Volume 21, Issue 12, Pages 1055-1060 (June 2011) Cell Plate Restricted Association of DRP1A and PIN Proteins Is Required for Cell Polarity Establishment in Arabidopsis  Jozef Mravec, Jan Petrášek, Na Li, Sjef Boeren, Rumyana Karlova, Saeko Kitakura, Markéta Pařezová, Satoshi Naramoto, Tomasz Nodzyński, Pankaj Dhonukshe, Sebastian Y. Bednarek, Eva Zažímalová, Sacco de Vries, Jiří Friml  Current Biology  Volume 21, Issue 12, Pages 1055-1060 (June 2011) DOI: 10.1016/j.cub.2011.05.018 Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Colocalization of PIN, DRP1A, and CLC Proteins (A) Time-lapse scanning during cytokinesis (20 min interval) in DRP1A-mRFP1/PIN2-GFP double-transgenic lines. Proteins largely colocalize on the forming cell plate (arrowheads). (B) Indirect immunofluorescence localization of CLC-GFP and DRP1A proteins showing partial colocalization on the cell plate (arrowheads). α-DRP1A and α-GFP antibodies were used for the immunolocalization. See also Figure S2. Current Biology 2011 21, 1055-1060DOI: (10.1016/j.cub.2011.05.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Analyses of the DRP1A-PIN Association (A) In planta analysis of PIN1-DRP1A association by bimolecular fluorescence complementation (BiFC). Top: control line coexpressing DRP1A fused with N- and C-terminal parts of the YFP (SYN or SYC) confirms dimerization and oligomerization of DRP1A proteins at the cell plate. Bottom: line coexpressing DRP1A-SYN and PIN1 hydrophilic loop fused with SYC. The signal is again visible at the cell plate. (B and C) Förster resonance energy transfer (FRET) analysis of PIN1-GFP/DRP1-mRFP1 in the stable double-transformed BY-2 cells. (B) Unmixed emission of GFP donor at 500–520 nm (green) and mRFP1 acceptor at 600–620 nm (red) from the emission spectrum detected after excitation with a 477 nm laser. (C) The fluorescence emission spectrum measured in the selected regions of interest (ROI, inset) numbered from 1 to 5 (1, middle of the cell plate; 2 and 3, growing ends of the cell plate; 4 and 5, transversal and longitudinal plasma membranes). Emission peak at 600–620 nm implies highest FRET at growing edges of the cell plate, lower in the middle of the cell plate, and no FRET detected at the plasma membrane. See also Figure S3. Current Biology 2011 21, 1055-1060DOI: (10.1016/j.cub.2011.05.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Localization of PIN2 in drp1a2 Mutant (A and B) Effect of drp1a mutation on the localization of PIN2. Confocal section of the epidermal layer. PIN2-GFP in the wild-type (A) and the drp1a2 mutant (B). (C–H) Various localization defects (marked by arrowheads) of PIN2-GFP observed in drp1a2 mutant with the percentage of observation among 367 cells in total. (C) Normal apical localization of PIN2-GFP. (D) Complete loss of polarity. (E) Accumulation of PIN2-GFP signal on newly formed septum. (F) Oblique and transversal membranes. (G) Cell wall stubs. (H) Round intracellular compartments. (I) Confocal section of PIN2-GFP on the newly formed septum in the wild-type and drp1a2. (J) Moderate accumulation of the PIN2-GFP signal on the septum in the wild-type when compared to some drp1a2 cells. See also Figure S4. Current Biology 2011 21, 1055-1060DOI: (10.1016/j.cub.2011.05.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 Auxin-Related Developmental Phenotypes in drp1 Mutants (A–C) Seedling phenotypes of drp1 mutants. Six-day-old seedlings of wild-type, drp1a2, and drp1c2 (A). Quantification of root elongation and gravitropism (error bars represent standard error of the mean [SEM]; n = 20; ∗p < 0.05) (B). Seedling of the drp1a2/drp1c2+/− mutant with minute-like phenotype (C). (D) Phenotype of 6-day-old seedlings of estradiol-inducible artificial microRNA (amiRNA) drp1a/c/e line transferred 3 days after germination on estradiol plate. Arrowhead shows agravitropic growth of the root. Scale bars represent 0.5 cm. (E and F) Embryonal phenotypes. Wild-type development (E) and phenotypes observed in drp1a2+/−/ drp1c2+/− plant (F) marked with arrowheads. (G) Shoot phenotypes in drp1a2 and drp1c2 mutants marked with arrowheads. Both mutants are semisterile. Arrowheads show nonelongated silliques. (H) Flower phenotypes. Mutant drp1a2 forms approximately 20% of flowers with five petals. Petals of drp1a2 mutant are unusually elongated. (I) Expression of the auxin-responsive marker DR5rev::GFP in the wild-type and drp1a2 mutant. Decrease of signal can be observed in the drp1a2 mutant. See also Figure S5. Current Biology 2011 21, 1055-1060DOI: (10.1016/j.cub.2011.05.018) Copyright © 2011 Elsevier Ltd Terms and Conditions