Volume 9, Issue 4, Pages 528-540 (April 2016) The Membrane-Associated Sec1/Munc18 KEULE is Required for Phragmoplast Microtubule Reorganization During Cytokinesis in Arabidopsis  Alexander Steiner, Lin Müller, Katarzyna Rybak, Vera Vodermaier, Eva Facher, Martha Thellmann, Raksha Ravikumar, Gerhard Wanner, Marie-Theres Hauser, Farhah F. Assaad  Molecular Plant  Volume 9, Issue 4, Pages 528-540 (April 2016) DOI: 10.1016/j.molp.2015.12.005 Copyright © 2016 The Author Terms and Conditions

Figure 1 Characterization of KEULE-GFP. (A) PKEU::KEULE-GFP with FM4-64 in non-dividing cells. Homozygous keulemm125 mutant rescued by the construct. Note cytosolic localization pattern. (B and C) Time lapses are shown, with minutes indicated in the right panel. Arrowheads point to the leading edges of the cell plate (CP). (B) PKEU::KEULE-GFP with PVHAa1::VHAa1-mRFP. (C) PKEU::KEULE-GFP with PSYP61::SYP61-CFP and FM4-64. Arrow points to the CP. (D) T7-KNOLLE in vitro pull-down with protein extracts from different tissues. Western blot probed with anti-KEULE peptide antibody (Assaad et al., 2001). Note that, although more KEULE is present in flowers and root tips than in leaves, more KEULE protein is bound to the KNOLLE affinity column in leaf extracts. Bars represent 10 μm. See Supplemental Figure 1 for the functionality of the PKEU::KEULE-GFP gene fusion. Molecular Plant 2016 9, 528-540DOI: (10.1016/j.molp.2015.12.005) Copyright © 2016 The Author Terms and Conditions

Figure 2 Localization Dynamics of Membrane and Microtubule Markers During Cytokinesis. (A–C) Time lapses are shown, with minutes indicated in the right panel. Microtubule array stages are indicated on the left. Arrowheads point to the leading edges of the cell plate. Bars represent 10 μm. (A) PKEU::KEULE-GFP with mCherry-TUA5. The signal accumulates at the cell equator as of the phragmoplast assembly stage (white-rimmed blue arrowhead). (B) PKN::KNOLLE-YFP with mCherry-TUA5. (C) PSYP121::SYP121-GFP with mCherry-TUA5. White-rimmed black arrowheads point to vacuoles. Note signal in vacuoles in (B) and the absence of a vacuolar signal in (A) and (C). (D) Line graphs of the cell plate at the ring-shaped phragmoplast stage from PKEU::KEULE-GFP, PKN::KNOLLE-YFP, PSYP121::SYP121-GFP depicting fluorescence intensity along the Y axis. Note that, in contrast to PKEU::KEULE-GFP, neither KNOLLE nor SYP121 reorganize to the periphery of the CP at the ring-shaped phragmoplast stage. Molecular Plant 2016 9, 528-540DOI: (10.1016/j.molp.2015.12.005) Copyright © 2016 The Author Terms and Conditions

Figure 3 The Role of Tethering Factors in Recruiting KEULE to the Cell Plate. (A, D, and E) Time lapses are shown, with minutes indicated in the right panel. Arrowheads point to the leading edges of the cell plate. Bars represent 10 μm. (B and C) Antibody stains of root tips. DAPI/nucleus (white); microtubules (red); anti-KNOLLE/anti-GFP (green). MT, microtubule. Bars represent 5 μm. (A) PKEU::KEULE-GFP in exo84b-2 exocyst mutant. Note the unimpaired localization dynamics. (B) PKEU::KEULE-GFP mis-localization in trs120-4 TRAPPII mutant. Note the absence of a cytosolic or membrane-associated signal in the mutant (lower panel) compared with the wild-type (upper panel). (C) Native KNOLLE in trs120-4 TRAPPII mutant, impaired in cell plate biogenesis. Recruitment to the cell plate, however, does not appear to be impaired. Arrow points to the cell plate. See Figure 5A for wild-type control. (D) PKEU::KEULE-GFP with P35S::SEC6-mRFP. See Supplemental Movie 1 for a full time lapse. White-rimmed blue arrowheads point to the first appearance of the markers at the cell equator and white-rimmed blue arrows to cross walls. The two markers differ in their localization dynamics. (E) PKEU::KEULE-GFP with PUBQ::TRS120-mCherry. The two markers co-localize throughout cytokinesis. Molecular Plant 2016 9, 528-540DOI: (10.1016/j.molp.2015.12.005) Copyright © 2016 The Author Terms and Conditions

Figure 4 Localization Dynamics of PKEU::KEULE-GFP During Cytokinesis, with Respect to Microtubule Markers and in Mutants with Defects in Microtubule Reorganization. (A) 3D heatmaps of PKEU::KEULE-GFP and PPLE::GFP-PLEIADE depicting fluorescence intensity along the z axis with a scale ranging from blue (low) to red (high). Note that the PPLE::GFP-PLEIADE signal is concentrated in sharp peaks at the leading edges of the cell plate (arrowhead), whereas PKEU::KEULE-GFP is more evenly spread (bracket), suggesting that microtubule reorganization precedes membrane reorganization at the cell plate. (B) Relative signal intensity at the middle compared with the leading edges of the cell plate (CP). Mean signal intensity was taken in line graphs of the earliest time points depicting phragmoplast microtubule reorganization from solid to ring-shaped in time lapses of the GFP markers in combination with mCherry-TUA5. Mean ± SEM. ****p < 0.0005; ***p = 0.001 for comparison with GFP-PLEIADE; n ≥ 4. (C and D) Time lapses are shown, with minutes indicated in the right panel. Arrowheads point to the leading edges of the cell plate. Bars represent 10 μm. (C) PKEU::KEULE-GFP in hikG235. (D) PKEU::KEULE-GFP in ple-4. See Supplemental Figure 2 for the functionality and localization dynamics of PPLE::GFP-PLEIADE gene fusion. Molecular Plant 2016 9, 528-540DOI: (10.1016/j.molp.2015.12.005) Copyright © 2016 The Author Terms and Conditions

Figure 5 Microtubule Reorganization and Cell Plate Formation in Microtubule-Related Mutants. Upper panels: Environmental scanning electron micrographs. Arrowheads point to bloated cells and arrows to cell wall stubs. Bars represent 0.5 mm overview; 20 μm close up. Lower panels: Antibody stains of root tips. DAPI/nucleus (white); microtubules (red); KNOLLE protein (green). Bars represent 10 μm. (A) Wild-type. (B) ple-4; note the enlarged microtubule (MT)-free phragmoplast midzone (asterisk), as has been described (Müller et al., 2004; Ho et al., 2012). (C) hikG235. In both ple-4 and hikG235 mutants, phragmoplast MT reorganization from solid to ring-shaped occurred in a majority of cases; in a minority of cases, partial reorganization of phragmoplast MTs was observed (% indicated in right panel). The overall appearance of the cell plate did not appear to differ from the wild-type. See Supplemental Figure 3 for mor1-1 phenotypes. Molecular Plant 2016 9, 528-540DOI: (10.1016/j.molp.2015.12.005) Copyright © 2016 The Author Terms and Conditions

Figure 6 Characterization of keule Mutants: 3D Reconstructions and Phragmoplast Microtubule Array Organization. (A and B) Focused ion beam/scanning electron micrographs of high-pressure frozen, freeze substituted 5-day-old root tips. Single slices are shown in the upper panel and 3D reconstructions of entire stacks in the lower panel. Nuclei, blue spherical structures; cross walls, green; cell outline, beige. Bars represent 2 μm. (A) Wild-type. Note the regular shape of the cell and the complete cross wall. (B) keuleT282. Note that there are four nuclei but no apparent cross walls in the 3D reconstruction (right panel). Three of these nuclei (N) can be seen in a single slice on the left. See Supplemental Figure 4 for a description of how the 3D reconstruction was carried out for (B). (C–E) Upper panels in (C) and (D): environmental scanning electron micrographs of wild-type and mutant seedlings. Arrowheads point to bloated cells and arrows to cell wall stubs. Bars represent 0.5 mm overview; 20 μm close up. Lower panels and (E): Antibody stains of root tips. DAPI/nucleus (white); microtubules (red); KNOLLE protein (green). Bars represent 10 μm. (C) massue-5. Note the complete cell plate and timely reorganization of phragmoplast microtubules, as in the wild-type (Figure 5A). (D) keuleT282. Note the unreorganized phragmoplast (open arrowhead). The cell plate appears to have assembled. It is clearly misoriented. (E) PPLE::GFP-PLEIADE in keuleT282. MT, microtubules. Molecular Plant 2016 9, 528-540DOI: (10.1016/j.molp.2015.12.005) Copyright © 2016 The Author Terms and Conditions