We expressed the Arabidopsis BOR1-GFP fusion construct ⁷ in tobacco BY-2 cells under the control of the cauliflower mosaic virus 35S RNA promoter. Examination of transformed cells at growing phase using an epifluorescence microscope indicated that the fluorescence localized at the most peripheral part of the cells as well as intracellular dots ( Figure 1A). In contrast, cells grown to the stationary-phase showed faint punctate distribution of the fluorescence in the cell ( Figure 1B). To examine whether the peripherally-localized BOR1-GFP in rapidly-growing cells actually localized to the plasma membrane we stained the cells with FM4-64 for 15 min on ice and compared the pattern of FM4-64 and the GFP fluorescence using a spinning disk confocal microscope ( Figure 1C, 0 min). The peripheral staining pattern of FM4-64 is almost completely identical to that of the fluorescence of GFP. When the FM4-64 incubated cells were further incubated at room temperature for 30 min, we observed internalization of the FM4-64 signal. Under this condition we did not observe the internalization of BOR1-GFP signal ( Figure 1C, 30 min). These observations suggest that BOR1-GFP is localized to the plasma membrane in transformed tobacco BY-2 cells.

We also analyzed the distribution of the fluorescence protein after fractionation of the cell lysate into membranous organelle and soluble fractions. As shown in Figure 1D, BOR1-GFP was enriched in the precipitated fractions. This confirmed that BOR1-GFP was targeted to the membranes in tobacco BY-2 cells. Taken together, we concluded that a significant portion of BOR1-GFP expressed in tobacco BY-2 cells is targeted to the plasma membrane as in the case of Arabidopsis root cells.

In order to address whether BOR1-GFP is degraded upon supply of high concentrations of borate in tobacco cells as observed in Arabidopsis, we first analyzed the effect of borate on the growth of tobacco cells that expressed the BOR1-GFP. Tobacco cells were grown for a week in medium containing varied concentrations of borate (0.1 to 20 mM) and the volume of cells was measured. No difference in the response to cell growth was observed between non-transformed and BOR1-GFP expressing cells. Medium containing up to 5 mM borate caused little defect in the growth of cells ( Figure 2).

We then treated the transformed cells with 5 mM borate for up to 90 min and the fluorescence of BOR1-GFP was monitored under an epifluorescence microscope ( Figure 3A). We found that the BOR1-GFP signal at the peripheral part of the cells decreased significantly during incubation. The same decrease in signal was also observed in the presence of 3 mM borate. Interestingly, lower concentrations of borate (0.1 mM) did not cause a loss of the BOR1-GFP signal. This decrease is specific for BOR1-GFP as the same treatment did not decrease the fluorescence of plasma membrane intrinsic protein (PIP)-GFP, which is a plasma membrane aquaporin fused with GFP.

We also analyzed the fate of BOR1-GFP after separation of cellular proteins by SDS/PAGE and recorded the fluorescence ( Figure 3B). We found that BOR1-GFP signal was decreased in the presence of higher concentrations of borate. This decrease was specific for BOR1-GFP as this protein was not decreased without exposure to borate and the level of PIP-GFP was not changed even with high concentrations of borate. Quantification of the intensities of the BOR1-GFP band allowed us to estimate the kinetic parameters of the decline in the signal intensity. The decrease ratio fitted well into an index recurrence curve with a lag time of 11.7 min and half life of 62 min ( Figure 3C), suggesting that the degradation kinetics is first order. The presence of a lag time suggested that several steps of cellular events must have taken place before degradation of the BOR1-GFP.

As described above, BOR1-GFP but not PIP-GFP was degraded upon addition of borate. In Arabidopsis roots BOR1-GFP behavior differs from some other plasma membrane proteins in both borate-dependent degradation and polarized localization in cells ^{7, 8} . To address whether localization of BOR1-GFP and PIP-GFP differs in tobacco BY-2 cells, we compared the localization of these proteins in tobacco BY-2 cells at the early log-phase stage ( Figure 4).

With our culture conditions, BY-2 cells at the stationary phase are largely unicellular. Cells at this stage are approximately three-times longer than cells at mid-log phase. When such cells are transferred to fresh medium, they start to divide at the center resulting in two daughter cells which elongate slightly and further divide at the center of each daughter cell without separation. At this stage, a linearly attached clump of cells contain four cells. The clump has four cell walls with a distinct location and age; the one between the two cells that generated at the first cell division, the one between the two cells that generated at the second cell division, the one exposed to the culture medium in the center of two cells, and the one exposed to medium of the two extreme cells containing tips of the cell wall clump. We name these four faces of cells as A1, A2, E1 and E2, respectively ( Figure 4B).

We measured the density of the green fluorescence of these four faces from cells expressing BOR1-GFP and PIP-GFP and the distribution of their relative density (relative to the E1 face). In both cases, the fluorescence density at E1 and E2 was almost the same but in the case of PIP-GFP, the densities at A1 and A2 were approximately 1.5- and 2-fold higher than that of E1, respectively ( Figure 4C). These fluorescence intensities between cells suggest that PIP-GFP is distributed nearly evenly in the plasma membrane as the A1 and A2 faces have two sheets of plasma membrane. In contrast, the density of BOR1-GFP at both A1 and A2 faces were approximately 5-fold higher than that of E1 ( Figure 4C). This observation indicated the polarized localization of BOR1-GFP and clearly showed that this fusion protein accumulates to the plasma membrane that is facing neighboring cells in growing tobacco BY-2 cells.

The observation that BOR1-GFP is degraded upon addition of borate in the medium ( Figure 3) suggest two possible scenarios for the borate-dependent decrease in BOR1-GFP. The first possible case is that the decrease of BOR1-GFP in the presence of high concentrations of borate is the result of inducible degradation. Another possibility is that the rate of the degradation of BOR1-GFP is rapid in BY-2 cells even in the absence of borate but a high rate of biosynthesis of BOR1-GFP in the normal medium maintained the level of the transporter.

To address the likely scenario causing the BOR1-GFP decrease in tobacco cells under high borate conditions, we treated the transformed cells with a protein biosynthesis inhibitor cycloheximide (CHX) in either normal or high-boron medium and monitored the level of BOR1-GFP ( Figure 5). In the normal borate medium, addition of CHX did not change the level of BOR1-GFP significantly over 2 hours. This observation indicated that the turnover of BOR1-GFP in normal medium is slow and most of the BOR1-GFP showing the green fluorescence is the result of the accumulation of this protein. This also indicated that the decrease in BOR1-GFP in high borate medium is a result of inducible degradation.

We observed partial inhibition of BOR1-GFP degradation in the presence of CHX ( Figure 5). This observation indicated that continuous synthesis of degradation machinery is necessary for the continuation of the degradation. The partial suppression of the degradation also indicated that the machinery for the degradation is already present in cells that were cultured in the normal medium.

It was shown previously that endocytosis is the first step of BOR1-GFP degradation in Arabidopsis ^{7, 8} . To assess whether similar machinery operates in tobacco BY-2 cells in the presence of high concentrations of borate, we first analyzed the BOR1-GFP signal in cells cultured for 1 to 2 hours under high borate conditions using a confocal laser scanning microscope (CLSM). We found intracellular puncta in cells treated with high borate ( Figure 6A). We then analyzed the colocalization of the BOR1-GFP signal with an early endosome that can be marked with FM4-64. Tobacco cells expressing BOR1-GFP were incubated for 2 hours in a medium containing 3 mM borate and then the cells were stained with FM4-64 on ice, washed with cold medium containing 3 mM borate, after which they were returned to room temperature to resume intracellular trafficking as described ¹⁴ . Five to 10 min after FM4-64 incubation, dotted structures emitting red fluorescence, which represent an early endosome distinct from the trans-Golgi network (TGN) in tobacco cells ¹⁴ , was observed ( Figure 6B, left). Most of the early endosome signal colocalized with intracellular dots of BOR1-GFP, suggesting that BOR1-GFP passes through early endosome during degradation as in the case of Arabidopsis cells.

It is also known that FM4-64 is transported from the early endosome to the TGN, and thereafter targeted to the vacuolar membrane. To evaluate whether BOR1-GFP passes through the TGN, we expressed SCAMP2-mRFP ¹⁴ in cells expressing BOR1-GFP. SCAMP2-mRFP localizes in the plasma membrane, the TGN and the secretory vesicle cluster, which is an exocytotic compartment derived from the TGN ¹⁴ . No colocalization of green and red intracellular dots was observed when cells were treated with 3 mM borate for 2 hours ( Figure 6C). These findings suggested that the BOR1-GFP is internalized to endosomes and then sorted to a degradation pathway from the endocytotic route, which is directed to the TGN.

It was previously reported that the treatment of Arabidopsis roots with brefeldin A (BFA), which is an inhibitor for both endocytosis and exocytosis, prevents the borate-dependent degradation of BOR1-GFP ⁷ . To address whether this compound is also effective in tobacco BY-2 cells and to assess the mechanism of the degradation, we analyzed borate-induced degradation in the presence of various inhibitors ( Figure 6). We used BFA, 2,3,5-triiodobenzoic acid (TIBA), a compound that stabilizes the actin bundle ¹⁵ ; tyrphostin A23, which is a tyrosine kinase inhibitor, which can inhibit endocytosis by competing with the interaction of the AP-2 adaptor complex and Tyr-containing endocytosis motifs in mammalian cells and also can inhibit the endocytosis of rice SCAMP1-YFP and FM4-64 in tobacco BY-2 cells ^{16, 17} ; ikarugamycin, which is an inhibitor of clathrin-mediated endocytosis in tobacco ¹⁸ ; colchicine, which is an inhibitor of microtubule depolymerization; latrunculin B, which inhibits actin polymerization; 2,3-butanedione monoxime (BDM), which is a general myosin ATPase inhibitor and inhibits the endocytosis of SCAMP2-mRFP in tobacco BY-2 cells ¹⁴ ; MG-132, which is an inhibitor of proteasomes at low concentrations and also inhibits other proteases; and K-252a, which is a general Ser/Thr type protein kinase inhibitor.

Three hours after application of inhibitor solution to the tobacco cell culture, borate was added at a final concentration of 5 mM and further incubated for 2 hours. Thereafter, the amount of BOR1-GFP was quantified ( Figure 7A). We found that BFA and TIBA inhibited degradation almost completely (>80% remained) at a concentration that inhibits the endocytosis of SCAMP2-YFP ¹⁴ . A slightly weaker effect of tyrphostin A23 was observed at 400 μM, which is a concentration similar to that used in the works of Leborgne-Castel et al. ¹⁶ and Lam et al. ¹⁷ . In this case, approximately 70% of BOR1-GFP remained in the cell. We also found partial inhibition of degradation by BDM at 5 mM, the concentration that inhibits the endocytosis of SCAMP2-YFP ¹⁴ . We also observed partial inhibition by MG-132 at 400 μM. In these two cases, approximately 40% of BOR1-GFP remained in the cell. Other inhibitors tested did not cause any detectable effect on the extent of degradation.

Time-course analysis of the decrease of BOR1-GFP in the presence of TIBA and tyrphostin A23 ( Figure 7B) clearly showed that most of the degradation was inhibited up to 3 hours. We next addressed whether the inhibition occurred at the point of endocytosis from the plasma membrane or at intermediate compartments. Fluorescence images were collected in transformed tobacco cells in the presence of both inhibitors and borate. We found that the images were not significantly different up to 3 hours in the presence of either BFA or TIBA ( Figure 7C). These observations suggested that both BFA and TIBA inhibited the early event of endocytosis. We could not analyze fluorescence images in the presence of tyrphostin A23 because treatment of BY-2 cells with this compound increased cellular fluorescence significantly and such fluorescence was independent of the expression of BOR1-GFP.

To address whether the inhibition of endocytosis required BY-2 cells to be pretreated with BFA, tyrphostin A23 or TIBA, we investigated the timing of adding the inhibitors ( Figure 7D). We compared the level of BOR1-GFP without inhibitors with BOR1-GFP levels when these inhibitors were added into the culture. We found that addition of inhibitors at the same time, in the lag time as well as during the degradation phase, prevented the decrease in BOR1-GFP almost completely. These observations indicated that these inhibitors have a direct effect against the endocytosis of BOR1-GFP and were not a side effect of these chemicals. These observations also suggested that actin dynamics, GDP-GTP exchange of Arf small GTPase, and either tyrosine phosphorylation or AP-2 adaptor interaction are necessary for the degradation of BOP1-GFP in tobacco BY-2 cells.

It was shown that both polarized localization and borate-dependent degradation are mediated by specific Tyr residues in BOR1-GFP in Arabidopsis root cells ⁸ . To address whether these Tyr residues also contribute to the localization and borate-induced degradation of BOR1-GFP in tobacco BY-2 cells, we expressed Y373A/Y398A/Y405A mutants of BOR1-GFP in BY-2 cells ( Figure 8). Confocal images of the mutant BOR1-GFP displayed higher intensity of fluorescence signals between attached cells as in the case of wild-type BOR1-GFP ( Figure 8A). Quantification of the intensities of different faces indicated that this protein also showed a polar localization, although the polarized nature seems somewhat weaker than the wild-type ( Figure 8B). This observation indicated that the polarized localization of BOR1-GFP in tobacco cells is largely independent of the tyrosine residues in the BOR1 protein.

When tobacco cells expressing the mutant protein were incubated in medium containing 5 mM borate, we still observed strong fluorescence of GFP signals. We did not detect any difference in the pattern and intensity of the fluorescence signals between cells incubated in normal medium and high borate medium ( Figure 8C). Under the same condition, most of the wild-type BOR1-GFP was degraded in the cell ( Figure 3, Figure 8C). This observation indicated that Y373, Y398, and/or Y405 residues of BOR1-GFP are involved in borate-induced degradation in tobacco BY-2 cells as in the case in Arabidopsis root cells.

Plasmids for the expression of wild-type and mutant BOR1-GFP, and SCAMP2-mRFP have been described previously ^{7, 8, 14} . The expression plasmid for PIP-GFP was a kind gift from Dr. M. Maeshima (Nagoya University). This plasmid contains radish plasma membrane type aquaporin PAQ1 ²⁹ and GFP fusion protein under an enhancer-duplicated cauliflower mosaic virus 35S promoter ³⁰ .

The following chemicals were used to test the inhibition of the degradation: BFA (Wako Pure Chemicals, Osaka, Japan), CHX (Wako Pure Chemicals, Osaka, Japan), TIBA (Aldrich Chem. Co. USA), Tyrphostin A23 (Sigma-Aldrich, Inc, Missouri, USA), colchicine (Tokyo Kasei Co., Tokyo, Japan), K-252a (Sigma-Aldrich, Inc, Missouri, USA), BDM (Sigma-Aldrich, Inc., Missouri, USA), Latruncurin A (Calbiochem, San Diego, USA), ikarugamycin (BIOMOL Research Labs Inc. USA), MG-132 (Calbiochem Inc. San Diego USA), wortmannin (Calbiochem Inc. San Diego USA), 3MA (Sigma-Aldrich, Inc., Missouri, USA) and E-64 (Peptide Institute, Osaka, JAPAN).

The culture of tobacco BY-2 cells (a kind gift from Drs. K. Nakamura (Nagoya University) and T. Nagata (University of Tokyo) and the transformation of this cell line were as described ³⁰ . Cells were cultured weekly and 3 or 4 day-old cultures were used unless otherwise stated. For transient transformation of cells expressing BOR1-GFP to express SCAMP2-mRFP in the same cell, Agrobacterium tumefaciens EHA105 ³¹ harboring a binary plasmid was co-cultured with BOR1-GFP expressing tobacco cells for 2 days, washed with fresh medium and further incubated in medium containing 200 mg/L cefotaxime for a day. Thereafter fluorescence of cells was visualized using CLSM.

Cells expressing GFP fusion proteins were lysed with equal volumes of buffer consisting of 0.4 M Sorbitol, 0.1 M KOAc, 6 mM EGTA, 4 mM EDTA, 40 mM HEPES-KOH, 2 mM DTT, 1 drop/100 ml protease inhibitor tablet (Complete EDTA-free, Roche), pH 7.3 at room temperature. Cells were lysed by sonication and the resulted cell lysate was centrifuged at 300 × g for 10 min. The resulting supernatant was used as the cell lysate. Proteins in the cell lysate was mixed with 1/5 volume of a buffer consisting 0.25 M Tris-HCl, 0.5 M dithiothreitol, 50 % glycerol, 10 % SDS, 0.025 % bromophenol blue, pH 6.8 and loaded on SDS-polyacrylamide gels without heating. Fluorescence images of the gels were recorded using Typhoon 9600 scanner (GE Healthcare, Tokyo, Japan) at the following setting; filter 520SPCy2, excitation 488 nm blue laser at PMT 650, medium sensitivity). The intensities of bands were quantified using Image Quant software (GE Healthcare).

For epifluorescence detection, cells were mounted on glass slides and observed using IX40 inverted microscope with an LCPlanFL 20X lens using NBY excitation/emission filter set and a DP-70 camera (Olympus Co. Tokyo Japan). For time-lapse image collection, cell suspension was placed into in a well of glass-bottom 96 well micro-plates (EZVIEW® Glass Bottom Culture Plates LB, Asahi Techno Glass Co. Tokyo, Japan). Cells in the plate wells were observed using an IX80 inverted fluorescence microscope equipped with DSU spinning disk confocal unit and an Uplan SApo 40×/0.90 lens using NBY or RFP excitation/emission filter sets (Olympus). Images were collected using an iXON+-DU888E-CS0-BV camera (Andor) under the control of Meta Imaging Series 7.6.3 software (Molecular Devices Inc. Sunnyvale, CA, USA).

For the colocalization analysis, cells were mounted on slides with culture medium. These cells were observed using a CLSM system (LSM510 META, Axioplan2 Imaging; Carl Zeiss) with a Plan-Apochromat lens (633 1.4 oil differential interference contrast; optical slices of 1 mm). We used a 25-mW argon laser (power, 5%) with 488-nm excitation and a 505–530-nm band-pass filter for GFP8. For FM4-64 (Molecular Probes, Eugen, USA) we used a 650-nm long-pass filter and 488-nm excitation. A 560-nm long-pass filter and 1-mW He-Ne laser at 80% power with 543-nm excitation was used for Alexa Fluor 568 and mRFP. Crosstalk was prevented using a multitrack configuration with line sequential scanning. Composite figures were prepared using Zeiss LSM Image Browser software.

For the measurement of intensity, confocal images at 256 bit tiff file was quantified using ImageJ software. If required, relative density to the E1 face was calculated in each cell clump.

An aliquot of 0.5 M boric acid in water was added to 96.5 ml of suspension culture of three-day old tobacco cells expressing BOR1-GFP in a 300 ml Erlenmeyer flask at a concentration of 3 or 5 mM (as indicated in the legends to figures) and further cultured with the same condition of regular culture. For the control, cells grown in a normal culture medium, which contains 0.1 mM borate, was used. At the indicated time, a 0.5 or 1 ml aliquot of the culture was removed from the flask and used for the analysis.

For the comparison of the two values student’ t-test was employed using Microsoft Excel software. For the comparison of difference with multi values, Tukey’s test was used.