Methods

Methods A pulse-chase strategy combining click-EdU and photoconvertible fluorescent reporter: tracking Golgi protein dynamics during the cell cycle Micka€el Bourge1, Cecile Fort2, Marie-No€elle Soler1, Beatrice Satiat-Jeunema^ıtre1,2 and Spencer C. Brown1 1

P^ole de Biologie Cellulaire, Imagif, Centre de Recherche de Gif (FRC3115), CNRS, Saclay Plant Sciences, 91198 Gif-sur-Yvette Cedex, France; 2Laboratoire Dynamique de la

Compartimentation Cellulaire, CNRS UPR2355, Institut des Sciences du Vegetal, Saclay Plant Sciences, Centre de Recherche de Gif (FRC3115), 91198 Gif-sur-Yvette Cedex, France

Summary Authors for correspondence: B
eatrice Satiat-Jeunema^ıtre Tel: +33 0169823798 Email: [email protected] Micka€ el Bourge Tel: +33 0 169823798 Email: [email protected] Received: 23 June 2014 Accepted: 13 August 2014

New Phytologist (2015) 205: 938–950 doi: 10.1111/nph.13069

Key words: cell cycle, click chemistry, 5ethynyl-20 -deoxyuridine (EdU), fluorescent proteins, G1 subcompartments, Golgi synthesis, Kaede pulse-chase, tobacco (Nicotiana tabacum) BY2 cells.

 Imaging or quantifying protein synthesis in cellulo through a well-resolved analysis of the cell cycle (also defining G1 subcompartments) is a methodological challenge. Click chemistry is the method of choice to reveal the thymidine analogue 5-ethynyl-20 -deoxyuridine (EdU) and track proliferating nuclei undergoing DNA synthesis. However, the click reaction quenches fluorescent proteins. Our challenge was to reconcile these two tools.  A robust protocol based on a high-resolution cytometric cell cycle analysis in tobacco (Nicotiana tabacum) BY2 cells expressing fluorescent Golgi markers has been established. This was broadly applicable to tissues, cell clusters, and other eukaryotic material, and compatible with Scale clearing. EdU was then used with the photoconvertible protein sialyl transferase (ST)-Kaede as a Golgi marker in a photoconversion pulse-chase cytometric configuration resolving, in addition, subcompartments of G1.  Quantitative restoration of protein fluorescence was achieved by introducing acidic EDTA washes to strip the copper from these proteins which were then imaged at neutral pH.  The rate of synthesis of this Golgi membrane marker was low during early G1, but in the second half of G1 (30% of cycle duration) much of the synthesis occurred. Marker synthesis then persisted during S and G2. These insights into Golgi biology are discussed in terms of the cell’s ability to adapt exocytosis to cell growth needs.

Introduction Cell growth, cell division, and cell responses to biotic or abiotic environments involve tight coordination between nuclear and cytoplasmic elements (Nurse, 2000). Understanding this cell orchestration implies the combined use of a proper assessment of cell cycle phases at the individual cell or cell population level and the analysis of dynamic events within the cytosol or the nucleoplasm. Two tools have been important in this field: fluorescent protein tags to track proteins of interest, and nucleotide analogues to delimit nuclear phases. In plant biology, a substantial toolbox is now available for studying DNA dynamics without applying chemical synchronization strategies that can perturb nucleo-cytoplasmic coordination (Coba de la Pe~ na & Brown, 2001; Brown et al., 2010b). This includes flow cytometry, fluorescent nuclear proteins and cell sorting (Galbraith et al., 2011; Bourdon et al., 2012; Wozny et al., 2012). Cytometry of cells stained for DNA (typically with propidium iodide or 40 ,6-diamidino-2-phenylindole 938 New Phytologist (2015) 205: 938–950 www.newphytologist.com

(DAPI)) is commonly used to specify the position of each cell in its cycle and to obtain the frequencies (or probabilities) of cells in each phase (Brown et al., 2010b; Ronot et al., 2010). To analyse their progression through the cycle, individual cells are classified as being in G1, S and G2/M phases and possible subphases. In plants, such analyses are usually performed on protoplasts prepared from cell suspensions or tissues in order to respect constraints imposed by the cytometer. Unfortunately, cell cycle analysis of protoplasts often lacks resolution because of screening effects of organelles, cytoplasmic DNA, nonspecific labelling and flow-orientation issues. A method for high-resolution cell cycle analysis of protoplasts is therefore needed, and this should ideally be compatible with fluorescent proteins. More complex methods than those that simply use the intensity of DNA dyes involve incorporation of detectable nucleotide analogues during DNA replication. The bromo-deoxy-uridine (BrdU) technique is based on incorporation of this thymidine analogue during S-phase (Leif et al., 2004). Following Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist immunolabelling, DNA synthesis can be quantified by cytometry. Alternatively, incorporation can be quantified by quenching of Hoechst 33 258 by BrdU (Kubbies et al., 1992; Coba de la Pe~ na & Brown, 2001). An easier and more efficient alternative to the use of BrdU is now available, 5-ethynyl-20 -deoxyuridine (EdU) incorporation. EdU is a thymidine analogue in which a terminal alkyne group replaces the methyl group in the 5 position. It is readily incorporated into nuclear DNA during replication via a scavenging mechanism present in eukaryot cells. The novelty is that the subsequent EdU detection is based on ‘click chemistry’ after fixation or permeabilization of cells, which is easier to use than immunolabelling. The copper(I) (Cu(I))-catalysed (3 + 2) cyclo addition reaction used in this EdU revelation is in fact broadly applied in biochemistry and cell biology. In the presence of Cu (I), the alkyne of EdU reacts with an azide-containing fluorochrome, forming a stable covalent bond. The click chemistry uses an in situ reducing agent such as sodium ascorbate, the reductant of choice for click reactions in organic synthesis (Bruckman et al., 2008; Hong et al., 2009). Importantly, the reaction between ethynyl groups on DNA and fluorescent azide does not require DNA denaturation, rendering EdU labelling more sensitive and specific than BrdU immunolabelling (Hein et al., 2008; Diermeier-Daucher et al., 2009; Kotogany et al., 2010; Darzynkiewicz et al., 2011). Revelation is possible even on fresh tissue with a cell-permeant reagent, carboxy-tetramethylrhodamine-azide (Salic & Mitchison, 2008). However, it has a major drawback, as the copper-catalysed click reaction quenches the emission of fluorescent proteins such as DsRed (Eli & Chakrabartty, 2006; Rahimi et al., 2008) and GFP (Hotzer et al., 2011). At best (and rather rarely), the GFP marker could be identified qualitatively (Vanstraelen et al., 2009). Given the importance of both tools for kinetic and development studies, their compatibility would be essential. Furthermore, various cytometric applications would require quantitative retention of fluorescent protein properties, permitting multiparametric analysis involving both parameters. Here we proposed to revisit the click reaction and reconcile the combined use of EdU and fluorescent-tagged proteins. We have established a generic protocol to restore quantitatively the fluorescence intensity of fluorescent proteins after a click reaction, in plant protoplasts, cells, or tissues and also in nonplant organisms. We also show its compatibility with the Scale clearing method, before deep 3D microscopy with fluorescent proteins (Hama et al., 2011), a technique pertinent for plant tissue imaging. Finally, this proposed generic strategy was used to follow the synthesis of the Golgi marker sialyl transferase motif (ST)-Kaede protein (Saint-Jore et al., 2002; Brown et al., 2010a) during the cell cycle of tobacco (Nicotiana tabacum) BY2 cells. Using the properties of Kaede photoconvertible fluorescent protein and EdU labelling, together with cytometry and cell imaging, new insights into the synthesis of Golgi proteins during the cell cycle were provided. With a pulse-chase configuration initiated by photoconversion of ST-Kaede from green to red, we aimed to quantify pre-existing protein (the red fraction) and de novo synthesis (green protein) while positioning cells within a highly resolved proliferation cycle. Given that G1 occupies two-thirds of Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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the cell cycle duration, photoconversion followed only later by EdU labelling will allow a unique temporal identification of subcompartments within G1, and should provide insights into the behaviour of Golgi markers in the G1 period.

Materials and Methods Biological material Roots Seeds of Arabidopsis thaliana (L.) Heynh, ecotype Columbia (Col-0) (wild type, or expressing mGFP5-ER under the promoter of SCARECROW fused with GFP, pSCR-GFP), were grown as in Vanstraelen et al. (2009). BY2 cultures Nicotiana tabacum L. cv Bright Yellow 2 (BY2) suspensions were subcultured weekly as described previously (Aubert et al., 2011). Most previous studies have used used 3-dold cultures in logarithmic growth. Transgenic BY2 cells expressing a transmembrane domain and short cytoplasmic tail (52 amino acids) of a rat a-2,6-sialyl transferase (ST) fused with either GFP under the control of the 6 9 tandemly repeated cauliflower mosaic virus (CaMV) 35S promoter (ST-GFP; Saint-Jore et al., 2002) or Kaede (ST-Kaede; Brown et al., 2010a) were used to monitor Golgi membranes. The ST-GFP cell line was maintained in a medium supplemented with 100 mg l1 kanamycin (Saint-Jore et al., 2002). BY2 cells expressing ST-Kaede were maintained in a medium supplemented with 50 mg l1 hygromycin. Protoplasts The suspension was centrifuged for 5 min at 80 g and resuspended in basal medium protoplasting buffer (BMP, pH 5.6), composed with Murashige–Skoog (MS) without hormone supplemented with 100 mg l1 sorbitol (550 mM). After a second centrifugation, the supernatant was removed and cells were resuspended in enzymes (Onozuka cellulase-RS 0.5% (Yakult Honsha, Tokyo, Japan), Macerozyme-R10 0.5% (Yakult Honsha) and Pectolyase-Y23 0.05% (MP Biomedicals, Santa Ana, CA, USA) in BMP) for 2.5 h at 25°C with rotary agitation. Protoplasts were washed twice in cool BMP, filtered through 50 lm, and kept at 6°C in darkness. EdU treatment and revelation EdU incorporation: cell cultures EdU (10 mM in DMSO; Invitrogen) was diluted to 20 lM in BY2 culture medium, vortexed, and added (v/v) to cultures. BY2 cells were then incubated in 10 lM EdU for 0.5–24 h (routinely 1.5–6 h), according to the aim of the experiment: a short incubation period was used to identify nuclei in S-phase, and a long incubation period to obtain a large fraction of EdU-positive nuclei. EdU incorporation: roots Arabidopsis thaliana seedlings that germinated in sterile conditions on 2% agar were transferred to a 12-well plate on a shaker with 1.5 ml of half-strength Murashige–Skoog (MS) medium containing 10 lM EdU and grown for 10 h at 26°C in the dark. New Phytologist (2015) 205: 938–950 www.newphytologist.com

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940 Methods

EdU revelation: cells or protoplasts Cells or protoplasts were fixed with 3% (w/v) paraformaldehyde (PFA) in phosphatebuffered saline (PBS) supplemented with 100 mg ml1 sorbitol for 30 min at room temperature and washed twice with PHB buffer (PBS, 10 mM HEPES, pH 7.4, and 30 mg ml1 bovine serum albumen (BSA)). To perform the click reaction, fixed protoplasts were incubated for 30 min at room temperature in reaction buffer (PHB, pH 7.4, from 80 lM to 2 mM CuSO4 (routinely 1.3 mM), 10 mM sodium ascorbate, and 4 lM AlexaFluor647-azide; Invitrogen) and washed once in PHB buffer. EdU revelation: seedlings Seedlings were rinsed once and fixed in 3.7% formaldehyde in PBS (pH 7.4) for 15 min at room temperature, and washed twice in PBS containing 3% BSA and once in PBS containing 0.5% Triton for 20 min. After two further washes in PBS containing 3% BSA, seedlings were transferred to a 12-well plate and the washing solution was replaced with 0.5 ml of freshly prepared click-reaction buffer for 30 min of incubation in the dark with gentle shaking. Samples were washed in 3% BSA in PBS and stored in PBS. Fluorescence restoration Cells or protoplasts were incubated for 90 min at room temperature in PMB (PBS, 10 mM MES, pH 5.0, and 30 mg ml1 BSA) with 40 mM EDTA.Na2. Cells or protoplasts were then washed once in PMB and resuspended in PHB (pH 7.4). Protoplasts were labelled with 5 lg ml1 DAPI. For A. thaliana roots, the restoration protocol consisted of two washes in acidic EDTA (PMB, pH 5), one overnight and one of 3 h during the day, and the roots were then rinsed twice in PHB. Cytometry Optical configuration Protoplasts were analysed on a MoFlo Astrios (Beckman-Coulter, Roissy CDG, France). DAPI was excited by a 355-nm laser (100 mW), taking emission at 432–482 nm. Edu-AlexaFluor647 was excited by a 640-nm laser (60 mW), emission 655–685 nm. Green Kaede or GFP was excited by a 488-nm laser (100 mW), emission 500–552 nm. Red Kaede was excited by a 561-nm laser (200 mW), emission 571–587 nm. The ratio ‘green ST-Kaede:red ST-Kaede’ was calculated for each cell. Pulse compensation was established with single-labelled aliquots. Each histogram comprised > 10 000 protoplasts. Gating strategy and statistics The statistics retained (see Fig. 6 below) were: cell number (and thus frequency), and the mean, median and modal values for cellular green Kaede intensity, red Kaede intensity, and the ratio ‘green ST-Kaede : red ST-Kaede’. Means were used in subsequent data processing. Most experiments involved triplicate samples. Red and green intensities were always normalized before pooling the results of triplicate experiments. For this, the average cellular value of a preparation was calculated as the geometric mean after summing the product ‘mean intensity observed in a subphase’ by ‘frequency of that phase’. For the final modelling of data (Table 1; see Fig. 7 below), the typical duration of each phase was calculated from their respective frequencies in three major experiments (each New Phytologist (2015) 205: 938–950 www.newphytologist.com

performed in triplicate) and cell doubling time (16 h), assigning 30 min for mitosis (Brown et al., 2010a). Microscopy Photoconversion of Kaede A 35-mm Petri dish containing 4 ml of culture was placed on aluminium foil (on a slide) and gently shaken by hand for 5 min under the light from the violet filter (395–446 nm; 200 W mercury arc) of a microscope with no objective (Brown et al., 2010a). Anti-UV protective glasses were worn. Imaging GFP or Kaede Fusion proteins were imaged on widefield (Leica DMI6000B) and confocal microscopes (Leica SP2, Nanterre, France; and Nikon A1R, Champigny-sur-Marne, France) using an HCX PL APO CS 963 1.40 oil objective (960 objective for Nikon). GFP and green Kaede were imaged using 488-nm excitation, and emission at 500–528 nm. Red Kaede, using either 543 or 561 nm excitation, was detected at 580– 630 nm. For security during imaging, a violet-blocking 450-nm long-pass filter was inserted into the microscope illumination pathway to avoid accidental photoconversion. EdU-AlexaFluor647 was imaged using 633-nm excitation, and emission at 670– 720 nm. Fluorescence microplate reader Red Kaede stability and green Kaede neosynthesis were quantified using a 24-well microplate reader and an ST-Kaede BY2 culture, with or without initial photoconversion in a Petri dish (see Fig. S4). Scale clearing Arabidopsis thaliana seedlings (5 d old)  10 lM EdU were fixed as described above in the ‘EdU revelation: seedling’ section, then either stored at 4°C in PBS (uncleared control) or cleared according to the Scale protocol (Hama et al., 2011). The Scale A2 solution of 4 M urea, 10% (w/w) glycerol and 0.1% (w/v) Triton was prepared at least 24 h earlier. After 2 wk, all plantlets were rinsed in PHB then processed with the click reaction. Part of each lot was then subjected to the restoration protocol with two washes in acidic EDTA (PMB, pH 5, with 40 mM EDTA.Na2) as described above in the ‘Fluorescence restoration’ section. After three rinses in PHB (pH 7.4), all plants were rinsed for 30 min in 0.5 M sucrose and mounted in Citifluor AF1 (Biovalley, Marne-la-Vallee, France) containing 2 lg ml1 DAPI. (Scale A2 or 0.5 M sucrose can be used instead of Citifluor.) Immunolabelling With appropriate protection from strong light, well-suspended protoplasts were fixed with gentle agitation at 20°C using a 10fold volume of 3% PFA in MSB (PBS, 10 mM EGTA and 10 mM MgSO4, pH 6.8) supplemented with 0.6 M sorbitol. After centrifugations (3 min at 200 g), a first wash of 10 min in Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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T0, cell division; negative values indicate times before cell division. For each of the seven cohorts, the 90-min 5-ethynyl-20 -deoxyuridine (EdU) labelling defines a front-porch and a back-porch, namely, the most advanced cell and the most lagging cell to be found; the mid-point of this range defines the median (most typical) cell. This is demonstrated for two cohorts in Fig. 7. Considering the first cohort G1, an EdU-negative cell could have spent 6 h in end of G1 (the front porch), or 6 h coming through S (before addition of EdU), G2 and M (the back porch). The panel If EdUx2 proposes several key additional G1 time-points that could be sampled if the initial culture was duplicated and EdU added at the outset, just after photoconversion of sialyl transferase (ST)-Kaede followed by the 6-h chase.

4.81 3.55 4.18 1.18 1.82 5.94 4.81 5.38 2.38 0.63 0.7 11.76–12.45 5.75 6.45 6.10 9.10 12.1 10.06 10.06 6.00 4.06 0.97 2.03 5.03 Real duration (h) Real time Back-porch (h) Front-Porch (h) Startpoint median (h) Midpoint median (h) Endpoint median (h)

0.55 10.07–10.61 4.06 4.61 4.34 7.34 10.34

0.58 10.62–11.19 4.61 5.19 4.90 7.90 10.9

0.56 11.20–11.75 5.19 5.75 5.47 8.47 11.47

0.84 12.46–13.29 6.45 7.29 6.87 9.87 12.87

2.23 13.30–15.52 7.95 9.52 8.74 11.74 14.74

0.48 15.53–16 9.52 10.00 9.76 12.76 15.76

16 16

G1 EdU+ If EdUx2 G1 EdU++ If EdUx2 Total (h) M G2° 7 G2e+ 6 Sd 5 Sc 4 Sb 3 Sa 2 G1° 1 Cohort name Cohort number #

Table 1 Schedule of cells found in various cohorts of proliferation in Nicotiana tabacum BY2 cells

3.55 4.06 0.25 3.26 6.26

G1°° If EdUx2

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MSB/sorbitol was followed by a second wash in MSB/sorbitol containing 0.5% BSA and 0.5% Triton. The preparation was finally resuspended in this last buffer, at 15 9 106 protoplasts ml1. A 60-ll aliquot was incubated with 40 ll of JIM84 (undiluted culture supernatant; Horsley et al., 1993) for 2 h at 30°C with gentle shaking. After two washes in MSB/sorbitol/ BSA/Triton, the sample was incubated with the secondary antibody, AlexaFluor488 goat anti-rat IgG (Invitrogen), at 1 : 40 dilution, then washed once. Mannosidase Protoplasts (105) were sorted into 50 ll of 5 9 extraction buffer at 4°C (25 mM MgCl2, 25 mM Na2EDTA, 0.5% (w/v) polyvinylpyrrolidone 10 000, 0.5% (w/v) Triton X100 and 25 mM HEPES-KOH, pH 7.2). Aliquots were made up to 600 ll with PBS, vortexed, sonicated for 10 s and centrifuged at 10 000 g and the supernatant was stored at 4°C. Protein was assessed with a Nanodrop spectrophotometer (Labtech, Palaiseau, France). Mannosidase (EC 3.2.1.24) activity was assayed as the nitrophenol released from 0.6 mM 4-nitrophenol-a-D-mannopyrannoside (Sigma) at pH 4.5 during 12 h at 37°C. A 200-ll aliquot was mixed with 44 ll of 5 9 reaction buffer (325 mM sodium acetate titrated to pH 4.5 with acetic acid). The reaction was started by the addition of 6.25 ll of nitrophenol-mannopyrannoside from a 24 mM stock in DMSO. It was terminated by the addition of 600 ll of Na2CO3, and absorbance of nitrophenol was read at 405 nm against an incubation containing water rather than protoplast extract. Replicated control incubations received Na2CO3 at the outset, and were negative. Activity was expressed per aliquot and then per protoplast.

Results EdU labelling improves cell cycle resolution in BY2 protoplasts First, we checked the utility of cell cycle analysis with EdU incorporation in addition to the conventional DAPI marker. DNA histograms with DAPI-stained isolated plant nuclei are usually sufficiently precise to determine frequencies of phases G1, S and G2 (Fig. 1a). However, DNA histograms with DAPI-stained fixed protoplasts were poorly resolved (Fig. 1b): only G1 was clearly observed; S and G2/M phases were not accurately discriminated. By contrast, when a BY2 cell culture was incubated for 90 min with EdU and the derived protoplasts were processed to obtain both DAPI and EdU-AlexaFluor647 labelling (Fig. 1c), G1, S and G2/M phases could be easily determined by bivariate analysis. In addition, early S and early G2 could be defined by the cell population having incorporated EdU for only part of the 90-min incubation. This is a vast improvement over the protoplasts represented in Fig. 1(b). It is also superior to any statistical model (Fig. 1a) where it is impossible to attribute any given cell (or nucleus) to late G1 rather than early S, or late S rather than G2. New Phytologist (2015) 205: 938–950 www.newphytologist.com

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942 Methods

(a)

(c)

(b)

Fig. 1 5-ethynyl-20 -deoxyuridine (EdU) click reaction improves Nicotiana tabacum BY-2 protoplast cell cycle resolution by cytometry. (a) Histogram of 40 ,6-diamidino-2-phenylindole (DAPI)-labelled nuclei isolated from BY2 cells showing the proportions of the three phases G1, S and G2 according to nuclear DNA content. Insert, DAPI-stained nuclei. (b) DNA histogram of BY2 fixed protoplasts with DAPI staining, showing very poor discrimination between the three phases. Insert, differential interference contrast (DIC) and blue fluorescence of DAPI-stained fixed protoplast. (c) Bivariate cell cycle analysis of BY2 protoplasts. After 90 min of EdU incubation of a BY2 culture, protoplasts were prepared and fixed, EdU was revealed by AlexaFluor647azide and DNA was counter-stained with DAPI. EdU-negative G1 and G2 phases are clearly delimited, while EdU incorporation identifies early S and full-S cohorts, plus those that completed S to arrive in G2 during the 90-min labelling. Inserts, DAPI-stained EdU+ protoplast; upper insert, AlexaFluor647revealed EdU; middle insert, DIC; lower insert, DAPI. (a–c) Bars, 10 lm.

Similar results were obtained using the combination propidium iodide (PI, with Rnase) and EdU or the tandem Hoechst 33342 and EdU (not shown). Hoechst fluorescence was, however, decreased by 9% specifically in EdU-positive cells, indicating that EdU quenched Hoechst fluorescence as does its analogue BrdU (Coba de la Pe~ na & Brown, 2001), a methodological constraint. Moreover, the major compartment, G1, is unfortunately still portrayed as a uniform population, a shortcoming we propose to address using a pulse-chase strategy as described below in the section ‘Combining photoconversion pulse-chase and EdU to study the evolution of Golgi proteins in the cycle’. Copper used in the EdU click reaction quenches fluorescent proteins To demonstrate the deleterious impact of click chemistry on protein fluorescence, BY2 cells expressing reporters for the plant Golgi apparatus (ST-GFP or ST-Kaede) (Saint-Jore et al., 2002; Brown et al., 2010a) were analysed by cytometry and microscopy. In control conditions, the fluorescence associated with ST-GFP or ST-Kaede (Fig. 2a) revealed a bright punctuate fluorescent pattern characteristic of the Golgi apparatus (Aubert et al., 2011). This was not altered by EdU treatment alone (not shown). However, after addition of the click reagents, both ST-Kaede and STGFP cells lost their punctate Kaede/GFP signal (Fig. 2b). Mean cellular fluorescence declined 100-fold (Fig. 2c). We therefore tested the impact of the different components of the click reaction on fluorescence intensity. The addition of 1 mM copper sulfate  10 mM ascorbate, namely the formation of ion copper I (CuI) and copper II (CuII), largely quenched the fluorescence of ST-Kaede protoplasts, by New Phytologist (2015) 205: 938–950 www.newphytologist.com

95% (Fig. 2c). The quenching was copper dose-dependent (Fig. 2d, dashed line), with an initial linear relationship between quenching and copper concentration. However, fluorescence was almost fully quenched at 0.4 mM copper, being halved at 0.2 mM. Decreasing the copper concentration may therefore be a way to alleviate GFP quenching. The threshold copper concentration for a click reaction that was still efficient had then to be determined. The protocol for EdU revelation was applied to aliquots of EdU+_labelled protoplasts, varying only the concentration of copper in the reaction (Fig. 2d, solid curve). Maximal efficiency of the click reaction was ensured by 0.7 mM copper, a value at which Kaede fluorescence was already quenched. The addition of 0.45 mM copper, where half of the fluorescence was quenched, gave only 50% click-reaction efficiency (evaluated as AlexaFluor647 labelling). Similar results and thresholds were obtained by cytometry for the red form of ST-Kaede, and ST-GFP, indicating that the copper effect on protein fluorescence was general. These observations indicated that an acceptable compromise between the copper concentration needed for an efficient click reaction and its negative impact on fluorescent proteins was going to prove difficult. Therefore, an alternative strategy – to recover the lost fluorescence whatever its quenching – was adopted. Restoration of fluorescence intensity after copper chelation Various assays were performed with one objective: to extract copper to restore fluorescence yield. Thorough washing alone was ineffective. The strategy was therefore to strip copper with chelators such as EDTA, bathocuproine-disulfonate di-sodium, EGTA or TGTA (B. Vauzeilles, Universite Paris-Sud, Orsay, France). The best results were obtained using EDTA, as described in the next paragraph. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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(a)

(b)

(c)

(d)

The effect of an EDTA concentration series on Kaede or GFP fluorescence recovery is summarized in Fig. 3. The mean fluorescence intensity of fixed unprocessed protoplasts analysed by cytometry was taken as the reference (100%). The protoplasts were then treated with the click reaction (using 1.3 mM copper) for 90 min, and washed in PHB buffer without EDTA. Only 30% of the initial fluorescence remained. Washes were performed again, adding EDTA to the washing medium in a concentration range from 5 to 90 mM. Fluorescence recovery was observed, partly in a concentration-dependent manner (10– 20 mM EDTA), to reach finally a plateau of 70% at 20 mM. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

Fig. 2 Green sialyl transferase (ST)-Kaede fluorescence is quenched by copper during the click reaction in Nicotiana tabacum BY2 cells. (a, b) STKaede BY2 cells plasmolysed and fixed, and imaged with the same camera settings (a) before or (b) after the click reaction at 1.3 mM copper. Bars, 10 lm. (c) ST-Kaede fluorescence intensity in BY2 protoplasts before (black histogram) and after (grey histogram) 1 mM copper(II) addition, analysed by cytometry: note the 95% decline. (d) The effect of copper concentration upon ST-Kaede fluorescence (dashed line, added directly) and click-reaction efficiency (solid line, in the reaction buffer), analysed by cytometry ( SD). Conservation of Kaede fluorescence is expressed as percentage of initial fluorescence (no copper). To measure click-reaction efficiency, BY2 cells were incubated for 24 h with 10 lM 5-ethynyl-20 deoxyuridine (EdU) in order to have most cells positive for EdU, then processed with the click reaction using a copper concentration series. An index of click-reaction efficiency was calculated: (FEdU+/FEdU-) 9 (frequency EdU+ cells), where F is mean cellular fluorescence at 670/30 nm, and normalized (100 = index observed with routine 1.3 mM copper). Note that fluorescence was almost fully quenched at 0.4 mM copper, while 0.7 mM copper was necessary for full click-reaction efficiency.

Higher EDTA concentrations (20–90 mM) or longer (overnight) washes did not improve the response. Similar results were obtained with ST-GFP, but the EDTA incubation was less effective for the red form of Kaede, where fluorescence intensity was only 20% restored (not shown). These observations indicated that the quenching could be alleviated, but fluorescence was never fully restored. More critically, the protocol was not robust, the level of initial quenching and subsequent restoration varying dramatically from one experiment to another and from one tissue to another. This was already evident in the large error bars for the triplicate samples of the protoplast analyses presented in Fig. 3. Despite the high affinity of EDTA for copper and the large wash volumes, copper removal was incomplete and variable. So other parameters needed to be considered. The copper valence was then modulated in an attempt to alter copper binding or fluorescence quenching using various compounds with distinctive redox properties to test their cooperative effects with EDTA in washes, assessing quenched cells and protoplasts: ascorbate, dithiothreitol, cysteamine, glutathione, trace peroxide, Tween and counter ions (cobalt and nickel). None of these favoured fluorescence recovery. The accessibility of copper should depend upon the protonation state of surface amino acids. We therefore tested the effect of pH on the ability of EDTA to strip copper and restore fluorescence. After the click reaction, cells or protoplasts were incubated for from 30 to 180 min with 40 mM EDTA (a plateau concentration in Fig. 3) in an acidic buffer (PMB with 10 mM MES, pH 5) to test the stripping of copper. Cells were subsequently resuspended in medium at pH 7.4 to favour Kaede/GFP fluorescence. After 30 min of incubation, acidic EDTA restored most of the green Kaede fluorescence of protoplasts (88% of initial fluorescence) which reached a maximum after 90 min (> 95% of initial fluorescence) (Fig. 4). Importantly, this restoration was far more uniform than found in earlier trials: the error bars of triplicate samples (Fig. 4) were very tight. With the same protocol, red Kaede fluorescence intensity was also restored, uniformly but less completely (only 50% of initial fluorescence), suggesting that copper was more difficult to extract from the red chromophore. It was then possible to image AlexaFluor647-derived EdU with New Phytologist (2015) 205: 938–950 www.newphytologist.com

944 Methods

Fig. 3 Copper chelation by EDTA partially restores sialyl transferase (ST)Kaede fluorescence in Nicotiana tabacum BY2 cells. After the click reaction and one wash, ST-Kaede BY2 protoplasts were incubated for 3 h in increasing concentrations of EDTA in PHB, pH 7.4. After a further wash, protoplasts were analysed by cytometry. Recovery of green ST-Kaede fluorescence was expressed relative to that of fixed protoplasts from before the click reaction (100%). The large error bars ( SD) from triplicate samples reflect the variability of this inconsistent recovery.

New Phytologist process. Our protocol was tested on seedlings of A. thaliana pSCR-GFP displaying a root endoderm tagged with mGFP5ER. In these roots, having been subjected to the acid EDTA restoration protocol, both mGFP5 and AlexaFluor647 could readily be imaged (Fig. 5d–f). These results show that click reactions and fluorescence restoration protocols should be independent of the cell type and could be used directly on tissue. Similar trials were indeed successful with Medaka embryos expressing CFP in the optic crystalline (not shown). We investigated the compatibility of the click EdU reaction with the Scale clearing technique conceived to optimize photonic imaging in depth while retaining the quality of fluorescent proteins after fixation (Hama et al., 2011). Following 7–14 d of urea/glycerol treatment, tissues became highly transparent after Scale, and differential interference contrast (DIC) images could be taken deep into the roots (60–70 lm), at a greater depth than that achieved in uncleared controls (compare Fig. S1a,f). Clearing with Scale in no way interfered with DAPI labelling and with click EdU revelation and our restoration protocol (Fig. S1f–j). However, while retaining the cell-specific GFP label, the Scale protocol raised autofluorescence from endogenous plant compounds, in both GFP+ and GFP tissues (Fig. S1 h). Combining photoconversion pulse-chase and EdU to study the evolution of Golgi proteins in the cycle

Fig. 4 Copper chelation by EDTA in acidic medium consistently restores sialyl transferase (ST)-Kaede fluorescence in Nicotiana tabacum BY2 cells. After the click reaction, rinsed fixed ST-Kaede BY2 protoplasts were incubated in 40 mM EDTA in PMB, pH 5.0, for various periods (0, 30, 90 and 180 min). Protoplasts were then rinsed and resuspended in PHB, pH 7.4, to favour fluorescence. Analysed by cytometry, cellular intensity was expressed relative to that of fixed protoplasts from before the click reaction (100%). The error bars are shown, essentially the same size as the symbols ( SD). Closed diamonds, green Kaede; open circles, red Kaede.

green Kaede (Fig. 5a–c). Fluorophore characteristics were not altered as Kaede remained photoconvertible (not shown). These results show that restoration of fluorescence based on acid EDTA washes is the key to reconciling the click reaction and use of fluorescent proteins. Restoration is compatible with plant tissues and Scale clearing As the click reaction is used in various tissues and organisms, we examined how generic might be this fluorescence restoration New Phytologist (2015) 205: 938–950 www.newphytologist.com

To exploit the potential of our generic EdU-fluorescent protein protocol, we developed a ‘proof of concept’ through the study of Golgi membrane synthesis during the cell cycle of asynchronous BY2 cells, comparing the results with data obtained by unsatisfactory conventional bivariate cytometry analyses. The experimental strategy was based on the expression of ST-GFP/ST-Kaede proteins (Fig. S2a) or on immunolabelling of endogenous Golgi associated-proteins by a monoclonal antibody, JIM84, a wellknown marker for Golgi membranes (Horsley et al., 1993). The first step was to validate the use of these markers as reporters for Golgi membrane mass. Fluorescence related to Golgi membranes is linked to Golgispecific properties and not to variation in total protein First, BY2 protoplasts with (red) ST-Kaede and JIM84-AlexaFluor488 were analysed by cytometry, determining the ratio JIM84: ST-Kaede cell by cell. This ratio did not change when comparing very different populations such as G1 and G2 (the ratio differed by only 2.3% (SD 2%) for n = 10 from three experiments), indicating a similar behaviour of the two Golgi markers. This observation confirmed that the ST-Kaede transgene with its constitutive CaMV 35S promotor was reporting much like the JIM84-labelled endogenous proteins associated with Golgi membranes. Fixed BY2 protoplasts with (green) ST-Kaede or JIM84-AlexaFluor488 were then labelled with sulforhodamine101 (whole protein labelling; Sangwan et al., 1992) for cytometry (Fig. S3). In relation to total protein revealed by sulforhodamine101, our analyses showed that the two Golgi markers were not directly Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 5 Efficiency of the 5-ethynyl-20 deoxyuridine (EdU) restoration protocols with Nicotiana tabacum BY2 cells and Arabidopsis thaliana roots. (a–c) Of the four sialyl transferase (ST)-Kaede BY2 cells in this cluster, two have strongly incorporated EdU and a third (arrow) was in DNA synthesis for part of the 90-min incubation. (d–f) Arabidopsis thaliana SCR-GFP displays a root endoderm tagged with mGFP5. Following 24 h of EdU incubation, roots were fixed, permeabilized, processed with the click reaction, restored with acidic EDTA, neutralized and imaged. Nuclear DNA synthesis is marked in red. Inserts, miniatures in light transmission mode.

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correlated to cellular proteins levels. Modulation of these markers seemed to be independent of general protein synthesis (Fig. S3b). For instance, comparing several populations, doubling of protein content was associated with only an 8% increment in JIM84 labelling or a 6% increment in ST-Kaede (see coordinates identified by dashed lines in Fig. S3b), suggesting phases of synthesis for these two markers distinct from general cellular protein synthesis. To complete this validation, a third Golgi membrane protein, a-mannosidase, was analysed in relation to ST-Kaede intensity (Saint-Jore-Dupas et al., 2005). Live BY2 protoplasts were sorted into two populations according to ST-Kaede fluorescence intensity, and their respective a-mannosidase (EC 3.2.1.24) activity was assayed with 4-nitrophenol-a-D-mannopyrannoside as a substrate. An increment of 11% in ST-Kaede expression was mirrored by 13% more enzyme activity per unit protein (SD 5%; n = 12 from three experiments), corroborating the increase of STKaede protein with a synthesis of Golgi membranes. These data indicate that the Golgi-associated fluorescence of ST-GFP or STKaede correlated with endogenous Golgi membrane markers.

G1 population (using immunolabelling) or 15  2% when analysing ST-Kaede (n = 10 from three independent cultures for each marker). Besides the fact than the endogeneous and the heterologous markers again behaved in the same way, this result does not point to strong synthesis during G2. However, the temporal resolution represented by the three simple phases G1, S and G2 is too vague to precisely relate Golgi protein synthesis to a biological process – indicators of early, mid, and late phases are required. To improve resolution, we undertook to combine EdU labelling and the Kaede green/red photoconvertible Golgi marker (Brown et al., 2010a) for a pulse-chase strategy in a cytometry study. As for any ratiometric method, calculating the green:red ST-Kaede ratio cell by cell compensates for cellular variability (in size and expression level) with respect to the desired parameter ‘Golgimembrane synthesis per cell’, permitting neosynthesis to be related to the initial cellular expression level. Furthermore, as EdU identifies cohorts through S and G2, by using a time-shift between ST-Kaede conversion and EdU incubation, we should be able to attribute (red) ST-Kaede values back to subpopulations that were initially in late G1.

Bivariate analysis revealed an increase in fluorescence related to Golgi membranes mostly in G1 and to some extent in G2/M

EdU labelling revealed massive Golgi protein synthesis in early G1

Bivariate analyses of propidium-stained protoplasts either expressing ST-GFP protein (Fig. S2a) or immunolabelled with JIM84 (Fig. S2b) revealed G1:S:G2 populations. The relationship between the fluorescence of Golgi proteins and the cycle phase indicated a substantial increase during G1, a constant intensity during S, similar to that at the entry point from G1, and then a marginal increase in GFP intensity in G2/M relative to S. Evolution of JIM84-FITC intensity in BY2 cells showed features similar to those observed with the ST-GFP cell line. In replicated experiments, the mean JIM84 fluorescence of the G2 population increased only marginally: 10  2% relative to the Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

First, the stability of green and red forms of ST-Kaede in BY2 cell cultures was assessed with a multi-well plate reader (Fig. S4). Following photoconversion, ST-Kaede neosynthesis (green) was linear over 20 h, doubling in the cycle duration of 16 h. Next, a BY2 culture was irradiated with violet to photoconvert the resident ST-Kaede entirely to its red form (pulse), and then maintained for 6 h of de novo synthesis of green ST-Kaede (chase). EdU was added for the last 90 min of this chase. Cells were processed for EdU with click chemistry, washed with acidic EDTA for restoration of protein fluorescence, and then analysed by cytometry. The cytometric format is summarized in Fig. 6 and in the Materials and Methods section. New Phytologist (2015) 205: 938–950 www.newphytologist.com

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Fig. 6 Typical cytometric histograms. Regions of interest (ROI), and ROI combinations used to obtain statistics for identified cells populations in a multiparametric analysis of 40 ,6-diamidino-2-phenylindole (DAPI) and 5-ethynyl-20 -deoxyuridine (EdU)-AlexaFluor647 sialyl transferase (ST)-Kaede Nicotiana tabacum BY2 protoplasts. (a) A first ROI was determined on a dot plot with laser side-scatter (SSC) versus DAPI. (b) Using DAPI pulse area and height, doublets were discarded from the target analysis. (c) Using gating logic R2 combining ROI ‘cells’ and ‘singlets’, cells negative for Kaede fluorescence were eliminated. (d) Focusing on singlet cells positive for Kaede fluorescence (R3), the cell cycle was divided into seven parts (G1, Sa, Sb, Sc, Sd, G2e+ and G2°) identified by EdU incorporation and DAPI staining. (e) Green Kaede intensity; (f) red Kaede intensity and (g) cell-by-cell ratio of green:red (G:R) fluorescence were analysed on histograms, for each of the seven parts of the cell cycle. Only histograms from one part (G1) are shown. From 10 experiments, mean frequencies of G1:S:G2 were 65:15:20, respectively. For a total cycle time of 16 h, one deduces 10 h for G1, 2.5 h for S, and 3.5 h for the G2/M phase.

EdU revealed the position of each cell in its cycle at the end of its 6-h Kaede chase, defining seven cohorts of cells (#1–7) related to distinct positions along the cell cycle (G1, Sa, Sb, Sc, Sd, G2e+ and G2°; Table 1). At the same time, Kaede intensities were also determined for these seven cohorts identified (Figs 6d, S5). Red ST-Kaede intensity had increased modestly in cells progressing along the cycle, with much higher intensities in cells sampled late in the cycle (G2) but in fact converted (pulsed) in late G1. The green:red ratio for each point should give an estimate of the de novo synthesis of the Golgi protein for each position in the cell cycle. However, to determine this New Phytologist (2015) 205: 938–950 www.newphytologist.com

synthesis rate, two corrections of these raw data have to be made. First, a fixed denominator (R0) has to be set, as the intensity of red ST-Kaede evolved between cell cohorts (Table 1). Secondly, several of the cell cycle subphases only last c. 30 min, whereas the chase had a duration of 6 h. This end-point terminology must be subordinated to the path of each cohort during this 6-h period, which retroactively provides unique insights into late G1. Figure 7 takes into account these considerations described in Table 1. In the most advanced cohort (#7) – pulsed in late G1 – red ST-Kaede was already 160% relative to R0 (the intensity after Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist

Fig. 7 Data processed for development of the Golgi membrane throughout the Nicotiana tabacum BY2 cell cycle in a pulse-chase with sialyl transferase (ST)-Kaede (raw data, Supporting Information Fig. S5; schedule in Table 1). x-axis: time relative to cytokinesis (t0), using the mean frequencies of cycle phases observed during nine analyses, 30 min for mitosis (M) and 16 h for a full cycle. The cycle phases are indicated as in Fig. 6 (G2e+ = 5-ethynyl-20 -deoxyuridine (EdU+) early G2 cells; G2° = EdU-negative late G2 cells). y-axis (left), mean fluorescence intensity of fixed protoplasts in these phases (from three experiments, each performed in triplicate). Red ST-Kaede (red squares) is in arbitrary units where the value at time zero, R0, is the average cellular mass of this Golgi protein immediately after cell division. From microplate readings (Fig. S4), mass doubles during a full cycle to the hypothetical point t16; 2R0. y-axis (right, black triangles): the cellular ratio green:red obtained by cytometry. For the first (#1) and last (#7) of the cohorts, empty boxes represent the most advanced cell and the most lagging cell to be included at photoconversion; the mid-point of this range defines the beginning of the 6-h chase of the median cell (represented in grey). This median cell was used to represent the time coordinates (Table 1); mean red ST-Kaede values are graphed at its pulse position and the ratio is graphed at its midpoint. Red lines indicate this relation for the first and last cohorts. The typical chase durations of the other groups (#2–#6) are represented in the upper part.

cytokinesis). The accumulation of (red) Golgi protein mass rose abruptly in the last 2 h of G1. This observation is enough per se to deduce that Golgi mass increased massively in late G1. Further quantification of de novo (green) Golgi protein permits ratiometric analyses to integrate probable differences linked to cell size heterogeneity. The green: red ratio (where red is still assigned to R0) confirmed the former observations. Consistently, the green:red ratio (calculated cell by cell) rose throughout the cycle. During the 6 h of pulse-chase, it was clear that a higher rate of synthesis (higher green:R0 ratio) was attained for the cell cohort that passed through the last 2 h of G1. These data demonstrate that Golgi membrane synthesis is rather low during early G1, is massive in the latter third of G1, and persists during DNA synthesis and G2.

Discussion Click reaction revealed EdU: pertinent to cell cycle resolution The click reaction is being used in numerous protocols to derive an incorporated alkyne with an azide ligand: with DNA, lipids, Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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protein lipidation, etc. In particular, it is used to reveal EdU incorporated by cells undergoing DNA synthesis in whole organisms. EdU and BrdU analogues have proved useful for studying cell cycle kinetics, DNA replication and sister chromatid exchange, for assessing cell proliferation of normal or pathological cells or tissues (Leif et al., 2004), and for identifying stem cells in development (Alunni et al., 2010). In many such instances, fluorescent markers are used in parallel, for example to identify cell lineages (Vanstraelen et al., 2009). Our protocols permit the combination of EdU revealed using the click reaction with multicolour cytometry, in particular pulse-chase of a photoconvertible protein, to improve cell cycle analyses. Simpler and more robust than the BrdU method, EdU incorporation can be used to identify subphases within the cycle such as early, mid or late synthesis, and early or late G2 (Amiard et al., 2010; Kotogany et al., 2010). It can also be used to examine the minimal time between EdU incubation and positive-labelled mitoses, as a measure of G2 duration. But how might G1 phase be resolved into early and late subcompartments? Cell cycle resolution can be further improved by using two thymidine analogues sequentially (Alunni et al., 2010; Bradford & Clarke, 2011; Liboska et al., 2012). However, several commercial antibodies to BrdU cross-react with EdU, so they would need validation (Liboska et al., 2012), and the BrdU concentration should be low to avoid buffering later EdU incorporation. We show that associating photoconversion of a marker protein with subsequent EdU incubation is an elegant strategy to resolve G1 into subcompartments. The experimental design shown in Fig. 7 could still be enhanced by splitting the photoconverted culture into two and adding EdU immediately to the second half, for the duration of the 6-h chase. This parallel culture would allow identification of valuable new cohorts, with better insight into early and mid G1 (Table 1). Here we have combined DAPI, Hoechst 33 342 or propidium iodide with AlexaFluor647-azide to reveal incorporated EdU in plant cell cultures. Alternative fluorescent azide-ligands and other DNA dyes are available for spectral flexibility, but possible partial quenching by EdU (as observed here with Hoechst 33 342) should be checked, especially for any AT-binding dyes, EdU being a thymidine analogue. Compatibility between the click reaction and fluorescent proteins opens up new fields A commercial EdU kit (Click-IT; Invitrogen) uses a final concentration of 1.3 mM copper for catalysis, which completely quenches Kaede and GFP fluorescence. Quenching of GFP (Cubitt et al., 1995) and its analogues by metal ions, especially copper, has actually been used to develop biosensors active in the micromolar range (Eli & Chakrabartty, 2006; Rahimi et al., 2008; Isarankura Na-Ayudhya et al., 2009; Hotzer et al., 2011). Elaborate corrective immunoprotocols have been proposed to regain signals (chromotek.com/en/reagents/booster/gfp-booster/), but these are inappropriate for multiple proteins. Here we have demonstrated that a simple restoration step of acidic EDTA washes renders the click reaction compatible with GFP, Kaede New Phytologist (2015) 205: 938–950 www.newphytologist.com

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and CFP. This has been used with fixed protoplasts, cell clusters, intact roots, fish embryos and other fluorescent proteins. The EdU protocol and acidic EDTA restoration steps are also compatible with the popular Scale clearing technique of Hama et al. (2011). This practical result could be explained by changes either in the affinity of amino acids for copper or in the accessibility of copper to the chelator. The affinity of copper for fluorescent proteins is increased when amino acids are mutated into histidines (Richmond et al., 2000; Tansila et al., 2008). Histidine’s imidazole group with pKa  6.0 will be mostly protonated at more acid values, changing its electrical charge and decreasing its affinity for copper and/or change steric accessibility, favouring chelation by EDTA. Although EDTA has an amine with pK 6.13, its highaffinity copper coordination is also ensured by carboxylates with very acid pKa (Eli & Chakrabartty, 2006; Rahimi et al., 2008; Hotzer et al., 2011). The restoration of the red form of Kaede (50%) was less than that observed with the green form (95%), but was stable (Fig. 4). Indeed, photoconversion of Kaede involves irreversible covalent modifications, changing the groups neighbouring the histidine imidazole ring (Li et al., 2010). Even though fluorescent proteins showed different degrees of recovery, the restoration protocol following the click reaction was reproducible and stable with low variation in final fluorescence intensity between samples. This is a prerequisite for quantitative microscopy or cytometry and should enable new experimental strategies (Sparkes & Brandizzi, 2012), including association of EdU with the fluorescent ubiquitination-based cell-cycle indicator (FUCCI) dual-colour cycle phase markers (Kurzawa & Morris, 2010).

protein levels), validating its use as an indicator for Golgi membrane synthesis. Our ST-Kaede reporter indicates that Golgi membrane synthesis is actually a continuous process over the cell cycle, being low during early G1 and massive in the last third of G1, and persisting during DNA synthesis and G2. Indeed, half of the synthesis was in the last c. 3 h of G1. Segui-Simarro et al. (2006) found that the number of Golgi bodies increased during the G2 phase, reflecting the need for Golgi secretion during cell enlargement. In our case (BY2 suspensions), it was during G1 that cells mainly enlarged. We may then postulate that an increase in Golgi body number may simply be triggered by cell needs during any phase of the cell cycle (e.g. growth of plasma membrane and/or cell wall during G1, restoration of their number after reduction during mitosis, etc.), without any particular cell cycle checkpoint. This would then satisfy the secretory requirements of maturing or differentiating cells that are not going on to further division. To further elucidate Golgi stack genesis, whether this be by fission of pre-existing stacks, by maturation of endoplasmic reticulum (ER) domains or by de novo formation around Golgi matrix proteins (Hawes et al., 2010), the cytometer could be used to sort the fixed protoplasts based on EdU labelling for subsequent light or electron microscopy. In conclusion, the quantitative restoration of the emission of various fluorescent proteins following parallel use of click chemistry with EdU, relying on a simple acidic EDTA wash, proved to be a robust approach to track protein synthesis in plant tissues, especially when it was used concomitantly with a photoconversion pulse-chase experiment, providing insights into G1.

Marginal rate of synthesis of a Golgi membrane marker during the cell cycle

Acknowledgements

Applying this protocol to restore both forms of a photoconvertible protein, we undertook ratiometric fluorescence to assess the cell cycle marginal rate of synthesis of a Golgi membrane marker, ST-Kaede, in BY2 tobacco cells, where the green form indicated de novo protein synthesis and red the initial cellular Golgi mass. This is a photoconversion pulse-chase (PCPC) configuration in the context of an EdU-based cell cycle analysis. It is usually assumed that the Golgi apparatus must increase in mass during the cell cycle to ensure equal membrane distribution between the two daughter cells at the end of mitosis. However, the key points during the cell cycle when Golgi synthesis occurs are not clear. Contradictory reports solely based on microscopy suggest an increase of Golgi stack number during mitosis (Garcia-Herdugo et al., 1988), or during cytokinesis (Hirose & Komamine, 1989), or before mitosis (Nebenfuhr et al., 2000; Segui-Simarro & Staehelin, 2006). Our ST-Kaede-PCPC experiment using EdU quantifies the evolution of a transgenic nonfunctional marker of plant Golgi membranes during the cell cycle. The intensity of the ST-Kaede marker was strongly correlated with endogenous Golgi membrane components (Fig. S3a) recognized by the antibody JIM84 (Satiat-Jeunemaitre & Hawes, 1992) and with a-mannosidase activity (but not with cellular New Phytologist (2015) 205: 938–950 www.newphytologist.com

This work has benefited from the facilities and expertise of the Imagif Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) which is supported by the Infrastructures en Biologie Sante et Agronomie (IBiSA), the French National Research Agency under Investments for the Future programs ‘France-BioImaging infrastructure’(ANR-10-INSB-04-01), ‘Saclay Plant Sciences’ (ANR10-LABX-0040-SPS), and ‘Endorepigen’ (ANR-09-GENM105), and also the Conseil General de l’Essonne. M.B. received the Prix Jeune chercheur 2011 of the Association Francßaise de Cytometrie for this development, and thanks the association for their support. We also thank Lauranne Pujol for her benchwork and Olivier Catrice for initial cytometry (Fig. S2). Seeds of Arabidopsis thaliana SCARECROW were provided by C. Rechenmann (ISV, Gif) and embryos of Medaka, Oryzias latipes, by J-S. Joly (INAF, Gif). We thank Chris Hawes (Oxford Brookes University, UK) for providing JIM84 antibody and John Runions (Oxford Brookes University, UK) for useful discussion.

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Compatibility of the EdU and restoration protocols with the Scale clearing method.

Fig. S3 Correlation of two Golgi markers JIM84-AlexaFluor488 and ST-Kaede in BY2 cells. Fig. S4 Stability and synthesis of ST-Kaede in a BY2 cell culture. Fig. S5 Development of Golgi membrane throughout the BY2 cell cycle after photoconversion of ST-Kaede. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

Fig. S2 Bivariate analysis of DNA versus Golgi proteins in BY2 protoplasts.

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