ER-Tracker dye and BODIPY-brefeldin A differentiate ...

Journal of Microscopy, Vol. 197, Pt 3, March 2000, pp. 239±248. Received 2 July 1999; accepted 21 October 1999

ER-Tracker dye and BODIPY-brefeldin A differentiate the endoplasmic reticulum and Golgi bodies from the tubularvacuole system in living hyphae of Pisolithus tinctorius L. COLE, D. DAVIES, G. J. HYDE & A. E. ASHFORD School of Biological Sciences, The University of New South Wales, Sydney 2052, Australia

Key words. BODIPY-brefeldin A, endoplasmic reticulum, ER-TrackerTM Blue-White DPX, ¯uorescence microscopy, freeze-substitution, fungi, Golgi bodies, Pisolithus tinctorius, ultrastructure, vacuoles.

Summary TM

Two ¯uorochromes, ER-Tracker Blue-White DPX dye and the ¯uorescent brefeldin A (BFA) derivative, BODIPY-BFA, label the endoplasmic reticulum (ER) in hyphal tips of Pisolithus tinctorius and allow its differentiation from the tubular-vacuole system at the light microscope level in living cells. The ER-Tracker dye labels a reticulate network similar in distribution to ER as seen in electron micrographs of freeze-substituted hyphae. BODIPY-BFA stains a thicker axially aligned structure with an expanded region at the apex, which is similar to that seen when hyphae are stained with ER-Tracker dye in the presence of unconjugated BFA. This structure is considered to be ER modi®ed by BFA, a view supported by ultrastructural observations of the effect of BFA on the fungal ER. Both ¯uorescent probes also stain punctate structures, which are most likely to be Golgi bodies. Neither probe labels the tubular-vacuole system.

Introduction Epi¯uorescence and confocal microscopy, together with the production of ¯uorescent probes for labelling cell organelles, provide a range of options for studying the dynamics of different organelle systems and their interrelationships in living cells. However, many of these probes are non-speci®c and it is often necessary to con®rm light microscope (LM) observations at the electron microscope (EM) level. Here, we investigate two ¯uorochromes that can be employed to label the endoplasmic reticulum (ER) and Golgi bodies, and to differentiate the ER from the tubular-vacuole system which often takes a reticulate form in living tip-cells of the ®lamentous fungus Pisolithus tinctorius (Shepherd et al., 1993; Cole et al., 1997, 1998). Correspondence to: Dr Louise Cole. Tel: 61 2 93851602; fax: 61 2 93851558; e-mail: [email protected] q 2000 The Royal Microscopical Society

A number of markers have been used to label the ER in animal and plant cells at the LM level. These include the dicarbocyanine dye, DiOC6(3) (Terasaki et al., 1984; Quader & Schnepf, 1986; Quader et al., 1987; Terasaki & Reese, 1992), antibodies which recognize an ER retention signal such as an HDEL or KDEL sequence (Napier et al., 1992; Henderson et al., 1994) or ER-speci®c proteins fused to green ¯uorescent protein (GFP; Boevink et al., 1996, 1998). DiOC6(3) is, however, generally considered to be a nonspeci®c stain for all intracellular membranes and therefore not suitable for differentiating the ER from other reticulate organelles (Terasaki & Reese, 1992). Furthermore, whilst DiOC6(3) is reported to be a useful probe for labelling the ER in fungi (Butt et al., 1989), in P. tinctorius it preferentially labels mitochondria (Cole et al., 1997). Anti-HDEL antibodies have been used to label the ER in the oomycete Phytophthora cinnamomi (Hardham & Mitchell, 1998) but were unsuccessful in labelling any component of P. tinctorius hyphae (W. G. Allaway, 1994, personal communication). Furthermore, the wild-type GFP gene does not function in many organisms including ®lamentous fungi (FernaÂndezÂ balos et al., 1998). None the less, using modi®ed GFP A genes, GFP has been successfully expressed in a few fungi (Spellig et al., 1996; Cormack et al., 1997) and also used as Â balos a tool for protein localization in the ER (FernaÂndez-A et al., 1998). Thus, to date, LM studies showing the distribution of ER in living fungal cells are scant. Similarly, Golgi-markers, such as N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl sphingosine (C6-NBD-ceramide), ceramides labelled with the ¯uorophore boron dipyromethene di¯uoride (BODIPY) or JIM 84 antibody that have been developed for use in animal and plant cells (Lipsky & Pagano, 1985; Pagano et al., 1991; Horsley et al., 1993) have to our knowledge not been proven useful as probes in studies of the fungal Golgi. For example, we have attempted to label the Golgi bodies in living P. tinctorius hyphae, using 239

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F L U O R E S C E N T PROB E S D I F F ER EN T I AT E F U N G A L E R A N D G O L G I B O D I E S F RO M T U BU LA R VAC U O L E S

both C6-NBD- and BODIPYÒ FL C5-ceramide (Molecular Probes Inc.) without success (D. Davies, unpublished results). In this paper, we explore the use of two ¯uorochromes: ER-TrackerTM Blue-White DPX (Haugland, 1996) and ¯uorescent brefeldin A (BFA) derivative, BODIPY-BFA (BFA esteri®ed to BODIPY 558/568; Deng et al., 1995). ERTracker Blue-White DPX has been used successfully to label the ER, and BODIPY-BFA, the ER and Golgi apparatus in animal cells. These data suggest that those probes may be useful as markers for the ER and Golgi bodies in P. tinctorius and, moreover, that they will allow differentiation of these structures from other organelle systems such as the tubularvacuole network. BFA is a macrocyclic lactone and is synthesized by various fungi (HaÈrri et al., 1963; Betina, 1992). It has been shown to block secretion in animal, plant and yeast cells by interfering with ER-to-Golgi and/or post-Golgi transport (Klausner et al., 1992; Driouich et al., 1993; Rambourg et al., 1995). In this study, we investigated the possibility that BODIPY-BFA can be used to determine the sites of action of unconjugated BFA (i.e. the ER and Golgi bodies) at the LM level. We employed the free metabolite BFA to compare its effect on the localization of the ERTracker dye with the distribution of BODIPY-BFA in P. tinctorius hyphae. In addition, we observed the spatial distribution of the ER in freeze-substituted hyphae in the presence and absence of BFA. Close examination of Golgi bodies was also carried out at the EM level for comparison with LM studies. Other ¯uorochromes such as the vacuole tracer Oregon Green 488 carboxylic acid diacetate (carboxy-DFFDA; Cole et al., 1997) and DiOC6(3) were employed in dual-labelling studies using BODIPY-BFA to con®rm the localization of these probes in different organelle systems in living P. tinctorius hyphae.

Materials and methods Abbreviations 4,4-di¯uoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3propionic acid (BODIPY); brefeldin A (BFA); differential interference contrast (DIC); 3,30 -dihexyloxycarboxyanine iodide (DiOC6(3)); dimethyl sulfoxide (DMSO); Oregon Green 488 carboxylic acid diacetate (carboxy-DFFDA); reverseosmosis water (RO water); room temperature (RT).

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Fungal cultures Cultures of P. tinctorius (Pers.) Coker and Couch, strain DI15, isolated by Grenville et al. (1986), were grown on modi®ed Melin-Norkrans (MMN) agar (Marx, 1969) using a modi®ed cellophane-sandwich technique (Campbell, 1983) as described previously (Cole et al., 1997). Cultures were kept in the dark at 23 8C for 8±14 days.

Reagents and ¯uorochromes BFA was purchased from Sigma Chemical Co. Ltd (St. Louis, MO, U.S.A.), dissolved in ethanol (0.5 mg mL 1) and stored at 20 8C. Fluorochromes were purchased from Molecular Probes Inc. (Eugene, Oregon, U.S.A.) and stored as stock solutions in DMSO, unless otherwise stated, at 20 8C at the following concentrations: BODIPY 558/568 propionic acid (Lot no. 4121±1), 20 mmol L 1; BFA, BODIPY 558/ 568 conjugate `isomer 1' (BODIPY-BFA; Lot no. 4141±2), 100 mmol L 1; DiOC6(3) (Lot no. 0121), 10 mg mL 1 in methanol; ER-TrackerTM Blue-White DPX (Lot no. 3881±1), carboxy-DFFDA (Lot no. 3961±2), 1 mmol L 1; 10 mg mL 1.

Fluorochrome loading and drug treatment Wedges of mycelium (maximum width of 5 mm) supporting actively growing hyphal tips were excised and incubated directly in either (i) 10 mmol L 1 ER-TrackerTM Blue-White DPX, (ii) 0.5 mmol L 1 BODIPY-BFA in the presence or absence of 10 mg mL 1 carboxy-DFFDA, (iii) 0.5 mmol L 1 BODIPY-BFA containing 2 mg mL 1 DiOC6(3) or (iv) 0.5 mmol L 1 BODIPY 558/568 containing 20 mg mL 1 carboxy-DFFDA, for 15±30 min in the dark. The ERTracker dye was ®ltered prior to use to remove any dye aggregates. Hyphae were also treated with ¯uorochromes (as above) in the presence of 3.6 mmol L 1 BFA for 15 min. In the case of BODIPY-BFA, hyphae were pretreated with BFA for 15 min prior to BODIPY-BFA labelling. All incubations were carried out at RT. Samples were either mounted directly onto a microscope slide and observed immediately by epi¯uorescence microscopy or washed brie¯y in RO water prior to observation. In the case of the ER-Tracker dye, all observations were carried out with the specimen mounted in the probe solution, as staining was reduced considerably on its removal.

Fig. 1. Localization of ER and Golgi bodies in P. tinctorius hyphae. (A, B) Epi¯uorescence micrographs showing hyphae stained with ERTracker dye. (A) Fluorescence was located in a reticulate network (small arrows) which extended throughout the hypha. Hyphal apex (large arrow). Bar 5 mm. (B) Fluorescent punctate structures were also visible (arrowheads). Bar 5 mm. (C) Electron micrograph showing ER (er), tubular-vacuole system (v) and mitochondria (m). Note that these structures can clearly be distinguished from each other inside the cell. The ER cisternae were generally parallel to each other and were found throughout the cell. Bar 500 nm. (D) Electron micrograph showing Golgi bodies (g) with associated vesicles. Bar 500 nm. q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 239±248

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Epi¯uorescence microscopy Samples mounted on slides were observed by conventional epi¯uorescence microscopy using a Zeiss Axiophot epi¯uorescence microscope ®tted with Nomarski DIC optics and ´40, NA 0.75 and ´63, NA 1.4 objectives. The ®lter set BP365, DIC395 and LP420 was employed for observation of ER-Tracker Blue-White DPX ¯uorescence; BP450±490, FT510 and LP515±585 for carboxy-DFFDA and DiOC6(3) ¯uorescence and BP546, FT580 and LP590 for BODIPY 558/568 ¯uorescence. Individual images or sequences of images were captured with a real-time digital imaging set-up comprising an Image Point CCD camera (Photometrics, Tucson, AZ, U.S.A.), a PCI-compatible LG3 framegrabber (Scion Corp., Fredrick, MD, U.S.A.) and Scion version of NIH Image (public domain image analysis software) installed on a Power Macintosh 9500/132 computer. Images were arranged with Adobe Illustrator version 5.02 and printed with a Epson Stylus Color Photo EX printer. Laser scanning confocal images were collected using a BioRad MRC 1024 confocal microscope attached to a Leica DMRB microscope and ®tted with a ´63 objective.

Freeze-substitution Small wedges (maximum width of 5 mm) of mycelium supporting leading edge hyphae were excised from colonies grown between cellophane, one layer of cellophane was peeled off and the hyphae were then placed face-down onto MMN agar for at least 15 min prior to cryo®xation. For BFA treatment wedges were incubated in a solution of 3.6 mmol L 1 BFA for 15 min prior to cryo®xation. Hyphae were cryo®xed in liquid propane [high grade (99.5%); Linde Gas PTY Ltd, NSW, Australia] at 187 8C, freeze-substituted in 2% osmium tetroxide (ProSciTech, Queensland, Australia) in anhydrous acetone at 85 8C for 3 days and gradually brought to RT using a Leica AFS freeze substitution apparatus. The warming cycle was carried out at 5 8C h 1 and samples were held at 20 8C, 4 8C and RT for 2 h each. Hyphae were rinsed at RT with anhydrous acetone (4 times, 15 min) and gradually in®ltrated with Quetol 651 resin [Alltech Associates (Aust.) P/L, NSW,

Australia; Abad et al. (1988)], as suggested by Howard & O'Donnell (1987). Anhydrous acetone was prepared using molecular sieve (Type 3A; Crown Scienti®c PTY Ltd, NSW, Australia). Samples were polymerized (14 h at 60 8C) in fresh resin between two glass microscope slides coated with polytetra¯uoroethylene (The Chemilube Company, Victoria, Australia) using single glass coverslips as spacers.

Ultramicrotomy and transmission electron microscopy Samples in resin were selected and excised using a Zeiss diamond scriber ®tted to a Zeiss microscope and then mounted on resin stubs prior to ultramicrotomy as described previously (Hyde et al., 1999). Sections (70± 100 nm) were cut using a Reichert Ultracut microtome, collected onto Formvar-coated copper slotted grids (ProSciTech) and poststained using 2% uranyl acetate in methanol (8 min) and lead citrate (10 min; Reynolds, 1963). Sections were observed using a Hitachi H-7000 transmission electron microscope at 100 kV. Images were recorded on Kodak 4489 electron microscope ®lm and micrographs were printed using Multigrade Ilford paper.

Results Distribution of ER-Tracker dye and fungal ER and Golgi bodies In living hyphal tips of P. tinctorius, the ER-Tracker dye was localized in a reticulate network which extended along the length and appeared to span the entire width of tip-cells (Fig. 1A). In many cases, this ¯uorescent network appeared to be more abundant near the apex. The network was interconnected and showed limited motility. Occasionally, small ¯uorescent punctate structures could also be seen in the cytoplasm of the tip-cell (Fig. 1B). Neither the reticulate network nor punctate structures could be distinguished in hyphae observed by DIC microscopy, even using the ´63 objective. The reticulate network stained by the ER-Tracker dye was similar in distribution to the ER, as seen in electron micrographs of freeze-substituted hyphae (Fig. 1C). These showed that the ER was located throughout the hyphal tip

Fig. 2. Conventional epi¯uorescence microscopy of BODIPY-BFA labelling in P. tinctorius hyphal tip cells. Hyphae stained with BODIPY-BFA were observed before (A) and after (B) a brief rinse with RO water. In both cases, ¯uorescence was seen within an axially aligned structure located at the centre of the cell (small arrows) and accumulated in the apical region (asterisk). Small punctate ¯uorescent structures (arrowheads) can also be seen in A. (C) DIC image of B showing the axially aligned structure (small arrows). (D and E) Hypha treated with BFA then labelled with BODIPY-BFA. (D) Note that a similar pattern of staining is visible as shown in A and B. However, in this case the axially aligned structure (arrows) extended further back from the tip. (E) DIC image of (D) showing the axially aligned structure (arrows). (F and G) Hypha labelled with free BODIPY 558/568 only. Note that the dye accumulated in both tubular (arrows) and spherical (asterisks) vacuoles seen to form clusters here. (G) Carboxy-DFFDA stained the tubular-vacuole system to reveal both ®ne tubules (arrows) and clusters of spherical vacuoles (asterisks). Bar 10 mm. q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 239±248



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cell, that its membranes exhibited a smooth pro®le, and that its cisternae were generally parallel to each other and usually orientated parallel to the long axis of the hypha. The rough ER was characteristically identi®ed by the presence of ribosomes on its outer membrane surface. Ribosomes also occurred free in the cytoplasm. Other organelles, in particular mitochondria, vacuoles, and Golgi bodies and associated vesicles, were also localized in the sub-apical region (Fig. 1C,D). Electron microscopy revealed that the Golgi bodies consisted of a single cisternum or several cisternae of varying shapes and electron opacities with associated vesicles (e.g. Fig. 1D). These Golgi bodies were similar in size [(mean 6 SE), width, 338 6 16 nm; length, 498 6 27 nm, n 21] to the punctate structures [diameter (mean 6 SE), 630 6 19 nm, n 39] observed at the LM level following labelling with the ER-Tracker dye, and their distribution also agreed well with that of the punctate structures.

Distribution of BODIPY-BFA When living P. tinctorius hyphae were stained with BODIPYBFA, the probe was localized to an axially aligned structure in the central region of the tip-cell (Fig. 2A,B). In the apical region, this structure was less well de®ned; staining was more abundant and spanned the entire width of the cell (Fig. 2A,B). Stained punctate structures were also visible in the cytoplasm (Fig. 2A). However, such structures were often dif®cult to visualize as a result of the intense ¯uorescent staining throughout other parts of the hyphae. Samples mounted in probe solution showed more intense BODIPY-BFA staining (Fig. 2A) than those viewed after rinsing in RO water (Fig. 2B). None the less, the distribution of BODIPY-BFA ¯uorescence was similar in both cases. BODIPY-BFA staining was observed in some hyphae as quickly as 3±5 min after application of the probe. The axially aligned structure stained by BODIPY-BFA was also

Fig. 3. Effect of free BFA on ER-Tracker labelling and ultrastructure of the ER. (B) Epi¯uorescence micrograph showing hypha stained with ER-Tracker dye and BFA for 15 min. A ¯uorescent staining pattern similar to that observed following BODIPY-BFA treatment (cf. Fig. 2A,B) can be seen. Fluorescence was seen in an axially aligned structure (arrows) that was located at the centre of the cell and accumulated at the apex (asterisk). Bar 10 mm. (B) Electron micrograph showing the effect of BFA on the ER (er). The ER cisternae were distinct and redistributed along the central axis of the cell. Bar 500 nm. q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 239±248


visible by DIC microscopy (cf. Fig. 2B with C). After prolonged exposure (> 30 min) to BODIPY-BFA, most hyphae displayed signs of dye toxicity; for example, the tubular vacuoles ruptured and the cytoplasm appeared granular when observed by DIC microscopy (not shown here). When hyphae were pretreated with unconjugated BFA and subsequently labelled with BODIPY-BFA, the axially orientated structure could be seen more clearly and it appeared to extend further back from the tip (Fig. 2D,E). Stained punctate structures could also be seen in these hyphae (see Fig. 2D). Occasionally, faint labelling of an axially orientated internal structure was apparent following exposure of BFA-treated hyphae to free BODIPY, but this staining was lost almost immediately after the probe was washed out (data not shown). When untreated hyphae were exposed to free BODIPY only, little or no staining of an axially orientated internal region or concentration of ¯uorescence at the hyphal tip was observed. Instead, this probe was localized in the tubular-vacuole system (Fig. 2F). This was con®rmed by dual-labelling with the vacuole tracer, carboxy-DFFDA (Fig. 2G). Following dual-labelling with free BODIPY and carboxy-DFFDA, both ¯uorochromes were localized in the tubular-vacuole system (cf. Fig. 2F,G). It was evident that the labelling patterns observed following treatment of hyphae with the ER-Tracker dye (Fig. 1A) or BODIPY-BFA (Fig. 2A,B), seen as a reticulate network or a distinct axially aligned internal structure, respectively, were very different from that seen after loading of the tubularvacuole system with carboxy-DFFDA (Fig. 2G).

Con®rmation that BODIPY-BFA labels the ER Con®rmation that BODIPY-BFA labels the ER was obtained by comparing the localization of (i) BODIPY-BFA to that of the ER-Tracker dye in the presence of BFA and (ii) BODIPYBFA with the distribution of the ER seen in electron micrographs of freeze-substituted, BFA-treated cells. The distribution of the ER-Tracker dye in the presence of BFA is shown in Fig. 3A; ¯uorescence was located in an axially orientated internal structure with a morphology very similar to that seen with BODIPY-BFA (Fig. 2A,B). Stained

Fig. 4. Confocal laser scanning microscopy of hyphal tip-cells labelled either with (A) BODIPY-BFA and carboxy-DFFDA or (B) BODIPY-BFA and DiOC6(3) for 30 min. In both (A) and (B) the axially aligned structure shown by BODIPY-BFA (red) labelling is located at the centre of the cell and is abundant at the apex. In (A), carboxy-DFFDA (green) labels the tubular-vacuole system. Note that where BODIPY-BFA staining occurs, spherical vacuoles (v) are visible. Further back from the tip, where no BODIPYBFA staining occurs, tubular vacuoles (t) can be seen. In (B), DiOC6(3) (green) labels the elongate rod-shaped mitochondria (m) located at the cell periphery. Bar 5 mm. q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 239±248

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punctate structures could also be seen in hyphae labelled with ER-Tracker dye in the presence of BFA (data not shown). The distribution of the axially aligned structure seen following BODIPY-BFA or ER-Tracker labelling in the presence of free BFA agreed well with that of the ER observed in BFA-treated cells at the EM level (cf. Figs 2A±C and 3A with 3B). At the EM level, the ER was more distinct and appeared to be redistributed along the central axis of the hypha in the presence of BFA (Fig. 3B) compared with the controls (Fig. 1C).

Dual-labelling studies distinguish the ER network from other reticulate organelles Dual-labelling using BODIPY-BFA and carboxy-DFFDA, or BODIPY-BFA and DiOC6(3), con®rmed that the structures identi®ed by BODIPY-BFA labelling were neither elements of the tubular-vacuole system nor mitochondria (Fig. 4A,B). Where BODIPY-BFA staining occurred, the tubular-vacuole system, shown by carboxy-DFFDA labelling, was converted from a tubular network into aggregates of free vacuoles (Fig. 4A). Further back from the tip, where there was no BODIPY-BFA staining, it retained a tubular form (Fig. 4A). The mitochondria could also be readily distinguished from the BODIPY-BFA labelled structure (Fig. 4B). Dual-labelling with the ER-Tracker dye and any of the other ¯uorochromes proved dif®cult as a result of bleedthrough of their ¯uorescence through all ®lter sets tested.

Discussion ER-TrackerTM Blue-White DPX, a member of the dapoxyl dye family (Diwu et al., 1997), was introduced as a highly selective and photostable probe for ER in live animal cells (Haugland, 1996). In contrast to DiOC6(3), it does not stain mitochondria and is not apparently toxic to cells when applied at low concentrations. Our data show that ERTracker Blue-White DPX is localized to the ER in fungal cells. The reticulate network stained by the probe agrees with the distribution of the ER network, as seen in electron micrographs of freeze-substituted hyphae. Furthermore, it is clearly distinct from mitochondria and the tubular-vacuole system, which may also take on a reticulate form in fungi (Shepherd et al., 1993; Cole et al., 1997, 1998). However, the ER-Tracker dye, at least in these fungal cells, does not appear to be speci®c for the ER, as it also stained punctate structures tentatively identi®ed as Golgi bodies. Of all the organelles identi®ed at the ultrastructural level in P. tinctorius hyphae, it is considered that the punctate structures seen in the cytoplasm of living hyphae following labelling with the ER-Tracker dye are most likely to be Golgi bodies. Furthermore, these punctate structures are similar in size, appearance and location to the Golgi bodies seen in freeze-substituted P. tinctorius hyphae, which are typical of

other ®lamentous fungi (Hoch & Howard, 1980; Hoch, 1986; Roberson & Fuller, 1988; Sewall et al., 1989; Bourett & Howard, 1994). BODIPY-BFA stained both the ER and punctate structures also identi®ed as Golgi bodies. This is in agreement with the staining found in several animal cell lines, where it is reported that both ER and Golgi bodies are stained by this probe (Deng et al., 1995). In P. tinctorius, BODIPY-BFA also modi®ed the appearance and distribution of the ER. These changes were similar to those obtained with the ER-Tracker dye in the presence of unconjugated BFA and correlated well with the observed effects of BFA on the ER at the ultrastructural level. This strongly supports identi®cation of the target organelle as ER. There is less evidence, however, supporting localization of BODIPY-BFA (and ERTracker dye) in the Golgi bodies, as classic ¯uorescent Golgiprobes such as C6-NBD-and BODIPY C5 ceramide, which label Golgi bodies in animal cells (Lipsky & Pagano, 1985; Pagano et al., 1991), did not label any punctate structures in P. tinctorius hyphae (D. Davies, unpublished data). However, we know that BFA is also targeted to and modi®es Golgi bodies in P. tinctorius (L. Cole, unpublished data) as well as in other fungal cells (Rambourg et al., 1995; Rupes et al., 1995; Bourett & Howard, 1996; Satiat-Jeunemaitre et al., 1996). It therefore seems very likely that the punctate structures seen by BOPIDY-BFA are also fungal Golgi bodies. It is dif®cult to foresee what other organelle could be stained by these probes. Whilst BODIPY-BFA can be used to differentiate the ER and Golgi bodies from other organelles such as the tubular-vacuole system, it is clear in this study that BODIPY-BFA affected both the form and distribution of the ER and the tubular-vacuole system. The effects of BODIPY-BFA and unconjugated BFA on the tubular-vacuole system is the subject of another paper and will not be discussed here. Our data suggest that BODIPY-BFA has similar sites of action to unconjugated BFA in P. tinctorius hyphae. In animal cells, the biological activity of BODIPY-BFA is signi®cantly lower than that of unconjugated BFA and it has been suggested that the BFA component of BODIPY-BFA may not become biologically active until the ¯uorophore BODIPY has been cleaved from the molecule by esterases (Deng et al., 1995). This is clearly not the case in P. tinctorius cells. If BODIPY is cleaved from BODIPY-BFA inside the fungal cells we would expect some accumulation of BODIPY in the vacuole system, as seen when BODIPY alone is supplied, but this does not occur. In conclusion, both ER-Tracker Blue-White DPX and BODIPY-BFA are useful probes for localization of ER and Golgi bodies in living fungal cells. The dye-staining protocols are simple, quick and produce clear images in whole cells. They allow differentiation of ER and Golgi bodies from other organelle systems such as the tubularvacuole network and mitochondria. The probes may also q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 239±248


prove to be useful for studying the interrelationships of the ER and Golgi bodies in ®lamentous fungi. It is suggested further that the ER-Tracker dye may be applied to investigations of the role and spatial dynamics of the ER during other cell processes such as tip growth and septum formation. In the case of BODIPY-BFA, it is clear that this probe can be used to investigate the direct effects of BFA on the endomembrane system and hence to examine the secretory pathway in fungal cells using ¯uorescence microscopy.

Acknowledgements This research was funded by an ARC grant. We would like to thank James Pearse for general maintenance of fungal cultures and Associate Professor Bill Allaway for his comments on the manuscript.

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