SUPPLEMENTARY METHODS. PIN2-Dendra2 transgenic line creation

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1 SUPPLEMENTARY METHODS PIN2-Dendra2 transgenic line creation The PIN2-Dendra2 construct was described previously (Jásik et al., 2013). Shortly, for generation of the expressing vector encompassing the PIN2-Dendra2 fusion, DNA fragments of PIN2 including upstream and downstream regulatory sequences were multiplied by PCR from genomic DNA of Arabidopsis thaliana ecotype Columbia (Col-0), Dendra2 was amplified by PCR from a Gateway Dendra2-At-N entry clone (Evrogen, Moscow, Russia). Fragments were cloned sequentially into the pampat-msc vector (Genank: AY ) such that Dendra2 was positioned in the PIN2 intracytoplasmic loop downstream of Alanine 403. The construct was transformed into the Arabidopsis thaliana Col-0 accession using Agrobacterium tumefaciens GV3101(pMP90RK) by the floral-dip method. Selection of homozygous plants was performed by growing seedlings on medium supplemented with 7.5 mg/l phosphinothricin (PPT, Duchefa, Haarlem, The Netherlands). Plant material, cultivation conditions, media and chemicals Seeds were surface sterilized with 1% (w/v) sodium hypochlorite and germinated in Petri dishes with MSMO medium, diluted 1:1 (Sigma, #M6899) supplemented with 1% sucrose and solidified by 0.8% agar. Medium ph was adjusted to 5.8 before autoclaving. Dishes were kept in vertical position in a growth chamber at 21 ºC under continuous light (100 μmol m -2 s -1 ). efore performing the experiments, seedlings were transferred onto fresh medium for 12 h. Treatments of seedlings was performed on microscope slides covered with 1.5 ml solid culture medium containing drugs of interest. Four to five seedlings were used per slide and experiments were repeated three times. Slides were placed on the culture medium in Petri dishes and stored vertical under light and at 21 C. Antibiotics and refeldin A (FA) were dissolved in ethanol, Tyrphostin A 23 in DMSO. Concentration of ethanol in the medium did not exceed 0.1%, DMSO 0.05% (v/v). Cycloheximidine (CHX) and FA were used in concentration of 50 μm (if not indicated differently), Tyrphostin A23

2 in concentration of 100 μm, actinomycin A in concentration of 50 mg/l. Control treatments were performed with equal amounts of solvent. Medium ph was rechecked after drug supplementation and adjusted if required to the same value as control medium. Tyrphostin A23 was from Calbiochem (San Diego, USA), other chemicals were from Sigma-Aldrich (St. Louis, USA). Photoconversion and microscopy efore photoconversion and imaging, seedlings were covered by a coverslip which was removed gently again after imaging. The general scheme for time course analysis was as follows: roots were imaged in both channels (green and red fluorescence), photoconverted, again imaged and then reimaged at different time points during the experiment under the same microscope settings. Photoconversion of roots was performed for 15 s with a 100W mercury lamp, a 20 x /0.75 Plan Apochromat objective and an excitation filter P 405/5 nm of the inverted Zeiss Axiovert 200M microscope of the confocal laser scanning system (LSM 510 Meta, Carl Zeiss Jena, Germany) For quantitative analysis of PIN 2 abundance images were acquired with 8-bit color depth using a Zeiss LSM-510 META with the 20 x /0.75 Plan Apochromat objective and pinhole setting at 140 μm (4 μm optical slice). Red and green signals were acquired in a multi-track mode. For green signal capture, samples were excited with the 488-nm line of the argon laser and the signal was collected with a 505 to 530 nm band-pass filter. For red signal, samples were excited by the 543-nm line of the HeNe laser and fluorescence was collected with a 565 to 615 nm band-pass filter. For both channels the HFT UV/488/543/633 was used as main dichroic mirror. Gain and offset parameters of the LSM detector and laser line power in the photoconverted experiments were set such way that the green signal of the unconverted PM and the red signal of the photoconverted PM gave approximately the same values of around 70 arbitrary units of the 0 to 256 scale of the 8-bit color depth mode. Identical settings were kept to collect images at all time points within experiments. For emission spectra analysis, different excitation laser lines in combination with appropriate main dichroic mirrors were applied and fluorescence emissions were collected with the spectral Meta detector (lambda-mode).

3 Data evaluation and presentation Mean fluorescence intensities of the transversal PM in root epidermis were quantified by ImageJ software (NIH, ethesda, USA) using the straight line selection mode. Fifteen PMs were investigated at every time point in every root of four to five seedlings used per slide. Results in graphs are presented as normalized data if not otherwise stated. In Figure 1A, D and F at every time point the means of green signal intensities of PM in individual roots were normalized to the mean of green signal intensities emitted by PM of the same root before conversion. Time point 0 represents the relative green signal intensity recorded immediately after conversion. The means of red signal intensities of PM in individual roots at every time point were normalized to the mean red fluorescence intensity measured soon after conversion (time point 0). Values of the time points 0 were set to 1. The means of green signal intensities of FA-Cs in Figure 1A at every time point were normalized to the mean of green signal intensities emitted by PM of the same root before conversion. In a similar way the red signal intensities of FA-Cs at every time point were related to the mean red fluorescence intensity of PM measured immediately after conversion. In Figure 1C (right panel) in single roots the means of green signal intensities of FA-Cs at every time point during the experiment were normalized to the mean of green signal intensities emitted by FA-Cs of the same root before photoconversion. Time point 0 represents relative green signal intensity recorded immediately after photoconversion. The means of red signal intensities of FA-Cs were related to the mean red fluorescence intensity of FA-Cs measured immediately after photoconversion (time point 0). The value of the time points 0 was set to 1. In Figure 1G samples were imaged in both channels after 2.5 h treatment with FA and washing (time point 0). In single roots the means of green and red signal intensities of PM at every time point during the experiment were normalized to the means of green and red signal intensities emitted by the PM at time point 0. Values for green and red signal intensities of the time points 0 were set to 1. Graphs were prepared in Microsoft Excel (Microsoft, Redmont, USA). Points displayed at graphs represent means of normalized values of 12 to15 roots; bars correspond to standard errors of the means (SE). Images were processed with Adobe Photoshop CS2 (Adobe Systems, Mountain View, USA) and Microsoft Publisher (Microsoft, Redmont, USA) softwares.

4 SUPLEMENTARY FIGURES Supplementary Figure 1. Time course pattern of PIN2 populations in PM and FA-Cs. Images taken in green and red channels show decreasing red signal intensity and increasing green signal intensity in PM during the experiment. Note the prevalence of the green signal in FA-Cs at every time point from 2 to 12 h. Roots were imaged before and after photoconversion, then moved onto medium supplied with 50 μm FA and reimaged several times within 12 h under the same microscope settings. Scale bar = 20 μm.

5 Supplementary Figure 2. Comparison of spectra emitted by PM and FA-Cs. Spectra were measured with a Zeiss LSM- 510 microscope using the meta detector and the lambda acquisition mode in roots treated with 50 μm FA for 2 h. Roots were either photoconverted (marked as converted ) or not photoconverted (marked as unconverted ) and excited by the different laser lines. A region of interest 1 (ROI1) drawn in the red color in coded images represents FA-Cs; a region of interest 2 (ROI2) drawn in green color corresponds to PM. Red lines in related graphs show the emission spectrum patterns obtained by analysis of ROI1, the green lines represent ROI2.

6 C D Supplementary Figure 3. Monitoring of FA-Cs development with unconverted PIN2-Dendra2. Note that FA-Cs in seedlings grown on medium with 50 μm FA were initially small and reached full size within 2 h (A). The number of FA body s per cell slightly decreased during the extended cultivation of seedlings on FA () while FA-Cs size (C) and fluorescence signal intensity emitted by FA-Cs (D) increased dramatically within the first 2 h. Later on these parameters were more or less constant. In D at every time point the means of signal intensities of FA-Cs in individual roots were normalized to the mean of green signal intensities emitted by PM of the same root before conversion. In A the scale bar = 5 μm

7 time (h) Supplementary Figure 4. Effect of CHX and actinomycin A on PIN2 abundance in FA-Cs and PM. After pretreatment of seedlings by antibiotics and photoconversion, FA-Cs still contained the red PIN2-Dendra2 form but not the green PIN-Dendra2 variety (A). Quantitative data for actinomycin A and CHX effects are given in. Samples marked as converted were pretreated either with 50 mg/l actinomycin A or 50 μm CHX for 1.5 h, then placed on medium with the appropriate antibiotic and 50 μm FA, imaged, photoconverted, again imaged and then reimaged at different time points. In the unconverted experiment samples were also pretreated by antibiotics but not photoconverted. FA-Cs means of red signal intensities in the converted experiment at every time point during the experiment were normalized to the mean of red signal intensities emitted by PM of the same root immediately after conversion. In an unconverted approach FA-Cs means of green signal intensities were related to the mean of green signal intensities emitted by PM of the same root after 1.5 h antibiotic pretreatment. Scale bar = 10 μm.

8 C D Supplementary Figure 5. Disappearance of FA-Cs from cells after FA washingout. The number of cells with FA-Cs (A), number of FA-Cs per cell (), size of FA-Cs (C) and red signal intensity in FA-Cs (D) decreased rapidly after FA was washed out. Seedlings were kept on medium supplemented with 50 μm FA for 2.5 h, then rinsed for 10 s with liquid culture medium, placed on FA-free medium, photoconverted, imaged in the red channel (time point 0) and then reimaged at different time points. Control seedlings were not washed and were kept permanently on medium with FA. Their roots were also converted after 2.5 h, imaged (time point 0) and reimaged at the same time points as rinsed roots. In C the means of FA- Cs sizes in individual roots at each time point were normalized to the mean size of FA-Cs of the same root measured immediately after conversion (time point 0). The values of the time points 0 were set to 1. In D the means of red signal intensities emitted by FA-Cs in individual roots at every time point were related to the mean of red fluorescence intensity of FA-Cs measured in the same roots immediately after conversion (time point 0). The values of the time points 0 were set to 1.

9 Supplementary Figure 6. PIN2 abundance in PM after FA washing-out. When seedlings pretreated with 50 μm FA were rinsed and placed on FA-free medium, FA-Cs disappeared and the intensity of fluorescence signal in PM increased (A, marked as washed ), on the other hand samples permanently growing on FA medium showed no obvious differences (marked as not washed ). Graph in shows data obtained by the measurement of membrane intensities at different time points. PIN2-Dendra2 seedlings were grown on medium with 50 μm FA for 2.5 h and then after 10 s washing with liquid culture medium were placed on medium without FA, imaged in green channel (time point 0) and reimaged at different time points. Control seedlings grown permanently either with or without FA were imaged the first time after 2.5 h (time point 0) of cultivation and reimaged as shown in the graph. In single roots the means of green signal intensities at every time point during the experiment were normalized to the mean of green signal intensities emitted by PM of the same root at time point 0. Values of the time points 0 were set to 1. Scale bars = 10 μm

10 Supplementary Figure 7. The abundance of PIN2 in PM in the presence of CHX after FA washing-out. FA-Cs developed after photoconversion in the presence of CHX contained red signal and disappeared from the cells after washing. However, this vanishing did not increase the red signal intensity in PM (A). Quantitative data are given in. Seedlings were pretreated with 50 μm CHX for 1 h, photoconverted, treated with 50 μm FA in the presence of CHX for 2.5 h and then after 10 s washing with liquid culture medium they were placed on medium with CHX but without FA, imaged in red channel (time point 0) and reimaged at different time points. In single roots the means of red signal intensities at every time point during the experiment were normalized to the mean of red signal intensities emitted by the PM of the same root at time point 0. Values of the time points 0 were set to 1. Scale bars = 10 μm

11 Supplementary Figure 8. Effect of tyrphostin A23 (A23) on PIN2 turnover in PM and FA-Cs. A23 inhibits recovery of PIN2-Dendra2 green fluorescent pool in PM and inhibits PIN2 internalization (A) and affects the proportion of secreted and internalized PIN2-Dendra2 in FA-Cs (). Seedlings were treated with100 μm A23 for 30 min, then placed on medium either with (A23 pretreated + FA) or without 50 μm FA (A23 pretreated), imaged, photoconverted and reimaged. The control was kept on medium without A23 and FA; samples A23 continuously were cultivated permanently on medium with 100 μm A23. In both cases roots were imaged, after 30 min of cultivation, photoconverted and reimaged. In single roots the means of green fluorescence signal intensities of PM at every time point during the experiment were normalized to the mean of green signal intensities emitted by PM of the same root before conversion. Time points 0 represent relative signal intensities recorded after conversion. Red signal intensities were related to the mean red signal intensities measured immediately after conversion (time point 0). Values of the time points 0 were set to 1.In samples were treated with A23 for 30 min, than placed on medium supplemented with 50 μm FA, imaged, photoconverted and again imaged. Control samples were not pretreated with A23. In single roots, the means of green signal intensities of FA- Cs at every time point during the experiment were related to the mean of green signal intensities emitted by PM of the same root before photoconversion. Red signal intensities at every time point were related to the mean red fluorescence intensity of PM measured immediatelly after conversion.