An IP3-activated Ca2+ channel regulates fungal tip growth

Lorelei B. Silverman-Gavrila and Roger R. Lew*

Biology Department, York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada

* Author for correspondence (e-mail: planters{at}yorku.ca)

Accepted 23 September 2002


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hyphal extension in fungi requires a tip-high Ca2+ gradient, which is generated and maintained internally by inositol (1,4,5)-trisphosphate (IP3)-induced Ca2+ release from tip-localized vesicles and subapical Ca2+ sequestration. Using the planar bilayer method we demonstrated the presence of two types of IP3-activated Ca2+ channels in Neurospora crassa membranes with different conductances: one low (13 picosiemens), the other high (77 picosiemens). On sucrose density gradients the low conductance channel co-localized with endoplasmic reticulum and plasma membrane, and the high conductance channel co-localized with vacuolar membranes. We correlated the effect of inhibitors on channel activity with their effect on hyphal growth and Ca2+ gradients. The inhibitor of IP3-induced Ca2+ release, 2-aminoethoxidiphenylborate (2-APB), inhibits both channels, while heparin, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8) and dantrolene inhibit only the large conductance channel. Because 2-APB inhibits hyphal growth and dissipates the tip-high cytosolic [Ca2+] gradient, whereas heparin microinjection, TMB-8 and dantrolene treatments do not affect growth, we suggest that the small conductance channel generates the obligatory tip-high Ca2+ gradient during hyphal growth. Since IP3 production must be catalyzed by tip-localized phospholipase C, we show that a number of phospholipase C inhibitors [neomycin, 1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]- 1H-pyrrole-2,5-dione (U-73122) (but not the inactive pyrrolidine U-73343), 3-nitrocoumarin] inhibit hyphal growth and affect, similarly to 2-APB, the location of vesicular Ca2+ imaged by chlortetracycline staining.

Key words: Fungal tip growth, IP3 receptor, Ca2+ gradient, Phospholipase C, Ca2+ channels, Planar lipid membrane


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fungi, the major decomposers in terrestrial environments, explore new territory by polarized cellular growth (hyphal elongation). This growth trait is shared by members of other kingdoms, but unlike other organisms, tip growth is the dominant form of growth in fungi.

Although Ca2+ gradients are an important component in all tip-growing organisms examined so far, fungi are notable in utilizing a different mechanism to generate and maintain the tip-high cytoplasmic Ca2+ gradient. Measurements of ion fluxes have identified a tip-localized Ca2+ influx from the external environment during growth in the oomycete Saprolegnia ferax (Lew, 1999Go), root hairs (Felle and Hepler, 1997Go) and pollen tubes (Pierson et al., 1996Go), but not in the fungus Neurospora crassa (Lew, 1999Go; Silverman-Gavrila and Lew, 2000Go). Therefore, plasma membrane stretch-activated Ca2+ channels, identified as the mechanism of Ca2+ entry in S. ferax (Garrill et al., 1993Go), or another type of Ca2+ channel (Very and Davies, 2000Go) cannot be the mechanism generating the tip-high Ca2+ gradient in N. crassa. Because microinjection of a Ca2+ chelator 1,2 bis(ortho-aminophenoxy)ethane-N,N,N',N'-tetrapotassium acetate (BAPTA) into growing N. crassa hypha inhibits growth, and dissipates the tip high cytoplasmic Ca2+ gradient, there is a clear requirement for a tip-high Ca2+ gradient during growth. The gradient must be generated and maintained by internal mechanisms that are likely to be shared by other fungi, and may be phylogenetically unique to the fungal kingdom (Silverman-Gavrila and Lew, 2000Go).

Evidence is accumulating that supports the hypothesis that the fungal tip-high Ca2+ gradient is generated and maintained internally by inositol (1,4,5)-trisphosphate (IP3)-induced Ca2+ release from tip-localized vesicles and Ca2+ sequestration in the ER behind the tip (Silverman-Gavrila and Lew, 2001Go) (Fig. 1A). This is based upon inhibitor effects on hyphal growth and morphology, correlated with cytoplasmic Ca2+ distributions that were imaged using dual dye ratioing.



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Fig. 1. (A) Internal generation and maintenance of the Ca2+ gradient in growing hyphae of N. crassa. This model is based upon extensive screening of inhibitors of Ca2+ signaling and PLC effects on hyphal growth, morphology, cytoplasmic and vesicular Ca2+ gradients, as well as direct evidence of IP3-activated Ca2+ channels using the BLM technique. Treatment with the Ca2+ ATPase inhibitor cyclopiazonic acid inhibited growth (hyphal widening was observed) and increased cytoplasmic [Ca2+] behind the apex, consistent with Ca2+ sequestration into endoplasmic reticulum behind the growing apex (Silverman-Gavrila and Lew, 2001Go). Microinjection of IP3 receptor agonists (IP3 and Adenophostin A) behind the tip inhibited growth transiently, caused subapical branching, and affected the tip-high cytoplasmic Ca2+ gradient; these effects were not observed after microinjection of the biologically inactive L-IP3 (Silverman-Gavrila and Lew, 2001Go). IP3-induced subapical branching was similar to subapical branching induced by ionophoretic injection of Ca2+ (Silverman-Gavrila and Lew, 2000Go), suggesting that IP3-activated Ca2+ release occurs in growing hyphae. An inhibitor of the IP3 receptor, 2-APB inhibited hyphal elongation and dissipated the tip high cytoplasmic [Ca2+] gradient. Thus, tip-localized IP3 production due to a stretch-activated phospholipase C could activate vesicular Ca2+ channels at the growing apex to generate the tip-high [Ca2+] gradient. The Ca2+ would induce fusion of wall vesicles at the apex, before being sequestered behind the tip via the Ca2+ ATPase. (B) The BLM technique used to identify and measure IP3-activated Ca2 channel activity in membrane vesicles isolated. from N. crassa. Hyphae are first homogenized to release the endomembranes. Following a series of centrifugations, subcellular fractions of membranes are isolated. Vesicles are added to the cis-chamber and fused to the lipid bilayer formed across an aperture in a septum that separates two chambers: cis and trans. The electronics are configured as a high gain current to voltage converter capable of measuring picoAmpere currents through ion channels. With only one permeant ion, Ca2+, only Ca2+ channels will be observed. Agonist addition to the cis compartment would activate channels oriented with their ligand binding site facing the cis compartment.

 

In animal cells, the major site for IP3 receptors is the ER (Berridge and Irvine, 1989Go). In plants (and fungi), the vacuolar membranes appear to contain Ca2+ channels, which can be activated by IP3. For example, IP3 induces Ca2+ release from plant hypocotyl microsomes (Drobak and Ferguson, 1985Go), and from vacuoles and tonoplast vesicles (Alexandre et al., 1990Go; Allen et al., 1995Go). The plant IP3 receptor was characterized in detail by Biswas (Biswas et al., 1995Go). In fungi, IP3 activates Ca2+ release from vacuoles of Candida albicans (Calvert and Sanders, 1995Go), Saccharomyces cerevisiae (Belde et al., 1993Go) and N. crassa (Cornelius et al., 1989Go). In N. crassa, phosphate groups at the 1 and 5 positions of the inositol ring are the only essential arrangement required for receptor binding to release Ca2+ from vacuoles, a unique characteristic compared with animal IP3 receptors (Schultz et al., 1990Go). There are reports of IP3-sensitive Ca2+ stores other than vacuoles in cauliflower inflorescences (Muir and Sanders, 1997Go) and in growing pollen tubes: in the nuclear-rough ER region (Franklin-Tong et al., 1996Go; Malho, 1998aGo) and from vesicles or ER (Zheng and Yang, 2000Go). These observations suggest that plant and fungal vacuoles function as a major IP3-regulated intracellular Ca2+ store equivalent to ER and sarcoplasmic reticulum in animal cells, but IP3-activated Ca2+ release may also involve other organelles.

Enzymes and metabolites of the phosphoinositide cycle are present in plants (Stevenson et al., 2000Go), yeast and filamentous fungi. There are indications that one function of IP3 signaling is regulation of morphological transitions. IP3 is involved in germ tube formation in the yeast-mycelium transition in Ophiostoma ulmi (Brunton and Gadd, 1991Go). In C. albicans, there is an increase in IP3 during germ tube formation of the yeast to hyphal morphological transition (Gadd and Foster, 1997Go). Ins(1,4,5)P3 kinase activity is higher during germ tube formation compared with the yeast morph. Prior et al. (Prior et al., 1993Go; Prior et al., 1994Go) and Lakin-Thomas (Lakin-Thomas, 1993aGo; Lakin-Thomas, 1993bGo) identified the key components of the phosphoinositide signal transduction in N. crassa, showing that the number of isomers of bis and tris inositol phosphates present in fungi is far more complex than in animals and plants. They failed to demonstrate stimulation of phosphoinositide turnover by a variety of external stimuli. However, Kallies (Kallies et al., 1998Go) found that normal IP3 concentration within growing hyphae of N. crassa increased two to fivefold to trigger Ca2+ release during the heat shock response. IP3 signaling may be involved in the regulation of endogenous metabolism and differentiation since inhibitors of phosphoinositide turnover led to lower extension rates and increased hyphal branching (Hosking et al., 1995Go), including Li+ inhibition of inositol metabolism and hyphal growth (Hanson, 1991Go). Besides fungal organisms, there is evidence of IP3-mediated morphogenesis in tip-growing pollen tubes (Malho, 1998aGo; Kost et al., 1999Go).

Our evidence for the presence of an IP3 receptor in fungi was based upon an in vivo pharmacological signature: increase in cytoplasmic Ca2+ caused by IP3 and depletion of tip-localized [Ca2+]cyt by 2-aminoethoxidiphenylborate (2-APB), while other known inhibitors of IP3 receptors [8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), dantrolene, heparin] had no effect on hyphal growth (Silverman-Gavrila and Lew, 2001Go). This inhibitor signature was used to assess ion channel activities from endomembranes and to establish the presence of an IP3 receptor required for hyphal growth. Such an IP3 receptor would be novel and unique to fungi as neither the N. crassa nor yeast genomes have sequences homologous to animal IP3 receptors based on BLAST searches. To demonstrate the presence of a functional IP3 receptor in N. crassa, we used the bilayer lipid membrane (BLM) technique (Fig. 1B).

The BLM technique is an invaluable method for the investigation of the properties of ion channels from intracellular membranes that are inaccessible to patch clamp electrodes. In this technique, channel-containing vesicles are incorporated into a bilayer membrane so that ion flux through the channel can be studied under voltage clamp conditions, permitting the measurement of the channel conductance. The electrical activity of the channel is measured under well-controlled aqueous conditions. Thus, in solutions containing only Ca2+ as a permeant ion, only Ca2+ permeant channels will be observed. In addition to biophysical characterization of ion channels, the technique can be used to determine the direct effect of agonists and antagonists on ion channel conductance and kinetics. Thus, IP3 activation of Ca2+ channels can be observed directly, as well as inhibitor effects. With this technique, we were able to demonstrate IP3-activated Ca2+ channels in N. crassa membranes.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Germling preparation
Large scale conidial harvests were prepared by inoculating Ehrlenmeyer flasks containing 20 ml Voegel's medium (Voegel, 1959Go) plus 2% agar and 2% sucrose with N. crassa RL 21a (FGSC no. 2219) wild-type conidia. The cultures were grown for 2 days at 30°C in the dark, then at room temperature in continuous light for at least 3 days. Conidia were harvested and resuspended in Voegel's medium plus 2% sucrose at a final concentration of 106 conidia/ml. Conidia were germinated by incubation at 37°C on a shaker rotating at 250 rpm. To maximize isolation of membranes from growing hyphal tips compared with subapical regions, the germlings were collected after 5-6 hours of growth (germ tube length of 100 to 200 µm) by centrifugation at 160 g for 10 minutes.

Microsomal membrane preparation
Germlings were disrupted manually by vigorous grinding at 4°C in a mortar and pestle using glass sand (150-212 µm) in ice-cold homogenizing buffer (250 mM sucrose, 10 mM Na2EDTA, 5 mM MgSO4, 25 mM MES, 2.5 mM dithiothreitol, pH adjusted to 7 with 1 N KOH). BSA (1% w/v) was added just prior to homogenization. The homogenate was decanted and left on ice for about 1 minute to sediment the sand and cell debris. The supernatant was centrifuged at 40,000 g for 15-30 minutes to spin down the mitochondria, cell walls, nuclei and remove traces of sand using a Beckman SW-27 swinging bucket rotor in a Beckman ultracentrifuge or a Sorvall SS-34 fixed angle rotor in a Sorvall RC 5B plus centrifuge. Then the supernatant was centrifuged in a SW-28 swinging bucket rotor using a Beckman Optima ultracentrifuge for 1 hour at 80,000 g. The microsomal pellet was resuspended in 200 µl resuspension medium: either 1 M sucrose, the homogenizing medium without BSA or in 40% (1.17 g ml-1) sucrose and 10 mM MOPS (pH 6.4). The resuspension (about 1 mg/ml protein) was aliquoted and stored at -20°C. The aliquots (8 µl) were sufficient for 2-3 experiments; by using small aliquots we avoided membrane damage caused by freeze-thaw cycles. Protein concentration was determined colorimetrically using a modified Bradford protein assay (Bradford, 1976Go) with a BSA standard.

Subcellular fractionation in discontinuous sucrose density gradients
Specific membrane fractions from N. crassa were isolated by subcellular fractionation using density gradient centrifugation. Germlings were disrupted by grinding in homogenizing buffer (250 mM sucrose, 10 mM EGTA, 10 mM MgSO4, 25 mM MES, 2.5 mM dithiotreitol pH 7.0 with KOH). The 80,000 g microsomal pellet was resuspended in 40-200 µl resuspension buffer (250 mM sucrose, 2.5 mM MES, 2.5 mM dithiotreitol adjusted to pH 7 with solid BTP) and layered onto a discontinuous three step sucrose gradient [5 ml of 16%, 29%, 39% (w/w) sucrose buffered to pH 7.2 with 5 mM MOPS/BTP] and centrifuged for 2 hours at 100,000 g at 4°C. This method was designed to partition the microsomal fraction into `vacuolar' (16%) (Vaughn and Davis, 1981Go), ER (16/29%) (Borgeson and Bowman, 1983Go) and plasma membrane (29/39%) (Bowman et al., 1981Go) interfaces. The actual vacuolar membrane density has been reported to vary from 1.06 to 1.30 g cm-3 (Vaughn and Davis, 1981Go), depending on the details of homogenization and osmolarity. Because we used young germlings, which should not have had time to accumulate vacuolar polyphosphates and arginine, and homogenization at relatively low osmolarity (Vaughn and Davis, 1981Go), with EGTA present (Bowman and Bowman, 1982Go), we expect the vacuoles to have a low density, similar to plant vacuoles. To assure minimal contamination with the ER, we chose a cut-off density of 1.06 g cm-3 (16% w/w sucrose). The membranes from interfaces were collected and aliquoted in small Eppendorf tubes. Membranes were fused to planar lipid bilayers and channels assayed for IP3 activation, conductance and inhibitor effects.

Planar bilayer measurements of channel activity
Planar lipid bilayers were formed from a lipid mixture of 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphatidylcholine: 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphatidylethanolamine: 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphatidyl-L serine (sodium salts):cholesterol (50:10:30:10 parts, w/w) (Avanti Polar Lipids, Alabaster, AL), stored in benzene under N2 at -20°C. The lipid mixture was chosen to ensure a lipid environment similar to the naturally occurring fatty acids, phospholipids and sterol:phospholipid ratio (Aaronson et al., 1982Go), except that cholesterol was used instead of ergosterol. Vesicles and reagents (IP3 for receptor activation, inhibitors) were added to the cis compartment (Fig. 1B). The sign of the potential difference refers to the trans compartment; a positive current corresponds to a cation flux from trans to cis or an anion flux from cis to trans compartment and is shown as an upward deflection from zero current. Thus, a positive current is positive charge flux from the lumenal side to the cytoplasmic side, the direction of Ca2+ flux that is physiologically relevant.

Vesicle fusion with the bilayer
After a stable bilayer membrane had formed, 4-32 µl microsomal vesicles (1 mg/ml protein) were added to the cis chamber. The cis solution (1 M KCl, 10 mM CaCl2, 200 mM MOPS/BTP, pH 7.2) was hyperosmotic to the trans solution {either 50 mM Ca(OH)2 [an activity of 30 mM based on the activity coefficient for Ca(ClO4)2 (Zaytsev and Aseyev, 1992Go)] or Ba(OH)2 or D-Gluconic acid (hemi-magnesium salt) and 200 mM MOPS/BTP, pH 7.2}, and the vesicles were hyperosmotic to the cis compartment solution to enhance the frequency of vesicle fusion. In other experiments we used 100 µM Ca(OH)2 and 200 mM MOPS/BTP (pH 7.2) (calculated activity 95.9 µM) in trans. The current was monitored at various transmembrane holding potentials (±50 to 100 mV) for the appearance of K+ and/or Cl- channel activity as indicator of vesicle fusion to the bilayer.

Activation of the channel by IP3
Once channel incorporation into the bilayer membrane was detected, the cis solution was perfused away with 1 mM EGTA, 200 mM MOPS/BTP pH 7.2 to prevent further vesicle fusion and to leave Ca2+ on the trans side as the only ion present in the system. Free Ca2+ was calculated based on a Turing program implementation of the algorithm and binding constants from Goldstein (Goldstein, 1978Go). In the absence of any permeant ion except Ca2+, only Ca2+ channels can be observed. We did not observe spontaneous Ca2+ channel openings. Ca2+ channels were observed only after the addition of IP3, Na2ATP and CaCl2 to the cis compartment sequentially or together to attain a final concentration of 16 µM IP3, 100 µM Na2ATP and 1 mM CaCl2.

After IP3 activation of the Ca2+ channels, the effects of the inhibitors heparin, 2-APB, TMB-8 and dantrolene (Calbiochem, La Jolla, CA) on channel activity were examined.

Channel conductance
Current-voltage measurements were made with a BC-525 C bilayer clamp amplifier (Warner Instruments Corp); holding potentials ranged from -100 to +100. Channel currents were filtered at 1 KHz (corner frequency) with an 8 pole low-pass Bessel filter and recorded with a modified (pulse code modulation) VCR (DAS/VCR 900; Dagan Corp, Minneapolis, MN). All recordings were carried out at room temperature. Data acquisition and analysis were performed using AxoScope pCLAMP software version 1.1 (Axon Instruments, Burlingame, CA). Recording playbacks were filtered at 0.5 kHz with a 4-pole Bessel filter (LPF 100B, Warner Instruments) and digitized at 1 kHz. For computer analysis, the recorded voltage clamp signals were not filtered prior to recording and digitization. Current amplitudes were measured manually using cursors, choosing clear channel opening/closing events. The conductance and the reversal potential were determined from the slope and X-intercept of the current-voltage curve.

PLC inhibitors: treatments and growth measurements
N. crassa was grown in 35 mm tissue culture dishes on solid substrate (2% w/v gellan gum) containing 2% sucrose and Voegel`s minimal medium. After incubation at 28-30°C for 14 hours, the culture was flooded with buffer solution (BS) [10 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM PIPES (pH adjusted to 5.8 with KOH) and the osmolality adjusted to 260 mosmol/kg with sucrose] (Levina et al., 1995Go).

Individual growth rates were measured every minute for 10 minutes in BS with a Zeiss Axioskop microscope and a 60x water immersion objective. Then, PLC inhibitors {neomycin, 3-nitrocoumarin, 1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122)} or, as a control, the biologically inactive analog 1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-pyrrolidine-2,5-dione (U-73343) were perfused through the chamber (2 minutes were required for solution exchange) and growth rates measured for 10 minutes, followed by wash out and measurement of growth rates in BS alone for another 10 minutes. For inhibitors that required DMSO solubilization (U-73122, U-73343 and 3-nitrocoumarin) pre-and post-treatments in BS included DMSO at the final concentration used during the treatment. Growth rates in BS plus DMSO were very similar to growth rates in BS alone. The number of experiments for any given treatment ranged from 7 to 9. When morphological changes at the growing tip were observed during or after treatment, other hyphae from the plate were also examined to confirm that the effect was general.

Chlortetracycline imaging
In preliminary experiments we determined the optimal concentration of chlortetracycline (CTC) (Sigma), which did not affect hyphal growth but still provided good fluorescence signal staining. Similar growth rates were found for control untreated hyphae and hyphae-treated with CTC at 25-50 µM concentration range. After CTC addition, hyphal growth and organellar Ca2+ fluorescence were measured, and then the hyphae were treated with 2-APB, cyclopiazonic acid, neomycin or U-73122 (Calbiochem, La Jolla, CA) and growth and fluorescence measured. The fluorescence was detected using the same settings before and after inhibitor treatments. Both confocal (krypton-argon laser Bio Rad MRC, Nikon Optiphot microscope, 40x water immersion lens, BHS, filter power NDF=0, excitation at 488, <515 nm, direct and Kalman filtering) and conventional fluorescence (Zeiss 60x water immersion objective, FITC filter cube, Hammamatsu C4742-95 digital camera) microscopy were used and gave similar results.


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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibitor effects on growth and the Ca2+ gradient implicated IP3-activated Ca2+ release from internal stores as the generator of the gradient. IP3 microinjection inhibits growth transiently and modifies the tip-high Ca2+ gradient in growing fungal hyphae. It does not affect the trans-plasma membrane electrical potential (data not shown). Thus the putative IP3 receptor was expected to be localized to endomembranes, so we used cellular membrane fractionations and the BLM technique (Fig. 1B) to identify directly IP3-activated Ca2+ channels.

IP3 activates small and large conductance Ca2+ channels in Neurospora crassa
To obtain tip-enriched membranes we used conidial germlings. From a starting material of 30 g germlings, we obtained about 0.24 mg total membrane protein. The appearance of channel activity was used to confirm successful fusion of membrane vesicles to the planar bilayer (Fig. 2A). In initial experiments with total membranes, successful fusions were observed in 101/138 trials (73.2%) (Table 1). Following the incorporation of vesicles into the planar lipid bilayer, the KCl-containing cis solution was replaced with EGTA and MOPS/BTP so that trans Ca2+ was the only channel-permeant ion. No spontaneous Ca2+ channel activity was observed, with very rare exceptions (4 out of 79), which could be due to incomplete wash-out of the KCl. Subsequently, D-IP3, ATP and CaCl2 were added to the cis compartment and activation of the receptor was observed in 38 out of 101 successful fusions (37.6%) (Table 1). As a control, the biologically inactive IP3 enantiomer, L-IP3 did not activate the channel (n=2) (Fig. 2A). Other control experiments were performed by adding Ca2+ and/or ATP before IP3; in the absence of IP3 they did not activate the channel (n=2, data not shown). ATP was usually added along with IP3 because it is required for full activation (increased open probability, unchanged conductance) of the animal IP3 receptor (Bezprozvanny and Ehrlich, 1993Go). To assess the requirement for ATP for channel activity, IP3-induced Ca2+ release was studied in the absence and presence of varying [ATP]. ATP is not required for channel activation, nor does subsequent ATP addition increase significantly channel activity (n=4, data not shown).



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Fig. 2. (A) Activation of the channel by IP3, but not by the biologically inactive enantiomer L-IP3. After the formation of a lipid bilayer membrane across the aperture of the bilayer chambers, vesicles are added to the cis chamber. Vesicle fusion events with the bilayer are monitored by observing the appearance of channel activity (a), in this case at an applied voltage of +60 mV. The cis solution (1 M KCl, 10 mM CaCl2, 200 mM MOPS/BTP, pH 7.2) is replaced with 1 mM EGTA, 200 mM MOPS/BTP, pH 7.2 to remove all permeant ions except Ca2+. Trans-solution remained the same (50 mM Ca(OH)2, 200 mM MOPS/BTP, pH 7.2). No spontaneous Ca2+ channel activity is present after wash out (+60 mV clamp) (b). To demonstrate the presence of IP3-activated Ca2+ channel activity, the receptor agonist D-IP3 is added to the cis chamber. Addition of IP3 induces Ca2+ channel activity, with a conductance of about 10 pS (+60 mV clamp) (c). Channel activity is not present after removal of D-IP3 by wash-out (+40 mV clamp) (d). Ca2+ channels are not activated by addition of L-IP3 (+40 mV clamp) (e). The level marked closed on current traces represents zero current. The bars represent the current in pA (vertical) and time in seconds (horizontal). (B) The small conductance Ca2+ channel is inhibited by 2-APB but not by TMB-8. IP3 activates a small conductance Ca2+ channel (+50 mV clamp) (a). TMB-8 (200 µM) does not inhibit the channel (+70 mV clamp) (b), but 2-APB (25 µM) does (+50 mV clamp) (c).

 

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Table 1. Relative abundance of small and large conductance channels in microsomal membrane isolations

 

In asymmetric cis/trans solutions with 50 mM Ca(OH)2 (30 mM activity) and 200 mM MOPS/BTP pH 7.2 in the trans compartment, and 1 mM [Ca2+] in the cis compartment (calculated Ca2+ activity of 7.1 µM), IP3 activated two channels with mean conductances of 11.9±5.1 pS (n=16) and 82.9±69.7 pS (n=5) measured at potentials clamped between -100 and +100 mV (Table 1).

Inhibitor characterization of the small and large conductance channels and correlation with hyphal growth
Hyphal growth and cytosolic Ca2+ gradients are affected by 2-APB, but not by heparin, TMB-8 or dantrolene. This inhibitor signature was used in preliminary characterization of the IP3-activated Ca2+ channels. The small conductance channel was inhibited by 2-APB (25 µM) (Fig. 2B), but was unaffected by 200 µM TMB-8 (Fig. 2B), 50 µM heparin and 100 µM dantrolene (Fig. 3A,B). After 2-APB inhibition, addition of IP3 (16 µM) and ATP (0.1 mM) did not restore channel activity (data not shown). The large conductance channel was inhibited by 2-APB (data not shown), TMB-8 (Fig. 4A), heparin (Fig. 4B) and dantrolene (Fig. 4C). The latter three inhibitors do not affect hyphal growth or inhibit the small conductance channel. Thus, the small conductance channel is implicated as the generator of the tip-high Ca2+ gradient required during hyphal growth (Table 2).



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Fig. 3. (A) Heparin does not inhibit the small conductance channel. After vesicle fusion, no spontaneous Ca2+ channels are observed in the absence of IP3 (40 mV clamp) (a). D-IP3 activates Ca2+ channels (12 pS) (+40 mV clamp) (b). The competitive IP3 channel blocker heparin (50 µM) has no effect on channel activity even after a second treatment (40 mV clamp) (c).(B) Dantrolene does not inhibit the small conductance channel. Prior to IP3 addition, no spontaneous Ca2+ channels are present (+60 mV clamp) (a). IP3 opens a small conductance channel (11 pS) (+100 mV clamp) (b). Dantrolene does not inhibit the channel (+100 mV clamp) (c).

 


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Fig. 4. (A) TMB-8 inhibits the IP3-activated large conductance Ca2+ channel. Channel activity signals vesicle fusion (+100 mV clamp) (a). No spontaneous Ca2+ channels are observed after wash out (+45 mV clamp) (b). IP3 activates a large conductance channel (41 pS) (+70 mV clamp) (c). TMB-8 (200 µM) inhibits channel activity (+60 mV clamp) (d). (B) Heparin inhibits the large conductance channel. Representative channel current recordings show Ca2+ channel activation in response to the application of IP3 (+50 mV clamp) (a). The channels are inhibited by heparin (50 mV clamp) (b). (C) Dantrolene inhibits completely the large conductance channel. IP3-activated Ca2+ channel (131 pS) (+80 mV clamp) (a). Inhibition by 100 µM dantrolene (+80 mV clamp) (b).

 

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Table 2. Effects of Ca2+ regulators on IP3-activated Ca2+ channels, hyphal growth and morphology in N. crassa

 

Ionic selectivity of IP3 channels
Cationic selectivity was determined for Ba2+, Ca2+ and Mg2+ using Ba(OH)2, Ca(OH)2 and Mg gluconate, respectively, in the trans compartment. For the small conductance IP3 channel similar conductances were observed with either 50 mM Ba(OH)2 or Ca(OH)2, but the channel was not observed with Mg gluconate (n=3) (Fig. 5). The large channel transports Ba2+ and Ca2+, and Mg2+ but with a smaller channel amplitude (n=2, not shown). Using constant 50 mM [Ca2+] in trans, the channel conductance was determined at different [Ca2+] in cis. The conductance decreased slightly with increased [Ca2+]. A cis [Ca2+] of 40 mM does not inhibit the channel (data not shown).



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Fig. 5. The small conductance channel conducts Ca2+, but not Mg2+. After vesicle fusion, no spontaneous Ca2+ channels are present (+60 mV clamp) (a). After IP3 addition, the small conductance channel does not conduct Mg2+ (+60 mV clamp) (b), but does conduct Ca2+ (12 pS) (+60 mV clamp) (c). 2-APB inhibits completely the channel (+50 mV clamp) (d).

 

Reversal potentials and channel conductance measurements at defined Ca2+ activities
In initial experiments we chose trans and cis [Ca2+] to maximize our ability to observe measurable currents through IP3-activated Ca2+ channels that release Ca2+ from the lumenal side of fused vesicles (the trans compartment). The concentration in trans was 50 mM (calculated activity 30 mM). With cis Ca2+ activity at 7.09 µM, the Nernst potential for Ca2+ was -105 mV. Under these conditions, the reversal potentials for channels were difficult to measure because the membrane was not stable at negative voltages, and negative channel current amplitudes could not be measured reliably. To assess ion-selectivity of the channels, the [Ca2+] in the trans compartment was decreased to 100 µM (calculated Ca2+ activity of 96 µM) to bring the expected reversal potential to less negative values and to study channel activity under more physiological conditions. In N. crassa, Ca2+-containing vesicles are estimated to have a concentration of at least 26 µM (Torralba et al., 2001Go) and the tip-localized cytoplasmic Ca2+ activity was found to be in the nanomolar range (400-600 nM) (Silverman-Gavrila and Lew, 2000Go; Silverman-Gavrila and Lew, 2001Go). We used a higher cis Ca2+ activity of 7.09 µM. With Ca2+ as the only charge carrier, the average conductances (small channel: 15±3 pS (n=8); large channel: 153.5±90.7 (n=6) were similar to the conductances observed with cis and trans Ca2+ activities of 7.09 µM and 30 mM (13.4±5.6 pS (n=24) and 77.0±61.1 pS (n=8)), respectively (Fig. 6, Table 3). The theoretical reversal potential for Ca2+ was calculated to be -33 mV and for Cl- ~-200 mV. The observed reversal potential was -26.30±14.18 (n=8) for the small conductance channel and -18.59±5.72 (n=6) for the large conductance channel. As the reversal potential was close to the Nernst potential for Ca2+, we can consider the channels to be Ca2+ selective with no Cl- permeability. Voltage-dependent activation was not observed at cis and trans Ca2+ activities of 7.09 µM and 96 µM, respectively.



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Fig. 6. Current voltage curves of IP3-activated Ca2+ channels in N. crassa membranes. Current-voltage measurements reveal two distinct Ca2+channels: a small conductance channel (circles) and a large conductance channel (triangles). The current is recorded at different membrane potentials, then the voltage dependence of channel current is used to obtain current-voltage relations. Conductance and reversal potential are determined by linear regression. The calculated Nernst potential for Ca2+ is -33 mV for 100 µM Ca(OH)2 (calculated activity of 96 µM) in trans and 1 mM CaCl2 (calculated activity 7.09 µM) in the cis compartment. Note the difference in scale for small (left) and large (right) conductance channels. In this example, the channel conductance determined from the slope of current-voltages curves was 13 pS for the small channel and 111 pS for the large channel. Amplitude versus voltage was linear and reversed near -25 mV for the small channel and -16 mV for the large channel. Compiled data are presented in Table 3.

 

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Table 3. Reversal potential and conductance of small and large conductance channels with low Ca2+ in trans chamber

 

The small conductance channel correlates with ER and plasma membrane, the large conductance with vacuolar membrane in subcellular gradient fractionations
To identify possible origins of the IP3-activated Ca2+ channels, we fractionated microsomal endomembranes on sucrose density step gradients (Table 4). The mean conductance for the small channel was 15.11± 6.93 pS (n=6) and the large conductance channel 67.12±35.66 pS (n=3). Relative abundance of channels were 66:34% (27:14) small:large conductance channel, similar to the results obtained using microsomal endomembranes. Since Ca2+ containing vesicles destined for fusion at the tip are produced in the ER, and cause plasma membrane `expansion', we expect enrichment in these two fractions. Indeed, the small conductance channel is enriched in the plasma membrane (78%) and ER fractions (80%). The high conductance channel is enriched in the vacuolar membrane fraction (88%).


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Table 4. Relative enrichment of small and large conductance channels in sucrose density gradient subcellular membrane fractionations

 

Inhibitors of the IP3-activated Ca2+ channel and PLC affect similarly growth rates and Ca2+ containing vesicle distribution
If vesicular IP3-activated Ca2+ channels generate the tip-high Ca2+ gradient required for tip growth, IP3 production catalyzed by PLC must be localized at the hyphal tip. Three putative PI-specific PLC genes have been identified in N. crassa (Jung et al., 1997Go). Initially, we screened PLC inhibitors for their effect on growth rates and morphology. Neomycin inhibits growth completely at 400 µM and slightly at 100 µM. Growth rates are unaffected in control experiments with buffer solution (BS) (Fig. 7A). A known inhibitor of yeast phosphatidylinositol-specific PLC, 3-nitrocoumarin, inhibited growth (Fig. 7B, Fig. 8C) with a concentration dependence very similar to in vitro inhibition of PLC (half-maximal inhibition at 10 µg/ml) (Tisi et al., 2001Go). After washing out the inhibitor 10 minutes after treatment, hyphae recovered and restarted growth, showing that the inhibitor effect was reversible. The PLC inhibitor U-73122 was very potent, inhibiting growth at 10 µM. Growth was unaffected by the inactive U-73122 analog, U-73343 at 400 µM (Fig. 7C, Fig. 8A,B).



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Fig. 7. Effects of PLC inhibitors on N. crassa growth rates. (A) Neomycin treatment. (Upper panel) Control experiments for treatment with external added inhibitors show that solution exchange (BS was exchanged with BS) does not affect hyphal growth. Middle and lower panels illustrate growth rates of hyphae treated with neomycin (as marked). Neomycin inhibits hyphal growth, partially at 100 µM and completely at 400 µM. (B) 3-nitrocoumarin causes a dose dependent inhibition of hyphal elongation: complete at 40 µg/ml, very efficient at 20 µg/ml and slight at 4 µg/ml. (C) Growth inhibition with increasing concentration of U-73122. (Upper panel) Control experiments. No influence on growth rates is observed for hyphae treated with the inactive analog U-73343. (Middle and lower panel) U-73122 inhibits growth: efficiently at 10 µM and completely at 400 µM within 4 minutes after its addition. Thin lines show individual experiments. Thicker lines and symbols show means±s.e.

 


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Fig. 8. Effects of PLC inhibitors on hyphal growth in N. crassa. (A) U-73122 (400 µM) causes complete inhibition. (B) Control compound U-73343 did not affect hyphal elongation and morphology. (C) 3-nitrocoumarin (20 µg/ml) inhibited hyphal growth. The hypha recovered and restarted growth after washing out the inhibitor at 10 minutes. Pictures were taken at the time shown. Time 0 represents the addition of the inhibitor. Bars, 10 µm.

 

To demonstrate that the growth inhibition caused by PLC inhibitors was related to the IP3 production and affected the tip-high Ca2+ gradient we imaged their effects on organellar Ca2+ using chlortetracycline (CTC). In growing hyphae stained with CTC, strong fluorescence is detected associated with apical vesicles. When IP3-induced Ca2+ release is inhibited either directly by inhibiting IP3 channels with 2-APB (25 µM) or by inhibiting IP3 production by inhibiting PLC with 10 µM U-73122 or 400 mM neomycin, the tip-high Ca2+ fluorescence disappears and is replaced by increased Ca2+ fluorescence behind the tip (Fig. 9A-C). We compared this effect with that of an inhibitor of Ca2+ ATPase cyclopiazonic acid (100 µM), known to increase cytoplasmic [Ca2+] behind the hyphal tip (Silverman-Gavrila and Lew, 2001Go). No strong organellar Ca2+ fluorescence was observed behind the tip after cyclopiazonic acid treatment (Fig. 9D). We suggest that vesicular Ca2+ appeared subapically because Ca2+-storing vesicles, unable to release Ca2+, could not fuse at the tip, and retreated behind the tip during inhibition. Long-term, upon wash-out, vesicles would again move at the tip where a Ca2+ flush from vesicles will cause apical hyperbranching. Indeed, after 2-APB was washed out, hyphal growth resumed as multiple tips at the original apex (apical hyperbranching) (Fig. 10A). The same growth recovery phenotype was observed after washing-out the PLC inhibitor U-73122 (Fig. 10B). The unique hyperbranching phenotype is not observed after wash-out of the Ca2+-ATPase inhibitor cyclopiazonic acid (instead, the hyphal apex resumes normal elongation, data not shown).



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Fig. 9. Effects of Ca2+ and PLC inhibitors on organellar Ca2+ imaged with chlortetracycline. In normal growing hyphae significant fluorescence is detected associated with vesicles at the tip. (A) During Ca2+ imaging typical response (reduction of fluorescence at the tip, and an increase behind) is observed after the addition of 25 µM 2-APB at time 0 (n=18). At the same time hyphal elongation stops. (B) Similar results are obtained after treatment with 10 µM U-73122 (n=15). (C) Following the treatment with 400 µM neomycin similar effects occur (n=9). (D) In hyphae treated with cyclopiazonic acid the tip high Ca2+ is dissipated, but no increase in fluorescence behind the tip is observed (n=15). Bars, 10 µm.

 


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Fig. 10. Hyperbranching after washing out inhibitors of IP3 receptor and PLC. (A) Within 20 minutes of washing out 25 µM 2-APB, hyphal widening, which extended over 20-40 µm from the tip, and an apical hyperbranching phenotype was observed. (B) U-73122 (10 µM) wash-out also caused hyphal widening and multiple apical branches. The picture was taken 14 minutes after wash out. (C) Control hypha 16 minutes after wash out. Hyperbranching was not observed after wash-out of cyclopiazonic acid (not shown); in this case, hypha resumed normal hyphal elongation, similar to C. Bars, 10 µm.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We envisaged and tested a mechanism for the regulation of fungal expansion by IP3 channels in the model organism N. crassa (Fig. 1A). To our knowledge, this is the first direct demonstration of IP3-activated Ca2+ channels in fungi, including evidence that one channel type is involved in tip growth during hyphal expansion.

It may be significant that we did not observe spontaneous Ca2+ channel activity prior to IP3 addition. In preliminary experiments demonstrating the presence of a folic acid-activated Ca2+ channel of about 100 pS conductance in Dictyostelium discoideum, we did observe a spontaneous 12 pS Ca2+ channel prior to folate addition. Whether or not spontaneous Ca2+ channels are absent completely in fungi must await further analysis.

In the ascomycete N. crassa, we identified two types of IP3-activated Ca2+ channels that could be differentiated by their conductance: one small and one large. Reversal potential measurements confirm that both are Ca2+ selective. In addition to different conductances, ion permeability is also different, in that the small channel is impermeable to Mg2+.

We had already discovered a well defined inhibitor signature for hyphal growth, in which only a subset of known inhibitors of IP3 receptors affected growth and the cytoplasmic tip-high Ca2+ gradient required for growth (Silverman-Gavrila and Lew, 2001Go). We used this inhibitor signature to identify the channel that functions in tip growth. Inhibitors that inhibit only the large conductance channel do not affect tip-growth; for example, heparin microinjection, dantrolene, or TMB-8 treatments (Silverman-Gavrila and Lew, 2001Go). Thus, the large conductance IP3-activated Ca2+ channel does not play a role in hyphal growth. In contrast, 2-APB inhibits completely both the small and large conductance channel and hyphal growth and dissipates the tip-high Ca2+ gradient (Silverman-Gavrila and Lew, 2001Go). Both dissipation of the cytoplasmic calcium gradient and inhibition of hyphal growth occur within 1-2 minutes. The inhibition of growth, gradient dissipation and channel inhibition all occur at similar 2-APB concentrations (10-25 µM). In some organisms, the mode of action of 2-APB may not necessarily involve inhibition of IP3-activated Ca2+ channels. In animal cells, it is reported to inhibit Ca2+ entry (Kukkonen et al., 2001Go; Diver et al., 2001Go). In higher plants, capacitative Ca2+ entry has been postulated to play a role in pollen tube growth (Malho, 1998bGo). However, in the fungus N. crassa it is known that Ca2+ entry does not play a role in generation of the tip-high Ca2+ gradient. There are no tip-localized inward Ca2+ currents (Lew, 1999Go), modifying the electrical driving force for Ca2+ entry by voltage clamp has no effect on hyphal growth (Silverman-Gavrila and Lew, 2001Go), and IP3 microinjection does not affect the plasma membrane electrical potential, an expected consequence of Ca2+ entry (L.B.S.-G. and R.R.L., unpublished). We cannot discount completely the possibility of multiple sites of action by 2-APB. However, the direct demonstration of 2-APB inhibition of the IP3-activated Ca2+ channel, correlated with 2-APB inhibition of growth and dissipation of the tip-high Ca2+ gradient, suggest that the small conductance channel generates the tip-high Ca2+ gradient during growth. We propose that the small conductance IP3 —activated Ca2+ channel originates in the ER/Golgi body system. If the vesicles fuse with the plasma membrane at the apex the channel must be inactivated because IP3 microinjection has no effect on the plasma membrane potential.

The difference in inhibitor signatures indicates that the large conductance channel is localized in vacuoles as TMB-8, dantrolene and heparin are widely reported to inhibit the IP3-induced Ca2+ release from vacuoles in S. cerevisiae [TMB-8 and dantrolene (Belde et al., 1993Go)], in N. crassa [dantrolene (Cornelius et al., 1989Go)], in red beet root [heparin and TMB-8 (Brosnan and Sanders, 1990Go; Alexandre et al., 1990Go)], and C. albicans [partial inhibition by heparin (Calvert and Sanders, 1995Go)]. The localization of the large conductance channel on step gradients confirms its origin as vacuolar. The vacuolar IP3-activated Ca2+ channel may be important in signaling during environmental stress. Heat shock causes rapid increases in intracellular second messengers, cAMP and inositol phosphates (fivefold), as well as Ca2+ release from isolated N. crassa vacuoles (Kallies et al., 1998Go). In addition, vacuoles may regulate the homeostasis of cytosolic Ca2+ providing a storage reserve or detoxification system to prevent the effects of excessive Ca2+ in the cytoplasm. Cornelius and Nakashima isolated mutants with low rates of vacuolar Ca2+ uptake that were inhibited by normal [Ca2+] in medium (Cornelius and Nakashima, 1987Go). The vacuolar origin of this channel precludes a role in tip growth, since vacuoles are absent in the first 100 µm behind the hyphal apex (Zalokar, 1959Go).

Because there is no indication of sequences homologous to the multigenic IP3 receptor families in animals, we do not expect close relatedness to animal IP3 receptors. However, the channel does share similarities with type 3 IP3 receptors compared with types 1 and 2. Type 1 and 2 receptors are stimulated by low [Ca2+] and inhibited by high [Ca2+], we did not observe Ca2+ inhibition, similar to type 3 receptors (Thrower et al., 2001Go). Ca2+ release by type 3 is much less affected by ATP compared with type 1 (Thrower et al., 2001Go), similar to our observations. These observations are based on BLM measurements of channel activity, and may not hold true in the complex cytoplasmic milieu, in which other regulatory proteins may affect channel activity.

Since generation of the cytosolic tip-high Ca2+ gradient occurs at the growing apex, IP3 must be produced apically. PLC is the upstream step in IP3 signaling that catalyzes the conversion of phosphatidylinositol (4,5)-bisphosphate to IP3 and DAG. PLC activity is reported to be higher in hyphal versus yeast forms of C. albicans (Bennett et al., 1998Go). To confirm an apex-localized role of PLC, we examined the effects of PLC inhibitors on growth and organellar Ca2+. All inhibitors we examined inhibited growth followed by hyphal widening. It is notable that the half maximal inhibition of PLC activity by 3-nitrocoumarin, an efficient inhibitor of yeast PLC (Tisi et al., 2001Go), was similar to its inhibition of growth in vivo in N. crassa, strongly suggesting that PLC is essential for growth. Although any one of the inhibitors could inhibit other processes besides PLC, the fact that all affect hyphal growth implicates PLC activity as a central component of hyphal growth, producing IP3 at the apex. We then examined whether the PLC inhibitors affect organellar Ca2+.

The vesicular Ca2+ gradient has been visualized by CTC fluorescence in growing tips of pollen tubes (Reiss et al., 1985Go), the oomycetes S. ferax (Yuan and Heath, 1991Go) and N. crassa (Schmid and Harold, 1988Go). In N. crassa the CTC gradient is very similar to the steep gradient of apical vesicles (Collinge and Trinci, 1974Go) (L.B.S.-G. and R.R.L., unpublished) suggesting that vesicular Ca2+ originates from vesicles at the growing apex. Because 2-APB inhibits IP3-activated Ca2+ channels, and thus Ca2+ release from vesicles, and dissipates the cytosolic [Ca2+] gradient at the tip, we expected increased vesicular Ca2+ fluorescence at the tip. Instead vesicular Ca2+ appeared sub-apically. It is possible that Ca2+-containing vesicles, formed via the ER/Golgi body system, were unable to release Ca2+, could not fuse at the tip, and retreated subapically. Upon inhibitor wash out, they would again move forward, causing an apical hyperbranching phenotype. We observed the same subapical retreat during PLC inhibitor treatment and subsequent apical hyperbranching after wash out. As a control, cyclopiazonic acid, an inhibitor of ER Ca2+ ATPase, inhibits growth and increases basal cytoplasmic [Ca2+]. The loss of vesicular Ca2+ fluorescence in hyphae treated with cyclopiazonic acid is consistent with decreased Ca2+ sequestration behind the apex. Cyclopiazonic acid has also been reported to decrease vesicular Ca2+ at hyphal apices based on pyroantimoniate-staining of a vesicle sub-population in fixed tissues examined by electron transmission microscopy (Torralba et al., 2001Go).

In conclusion the present paper reports the first identification and characterization of IP3-activated Ca2+ channels in a lower eukaryote. One of these channels is implicated strongly as part of the mechanism responsible for the generation of a tip-high Ca2+ gradient required for fungal tip growth. Our present and evolving hypothesis is shown in Fig. 1A. Tip-localized PLC is activated, possibly by stretch (Kinnunen, 2000Go) and produces IP3. The IP3 activates Ca2+ channels from a subset of tip-localized Ca2+-containing vesicles that release Ca2+ into apical cytoplasm. The Ca2+ is required for fusion of wall vesicles with plasma membrane to cause tip expansion. The Ca2+ containing vesicles function in a `kiss and run' fashion, shuttling Ca2+ from ER to the growing apex. They accumulate Ca2+ through a Ca2+ ATPase. A `kiss and run' shuttle is consistent with the fact that Ca2+ efflux is not observed at growing tips (Lew, 1999Go).


    Acknowledgments
 
This work was supported by a NSERC grant to R.R.L. and an OGSST scholarship to L.B.S-G. Special thanks to Enzo Martegani (Milano University, Italy) for the generous gift of 3-nitrocoumarin.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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