Volume 38, Issue 3, Pages 305-315 (August 2016) Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose Synthesis in Arabidopsis  Zengyu Liu, Rene Schneider, Christopher Kesten, Yi Zhang, Marc Somssich, Youjun Zhang, Alisdair R. Fernie, Staffan Persson  Developmental Cell  Volume 38, Issue 3, Pages 305-315 (August 2016) DOI: 10.1016/j.devcel.2016.06.032 Copyright © 2016 Elsevier Inc. Terms and Conditions

Developmental Cell 2016 38, 305-315DOI: (10.1016/j.devcel.2016.06.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 CMU1 Is Localized as Static Puncta on Cortical Microtubules and Influences Cell Shape (A) Mutations in CMU1 and CMU2 caused cell twisting in etiolated Arabidopsis hypocotyls (indicated by the angle between the dashed lines; images by environment scanning electronic microscope). (B) Quantification of cell-twisting phenotype. Data represent mean ± SD. Different letters indicate p < 0.01, Fisher's least significant difference (LSD) test, n = 3 replicates (20–80 seedlings were checked in each replicate). (C) Hypocotyl cells expressing GFP-CMU1 and the tubulin marker mCherry (mCh)-TUA5. (D) Fluorescent intensity plot along the dashed line in (C) (Merge). Note the high correlation of signals. (E and F) Single images from time series of dual-labeled GFP-CMU1 mCh-TUA5 cells (E) and kymographs (F) of the two channels along yellow dashed line in (E). The yellow arrowheads are tracking the polymerizing microtubule end. Note that the GFP-CMU1 signal appears 25 s after the microtubule polymerization. (G) Microtubule binding assay via sedimentation. Note that the CMU1 is only detected in the pellet when the microtubules are present. MAPF, microtubule-associated protein fraction (positive control); Tub, tubulin; BSA was used as negative control. Pellet and soluble fractions are on the same gel; dashed black line indicates separation between the two fractions. See also Figures S1 and S2. Developmental Cell 2016 38, 305-315DOI: (10.1016/j.devcel.2016.06.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Loss of CMU Proteins Results in Lateral Displacement of Microtubules (A) Single frames of mCh-TUA5 in wild-type (left panel) and cmu1cmu2 (right panel). Yellow arrowheads indicate arched microtubules that coincide with microtubule lateral displacement. (B) Enlarged area of dashed box in (A) (right panel). Note the microtubule lateral displacement over time (red arrowheads). (C) Kymographs along red lines in (A). While oblique traces (yellow arrows) are prominent in the mutant, only vertical traces are found in the wild-type. (D) Distribution of microtubule lateral displacements in wild-type and cmu1cmu2, respectively. The lateral displacement was calculated from the lateral positional SD of the individual microtubule traces in the kymographs (as seen in C and exemplified in inset). The duration of the measurements was 10 min. (E) Dual-labeled GFP-CMU1 and mCh-TUA5 wild-type seedlings treated overnight with 50 μM morlin. The time series show an arching microtubule (dashed box). Note that GFP-CMU1 fluorescence is largely absent or diffuse in the indicated region while other microtubules are clearly decorated. (F) Decrease of GFP-CMU1 fluorescence further corroborated by a kymograph along the dashed line in (E) (Merge, middle panel). Developmental Cell 2016 38, 305-315DOI: (10.1016/j.devcel.2016.06.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 CMU1 Is Associated with Membranes (A) The GFP-CMU1 is absent from microtubules that are not in close contact with the plasma membrane (indicated by arrowheads). Single-frame images are from dual-labeled GFP-CMU1 and mCh-TUA5 etiolated hypocotyls. (B) Western blot analyses of microsomal membrane and soluble fractions reveal that GFP-CMU1 is associated with membranes. GFP (free) and GFP-ROP6 lines were used as negative and positive control, respectively. The Arabidopsis vacuolar pyrophosphatase (AVP1) is used as membrane marker detected by AVP1 antibody (tonoplast marker). Ponceau staining was used as loading control. The experiments were repeated three times with similar results. (C) GFP-CMU1 is only faintly detected at preprophase band, spindle, or early phragmoplast microtubule arrays (indicated by yellow arrows) in dividing cells of Arabidopsis roots. (D) GFP-CMU1 is localized to the cell plate (indicated by yellow wide arrows) and not phragmoplasts during cell division in Arabidopsis roots. Note the low GFP-CMU1 fluorescence associated with the phragmoplast (indicated by yellow arrowhead). See also Figures S3 and S4. Developmental Cell 2016 38, 305-315DOI: (10.1016/j.devcel.2016.06.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 CesAs Are Uncoupled from Microtubules in the cmu Mutant (A) Time-average images of dual-labeled GFP-CesA3 mCh-TUA5 hypocotyl cells in wild-type and cmu1cmu2 mutant. (B) Pearson correlation coefficient between the GFP-CesA3 trajectories and mCh-TUA5. Data are mean ± SD, ∗∗p < 0.01, Student's t test, n = 5 cells from five seedlings. (C) Individual frames from a time lapse showing individual CesA insertions after photobleaching (CesAs are indicated by yellow arrows) and cortical microtubules in wild-type (left) and cmu1cmu2 (right), respectively. (D) Kymograph along the dashed yellow lines in (C). (E) Single images from time series of etiolated seedlings expressing GFP-CesA3 and mCh-TUA5 showing GFP-CesA3 (both yellow and white arrowheads) movement along cortical microtubule in control cell (upper panel), or GFP-CesA3 movement that occurs across cortical microtubule that show lateral displacement in cmu1cmu2 mutant cell (lower panel). (F) Typical kymograph traces depicting CesA movement in wild-type (upper panels) and cmu1cmu2 (lower panels), respectively. Note the steady pace in the CesA traces (yellow dashed line in upper panel) in wild-type, while there is a deviation in CesA movement in the cmu1cmu2 mutant (dashed lines in lower panel). The yellow arrows are indicating the encounter or parting of CesAs with/from microtubules. The positions of kymographs are indicated by the yellow dashed lines in (E). (G) Effect of oryzalin treatment on the variation of CesA velocity. Data are means ± SD Different letters indicate p < 0.01, LSD test, n = 4–5 cells from five seedlings. At least 100 CesA kymograph traces were analyzed for each treatment. See also Figure S5. Developmental Cell 2016 38, 305-315DOI: (10.1016/j.devcel.2016.06.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 The Lateral Displacement of the Microtubules Is Caused by Catalytically Active CSCs (A) Single images of dual-labeled GFP-CesA3 mCh-TUA5 cmu1cmu2 cells before and after treatment with 200 nM isoxaben for 2 hr. Isoxaben treatment caused accumulation of CesAs in cytosolic compartments (arrowheads in right panel). (B) Kymographs of microtubules along dashed lines in (A). The arrowheads are indicating oblique microtubule traces, which suggest microtubule drift. Note vertical traces after isoxaben treatment. (C) Time-average images of dual-labeled GFP-CesA3 and mCh-TUA5 in cmu1cmu2pom2-4 mutant cells. (D) Kymographs along dashed lines in (C) (Merge). Note vertical traces of microtubules in the cmu1cmu2pom2-4 triple-mutant cells. (E) Quantification of microtubule lateral displacement shows that it is reduced in isoxaben-treated cmu1cmu2 and cmu1cmu2pom2-4 cells. Data are mean ± SD. Different letters indicate p < 0.01, LSD test, n = 5 or 6 cells from six seedlings. The duration of the measurements was 10 min. (F) Schematic model showing that CMUs prevent microtubule lateral displacements caused by aberrant CSC movements (red bold arrow), and effectively redirect the CSCs (black arrows). However, in the absence of CMU function (lower panel) the microtubules lack the capacity to redirect the CSCs. Here, the CSC-associated CSI1/POM2s associate with the microtubules and drag them with the moving CSCs. See also Figure S5. Developmental Cell 2016 38, 305-315DOI: (10.1016/j.devcel.2016.06.032) Copyright © 2016 Elsevier Inc. Terms and Conditions