number of longitudinal microtubules by microtubule severing. The blue overlay shows the paths of all microtubules that formed by microtubule severing from one single original microtubule (see fig. S7C).