The organic anion transport inhibitor, probenecid, inhibits the transport of

Lucifer Yellow at the plasma membrane and the tonoplast in suspension-

cultured plant cells

LOUISE COLE1, JULIAN COLEMAN2, ANNE KEARNS1, GARETH MORGAN1 and CHRIS HAWES1-*

1 School of Biological & Molecular Sciences, Oxford Polytechnic, Gipsy Lane, Headington, Oxford 0X3 OBP, UKdepartment of Plant Sciences, Oxford University, South Parks Rd, Oxford 0X1 3RA, UK

* Author for correspondence and reprints

Summary

In this paper we report on the uptake of themembrane-impermeant fluorescent probe LuciferYellow CH (LY-CH) into the vacuolar system of plantcell suspension cultures. LY-CH is internalised intovacuoles of maize cells at a faster 'rate' than carrotcells and in each case, the probe is also trapped at thecell wall. In the presence of the uricosuric drugprobenecid, the vacuolar uptake of LY-CH by carrotand maize cells is inhibited and in some cellsinternalisation of probe is blocked at the plasmamembrane. In electroporated carrot cells, LY-CH issequestered slowly from the cytoplasm into vacuoles

by a probenecid-inhibitable transport process. Theseresults are compared with the effects of probenecidon the sequestration of LY-CH from the cytoplasminto the lysosomal system of fibroblasts. In view ofthe above findings and recent evidence for theputative uptake of LY-CH by fluid-phase endocytosisin plant cells, the possibility that LY-CH is trans-ported across plant membranes via probenecid-inhibitable organic anion transporters is discussed.

Key words: endocytosis, electroporation, Lucifer Yellow CH,probenecid, plant cell suspension cultures.

Introduction

The most convincing evidence for endocytosis in plants hasbeen achieved by studies conducted at the electronmicroscope (EM) level involving the use of electron-opaquemarkers, e.g. cationised ferritin (Tanchak et al. 1984,1987,1988; Joachim and Robinson, 1984) and gold-conjugatedlectins (Hillmer et al. 1986). However, because the plantcell wall acts as a barrier to the passage of many electron-opaque probes, such endocytotic studies have been con-fined mainly to protoplasts.

Apart from some early reports on the uptake ofmacromolecules by plants (Brachet, 1954; Jensen andMcLaren, 1960) and more recent results on the internali-sation of heavy-metal salts into roots (Hiibner et al. 1985;Samuels and Bisalputra, 1990), there has been littleevidence to suggest that turgid plant cells can endocytose.It is only recently that results from experiments usingfluorescently labelled probes (e.g. FITC-labelled proteins,FITC-dextrans, Lucifer Yellow) have supported thecontention that fluid-phase endocytosis may take place inwalled cells (Oparka and Prior, 1988, Oparka et al. 1988;Hillmer et al. 1989,1990; Horn et al. 1989,1990; Cole et al.1990; Robinson and Hillmer, 1990; Owen et al. 1991).

The small, low molecular weight fluorescent molecule,Lucifer Yellow carbohydrazide (LY-CH, Afr=457.2), adisulphonated 4-aminonapthalimide, like all LuciferYellow derivatives, is intrinsically membrane-imper-meant, since it is highly negatively charged (being highlydissociated at physiological pH values: pKB values ofJournal of Cell Science 99, 546-555 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

disulphonates =£0.7) and thus extremely lipid-insoluble. Itpossesses a high quantum yield that is stable betweenpH 1 and 10, is extremely soluble in water and has a lowtoxicity to cells (Stewart, 1978, 1981).

Because of the properties listed above, LY-CH has beenused to demonstrate fluid-phase endocytosis in culturedanimal cells (Miller et al. 1983; Swanson et al. 1985, 1986,1987; Bernardini et al. 1986, 1987; McKinley and Wiley,1988; Cosson et al. 1989). In these studies, the probe wassequestered rapidly into the endosomal and lysosomalcompartments from the extracellular fluid and the dye wasretained in these compartments even after thoroughwashing of the cells. LY-CH has also been employed as afluorescent tracer to study endocytosis in yeast cells(Riezman, 1985; Riezman et al. 1986) and in the dimorphicfungus Candida albicans (Basrai et al. 1990).

As a result of these studies LY-CH is now being used toinvestigate fluid-phase endocytosis in plant systems. Thecompartmentalisation of LY-CH into plant vacuoles fromthe extracellular medium has been attributed to theinternalisation of the dye via a fluid-phase endocytoticpathway in .plant cells (Hillmer et al. 1989; Oparka andPrior, 1988; Oparka et al. 1988; Hillmer et al. 1989, 1990;Robinson and Hillmer, 1990; Owen et al. 1991). Incorroboration with these results, previous plant micro-injection studies involving LY-CH have also shown a lackof entry of the probe into vacuoles when the dye wasinjected into the cytoplasm directly (Erwee et al. 1985;Terry and Robards, 1987; Oparka and Prior, 1988; Hillmeret al. 1990). There have, however, been a few reports to

545

suggest that LY-CH, following its injection into thecytoplasm, can traverse the tonoplast (Fisher, 1988;Goodwin et al. 1990).

Research on mammalian cells in vitro has shown thatfollowing ATP*4" permeabilisation of the plasma mem-brane (PM), LY-CH is sequestered directly from thecytosol into the lysosomal system (Steinberg etal. 1987a,6,1988). At the same time, the drug probenecid (p-[dipropylsulphamoylj-benzoic acid), which inhibits therenal tubular secretion of organic anions and enhancesblood levels of antibiotics by decreasing the clearance ofdrugs, e.g. penicillin (Cunningham et al. 1981; Kahn andWeinman, 1985; Kahn et al. 1985), has been shown toprevent this sequestration of LY-CH into the lysosomes(Steinberg et al. 19876, 1988). Thus, these findings implythe presence of probenecid-inhibitable organic aniontransporters in the membranes of the endosomes/earlylysosomes.

In this paper we demonstrate the internalisation of LY-CH from the extracellular medium into the vacuoles ofsuspension-cultured cells of carrot and of maize. We showthat the LY-CH when inserted into the cytoplasm byelectroporation is subsequently sequestered into thevacuolar system. In addition, we show that probenecidinhibits the uptake of LY-CH at both the plasmamembrane and the tonoplast in the plant cells studied butonly at the endosome/lysosome membrane in fibroblasts.These results are discussed in the light of recent reports onthe putative endocytosis of LY-CH by plant cells and, inturn, question the reliability of the dye as a probe for fluid-phase endocytosis in plant systems.

Materials and methods

Cell suspension cultures of carrot (Daucus carota L.) were grownin Murashige & Skoog medium (M & S; Flow Laboratories;pH5.6) supplemented with 2.5% sucrose (BDH Chemicals Ltd),O.lmgdm 2,4-dichlorophenoxyacetic acid (2,4-D) (Sigma Ltd),O.lmgdm"3 t-zeatin (Sigma Ltd) and 6% coconut milk. Cellsuspension cultures of maize (Zea mays var. Black MexicanSweetcorn) were grown in M & S (pH5.3) supplemented with 2 %sucrose and 2mgdm~3 2,4-D. Cells (10 ml) were subculturedevery 14 days into 100 ml of sterile culture medium andmaintained as a fine suspension in a LHE orbital shakerincubator Mark 10 (140 revs min"1) at 26 °C, with a light intensityof 17/iEm"2s~1 for a 12 h day. Protoplasts were isolated fromcarrot cells three to five days after subculturing as described byColeman et al. (1987).

Estimates of cell and protoplast viability were determinedusing 0.01 % fluorescein diacetate (FDA) as described by Widholm(1972). Unless otherwise stated, the viability of plant cells andprotoplasts employed in all experiments was greater than 85 %. Inaddition, all plant experiments were performed Ln the dark at25 °C.

Mouse 3T3 fibroblasts were cultured on 13 mm round coverslipsin Iscove's Modified Dulbecco's medium (IMDM; pH 7.0) for 4 daysprior to use.

Epifluorescence microscopyEffect of probenecid. Cell suspension cultures of carrot (5-day-

old) and maize (5- to 6-day-old) were prewashed several times insterile 50 mM Mes (Sigma Ltd)-buffered (pH 6.3) M & S culturemedium (as appropriate) in the presence or absence of 5mMprobenecid (Sigma Ltd) prior to LY-CH treatment. Duringwashing, cells were pelleted by centrifugation at 1000 g for 2 minusing an MSE Centaur 2 bench top centrifuge. Cells wereincubated 1:1 (pcv(packed cell volume)/v) with LY-CH (Li+ or K+

salts, Sigma Ltd) at a final concentration of 0.5-1 mg LYml"1

Mes-buffered M & S culture medium±2.5mM probenecid in

sterile tissue culture dishes (5.0cm diam.x2.0cm deep, Sterilin)with constant agitation. Samples (1 ml) were removed after eachtreatment and washed by centrifugation (1000 g, 2 min) fourtimes with the appropriate medium (i.e. ±probenecid). Cells werethen mounted on slides and observed by epifluorescence mi-croscopy.

Carrot protoplasts were also treated 1:1 (pcv/v) with LY-CH(0.5 mg ml"1 50 mM Mes-buffered M & S culture medium (pH 6.4))in sterile tissue culture dishes (3.5 cm diam.xl.0cm deep, MetlabSupplies Ltd) in the presence and absence of probenecid.Protoplasts were washed by gentle centrifugation at 100 g for10 min prior to epifluorescence microscopy.

For a comparative study, the effects) of probenecid on theuptake and transport of LY-CH by mouse 3T3 fibroblasts wereinvestigated as follows: cells cultured on coverslips wereincubated in IMDM (pH 7.0) containing 0.5 mg ml"1 LY-CH in thepresence or absence of probenecid (5 mM) for 30 min at 37 °C. Cellswere washed three times with phosphate-buffered saline(PBS) ± probenecid and observed by epifluorescence microscopy.

Effect of low temperature. Suspension-cultured carrot cells (4-to 5-day-old) were prerinsed several times with Mes-buffered(pH5.6) M & S culture medium at 25°C and 0-4°C. Cells werethen incubated 1:1 (pcv/v) with LY-CH (final concentration of0.5 mgml"1) in Mes-buffered M & S culture medium at 0-4°C and25°C as appropriate. After 21 h, samples (lml) were removed,washed several times in buffered medium at the appropriatetemperature, mounted and observed by epifluorescence mi-croscopy.

Specimens were observed with a Zeiss standard epifluorescencemicroscope equipped with a BP450/490FT510 and BP520/560filter set and micrographs were recorded on Dford HP5 film.Confocal microscopy was carried out with a BIORAD LasersharpMRC 500 laser scanning confocal microscope.

ElectroporationSuspension-cultured carrot cells (3- to 7-day-old) prewashed incold (0-4 °C) M & S culture medium containing 10 mM Hepes(pH7.0) were electroporated (PG 200 Progenetor II, HoeferScientific Instruments) in the presence of lmgml"1 LY-CH(±probenecid) by a single electrical pulse (t=lms) with a fieldstrength equal to 1250Vcm"1 (capacitance=220jiF). Duringpermeabilisation, cells were maintained at a low temperature (onice) within the electroporation chamber. Viability of the cellsfollowing electroporation, as measured by FDA staining, wasapproximately 50 %. After electroporation, cell samples were kepton ice for 10-20 min and subsequently placed at 25 °C on a rockingtable for 45 min to allow sealing of plasma membranes. Cells werethen washed four times by centrifugation (1000 g, 2 min) withbuffered medium (± probenecid) at 25 °C prior to epifluorescencemicroscopy. At this stage, samples (0.75 ml) of the final cellsuspension were incubated further (1-5 h) and washed once inbuffered medium in the absence of probenecid, prior to epifluor-escence microscopy.

Mouse 3T3 fibroblasts were electroporated at 4°C in thepresence of 0.5mgml"1 LY-CH in a 10mM Hepes buffer (pH7.4)containing 150 mM NaCl. Electroporation was achieved by asingle pulse (i=lms) with a field strength of 1875 V cm"1

(capacitance=490/*F). Cells were subsequently incubated on icefor 10 min, washed three times in IMDM and observed after a 30-min incubation at 37 °C. Cells were also electroporated as above inthe presence of LY-CH plus 2.5 mM probenecid. After permeabili-sation these cells were incubated in IMDM plus 2.5 mMprobenecid (lh) and washed thoroughly with this medium. Insome cases, cells were then incubated further in IMDM only(30 min), prior to mounting and observation by epifluorescencemicroscopy.

Spectrofluorimetry: uptake of LY-CH versus timeEffect of probenecid. Suspension-cultured carrot cells (5-day-

old) were prewashed three times with 50 mM Mes-buffered(pH6.3) M & S culture medium in the presence and absence ofprobenecid (5mM), prior to LY-CH treatment. Cells wereincubated 1:1 (pcv/v) with LY-CH (lmgml"1) in Mes-buffered M& S culture medium±probenecid (2.5 mM) as described pre-

546 L. Cole et al.

viously. At appropriate intervals, 1 ml samples were removed andfiltered onto pre-wetted 25 mm diameter Whatman cellulosenitrate filters (0.45 ̂ m pore size) under low suction on a HoeferFH224 filtration unit. Cells were subsequently washed threetimes with Mes-bufTered medium, then the filter plus cells wereremoved and immersed completely in 2 ml of 5 % aqueous sodiumdodecyl sulphate (SDSXSigma Ltd) for lOmin. Samples (lml) ofSDS-treated cells were pelleted at 9600 # for 15 s using anHeraeus Sepatech Biofuge B. Samples (500 /d) of the supernatantwere made up to 3 ml with double-distilled water for spectrofluori-metric analysis. Quantitative measurements of LY-CH weremade with an Hitachi F-2000 fluorescence spectrophotometer atexcitation and emission wavelengths of 430 nm and 520 nm,respectively.

The effect of probenecid on the uptake of LY-CH versus time bycarrot protoplasts was also investigated: protoplasts were pre-rinsed twice with 50 mM Mes-buffered (pH6.4) M & S culturemedium prior to incubation with LY-CH (1 rngml"1 Mes-bufferedmedium)±probenecid (2.5 mM). Throughout the treatment period,samples (0.5 ml) were taken and washed thoroughly by centrifu-gation. The protoplast pellet was resuspended to 1.5 ml withaqueous 5% SDS for 2min, repelleted at 9600 g for 15 s andsamples (0.5 ml) of the supernatants were removed and made upto 3 ml with double-distilled water prior to spectrofluorimetricanalysis.

Effect of low temperature. Cells were prewashed three timeswith Mes-buffered (pH5.6) M & S culture medium at 0-4°C and25 °C prior to LY-CH treatment. At the corresponding tempera-ture, cells were incubated with LY-CH (O.Smgml"1) and atcertain time intervals, samples (1 ml) were washed in bufferedmedium at 0—4°C or 25 °C, as appropriate, prior to SDS treatmentand spectrofluorimetric analysis.

Results

Uptake of LY-CH by suspension-cultured plant cellsWhen suspension-cultured maize cells were incubatedwith LY-CH K+ for l h at 25°C, fluorescence was observedin vacuoles and at the cell wall (Fig. 1A,B). Further tothis, after a 16 h LY-CH incubation the vacuoles of maizecells fluoresced intensely (Fig. 1C,D). In comparison, whensuspension-cultured carrot cells were incubated with thesame probe for 4 h LY-CH fluorescence was observed at thecell wall but no fluorescence was observed inside the cell(Fig. 1E,F). However, after a 20 h LY-CH incubation, LY-CH was also visualised within the vacuolar system ofcarrot cells but it was excluded from the cytoplasm(Fig. 1G,H).

LY-CH accumulated in the vacuoles of carrot cellprotoplasts following prolonged LY-CH treatment (9-18 h)and no fluorescence was observed in the cytoplasm(Fig. 3G,H). Little uptake of LY-CH into vacuoles of carrotprotoplasts was observed prior to this.

When LY-CH-treated (18 h) suspension-cultured carrotcells were observed by confocal laser scanning microscopyin the fluorescence mode, intense fluorescence wasdetected in the vacuoles and at the cell wall (Fig. 2).

Effect of probenecid. When suspension-cultured carrotcells were incubated with LY-CH for 21 h in the presence ofprobenecid (2.5 mM), LY-CH fluorescence was observed atthe cell wall but not in the vacuoles. In many cells, the dyeappeared to accumulate in the cytoplasm (Fig. 3A-D).However, in some cells no internalisation of LY-CH wasobserved and only traces of fluorescence were detected atthe cell wall (Fig. 3E,F). When carrot cells that had beentreated with LY-CH in the absence of probenecid, werewashed in probenecid-medium for 10 min, the drug had noeffect on the compartmentalisation of LY-CH (not shownhere).

The uptake of LY-CH into vacuoles of carrot protoplastsfollowing a 9h LY-CH incubation was also blockedcompletely in the presence of 2.5 mM probenecid. In thiscase, LY-CH fluorescence was observed in the cytoplasmand appeared brightest at the nuclei (Fig. 3I,J comparewith Fig. 3G,H). In addition, in some protoplasts nointernalisation of LY-CH was observed (not shown here).

Further to this, the uptake and accumulation of LY-CHinto vacuoles of suspension-cultured maize cells after a16 h LY-CH incubation was also inhibited by 2.5 mMprobenecid (Fig. 4A,B). As expected, in many cells LY-CHfluorescence was observed in the cytoplasm and in thenucleus but in some cells no internalisation of the probewas detected. Traces of LY-CH fluorescence could also beseen in the cell wall. In no experiments were there anymajor morphological changes to the plant cells afterprobenecid treatment.

Effect of low temperature (0—4°C). When suspension-cultured carrot cells were incubated with LY-CH for 21 hat 0-4 CC intense LY-CH fluorescence was observed at thecell wall but no dye was detected inside the cell (not shownhere).

Transport of LY-CH following electroporation of fluor-escent probe into cytoplasm of suspension-cultured carrotcells. Following electroporation of suspension-culturedcarrot cells in the presence of LY-CH (lmgml"1), it wasobserved that LY-CH fluorescence was restricted predomi-nantly to the cytoplasm (Fig. 5A). However, after a 1 hincubation of electroporated cells in LY-CH-free medium(25 °C) there was a significant increase in the number ofcells showing LY-CH fluorescence in vacuoles (Fig. 5B).Little or no fluorescence was observed at the cell wall.

When cells were electroporated in the presence of bothLY-CH and probenecid, and incubated for up to l h inmedium with probenecid, LY-CH fluorescence wasexcluded from the vacuoles and remained diffuse in thecytoplasm. Following removal of probenecid and a furtherincubation of cells in probenecid-free medium(30-60 min), LY-CH appeared to be sequestered intovacuoles at a very low level and the remainder was lostfrom the cytoplasm.

Uptake of LY-CH by fibroblastsFollowing a 30 min-incubation (37 °C) of mouse 3T3fibroblasts with LY-CH (0.25mgml"1) and thoroughwashing of cells, LY-CH fluorescence was detected in theendosomal/lysosomal system (Fig. 6A,B). Similar LY-CHtreatment of fibroblasts in the presence of probenecid(2.5 mM) had no effect on the internalisation of the dye(Fig. 6C). After electroporation of LY-CH directly into thecytoplasm of cells, LY-CH was sequestered into theendosomal/lysosomal system. However, electroporation offibroblasts in the presence of probenecid followed by PMresealing and further incubation of cells in IMDMcontaining probenecid (30 min) resulted in LY-CH fluor-escence being restricted to the cytoplasm (Fig. 6E).Furthermore, on washing these cells in probenecid-freemedium, the majority of LY-CH fluorescence was lost fromthe cytoplasm but traces of the probe were detected in thelysosomal system (Fig. 6F).

Spectrofluorimetric analysis of LY-CH uptake bysuspension-cultured carrot cells and isolated protoplastsAt 25 °C, the kinetics of LY-CH uptake by suspension-cultured carrot cells with respect to time exhibited threecomponents (Fig. 7). The first two components representedthe initial phase of LY-CH uptake (1-4 h LY-CH treat-

Endocytosis in plant cells 547

Fig. 1. Internalisation of LY-CH K+ by cell suspension cultures of maize and carrot. Maize cells (5- to 6-day-old) showingfluorescence in vacuoles (v) and at the cell wall (B, arrows) following a l h incubation with l m g m P 1 LY-CH K+ (A,B) at 25°C.X410. After 16 h LY treatment, the majority of maize cells accumulate LY-CH in their vacuoles (C,D). X420. In comparison,suspension-cultured carrot cells (5-day-old) do not internalise LY-CH after a 4h LY-CH treatment (E,F) and only traces of the dyecan be observed at the cell wall (F, arrows). X630. After prolonged (20 h) LY-CH treatment, intense LY-CH fluorescence is observedin vacuoles of carrot cells (G,H). x630. A,C,E and G are phase-contrast micrographs; B,D,F and H are correspondingepifluorescence micrographs.

548 L. Cole et al.

Fig. 2. Laser scanning confocal fluorescence micrographshowing the accumulation of LY-CH Ld+ in vacuoles (v) and atthe cell walls (arrows) of suspension-cultured carrot cells (7-day-old) following an 18 h LYClmgmP1) treatment. X730.

ment), which was rapid and rose to a saturated level. After6h, this initial phase was superseded by the thirdcomponent, which increased exponentially.

At 25 °C, the uptake of LY-CH by carrot protoplasts wasbiphasic with respect to time. Initially, the uptake of LY-CH appeared to be both rapid and linear for the first 5 h ofLY-CH incubation. After 5h, the uptake of LY-CH byprotoplasts decreased gradually and approached a slowermore steady state (Fig. 8).

Effect of low temperature and probenecid on LY-CHuptake by suspension-cultured carrot cells. The initialsaturable phase of LY-CH uptake, apparent at 25°C,appeared to be relatively unaffected by low temperature(0-4°C). However, the latter exponential component(6-24h) was severely inhibited (Fig. 7).

In the presence of probenecid (Fig. 9), the pattern ofuptake of LY-CH was comparable to that observed at lowtemperature (Fig. 7), i.e. the initial saturable LY-CHuptake phase was apparent whereas the latter exponentialcomponent was severely inhibited.

Preliminary experiments using probenecid (2.5 mM)have also shown that the drug severely inhibits bothphases of uptake of LY-CH by carrot protoplasts (notshown here).

Discussion

Uptake of LY-CH by suspension-cultured plant cells andprotoplastsWe have shown that when cell suspension cultures ofcarrot and maize were incubated in growth medium in thepresence of the highly fluorescent probe LY-CH, the dyewas internalised and compartmentalised into vacuoles butat very different 'rates'. In view of the membrane-impermeant properties of LY-CH and its general accept-ance as a fluid-phase endocytotic marker in animal andyeast systems (Swanson et al. 1985; Riezman et al. 1986;Swanson, 1989), it could be assumed that in carrot and

maize cells the probe entered the vacuolar system viaconstitutive fluid-phase endocytosis. Thus, the rapidlygrowing maize cells appeared to exhibit a faster 'rate' offluid-phase endocytosis (LY-CH observed in vacuoles after1 h) than carrot cells at a similar stage of growth (LY-CHobserved in vacuoles after 20 h). Although, in our recentexperiments investigating LY-CH uptake by carrot cellsgrown in a medium supplemented with O.lmgdm"3

napthaleneacetic acid (NAA), instead of 2,4-D, there is anaccumulation of LY-CH in vacuoles after only a three hourLY-CH incubation (Hawes and Kearns, unpublishedresults). Different 'rates' of LY-CH uptake have also beenreported to occur in other plant systems, e.g. barley roots(Oparka et al. 1988), leaves of Commelina communis(Hillmer et al. 1990) and leaf protoplasts from differentplant species (Wright and Oparka, 1989).

The uptake of LY-CH into vacuoles of carrot protoplastsoccurred at a similar 'rate' (i.e. after 9h LY-CH incu-bation) to that observed in turgid plant cells. Our earlierresearch on endocytosis in plant protoplasts using purifiedfluorescent dextrans (FDs) as endocytotic markers (Cole etal. 1990) has indicated that plant protoplasts may indeedexhibit a slower 'rate' of endocytosis when compared withwalled cells. Our current results are therefore in contra-diction to the endocytotic properties of suspension-cultured carrot cells that are apparent when using FDs asfluid-phase endocytotic markers.

Effect of probenecid on the uptake of LY-CH from theextracellular medium into vacuoles of suspension-cultured plant cells and protoplastsWhen suspension-cultured carrot cells, carrot protoplastsand suspension-cultured maize cells were incubated withLY-CH in the presence of the organic anion transportinhibitor probenecid, the uptake of LY-CH into thevacuoles was blocked completely. In many cells, the dyeaccumulated in the cytoplasm, a result consistent withthose obtained when investigating the effect of probenecidon LY-CH sequestration from the cytoplasm into thelysosomal system in fibroblasts (see Results) and macro-phages (Steinberg et al. 19876). However, in other cells LY-CH uptake appeared to be blocked at the plasmamembrane. As in the former case, probenecid has a similareffect on the internalisation and compartmentalisation ofnon-conjugated fluorescein isothiocyanate (FITC) by sus-pension-cultured carrot cells and protoplasts (Cole et al.1990). It was suggested that the FLTC-anion could enterthe vacuoles from the cytoplasm via a probenecid-inhibitable anion transport mechanism present on thecarrot tonoplast. It may be that such a transportmechanism also facilitates the transport of LY-CH acrossthe tonoplast in the carrot, maize and perhaps other plantcell systems. This hypothesis is supported by evidence onthe effect of probenecid on the tonoplast of onion epidermalcells (Oparka et al. 1991).

Uptake of LY-CH by fibroblasts following introduction ofthe dye into the cytosol by electroporationIn order to compare the uptake of LY-CH into plant cellvacuoles with that previously reported for the uptake ofLY-CH by animal cells (Steinberg et al. 1987a, 19876,1988; Swanson et al. 1985, 1986, 1987; Swanson, 1989) wecarried out a series of experiments on LY-CH compartmen-talisation in 3T3 fibroblasts in the presence and absence ofthe drug probenecid.

Steinberg and his coworkers (19876,1988) observed thatprobenecid inhibited the uptake of LY-CH into lysosomes

Endocytosis in plant cells 549

Fig. 3. Effect of probenecid on the uptake of LY-CH by suspension-cultured carrot cells and protoplasts. (A-D) Carrot cells (5-day-old) incubated with LY-CH Li+ (lmgml"1) in the presence of probenecid (2.5 BM) for 21 h. After thorough washing of cells inmedium containing probenecid, LY fluorescence remains in the cytoplasm and nuclei (arrows) but is excluded from vacuoles (v).x630. However, in some cells (E,F) no uptake of LY-CH was observed and only traces of the dye could be seen at the cell wall (F,arrows). X650. (G,H) Protoplast showing fluorescence in vacuoles following an 18-h incubation with LY-CH Li+ (lmgml"1). X1030.(I,J) Protoplast incubated with LY-CH K+ (lmgml"1) for 9h in the presence of probenecid (2.5 mM). LY-CH accumulates in thecytoplasm and nuclei (arrows) but is excluded from the vacuoles (arrowheads). x630. A,C,E,G and I are phase-contrastmicrographs; B,D,F,H and J are the corresponding epifluorescence micrographs.

550 L. Cole et al.

Fig. 4. (A,B) Effect of probenecid (2.5mM) on the uptake of LY-CH K+ (1 mgml"1) by suspension-cultured maize cells (6-day-old)following a 16 h incubation with the dye. Note that LY-CH accumulates in the cytoplasm and at the nucleus, but is excluded fromvacuoles. (A) phase-contrast micrograph; (B) corresponding epifluorescence micrograph. X410.Fig. 5. (A,B) Epifluorescence micrographs showing compartmentalisation of LY-CH K+ by carrot cells following single-pulseelectroporation (field strength=1250Vcm~1, t=lms; and capacitance=220/iF) of dye into the cytoplasm. Electroporated cells wereincubated for 20min at 0-4 °C and 46min at 26 °C prior to thorough washing (3 times, 2min) with M & S containing 10 mM Hepes(pH7.0). (A) Cells, observed immediately following PM resealing and thorough washing, show an accumulation of LY-CH in thecytoplasm but no uptake of the dye into vacuoles. x680. (B) Cells observed following a further l h incubation in Hepes-bufferedmedium. Here, LY-CH is sequestered into vacuoles and little or no fluorescence can be seen in the cytoplasm. x680.

of, and the efflux of the dye from, macrophages and thusconcluded that the sequestration of LY-CH into lysosomesis mediated by probenecid-inhibitable organic aniontransporters present on the lysosomal membranes. Wehave observed a similar effect of probenecid on LY-CHuptake by fibroblasts, by using electroporation as a meansof introducing LY-CH into the cytoplasm. In the presenceof probenecid, the rapid sequestration of LY-CH intolysosomes was blocked completely and the dye remainedin the cytoplasm. On removal of probenecid, LY-CH waspartially internalised into the lysosomal system and theremainder of the dye was lost from the cytoplasm. Hence,it is evident that the presence of organic anion trans-porters (OATs) on lysosomal membranes is indeed acommon phenomenon in animal cells and that the activityof these OATs is probenecid-inhibitable. Furthermore, infibroblasts, contrary to our results using plant cells,probenecid treatment appeared to have no effect on thefluid-phase endocytotic uptake of LY-CH at the plasmamembrane of non-electroporated cells.

The trans-tonoplast transport of LY-CH by suspension-cultured carrot cellsFollowing the introduction of LY-CH into the cytoplasm ofcarrot cells by electroporation, the dye was sequesteredinto the vacuoles after 1-2 h, mimicking the resultobtained from the equivalent experiment with fibroblasts.Furthermore, this LY-CH sequestration into vacuoles wasalso inhibited by probenecid. Thus, it is possible that anorganic anion transport mechanism comparable to thatreported to occur on lysosomal membranes, operates at theplant tonoplast. Results from plant microinjection studiesusing LY-CH as a fluorescent tracer also support thishypothesis (Fisher, 1988; Goodwin et al. 1990). However,recently, Hillmer et al. (1990) have shown that with theintroduction of LY-CH into mesophyll protoplasts ofCommelina communis, using a patch-clamp pipette tech-nique, the probe was trapped in the cytoplasm and nottransported to the vacuole. Other microinjection studieshave also demonstrated that LY-CH remains predomi-nantly in the cytoplasm and is not internalised into the

Endocytosis in plant cells 551

Fig. 6. Effect of probenecid on the uptake of LY-CH K+ by normal and electroporated mouse 3T3 fibroblasta. Normal mouse 3T3nbroblasts were incubated with LY-CH (0.25 mgml"1) for 30min at 37°C in the absence (A,B) and presence of 2.5mM probenecid(C). (A) Phase-contrast micrograph of B. xlllO. (C) x970. In both cases (B and C), LY-CH was internalised rapidly into lysosomalcompartments and endosomes. (D-F) Electroporation of LY-CH into the cytoplasm of mouse 3T3 fibroblasta in the absence (D) andpresence (E and F) of 2.5mM probenecid (field strength=1875 Vcm~\ (=lms; and capacitance=490/<F) followed by a furtherincubation (30-60 min) at 37 °C and thorough washing of cells in the absence (D) and presence of probenecid (E and F).(D) Sequestration of LY-CH from the cytoplasm into endosomal/lysosomal system, x 1500. (E) In the presence of probenecid, LY-CHremained in the cytoplasm of electroporated cells and is absent from the endosomal/lysosomal system, x 1480. (F) Cells, treated asin E and then incubated further in probenecid-free medium (30 min), accumulated traces of LY-CH in the lysosomal system but themajority of LY-CH fluorescence was lost from the cytoplasm, x 1490.

plant vacuole (Erwee et al. 1985; Terry and Robards, 1987;Oparka and Prior, 1988; Hillmer et al. 1989, 1990). Thereare however, several reports of other membrane-imper-meant probes, such as the calcium-sensitive dyes fura-2(Clarkson et al. 1988) and Quin-2 (Gilroy et al 1989),accumulating in the vacuole after being introduced intothe cytoplasm of root hairs and protoplasts of suspension-cultured carrot cells. In addition, it has been demonstratedthat probenecid (2.5 mM) inhibits the sequestration offura-2 into the lysosomal system of macrophages (DiVirgilio et al. 1988).

Kinetics of LY-Cft uptake by suspension-cultured carrotcells and isolated protoplastsIn view of the results of our kinetics experiments it is

conceivable that the initial saturable phase of LY-CHuptake, which was relatively unaffected by low tempera-ture and probenecid, may represent the trapping of theprobe at the cell wall, as such an accumulation of the dyewithin the interstices of the cell wall would involvepassive diffusion. If this was so, then the latter exponen-tial uptake component that was both temperature-dependant and probenecid-inhibitable would indicate theactual rate of internalisation of LY-CH by carrot cells.

In protoplasts, a wall-free system, as expected, theinitial saturable phase of LY- CH uptake by suspension-cultured carrot cells was absent. During the first 5 h of LY-CH incubation the uptake of LY-CH by protoplasts wasboth rapid and linear. After 5 h, the uptake of LY-CH wasreduced and appeared to approach a more steady stateafter 9 h LY-CH incubation. This biphasic pattern of LY-

552 L. Cole et al.

CH uptake is similar to that reported in animal cells(Swanson et al. 1985), yeast (Riezman, 1985) and plantprotoplasts (Wright and Oparka, 1989) when treated withthe probe.

5.0

4.0

h

DO 3.0

25 °C

*• 0-4 °C

12 16Time (h)

20 24

Fig. 7. Effect of low temperature (0—4°C) on the kinetics ofuptake of LY-CH K+ by suspension-cultured carrot cells (3- to4-day-old). (O O) 26°C; ( • • ) 0-4°C.

0 1 2 3 4 5 6 7 8 9

It is conceivable that in carrot protoplasts the initialphase of LY-CH uptake may represent a rapidly turningover compartment, e.g. endocytotic vesicles that are notreadily detectable by fluorescence microscopy. It followsthen that the slower saturable phase of LY-CH uptakemay represent the accumulation of the dye into vacuoles (aslow-filling and -emptying compartment).

Preliminary experiments (Cole and Hawes, unpublishedresults) have also shown that both phases of LY-CHuptake by plant protoplasts are inhibited by the drug,probenecid. Thus, it is clear that the organic aniontransport inhibitor probenecid has a severe effect on theuptake of LY-CH into vacuoles of both suspension-culturedcarrot cells and their isolated protoplasts.

How does LY-CH enter the plant cell?In many probenecid-treated cells and protoplasts, nointernalisation of LY-CH was observed following exposureto the dye, a result corroborated by the overall reduction inuptake of LY-CH by both cells and protoplasts shown byspectrofluorimetry. Does this indicate that probenecidinhibits fluid-phase endocytosis or that there is a probene-cid-inhibitable anion transport mechanism on the plasmamembrane of suspension-cultured carrot cells? To date,the only suggested route for the transport of LY-CH acrossthe plasma membrane in animal, yeast and plant cells isvia an endocytotic pathway. However, such a route wouldappear to be unlikely in the plant cells studied here,considering that probenecid had no effect on fluid-phaseendocytosis of LY-CH by fibroblasts but prevented theuptake into vacuoles of the plant cells. Furthermore, insuspension-cultured cells of Morinda citrifolia, probenecidinhibits the uptake of LY-CH at the plasma membrane (D.O'Driscoll personal communication). In view of our resultsit is conceivable that there are probenecid-inhibitableorganic anion transporters on plant plasma membranes

Fig. 8. Uptake of LY-CH K+ by carrot protoplasts with respectto time.

12 16Time (h)

Fig. 9. Effect of probenecid on the kinetics of uptake of LY-CHK+ by suspension-cultured carrot cells (5-day-old). (O O)Cells incubated with LY-CH (lmgmT1) only. (•——•) Cellsincubated with LY-CH in the presence of probenecid (2.5 mM).

Endocytosis in plant cells 553

that may facilitate LY-CH uptake into the cell. Inaddition, although a similar anion transport mechanismhas been indicated at the tonoplast of suspension-culturedcarrot cells (this paper; and Cole et al. 1990), and onionepidermal cells (see accompanying paper; Oparka et al.1991), such a mechanism was not apparent at thetonoplast of suspension-cultured cells of Morinda citrifolia(O'Driscoll personal communication). Hence, probenecid-inhibitable anion transport mechanisms at the tonoplastand plasma membrane are by no means ubiquitous inplants. Furthermore, why probenecid inhibits the uptakeof LY-CH at the plasma membrane in some cultured carrotcells and not others has not been explained.

It is unequivocal from our studies that the use of thedrug probenecid has led to a better understanding ofmechanisms of uptake and compartmentalisation ofcertain fluorescent anions, e.g. FITC (Cole et al. 1990) andLY-CH (Oparka et al. 1991, O'Driscoll, personal communi-cation) in plant cell systems. We suggest that the uptake ofLY-CH into the plant vacuole may occur via a pathwaythat involves non-vesicle-mediated internalisation of thedye into the cytosol and its subsequent sequestration intothe vacuole. However, one cannot rule out the possibilitythat LY-CH concurrently enters the plant cell or proto-plast via constitutive fluid-phase endocytosis. In con-clusion, we suggest that the reliability of LY-CH, as afluid-phase endocytotic marker in plant systems should bereconsidered.

This work was supported by an AFRC grant to Dr Chris Hawes.We thank Dr David Shotton and Nick White for their help withthe confocal microscopy and are indebted to Dr Karl Oparka forcritically reading this manuscript.

References

BASBAI, M. A., NAIDER, F. AND BECKER, J. M (1990). Internalisation oflucifer yellow in Candida albicans by fluid phase endocytosis. J. gen.Microbwl. 138, 1059-1065.

BERNARDINO G., FERRAOUTI, M. AND PERES, A. (1986). The decrease ofXenopus egg membrane capacity during activation might be due toendocytosis. Gamete Res. 14, 123-127.

BIRNARDINI, G., FERRAGUTI, M. AND STIPANI, R. (1987). Fertilisationinduces endocytosis in Xenopus eggs. Cell Differ. 21, 255-260.

BRACKET, J. (1954). Effects of ribonuclease on living root-tip cells.Nature 174, 876.

CLARKSON, D. T., BROWNLEE, C. AND AYIING, S. M. (1988) Cytoplasmiccalcium measurements in intact higher plant cells: results fromfluorescence ratio imaging of fura-2. J. Cell Sci. 91, 71-80

COLE, L., COLEMAN, J., EVANS, D. AND HAWKS, C. (1990) Internalisationof fluorescein isothiocyanate and fluorescein isothiocyanate—dextranby suspension cultured carrot cells. J. Cell Sci. 96, 721-730.

COLEMAN, J., EVANS, D., HAWKS, C, HORSLKY, D. AND COLE, L. (1987).Structure and molecular organisation of higher plant coated vesicles.J. Cell Sci. 88, 35-45.

COSSON, P., DK CURTIS, I., POUYSEGUR, J., GRIFFITHS, G. AND DAVOUST, T.J. (1989). Low cytoplasmic pH inhibits endocytosis and transport fromtrans-Golgi network to cell surface. J. Cell Bwl. 108, 377-387.

CUNNINGHAM, R. F., ISRAILJ, Z. H. AND DAYTON, P. G. (1981). Clinicalpharmacokinetics of probenecid. Clin. Pharmacokinetics 6, 136-151.

Di ViBGruo, F., STEINBERG, T. H., SWANSON, J. A. AND SILVERSTEIN, S.C. (1988). Fura-2 secretion and sequestration in macrophages. J.Immun. 140, 915-920.

ERWBE, M. G., GOODWIN, P. B. AND VAN BKL, A. B. E. (1985). Cell-cellcommunication in the leaves of Commelina cyanea and other plants.PI. Cell Environ. 8, 173-178.

FISHER, D. G. (1988). Movement of lucifer yellow in leaves of Coleusblunei benth. PI. Cell Environ. 11, 639-644.

GILHOY, S., HUGHES, W. A AND TRKWAVAS, A. J. (1989). A comparisonbetween Quin-2 and Aequorin as indicators of cytoplasmic calciumlevelB in high plant cell protoplasts. PI. Physiol. 90, 482-491.

GooDwrN, P. B., SHEPHERD, V. AND ERWBE, M. G. (1990).Compartmentation of fluorescent tracers injected into the epidermalcells of Egena densa leaves. Planta 181, 129-136.

HFLLMER, S., DKPTA, H. AND ROBINSON, D. G. (1986). Confirmation ofendocytosis in higher plant protoplasts using lectin-gold conjugates.Eur. J. Cell Biol. 41, 142-149.

HILLMER, S., HEDRICH, R., ROBERT-NICOUD, M. AND ROBINSON, D. G.(1990). Uptake of Lucifer yellow CH in leaves of Commelinacommunis is mediated by endocytosis. Protoplasma 1S8, 142-148.

HnxMER, S., QUADER, H., ROBERT-NICOUD, M. AND ROBINSON, D. G.(1989). Lucifer Yellow uptake in cells and protoplasts of Daucus carotavisualized by laser scanning microscopy. J. exp. Bot. 40, 417—423.

HORN, M. A., HEINSTEIN, P. F. AND LOW, P. S. (1989). Receptor-mediatedendocytosis in plant cells. The Plant Cell 1, 1003-1009.

HORN, M. A., HEINSTEIN, P. F. AND LOW, P. S. (1990). Biotin-mediateddelivery of exogenous macromolecules into soybean cells. PI. Physiol.3, 1492-1496.

HUBNER, R., DEPTA, H. AND ROBINSON, D. G. (1985). Endocytosis inmaize root cap cells; evidence obtained using heavy metal saltsolutions. Protoplasma 129, 214-222.

JKNSKN, W. A. AND MCLAREN, A. D. (1960). Uptake of proteins intoplant cells — the possible occurrences of pinocytosis in plants. ExplCell Res 19, 414-417

JOACHIM, S. AND ROBINSON, D. G. (1984). Endocytosis of cationic ferritinby bean leaf protoplasts. Eur. J. Cell Bwl. 34, 212-216.

KAHN, A. M., SHELAT, H. AND WEINMAN, E. J. (1985). Urate and p-aminohippurate transport in rat renal basolateral vesicles. Am. J.Physiol. 249, 654-661.

KAHN, A. M. AND WEINMAN, E. J. (1986). Urate transport in theproximal tubule: in vivo and vesicle studies. Am. J. Physwl. 249,F789-F798.

MCKINLKY, D. N. AND WILEY, H. S. (1988). Reassessment of fluid-phaseendocytosis and diacytosis in monolayer cultures of human fibroblastsJ. cell Physiol. 136, 389-397.

MILLER, D. K., GRIFFITHS, E., LENARD, J. AND FIRESTONE, R. A. (1983).Cell killing by lysosomotropic detergents. J. Cell Bwl. 97, 1841-1851.

OPARKA, K. J., MURANT, E. A., WRIGHT, K. M., PRIOR, D. A. M. ANDHARRIS, N. (1991). The drug probenecid inhibits the transport offluorescent anions across the tonoplast of onion epidermal cells. J.Cell Sci. 99, 557-563.

OPARKA, K. J. AND PRIOR, D. A. M. (1988). Movement of Lucifer YellowCH in potato tuber storage tissues: a comparison of symplastic andapoplastic transport. Planta 176, 533-540.

OPARKA, K. J., ROBINSON, D , PRIOR, D. A. M., DERRICK, P. AND WRIGHT,K. M. (1988). Uptake of Lucifer Yellow CH into intact barley roots:evidence for fluid-phase endocytosis. Planta 176, 541-647.

OWEN, T. P., JR, PLATT-ALOIA, K. A. AND THOMSON, W. W. (1991).Ultrastructural localization of Lucifer Yellow and endocytosis in plantcells. Protoplasma 160, 115-120.

RIEZMAN, H (1985). Endocytosis in yeast: several of the yeast secretorymutants are defective in endocytosis. Cell 40, 1001-1009.

RIEZMAN, H , CHVATCHKO, Y AND DUUC, V (1986). Endocytosis inyeast. Trends bwchem. Sci. 11, 325-328.

RoBrNSON, D. G. AND HILLMER, S. (1990). Endocytosis in plants. Physiol.PI 79, 96-104

SAMUELS, A. L. AND BISALPUTRA, T. (1990). Endocytosis in elongatingroot cells of Lobelia erinus. J. Cell Sci. 97, 157-165.

STSINBERO, T. H., NEWMAN, A. S., SWANSON, J. A. AND SILVBRSTEIN, S.C. (1987a). ATP1" permeabilises the plasma membrane of mousemacrophages to fluorescent dyes. J. bwl. Chem. 262, 8884-8888.

STEINBERG, T. H., NEWMAN, A. S., SWANSON, J. A. AND SILVERSTEIN, S.C. (19876). Macrophages possess probenecid-inhibitable organic aniontransporters that remove fluorescent dyes from the cytoplasmicmatrix. J. Cell Bwl. 108, 2695-2702.

STEINBKRC, T. H., SWANSON, J. A. AND SILVERSTEIN, S. C. (1988). Aprelysosomal compartment sequesters membrane-impermeantfluorescent dyes from the cytoplasmic matrix of J774 macrophages. J.Cell Biol. 107, 887-896.

STEWART, W. W. (1978). Functional connections between cells asrevealed by dye-coupling with a highly fluorescent naphthalimidetracer. Cell 14, 741-759.

STEWART, W. W. (1981). Lucifer dyes - highly fluorescent dyes forbiological tracing. Nature 292, 17-21.

SWANSON, J. (1989). Fluorescent labelling of endocytic compartments. InMethods in Cell Bwlogy (ed. Wang, Y. L. and Lansing Taylor, D ), pp.137-151. Academic Press Inc., New York.

SWANSON, J., BURKE, E. AND SILVERSTEIN, S. C. (1987). Tubularlysosomes accompany stimulated pinocytosis in macrophages. J. CellBiol. 104, 1217-1222.

SWANSON, J., YIRINEC, B., BURKE, E., BUSHNBLL, A. AND SILVERSTEIN, S.C (1986). Effect of alterations in the size of the vacuolar compartmenton pinocytosis in J774 2 macrophages. J. cell. Physiol. 128, 195-201

SWANSON, J. A., YIRINEC, B. D. AND SILVERSTEIN, S. C. (1985). Phorbolesters and horseradish peroxidase stimulate pinocytosis and redirectthe flow of pinocytosed fluid in macrophages. J. Cell Biol. 100,851-859.

554 L. Cole et al.

TANCHAK, M. A., GRIFFING, L. R., MHRSBY, B. G. AND FOWKB, L. C. WIDHOLM, J. M. (1972). The use of fluoreacein diacetate and(1984). EndocytosiB of cationized ferritin by coated vesicles of soybean phenosafranine for determining viability of cultured plant cells. Stainprotoplasts. Planta 162, 481-486. Technol. 47, 189-194.

TANCHAK, M. A., RBNNIE, P. J. AND FOWKB, L. C. (1987). Ultrastructure WRIGHT, K. M. AND OPARKA, K. J. (1989). Uptake of Lucifer Yellow CHof the partially coated reticulum and dictyosomes during endocytosis into plant cell protoplasts: a quantitative assessment of fluid-phaseby soybean protoplasts. Planta 162, 433-441. endocytosis. Planta 179, 257-264.

TBRRY, B R. AND ROBARDS, A. W. (1987). Hydrodynamic radius alonegoverns the mobility of molecules through plasmodesmata. Planta171, 145-157. (Received 25 February 1991 - Accepted 17 April 1991)

Endocytosis in plant cells 555