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Fungal hyphae extend by polarized or tip growth. Although the mechanisms that culminate in hyphal extension remain poorly understood, there is an increasing body of evidence which implicates the formation and maintenance of a tip-high cytosolic free-Ca²⁺ gradient as a deterministic feature (for a review, see reference 8). Localized activity of Ca²⁺-permeable ion channels in the apical plasma membrane (PM) is held as the most likely mechanism for Ca²⁺ entry at the tip (8). However, attempts to employ patch clamp electrophysiology to characterize tip ion channel activity in heterokont and fungal hyphal protoplasts (produced by cell wall digestion) have been curtailed by an inability to form high-electrical-resistance seals (gigaohm range) on apical and subapical PM (e.g., in Saprolegnia ferax [4, 10] and Neurospora crassa [11]). This is an essential prerequisite for full examination of fundamental channel properties such as permeability and voltage gating (7). To date, the only report of gigaohm seal formation on filamentous fungal PM has been a study on enzymatically produced Uromyces germling protoplasts (17).

The use of laser microsurgery has expedited PM channel characterization for tip-growing rhizoids of the brown alga Fucus. After plasmolysis, a UV beam cuts the apical cell wall, allowing the release of a protoplast (by osmotic manipulation) with a PM essentially free from cell wall deposits and hence more likely to form a gigaohm seal (16). Moreover, this method permits retention of the inherent polarity of the system. Here we describe the application of laser microsurgery to N. crassa hyphae, with the objective of the formation of gigaohm resistance seals on the apical PM.

Culture and growth conditions. N. crassa (wild-type strain RL21; Fungal Genetics Stock Center number 2219; obtained from Yale University) was maintained at 23°C on plate cultures as described previously (9). Potato dextrose agar (Sigma, Poole, United Kingdom) in a patch clamp recording chamber was inoculated with a loop of mycelium from a plate culture. The chamber base incorporated a 1-cm-diameter borosilicate glass section (from zero-grade coverslips; BDH, Poole, United Kingdom) to allow laser beam access; potato dextrose agar covered this section to a depth of approximately 100 µm. The chamber was sealed with Parafilm (American National Can Corp., Chicago, Ill.) to reduce dehydration and was maintained at 23°C for 5 to 6 h, yielding sparse mycelial growth. This chamber growth method was found to be preferable, in terms of hyphal tip adherence, morphological integrity, and efficiency of apical plasmolysis, to either excision of hyphae grown on a membrane (4) or poly-L-lysine treatment of the chamber base (16).

Laser instrumentation. The pulsed UV beam (triggered remotely via a foot switch) was generated by a VSL 337-nm nitrogen laser (Laser Science Inc., Cambridge, Mass.) and then diverted by the dichroic mirror of a Nikon Optiphot microscope to enter the objective (Nikon UV 40× Fluor; Nikon Corporation, Tokyo, Japan) as described by Taylor and Brownlee (16). Laser alignment resulted in a beam diameter of approximately 1 µm at the point of focus.

Hyphal plasmolysis. All procedures were performed at the microscope stage at room temperature. Hyphae growing in the chamber were plasmolyzed by successive superfusion with 1.0, 1.5, and 2 M D-sorbitol (>98% purity; Sigma) or L-sorbose over a total period of approximately 15 min. Either osmoticum was equally effective. During the initial plasmolysis, hyphae were exposed additionally to 0.01% (wt/vol) calcofluor white (3) for 2 to 3 min to aid in the aiming of the laser beam (16). The retraction of the apical PM and cytoplasm was regular and extended 2 to 10 µm from the wall (Fig. 1); a retraction of 3 µm was sufficient to perform microsurgery without damaging the apical PM.

View larger version (82K): [in this window] [in a new window]   FIG. 1.   Isolation of the hyphal apical protoplast by laser microsurgery. (A) Plasmolysis of a hyphal tip; plasma membrane (PM) at the tip, retracted from the cell wall (CW) in 2 M L-sorbose. Bar, 5 µm. (B) Release of an apical protoplast. The tip cell wall of the plasmolyzed hypha was removed by laser microsurgery, and the apical protoplast was extruded in response to a hypotonic bath solution. PE, patch clamp electrode in contact with the apical PM. Bar, 20 µm. Images from a CCTV camera (RS Components, Corby, United Kingdom) were digitized and compressed by using Intel Smart Video Recorder hardware and software (version III; Intel Corporation, Swindon, United Kingdom).

Release of the apical protoplast. Following plasmolysis, hyphae were exposed to cutting solution (2 M D-sorbitol or L-sorbose, 5 mM CaCl₂). The cell wall of a surface-growing hypha was cut at its apex with 3 to 20 laser beam (single) pulses. Approximately 30% of the cut hyphae released an apical protoplast (Fig. 1) without further adjustment of the osmolarity or ionic composition of the bathing solution; however, the presence of calcium was essential to prevent bursting of the protoplast on extrusion. Superfusion with a hypotonic solution (0.8 to 1 M D-sorbitol, 5 mM CaCl₂) promoted release of the apical protoplasts from the remaining hyphae. Subapical protoplasts could sometimes also be released on prolonged exposure to this solution. Extruded apical protoplasts were rounded; the cytoplasm was dense in appearance and not vacuolated.

Patch clamp procedures and analysis. Standard patch clamp procedures were used (7). Bath and pipette solutions used to test the sealing efficiency consisted of either 50 mM CaCl₂ or 50 mM KCl plus 10 mM CaCl₂, both with 10 mM HEPES-Tris (pH 7.2) and 0.6 to 1.5 M D-sorbitol. The solutions used for channel analysis are indicated in the legend to Fig. 3. Pipettes were fabricated from Kimax-51 borosilicate glass tubes (Kimble, Vineland, N.J.). Microelectrode resistance was measured as 15 to 20 M in symmetrical 50 mM KCl plus 10 mM CaCl₂. A reference Ag/AgCl half-cell filled with 50 mM KCl, 10 mM CaCl₂, 10 mM HEPES-Tris (pH 7.2), and 1% (wt/vol) agar completed the circuit. The patch clamp amplifier was an L/M PCA amplifier (List Electronics, Darmstadt, Germany). Voltage-pulse protocols and data acquisition and analysis were performed with a CED 1401 analog-to-digital converter and software (Cambridge Electronic Design, Cambridge, United Kingdom). Data were filtered at 200 Hz and sampled at 500 Hz. Liquid junction potentials were determined to be less than or equal to ±5 mV in all experiments. In traces from excised patches, downward deflections indicate an outward current of negative charge or an inward current of positive charge through the channel.

Patch clamping the apical PM. High-electrical-resistance seal formation between the apical PM and the patch electrode could be achieved with a 36% success rate (n = 25) immediately after extrusion of the apical protoplast. With gentle suction, gigaohm resistance seals (2 to 10 G) formed in under 1 min and were stable for at least 10 min. However, the ability to form gigaohm seals declined sharply with time. In more than 80 trials, no gigaohm seals were obtained from 10 min after protoplast extrusion. Seal resistances were in the range of 50 to 100 M and were not improved by adjustment of the medium composition (osmolarity or ionic strength) or by coating pipette tips with poly-L-lysine. Staining of apical protoplasts with calcofluor white (visualized with a Vickers M17 fluorescence microscope [Vickers Instruments, Coulsden, United Kingdom]) strongly suggested that the cause was cell wall deposition (Fig. 2A to D). Since the 5-min "window of opportunity" for forming seals is not ergonomically desirable, attempts were made to increase the period of sealing by incorporation of cell wall synthesis and exocytosis inhibitors.

View larger version (118K): [in this window] [in a new window]   FIG. 2.   Kinetics of cell wall rebuilding in hyphal apical protoplasts treated or not treated with BFA. Hyphal apical protoplasts were isolated by laser microsurgery, as explained in the legend to Fig. 1. Cell wall rebuilding was assessed by calcofluor white staining (0.01% calcofluor white in 1 M L-sorbose). (A to D) Untreated protoplast. (A) White-light micrograph of a hypha with a released apical protoplast. The photograph was taken 1 h after the release of the apical protoplast. A very small subapical protoplast, which was released 30 min after the apical one, can also be seen. (B to D) Corresponding fluorescence micrographs taken 10 min, 30 min, and 1 h, respectively, after the extrusion of the apical protoplast. Bar, 50 µm. (E and F) BFA-treated protoplasts. Hyphae were pretreated with 30 µg of BFA ml1 approximately 20 min prior to protoplast release. The BFA concentration was reduced to 5 µg ml1 in the protoplast bathing solution. (E) White-light micrograph of one apical and one subapical protoplast released from a hyphal tip. (F) Corresponding fluorescence micrograph taken 1 h after protoplast release. Bar, 50 µm.

Effects of cell wall synthesis inhibitors. To increase the likelihood of cell wall synthesis inhibitors reaching an internal active site, they were incorporated into the plasmolysis and patch clamp bath solutions. The ability to form gigaohm resistance seals more than 10 min after protoplast extrusion was not improved by including the cellulose formation inhibitor 2,6-dichlorobenzonitrile (Fluka, Gillingham, United Kingdom), even when it was used at 10 µg ml¹ (n = 16), a 10-fold-higher concentration than that required to block plant cell wall regeneration (12). Two inhibitors of chitin synthesis, nikkomycin Z (Calbiochem, Nottingham, United Kingdom) (n = 6) and UDP (Sigma) (n = 10), were used at 1 and 10 mM, respectively, without success (estimated in vitro K_i values, 2 µM for nikkomycin Z and 0.8 mM for UDP [6]).

Inhibitors of exocytosis. It was reasoned that renewed vesicle fusion would reduce the sealing efficiency as exocytosis ultimately commands cell wall deposition in tip-growing systems. Respiratory blockade by azide (which rapidly depletes ATP in N. crassa [15]) was used to perturb vesicle movement. Addition of 10 mM sodium azide to the patch clamp bath solution allowed gigaohm seal formation (4 to 10 G, and of stability comparable to that of control conditions) up to 2 h after protoplast isolation but at a low success rate (around 10%) after the first 10 min (n = 80). Fluorescein 5-isothiocyanate (20 µM), which blocks exocytosis in pollen tube tips (14), was not effective here when incorporated into the patch clamp bath solution. Brefeldin A (BFA; Sigma) is a potent inhibitor of intracellular transport (13). It prevents pollen tube extension, causing accumulation of putative secretory vesicles rich in pectinaceous cell wall material (5), and inhibits apical extension in Candida albicans germ tubes (1) and in the rice blast fungus Magnaporthe grisea (2), causing a reduction in the number of apical vesicles (1, 2). Here, BFA (at 3 to 30 µg ml¹, depending on the batch) was incorporated into the cutting and patch clamp solutions. The presence of BFA during plasmolysis was found to be noncritical. BFA produced a dramatic improvement in sealing efficiency, supporting a 40% gigaohm seal formation (1 to 50 G) for up to 2 h after protoplast release (n = 166), with 80% of those seals being stable for >10 min. In contrast to untreated protoplasts, BFA-treated protoplasts showed no cell wall staining with calcofluor white up to 2 h after their release (Fig. 2E and F).

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Examples of single-channel and whole-cell recordings at the hyphal tip of N. crassa. Tip protoplasts were isolated by laser microsurgery. Protoplasts were treated with 30 µg of BFA ml1. (A and B) Single-channel recordings from outside-out patches. The bath solution contained 10 mM KCl, 5 mM CaCl2, 10 mM MES [2-(N-morpholino)ethanesulfonic acid]-Tris (pH 5.8), and 1 M sorbitol. The pipette solution consisted of 50 mM KCl, 2 mM MgATP, 5 mM EGTA, 0.5 mM CaCl2 (10 nM free Ca2+), 1 mM MgCl2 (0.65 mM total free Mg2+), 10 mM HEPES, 2 mM KOH (pH 7.2), and 0.95 M sorbitol. The K+, Cl, and Ca2+ equilibrium potentials under these conditions were as follows: EK = 41 mV, EC1 = +24 mV, and ECa = +158 mV. Shown are activity traces and current-voltage (I/V) relationships of a probable anion channel (A) and a potassium channel (B). The holding potential (HP) was 40 mV; the clamped potentials are indicated at the right of the current traces. >, current level at which the channel was in a closed state. Unitary channel conductance (G) was estimated as 17 pS (A) or 9 pS (B). (C) Whole-cell Ca2+-permeable conductance. The bath solution consisted of 50 mM calcium gluconate (free Ca2+ activity determined with a Ca2+-selective microelectrode, ~6 mM), 5 mM MgCl2, 10 mM MES-Tris (pH 6.0), and 0.85 M sorbitol. The pipette solution contained 4.6 mM MgATP, 4.6 mM EGTA, 1.9 mM CaCl2 (0.15 µM free Ca2+), 0.9 mM MgCl2 (0.8 mM total free Mg2+), 9 mM HEPES-Tris (pH 7.0), and 0.96 M sorbitol. The Ca2+ and C1 equilibrium potentials in these conditions were as follows: ECa ~ +125 mV and EC1 = 13 mV; the equilibrium potential of the summed divalent ions, ECa + Mg, was ~+25 mV, and EC1 + gluconate was more negative than EC1. The HP was +50 mV. The current trace was obtained by slowly hyperpolarizing the cell from the HP to 180 mV (10 mV/s).

Concluding remarks. The apical PM of a fungal hypha can be used for patch clamp single-ion-channel analysis when exposed via laser cell wall microsurgery. Blocking exocytosis with BFA appears to delay cell wall formation by the apical protoplast, allowing a prolongation of the seal formation period. The unequivocal identification of the apical protoplast combined with gigaohm seal formation can now allow full characterization of channels believed to be essential for tip growth, including the study of their regulation by cytosolic moieties.