Calcium gradient dependence of Neurospora crassa hyphal growth

Lorelei B. Silverman-Gavrila{dagger} and Roger R. Lew{dagger}

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

Correspondence
Roger R. Lew
planters{at}yorku.ca


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A tip-high cytoplasmic calcium gradient has been identified as a requirement for hyphal growth in the fungus Neurospora crassa. The Ca2+ gradient is less steep compared to wall vesicle, wall incorporation and vesicular Ca2+ gradients, but this can be explained by Ca2+ diffusion. Analysis of the relation between the rate of hyphal growth and the spatial distribution of tip-localized calcium indicates that hyphal growth rates depend upon the tip-localized calcium concentration. It is not the steepness of the calcium gradient, but tip-localized calcium and the difference in tip-localized calcium versus subapical calcium concentration which correlate closely with hyphal growth rate. A minimal concentration difference between the apex and subapical region of 30 nM is required for growth to occur. The calcium concentration dependence of growth may relate directly to biochemical functions of calcium in hyphal extension, such as vesicle fusion and enzyme activation during cellular expansion. Initiation of tip growth may rely upon random Ca2+ motions causing localized regions of elevated calcium. Continued hyphal expansion may activate a stretch-activated phospholipase C which would increase tip-localized inositol 1,4,5-trisphosphate (IP3). Hyphal expansion, induced by mild hypoosmotic treatment, does increase diacylglycerol, the other product of phospholipase C activity. This is consistent with evidence that IP3-activated Ca2+ channels generate and maintain the tip-high calcium gradient.


Abbreviations: BAPTA, 1,2-bis(ortho-aminophenoxy)ethane-N,N,N',N'-tetrapotassium acetate; CTC, chlortetracycline; fluo-3, 2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4'-methyl-2,2'-(ethylenedioxy)dianiline-N,N,N',N'-tetraacetic acid; IP3, inositol 1,4,5-trisphosphate

{dagger}Both authors contributed equally to the work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Polarized cell expansion, culminating in tubular extensions (tip growth), is a morphogenic process observed in all kingdoms, from bacteria to animals. In all the examples of tip-growing eukaryotic cells which have been studied so far, there is a relationship between tip extension and internal calcium gradients. For example, pollen tubes exhibit a cytoplasmic tip-high Ca2+ gradient during growth. The gradient is quite steep: [Ca2+] is about 3·0 µM at the tip, decreasing to 0·2 µM about 20 µm behind the tip (Pierson et al., 1994). Inhibition of pollen tube growth by BAPTA injection correlates with dissipation of the cytoplasmic tip-high Ca2+ gradient and inhibition of tip-localized Ca2+ influx (Pierson et al., 1994). In addition, pulsations of the tip-localized Ca2+ concentration are correlated with pulsatile growth in pollen tubes (Pierson et al., 1996; Messerli & Robinson, 1997). During pulsatile pollen tube growth, growth precedes increased Ca2+ influx and pulsatile cytoplasmic Ca2+ increases by a few seconds (Messerli et al., 1999, 2000). This suggests that Ca2+ influx ‘senses' tip expansion during growth; the response naturally lags behind tip expansion. Such a mechanism has been proposed to be mediated by stretch-activated Ca2+ channels localized at the tip. Stretch-activated Ca2+ channels have been characterized in the oomycete Saprolegnia ferax (Garrill et al., 1993), in which there is evidence for a correlation between growth rate and the magnitude of the Ca2+ gradient measured using ratio imaging of Ca2+ and pH-sensitive fluorescent dyes (Hyde & Heath, 1997).

Fungi also exhibit tip-high Ca2+ gradients during hyphal growth. Spatial cytoplasmic [Ca2+] has been measured using quantitative dual dye (fluo-3 and Fura Red) ratio imaging (Silverman-Gavrila & Lew, 2000). Analogous to pollen tubes, injection of BAPTA dissipates the gradient and stops growth (Silverman-Gavrila & Lew, 2000). Unlike pollen tubes (Pierson et al., 1994), root hairs (Felle & Hepler, 1997) or S. ferax (Lew, 1999), there is no indication that Ca2+ influx at the growing tip is responsible for generating the tip-high gradient. Although Neurospora crassa does have stretch-activated Ca2+ channels (Levina et al., 1995), there is no net Ca2+ influx during hyphal growth (Lew, 1999) and direct manipulation of the membrane potential to modify the driving force for Ca2+ influx does not affect growth rate (Silverman-Gavrila & Lew, 2000). The gradient is generated and maintained internally by the concerted action of inositol 1,4,5-trisphosphate (IP3)-activated Ca2+ release from tip-localized vesicles (Silverman-Gavrila & Lew, 2002) and Ca2+-ATPase-mediated sequestration into the endoplasmic reticulum behind the growing tip (Silverman-Gavrila & Lew, 2001). The location of the tip-localized vesicles is maintained by interaction with the actin cytoskeleton (Silverman-Gavrila & Lew, 2001).

Our objective in this paper is to explore the relation between the Ca2+ gradient and growth in the ascomycete N. crassa, and to identify a possible growth sensor responsible for generating the gradient to maintain continued growth. Does growth depend upon an absolute [Ca2+] at the tip, or is it the steepness of the gradient that is required during growth? Our assessment is done in the context of spatial regulation of the Ca2+ gradient, and its relation to other aspects of the polar cytology of N. crassa hyphae. Random fluctuations of the Ca2+ distribution may generate localized regions of elevated Ca2+ to initiate tip growth. Based on stretch-activated production of diacylglycerol, we propose that activation of a tip-localized phospholipase C may ‘sense’ growth, initiating a cascade of events that maintains the Ca2+ gradient during continued hyphal growth.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Culturing.
The wild-type Neurospora crassa strain RL21a (FGSC no. 2219) was grown in 35 mm tissue culture dishes on solid substrate (2 %, w/v, gellan gum) containing 2 % sucrose and Vogel's minimal medium (Vogel, 1956), and incubated at 28–30 °C for 14 h. Prior to experiments, the culture was flooded with 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-1 with sucrose] (Levina et al., 1995).

Ratiometric fluorescence imaging of cytoplasmic calcium.
Cytosolic [Ca2+] was measured by ratio imaging the emission intensities of the Ca2+-sensitive fluorescent dyes fluo-3 and Fura Red. They were loaded ionophoretically into the hypha. The electrophysiological techniques are described in detail elsewhere (Silverman-Gavrila & Lew, 2000, 2001). The micropipette was filled at the tip with 0·33 mM fluo-3 and 0·99 mM Fura Red (both as potassium salts; Molecular Probes) and backfilled with 3 M KCl. Hyphae were impaled about 35 µm behind the tip. Fluorescence imaging was performed using a Bio-Rad MRC-600 confocal apparatus with a krypton–argon mixed gas laser attached to a Nikon Optiphot 2 microscope (Silverman-Gavrila & Lew, 2000). Briefly, the dyes were excited at 488 nm using 10 % laser intensity (neutral density filter 1) and the emitted fluorescence was detected simultaneously at 522 (fluo-3) and 640 (Fura Red) nm using fast photon counting (10 scans). Ratio intensities were measured using 2·54 µm longitudinal transects within the cytoplasm of the hyphae in the software program NIH-Image (Rasband & Bright, 1995). As detailed in previous work (Silverman-Gavrila & Lew, 2000), the signal to noise ratio is high; autofluorescence contributes less than 11 and 6 % to the fluorescence intensities of fluo-3 and Fura Red, respectively.

Growth measurements of hyphae microinjected with fluo-3 and Fura Red.
Individual growth rates were observed with a Nikon Optiphot microscope and a x40 water immersion objective. Growth rates were measured from thermal prints, after hyphae had resumed growth following impalement with the micropipette and dye microinjection by ionophoresis. Growth rates measured either immediately before or after the ratiometric fluorescence imaging were used to correlate growth with the cytoplasmic [Ca2+] gradient.

Vesicular calcium imaging.
Organellar Ca2+ fluorescence was measured after chlortetracycline (CTC; Sigma) addition. The optimal concentration of CTC which did not affect hyphal growth but still provided good fluorescence signal was determined to be about 25–50 µM. The fluorescence was detected by confocal microscopy using a Bio-Rad MRC-600 apparatus equipped with a krypton–argon laser on a Nikon Optiphot microscope with a x40 water immersion objective. A BHS filter was used (excitation at 488 nm, emission>515 nm), with no neutral density filter. The acquired images were filtered using Kalman digital filtering to improve visualization. Plots of fluorescence intensity versus distance from the tip were obtained using 2·54 µm longitudinal transects along the hyphae in the software program NIH-Image. To correct for the smaller volume elements at the hyphal apex, volume was estimated from cylindrical volume elements 0·127 µm in length, with radius calculated from an exponential best fit to hyphal diameter. The CTC fluorescence intensity for each 0·127 µm sample was divided by the volume of the corresponding volume element; i.e. for distance from the tip, n, from 0 to 25 µm in 0·127 µm steps, CTCcorrected=CTCn/volumen.

Diacylglycerol measurements using HPLC.
To maximize the isolation of diacylglycerol produced at growing hyphal tips, we used conidial germlings. Large-scale conidial harvests were incubated in Vogel's minimal medium at 37 °C for 5–6 h, at which time the germlings had grown about 100–200 µm. Prior to isolation of diacylglycerol using chloroform/methanol extraction, the germlings were treated with phospholipase C inhibitors at concentrations that inhibited growth completely (Silverman-Gavrila & Lew, 2002) (neomycin, 400 µM; 3-nitrocoumarin, 40 µg ml-1; U-73122 or the inactive analogue U-73343, 400 µM), or subjected to hypoosmotic stress: either severe (a 1 : 19 dilution of Vogel's minimal medium with distilled H2O) or mild (a 1 : 1 dilution of Vogel's minimal medium with distilled H2O) stress. Immediately after treatment, the germlings were collected by filtration through a 0·22 µm filter, then scraped into a 1·5 ml Eppendorf tube containing 0·75 ml ice-cold chloroform/methanol (1 : 2, v/v), vortex-mixed and kept on ice for 15 min. Diacylglycerol extraction followed the protocol described in detail by Ramsdale & Lakin-Thomas (2000), adapted from Bligh & Dyer (1959). The diacylglycerol extracts were stored at -20 °C in chloroform containing 50 µg butylated hydroxytoluene ml-1. Mycelial dry weight was determined by washing mycelial debris from the initial extraction in methanol, drying overnight at 60 °C, then weighing. The lipids were measured using an HPLC technique modified from Bocckino et al. (1985). We used a Betasil silica-60 (5 µm particle size) (250x4·6 mm) column (ThermoHypersil-Keystone). Chromatography was performed using a BioCAD Sprint chromatography system (PerSeptiva Biosystems). The solvent was hexane/2-propanol/glacial acetic acid (250 : 2·5 : 0·025) (HIA) run at 3 ml min-1 at about 1200 p.s.i. (8280 kPa). Lipid samples (125 µl) were dried under N2 at 60 °C and redissolved in 500 µl HIA. After equilibration of the column with HIA, 100 µl samples were injected into the column. Lipids were detected by the A205. Diacylglycerol and ergosterol (Sigma-Aldrich) standards were used to identify HPLC peaks. All other reagents were obtained from Sigma-Aldrich and were HPLC grade.

Data analysis.
The experiments (77 in all) were sorted by growth rate and mean Ca2+ gradients were calculated for subsamples (n=11 or n=7). This assured an even spread of growth rates. A statistical software package (SYSTAT, version 5.0) was used for linear and nonlinear regression analysis of the relation between growth rate and various aspects of the Ca2+ gradient. Best fits for various mathematical models were obtained by minimization of least squares, , with either a quasi-Newton or Simplex method (Wilkinson, 1988). Linear or exponential models were used as described in Results. Goodness of fit was assessed quantitatively with correlation coefficients and two-tail probabilities.

Ca2+ random walks.
A computer program was written in C to produce a 64 by 64 array in which each array element contained 50 calcium ions initially. A uniform [0,1] random number generator based on a combination of two linear congruential sequences (L. Devroye; http://www-cgrl.cs.mcgill.ca/~luc/rng.html) was used to move 25 % of the calcium molecules to one of the four bounding array elements, depending on whether the random number fell in the range 0<x<=0·25, 0·25<x<=0·5, 0·5<x<=0·75 or 0·75<x<=1·0. The process was iterated 128 times. For visualization, the arrays were converted into images that were processed with a Gaussian filter and linear contrast stretch in NIH-Image (Rasband & Bright, 1995).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
An example of cytoplasmic [Ca2+] ratio imaging is shown in Fig. 1; quantification is shown in Fig. 2. The dyes co-localized in the cytoplasm-rich regions of the hypha. Occasional zones of exclusion, probably nuclei, were observed behind the hyphal apex. Dye sequestration was very rare; those experiments were not used in the analysis. The pseudocolour image shows the spatial distribution of the tip-high Ca2+ gradient. Longitudinal transects were used to produce quantitative measurements for analysis of the Ca2+ gradient dependence of the growth rate (Fig. 2). Individual experiments were sorted according to growth rate and compiled into samples of 11 experiments each (Table 1). Thus there is a mean growth rate for each mean gradient. Cytoplasmic [Ca2+] is compiled for regions of the hyphae extending from the tip to 20 µm, where subapical [Ca2+] approaches its basal level, [Ca2+]basal. To test the relation between the gradient and growth rate, we determined the correlations between growth rate and the steepness of the [Ca2+] gradient, tip-localized [Ca2+], and the difference between tip-localized [Ca2+] and basal [Ca2+].



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Fig. 1. Example of cytoplasmic free [Ca2+] imaging in a growing hypha using dual dye ratio imaging. (a) Fluo-3 fluorescence. (b) Fura Red fluorescence. (c) Pseudocolour ratiometric image of Ca2+ distribution. Quantification, based on in vitro calibrations, is shown in Fig. 2. Note that (a) and (b) have been contrast-enhanced to show the homogeneous distributions of the dyes in the cytoplasm-rich tip of the N. crassa hypha. Sequestration of the dyes is not apparent in this example, and was in fact an extremely rare event (Silverman-Gavrila & Lew, 2000). The dark halo around the pseudocolour ratio image is due to a border effect at the edge of the hypha when the ratio is calculated. Transects (2·54 µm wide) were placed longitudinally, abutted against the edge of the hyphae and completely within the cytoplasm (the haloes were not included) (Silverman-Gavrila & Lew, 2000). The hypha was growing at a rate of 6·9 µm min-1 when the fluorescence images were taken. For scale, the hyphal base is 7 µm wide (a).

 


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Fig. 2. Quantification of cytoplasmic free [Ca2+] imaging using dual dye ratio imaging. The ratio image in Fig. 1 was quantified by taking a longitudinal transect along the hypha. An in vitro calibration was used to convert the ratio to cytoplasmic [Ca2+]. The best fit is to an exponential function of the form , where [Ca2+]basal is the subapical [Ca2+], summed with [Ca2+]max to approximate tip-localized [Ca2+]. The growth rate of the hypha was 6·9 µm min-1; the steepness (tau) was 6·6 µm.

 

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Table 1. Data summary for cytoplasmic [Ca2+] gradients at the hyphal tip

Mean [Ca2+] from ratio images from different regions of the hyphae are tabulated for hyphae growing at the mean growth rates as shown. The difference between tip-localized and basal [Ca2+] and the gradient steepness (tau) are shown for the compiled growth rates.

 
Hyphal growth dependence on Ca2+ gradient steepness and magnitude
The steepness of the gradient can be quantified by using an exponential fit of [Ca2+] versus distance from the tip. We used an exponential equation of the form , where [Ca2+]basal is the basal [Ca2+], and is summed with [Ca2+]max to approximate tip-localized [Ca2+], d is the distance from the tip, and tau is a measure of the steepness of the gradient; a small tau corresponds to a steep gradient (Table 1). Growth rate was poorly correlated with the steepness of the gradient (Fig. 3); the Pearson correlation coefficient was very small, r2=0·000, P=0·462. Rather than steepness per se, it is possible that growth rate depends upon the magnitude of the Ca2+ gradient, either tip-localized [Ca2+], or the difference between tip-localized and basal free cytoplasmic [Ca2+] (Fig. 3). Similar correlations were found for both measurements of the [Ca2+] gradient; the correlations, while small, were statistically significant. To assure that the choice of sample size (n=11) did not cause a fortuitous correlation, the 77 individual experiments, sorted by growth rate, were recompiled into sample sizes of 7. No correlation was observed for growth versus gradient steepness (r2=0·078, P=0·172). The correlation coefficients for growth versus either tip-localized [Ca2+], or the difference between tip-localized and basal free cytoplasmic [Ca2+] were smaller due to increased variability with the smaller sample sizes (r2=0·23), but were still statistically significant (P<0·05) (data not shown). As an additional check, data were compiled on the basis of experimental runs, 5–8 days of intensive fluorescence image acquisition. In this case, growth rate variability was high, but both tip-localized [Ca2+] and tip-localized versus basal free cytoplasmic [Ca2+] were correlated significantly with growth rate (0·37>r2>0·52, P <0·002, data not shown); the gradient steepness was not (r2=0·147, P=0·213). Thus growth rate depends upon the magnitude of tip-localized [Ca2+], which must be higher than basal free cytoplasmic [Ca2+]. Growth is not correlated with the steepness of the Ca2+ gradient.



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Fig. 3. Dependence of growth rate on the [Ca2+] gradient steepness, tip-localized [Ca2+], and the difference between apical and basal [Ca2+] ([Ca2+]apical-[Ca2+]basal). Linear best fits are shown, along with correlation coefficients and P-values. Steepness of the gradient was measured using best fits to an exponential equation, . The higher the tau value, the less steep is the gradient. Tip-localized [Ca2+] ([Ca2+]apical) is the mean [Ca2+] 0–2·5 µm behind the hyphal tip. The difference between apical and basal [Ca2+] ([Ca2+]apical-[Ca2+]basal) was measured as the difference in [Ca2+] at 0–2·5 µm versus [Ca2+] at 10–20 µm. Growth rates are shown as mean±SD [n=11, except zero growth (n=19)].

 
Initial Ca2+ gradient generation
Inhibitor effects on hyphal growth and the [Ca2+] gradient suggest that the Ca2+ gradient is generated and maintained by the action of an IP3-activated Ca2+ channel releasing Ca2+ at the tip, and a Ca2+-ATPase sequestering Ca2+ behind the hyphal tip (Silverman-Gavrila & Lew, 2001). An IP3-activated Ca2+ channel with an inhibitor signature consistent with such a role has been characterized biochemically (Silverman-Gavrila & Lew, 2002). But how is the gradient generated de novo and how is it regulated during hyphal expansion? The initiation of hyphal extension from spherical spores, either asexual or sexual, could result from the spontaneous appearance of localized regions of elevated [Ca2+] as a consequence of the vagaries of the Ca2+ random walk. A simplified test of this hypothesis is shown in Fig. 4. Starting from a homogeneous array, a random walk causes the appearance of a typical heterogeneous distribution of Ca2+, a model for the expected random distribution of Ca2+ within the cell. Within the array, regions of elevated Ca2+ do appear (Fig. 4a), some localized near the outer border of the array. Histogram analysis of the distribution reveals a subset of array elements containing 1·5–2-fold elevations of Ca2+ (Fig. 4b). Similar heterogeneous distributions, varying with time, are observed in the vesicular Ca2+ of a conidium (Fig. 4c) and cytoplasmic [Ca2+]basal in a hypha (Fig. 4d). Thus spontaneous localized Ca2+ elevations could be sufficient to trigger the cascade of events that result in hyphal initiation.



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Fig. 4. Heterogeneous Ca2+ distributions. (a) A Ca2+ random walk was used to generate a model of the typical random distribution of calcium within the cell. Localized regions of elevated Ca2+ appear throughout the two-dimensional 64 by 64 array. For visualization, the image was blurred (Gaussian), and a linear contrast stretch was applied. (b) The histogram shows frequency versus array element intensity. A subset of the arrays contain elevated Ca2+ (lighter regions), as expected. Time-dependent changes in Ca2+ distribution are observed in CTC fluorescence imaging of vesicular Ca2+ in conidium (from top to bottom: 0, 40 and 125 s) (c) and basal cytosolic Ca2+ in a hypha imaged with fluo-3 and Fura Red (d). They too could result in localized regions of elevated [Ca2+]. For scale, the conidium is 4 µm in diameter, and the hyphal cytoplasmic free Ca2+ images are 4 µm wide.

 
Spatial correlations of vesicular and cytoplasmic Ca2+ gradients and hyphal cytology
Once hyphal growth is initiated, Ca2+ release from internal stores is the likely source of tip-localized cytoplasmic Ca2+ during continued hyphal growth. Therefore, we examined the spatial correlation between Ca2+ stores and cytoplasmic Ca2+ (Fig. 5). CTC fluorescence was used to determine the spatial distribution of Ca2+-containing vesicles. The presence of Ca2+-containing vesicles at the hyphal apex (Dicker & Turian, 1990) has been confirmed after fixation and electron microscopy (Torralba et al., 2001). The vesicles can be monitored during hyphal growth using CTC; their distribution is affected by treatment with inhibitors of either IP3-activated Ca2+ channels or phospholipase C (Silverman-Gavrila & Lew, 2002). To obtain a quantitative distribution of CTC fluorescence, it must be corrected for the smaller cell volume near the apex. The corrected Ca2+-containing vesicle density exhibited a ‘steepness’, tau of about 1·2 µm. Using published data on vesicles destined for fusion at the expanding tip (Collinge & Trinci, 1974) and regions of maximal wall synthesis (Gooday, 1971), we calculated best fits to exponential functions to determine tau, a measure of steepness. The value was 1·6 µm, very similar to the ‘steepness' of hyphal diameter, tau=2·2 µm, and CTC fluorescence steepness (1·2 µm). By comparison, the tip-high cytoplasmic [Ca2+] gradient was not as steep. A subsample of total experiments with a ‘mean’ growth rate (6·3–14·0 µm min-1, mean±SD of 9·51±2·64 µm min-1, n=30) was used to examine spatial correlations. The tau value was 10·1 µm. One possible cause for a gradient of cytoplasmic free [Ca2+] gentler than the vesicular Ca2+ gradient could be diffusion of Ca2+ away from the growing tip after Ca2+ release from the vesicular stores. To examine whether diffusion was a reasonable explanation for the gentler Ca2+ gradient, the gradient was fit to a model for the time dependence of concentration changes due to diffusion, of the general form (Crank, 1975), where M is the initial concentration, D is the diffusion coefficient and t is the time. In aqueous solutions, the diffusion coefficient for Ca2+ varies with [Ca2+], but is about 775 µm2 s-1 in dilute CaCl2 (Wang, 1953). Intracellular diffusion coefficients for Ca2+ are in the range 2–15 µm2 s-1 (Al-Baldawi & Abercrombie, 1995; Nakatani et al., 2002). Ca2+ diffusion intracellularly is complicated by the complexity of the cytoplasm, especially the presence of Ca2+ buffers and transporters (Smith et al., 1998). To assess whether diffusion could explain the gentler Ca2+ gradient, the gradient from the subsample with ‘mean’ growth rate was initially fit to obtain an estimate of the diffusion coefficient at time 4 s (sufficient time for the hypha to extend about 0·6 µm). Then the diffusive gradients were calculated 1, 2, 4 and 8 s after the initial state, time zero, when all the calcium was located at the extreme tip (Fig. 6). The estimated diffusion coefficient was 6·3 µm2 s-1, in the range of reported intracellular diffusion coefficients, and lower than published values for Ca2+ in aqueous solution. A smaller diffusion coefficient is reasonable, given the structural complexity of the growing apex, in which a variety of physical obstacles, proteins, vesicles and cytoskeleton would impede the free movement of Ca2+. Thus the gentler Ca2+ gradient can be explained on the basis of Ca2+ diffusion away from the tip after release from the vesicular Ca2+ stores, which would require 1–8 s.



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Fig. 5. Calcium gradients in N. crassa hyphae: spatial correlations. (a) Cytosolic free [Ca2+] (circles, nM) and hyphal diameter (squares, µm). For the Ca2+ gradient, an exponential best fit (thin line) yielded a tau of 10·1 µm. The thick line is a best fit to the diffusion equation (time dependence of concentration) (Fig. 6). Essentially, diffusion can explain the gentler gradient for calcium compared to hyphal diameter. Hyphal diameters were measured from images captured on a digital camera on a Zeiss Axioscope using a x100 water immersion objective. Note that the y-axis is reversed. The best-fit exponential yielded a tau of 2·2 µm. (b) CTC fluorescence intensity (circles, in arbitrary units) and fluorescence intensity corrected for hyphal volume (squares, arbitrary fluorescence units per unit hyphal volume). Fluorescence intensity was measured from confocal images of medial sections, similar to measurements of fluo-3/Fura Red images (Silverman-Gavrila & Lew, 2000). Longitudinal transects (2·54 µm wide) along hyphae (23 experiments) were averaged versus distance from the apex. Note the close correspondence between hyphal diameter (first panel) and CTC fluorescence corrected for hyphal volume. The tau value from an exponential best fit was 1·3 µm. For gradient steepness comparisons, exponential fits of wall vesicle density based on percentage volume (reported by Collinge & Trinci, 1974) and wall synthesis based upon radioautography of hyphae grown in the presence of a radioactively labelled precursor of walls (Gooday, 1971) yield tau values of about 1·6 µm. Gradient steepness has also been reported for SNAREs based upon immunocytochemistry with an antibody to the yeast t-SNARE, Sso2p (Gupta & Heath, 2000). SNAREs are believed to play a role in ‘docking’ of vesicles at a site of fusion in expansion zones of plasma membrane. The best-fit exponential yielded a tau of 8·9 µm.

 


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Fig. 6. Time dependence of Ca2+ diffusion at the tip-high Ca2+ gradient of a growing hypha. The time dependence of [Ca2+] is shown at 1, 2, 4 and 8 s. The gradient was fit to a model for the time dependence of concentration changes due to diffusion, , where M is the initial concentration (the best fit yields a value of about 4·6 µM), D is the diffusion coefficient, [Ca2+]basal is the subapical [Ca2+] (best fit value of about 230 nM) and t is time. The mean gradient for a subsample of hyphae with a mean growth rate was initially fit to obtain an estimate of the diffusion coefficient (6·3 µm2 s-1) using the 4 s time interval, when the hypha would have grown about 0·6 µm. The time dependence of diffusion was calculated using the estimated diffusion coefficient and plotted for time intervals ranging from 1 to 8 s, as shown. Within the time frame 1–8 s, diffusion causes the gentler gradient compared to other cytological features of the growing hypha.

 
Hyphal growth ‘sensing’ and Ca2+ gradient maintenance
During hyphal elongation, continued generation of the Ca2+ gradient must rely upon some mechanism which senses hyphal expansion. Tip-localized IP3 production has been implicated as the cause of IP3-induced Ca2+ release from vesicular Ca2+ stores. Since IP3 is produced by the action of phospholipase C, we tested whether phospholipase C could be activated by membrane stretching, a natural consequence of the process of hyphal elongation. Rather than assay for IP3, we chose to assay the other product of phospholipase C activity, diacylglycerol, since extraction can be performed rapidly with minimal degradation of the diacylglycerol product. Diacylglycerol was identified based on the same retention time (15·5±2·4 min; n=29) as diacylglycerol standards [1,2-dipalmitoyl-sn-glycerol, 15·3±0·4 min (n=2); 1,2-dioleoyl-sn-glycerol, 14·6±1·2 min (n=5)] (Fig. 7). An ergosterol peak was also identified based on the same retention time as a standard [10·3±1·5 min (n=29) compared to 10·1±0·1 min (n=3)], and a single peak when lipid extracts were mixed with the ergosterol standard. Ergosterol served as an internal control for normalization of diacylglycerol levels to mycelial dry weight. Phospholipase C inhibitors known to inhibit hyphal growth (Silverman-Gavrila & Lew, 2002) depleted diacylglycerol significantly relative to the control (Table 2). Mild hypoosmotic shock (a 1 : 1 dilution with H2O), which causes the growing tips to bulge and continue growth (data not shown), caused elevated diacylglycerol levels. Severe hypoosmotic shock (perfusion with H2O), which causes growth to stop immediately (growth resumes eventually), caused diacylglycerol depletion.



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Fig. 7. HPLC elution profiles for diacylglycerol and ergosterol. (a) Control perfusion. (b) Mild hypoosmotic shock. (c) Phospholipase C inhibitor (3-nitrocoumarin). Each trace is representative of three to eight measurements. The diacylglycerol peaks are identified by arrows. The peak to its left is ergosterol, based on the same retention time as that of an ergosterol standard. The ergosterol peak was used as an internal control for normalizing to mycelial dry weight. Diacylglycerol levels relative to control measurements are shown in Table 2.

 

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Table 2. Diacylglycerol levels

The diacylglycerol (DAG) levels, normalized to the ergosterol level, are shown as a percentage of the control (a control was run for each experiment). The treatments were mild or severe hypoosmotic stress, or inibitors of phospholipase C, as shown. The values are mean±SD (n).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ratiometric fluorescence imaging is crucial for spatial measurements of the cytoplasmic [Ca2+] gradient in tip-growing organisms (Camacho et al., 2000). Dextran-conjugated dyes are reported to be less likely to become sequestered. However, they must be pressure-injected into the hyphae, which requires large aperture micropipettes and a higher probability that the hypha will be damaged. By ionophoresing free-acid dyes into the hyphae, we could use a smaller aperture micropipette, were able to control dye loading very efficiently, and obtain good quantification (Silverman-Gavrila & Lew, 2000). Dye sequestration was very rare, observed only in hypha which had been damaged. To examine the relation between cytoplasmic free [Ca2+] and hyphal growth, we compiled the data into subsamples, each with a mean growth rate and gradient. We have demonstrated that the hyphal growth rate depends upon the tip-localized [Ca2+], which must be elevated above basal [Ca2+] behind the tip. Hyphal growth does not depend upon the steepness of the Ca2+ gradient. To initiate tip growth, random molecular motion may be sufficient to generate the gradient. Stretch-activated phospholipase C may act as the growth sensor, maintaining the gradient as hyphal growth continues.

Ca2+ requirement for growth in fungi
Fungal growth requires extracellular calcium. Ca2+ concentrations greater than 10–100 nM are required for hyphal extension to occur in N. crassa (Schmid & Harold, 1988) and Fusarium graminearum (Robson et al., 1991). Similar extracellular [Ca2+] dependencies of growth are also observed for the oomycete S. ferax (Jackson & Heath, 1989) and root hairs (Schiefelbein et al., 1992). At low Ca2+ concentrations, hyphal morphology is aberrant: irregular hyphal width or bulbous spherical cells are observed. Ca2+ dependence of growth and morphology could be due to many different effects: some physical, such as Ca2+ cross-linking of wall components, some biochemical, such as Ca2+-dependent enzymic activities and cytoskeletal rearrangement, and some physiological, such as signalling. Since basal cytoplasmic [Ca2+] is similar to the minimal extracellular [Ca2+] required for growth, a role in biochemistry, signalling or both is likely. Whether the tip-localized cytoplasmic [Ca2+] is directly related to extracellular Ca2+ is not clear. In the spray mutant of N. crassa, the tip-high cytoplasmic [Ca2+] gradient is the same as that in the wild-type (Bok et al., 2001), but vesicular Ca2+, measured with CTC, is absent (Dicker & Turian, 1990). The rescue of the slow growth phenotype by elevated extracellular Ca2+ has no effect on the electrical properties of the plasma membrane (Bok et al., 2001), yet causes the reappearance of vesicular calcium (Dicker & Turian, 1990). This implies that vesicular Ca2+ normally functions as an intermediate step in generation of the tip-high Ca2+ gradient, and that vesicular Ca2+ storage, but not cytoplasmic Ca2+, is more closely related to extracellular [Ca2+]. There is evidence for this in other organisms. In the oomycete S. ferax, Jackson & Heath (1989) reported elevated CTC fluorescence when hyphae were grown in high extracellular [Ca2+]. By contrast, yeast cytoplasmic [Ca2+] is insensitive to extracellular [Ca2+] from 0·1 µM to 10 mM (Halachmi & Eilam, 1993).

Internal generation of the Ca2+ gradient
Tip-localized inward Ca2+ currents play a role in generation of the cytoplasmic tip-high Ca2+ gradient in pollen tubes (Pierson et al., 1994), root hairs (Schiefelbein et al., 1992; Felle & Hepler, 1997) and the oomycete S. ferax (Lew, 1999). Fungi (N. crassa) rely solely upon internal generation of the tip-high Ca2+ gradient (Lew, 1999; Silverman-Gavrila & Lew, 2000). Two distinct intracellular transporters maintain and generate the gradient: an IP3-activated Ca2+ channel (Silverman-Gavrila & Lew, 2002) localized to vesicles at the extreme apex of the growing hypha, releasing Ca2+ into the tip, and a Ca2+-ATPase sequestering Ca2+ behind the growing apex, into the endoplasmic reticulum (Silverman-Gavrila & Lew, 2001). One explanation for internal generation is that N. crassa is a terrestrial fungus, commonly found in burned over areas (Turner et al., 2001). The presence of sufficient external Ca2+ to maintain the tip-high Ca2+ gradient may not be assured, especially in aerial hyphae. Thus internal Ca2+ alone may be used to generate and maintain the Ca2+ gradient. The role of vesicular Ca2+ stores as the source of the elevated tip-localized [Ca2+] is supported by its direct dependence on extracellular [Ca2+], and the spatial correlation between Ca2+-containing vesicles, wall vesicles and wall synthesis. Once Ca2+ is released into the cytoplasm, it diffuses away from the tip.

Comparison with other organisms
For the oomycete S. ferax, analyses of the dependence of growth on the Ca2+ gradient relied upon ratio imaging of Ca2+-sensitive (fluo-3) and pH-sensitive (SNARF) fluorescent dyes (Hyde & Heath, 1997). The qualitative Ca2+ gradient was linear from 0 to 40 µm behind the tip. Growth was correlated with the difference between tip-localized Ca2+ and basal Ca2+. However, at higher growth rates, the growth rate became independent of the Ca2+ gradient. Ca2+ fluxes at the growing apex of S. ferax are independent of growth rate, although this may be due to interplay between Ca2+ influx and Ca2+ exocytosis (Lew, 1999). In a comparison of the Ca2+ gradient and root hair growth by Wymer et al. (1997), the steepness of the Ca2+ gradient was similar to that in N. crassa (tau values of 4–12 µm), and the growth rate was about 10-fold less. Both the tau values and the difference between apical and basal [Ca2+] were correlated with growth rate, based on datasets comparing 0, 0·5 and 1·5 µm min-1 growth rates. Thus either the gradient steepness or the tip-localized [Ca2+] could account for root hair growth, while in fungi it is the elevated tip-localized [Ca2+] which is important.

Tip-growing organisms grow at very different rates. Root hairs grow at about 1 µm min-1 while fungi and pollen tubes grow about 10-fold faster. Since the magnitudes of the cytoplasmic [Ca2+] gradient are similar, the kinetics of Ca2+ supply, either from internal stores or from the extracellular medium, or both, must vary to maintain a steady state Ca2+ gradient. Clearly, one important determinant of growth rate will be the rate of vesicle supply to the growing tip. In fungi, this would result in increased [Ca2+] at the tip, but only if Ca2+ release was activated by IP3 production. In other organisms, Ca2+ influx would elevate [Ca2+] directly.

Spontaneous generation of gradient to initiate growth
If elevated tip-localized [Ca2+] relative to basal [Ca2+] is the key factor regulating growth rate, initiation of the Ca2+ gradient will precede polar organization of cytological structures. Ca2+ is known to play a role in conidial germination in some fungal species (Osherov & May, 2001), but it is not known whether a [Ca2+] gradient precedes germination. In some organisms, Ca2+ elevation does precede the appearance of tip growth. For example, a localized region of elevated Ca2+ predicts the site of rhizoid formation in Pelvetia compressa (Pu & Robinson, 1998). In other organisms, the Ca2+ gradient appears after initiation of tip growth: increased Ca2+ appears only after bulge formation in root hair development (Wymer et al., 1997). Microinjection of Ca2+ into N. crassa hyphae is known to initiate branching (Silverman-Gavrila & Lew, 2000), and therefore should function in the initiation of tip growth. From a biochemical perspective, it may be reasonable that elevated [Ca2+] would be important for both initiation of tip growth and continued hyphal growth. For any enzymic activity regulated by Ca2+, we expect [Ca2+] dependence to correspond closely with cytoplasmic [Ca2+]. The higher the tip-localized [Ca2+], the greater the enzyme activity at the tip, resulting in faster growth. Thus enzymic activities important in hyphal growth should be activated by [Ca2+] 30–160 nM higher than the basal [Ca2+] of about 220 nM 10–20 µm behind the tip. This predicted [Ca2+] dependence may be useful as the enzymic mechanisms causing initiation of hyphal extension are examined in more detail. In N. crassa, Ca2+-calmodulin activates chitin synthase (Suresh & Subramanyam, 1997), cAMP phosphodiesterase (Tellez-Inon et al., 1985) and calcineurin (PP2B) (Prokisch et al., 1997) and binds to microtubule-associated proteins (Ortega-Perez et al., 1994), all potential elements of polar organization and growth. Calcineurin is of especial interest, since it appears to function in morphogenesis (Fox & Heitman, 2002) and generation or maintenance of the vesicular Ca2+ gradient imaged with CTC (Prokisch et al., 1997). Calcineurin forms an immunoprecipitable complex with COT1 (Gorovits et al., 1999), a serine threonine kinase known to function in normal hyphal growth (cf. Dickman & Yarden, 1999). In addition to a role in polar organization (Torralba & Heath, 2001), the Ca2+ gradient would cause localized vesicle fusion, either on its own (Hall & Simon, 1976), or in association with a plethora of vesicle fusion mediators (Gupta & Heath, 2000, 2002).

The initiation of the Ca2+ gradient could be spontaneous. That is, in the spherical conidium or ascospore, random redistribution of the Ca2+ molecules could transiently create a Ca2+ gradient. A simplified simulation of a Ca2+ molecule random walk (Fig. 4) does suggest that initiation of hyphal growth could be a consequence of random molecular motions.

Once generated, the Ca2+ gradient must be maintained during continued hyphal growth. We explored the possibility that stretch-activated phospholipase C could sense hyphal expansion, and increase tip-localized [Ca2+] to maintain hyphal growth.

Phospholipase C may be the growth sensor
Fungal growth normally relies upon an internal hydrostatic pressure which would generate a constant tension on the hyphal plasma membrane/wall interface. As the hypha expands, the tension would increase. A natural candidate for sensing of hyphal expansion would be tip-localized stretch-activated Ca2+ channels, as occurs in S. ferax (Garrill et al., 1993). However, we have been unable to demonstrate any role for stretch-activated Ca2+ channels in hyphal growth in N. crassa (Lew, 1999; Silverman-Gavrila & Lew, 2000), even though they do exist in the plasma membrane (Levina et al., 1995), distributed evenly along the hypha. Because IP3 plays a role in generation of the Ca2+ gradient from internal stores, it is possible that a stretch-activated phospholipase C (Kinnunen, 2000) could sense hyphal expansion, and cleave PIP2 to IP3 and diacylglycerol. Phospholipase C inhibitors do inhibit hyphal growth and modify the vesicular Ca2+ gradient similarly to inhibitors of the IP3-activated Ca2+ channel (Silverman-Gavrila & Lew, 2002). In fact, mild hypoosmotic stress does elevate rapidly diacylglycerol levels in conidial germlings (Table 2). Hypoosmotic stress activates phospholipase C in the plasma membrane of Dunaliella salina, as indicated by elevated diacylglycerol levels within 30 s (Ha & Thompson, 1991). In this case, phospholipase C activation probably plays a role in volume regulation of this wall-less green alga. Mechanical stretching is also reported to increase IP3 via phospholipase C activation in coronary artery (Tanaka et al., 1994). Thus a stretch-activated phospholipase C is a possible mechanism for sensing hyphal expansion. If this is the case, it is possible that other aspects of the polar cytology of the growing fungal tip may be regulated by other intermediates of the phosphatidylinositol and inositol phosphate metabolic pathways. Certainly, phosphoinositides have been implicated in regulation of the cytoskeleton (Yin & Janmey, 2003) and cellular polarity (Kost et al., 1999).


   ACKNOWLEDGEMENTS
 
This research was funded by a discovery grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) (R. R. L.) and an Ontario Graduate Scholarship in Science and Technology (OGSST) and Ontario Graduate Scholarship (OGS) (L. B. S.-G.). Special thanks to Dr P. L. Lakin-Thomas for her advice and assistance in diacylglycerol extractions and HPLC analysis, and to the two anonymous reviewers for their thoughtful criticisms and helpful comments.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Al-Baldawi, N. F. & Abercrombie, R. F. (1995). Calcium diffusion coefficient in Myxicola axoplasm. Cell Calcium 17, 430–438.

Bligh, E. G. & Dyer, W. J. (1959). A rapid method of lipid extraction and purification. Can J Biochem Physiol 37, 911–917.

Bocckino, S. B., Blackmore, P. F. & Exton, J. H. (1985). Stimulation of 1,2-diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II. J Biol Chem 260, 14201–14207.[Abstract/Free Full Text]

Bok, J. W., Silverman-Gavrila, L. B., Lew, R. R., Sone, T., Bowring, F. J., Catcheside, D. E. A. & Griffiths, A. J. F. (2001). Structure and function analysis of the calcium-related gene spray in Neurospora crassa. Fungal Genet Biol 32, 145–158.[CrossRef][Medline]

Camacho, L., Parton, R., Trewavas, A. J. & Malho, R. (2000). Imaging cytosolic free-calcium distribution and oscillations in pollen tubes with confocal microscopy: a comparison of different dyes and loading methods. Protoplasma 212, 162–173.

Collinge, A. J. & Trinci, A. P. J. (1974). Hyphal tips of wild-type and spreading colonial mutants of Neurospora crassa. Arch Microbiol 99, 353–368.[Medline]

Crank, J. (1975). The Mathematics of Diffusion, 2nd edn, p. 13. Oxford: Clarendon Press.

Dicker, J. W. & Turian, G. (1990). Calcium deficiencies and apical hyperbranching in wild-type and ‘frost’ and ‘spray’ morphological mutants of Neurospora crassa. J Gen Microbiol 136, 1413–1420.

Dickman, M. B. & Yarden, O. (1999). Serine/threonine protein kinases and phosphatases in filamentous fungi. Fungal Genet Biol 26, 99–117.[CrossRef][Medline]

Felle, H. H. & Hepler, P. K. (1997). The cytosolic Ca2+ concentration gradient of Sinapsis alba root hairs as revealed by Ca2+-selective microelectrode tests and fura-dextran ratio imaging. Plant Physiol 114, 39–45.[Abstract/Free Full Text]

Fox, D. S. & Heitman, J. (2002). Good fungi gone bad: the corruption of calcineurin. Bioessays 24, 894–903.[CrossRef][Medline]

Garrill, A., Jackson, S. L., Lew, R. R. & Heath, I. B. (1993). Ion channel activity and tip growth: tip-localized, stretch-activated channels generate a Ca2+ gradient that is required for tip growth in the oomycete Saprolegnia ferax. Eur J Cell Biol 60, 358–365.[Medline]

Gooday, G. W. (1971). An autoradiographic study of hyphal growth of some fungi. J Gen Microbiol 67, 125–133.

Gorovits, R., Propheta, O., Kolot, M., Dombradi, V. & Yarden, O. (1999). A mutation within the catalytic domain of COT1 kinase confers changes in the presence of two COT1 isoforms and in Ser/Thr protein kinase and phosphatase activities in Neurospora crassa. Fungal Genet Biol 27, 264–274.[CrossRef][Medline]

Gupta, G. D. & Heath, I. B. (2000). A tip-high gradient of a putative plasma membrane SNARE approximates the exocytotic gradient in hyphal apices of the fungus Neurospora crassa. Fungal Genet Biol 29, 187–199.[CrossRef][Medline]

Gupta, G. D. & Heath, I. B. (2002). Predicting the distribution, conservation, and functions of SNAREs and related proteins in fungi. Fungal Genet Biol 36, 1–21.[CrossRef][Medline]

Ha, K. S. & Thompson, G. A., Jr (1991). Diacylglycerol metabolism in the green alga Dunaliela salina under osmotic stress. Possible role of diacylglycerols in phospholipase C-mediated signal transduction. Plant Physiol 97, 921–927.

Halachmi, D. & Eilam, Y. (1993). Calcium homeostasis in yeast cells exposed to high concentrations of calcium. Roles of vacuolar H+-ATPase and cellular ATP. FEBS Lett 18, 73–78.[CrossRef]

Hall, J. E. & Simon, A. (1976). A simple model for calcium induced exocytosis. Biochim Biophys Acta 436, 613–616.[Medline]

Hyde, G. J. & Heath, I. B. (1997). Ca2+ gradients in hyphae and branches of Saprolegnia ferax. Fungal Genet Biol 21, 238–251.[CrossRef]

Jackson, S. L. & Heath, I. B. (1989). Effects of exogenous calcium ions on tip growth, intracellular Ca2+ concentration, and actin arrays in hyphae of the fungus Saprolegnia ferax. Exp Mycol 13, 1–12.

Kinnunen, P. J. K. (2000). Lipid bilayers as osmotic response elements. Cell Physiol Biochem 10, 243–250.[CrossRef][Medline]

Kost, B., Lemichez, E., Spielhofer, P., Hong, Y., Tolias, K., Carpenter, C. & Chua, N.-H. (1999). Rac homologues and compartmentalized phosphatidylinositol 4,5-bisphosphate act in a common pathway to regulate polar pollen tube growth. J Cell Biol 145, 317–330.[Abstract/Free Full Text]

Levina, N. N., Lew, R. R., Hyde, G. J. & Heath, I. B. (1995). The roles of Ca2+ and plasma membrane ion channels in hyphal tip growth of Neurospora crassa. J Cell Sci 108, 3405–3417.[Abstract/Free Full Text]

Lew, R. R. (1999). Comparative analysis of Ca2+ and H+ flux magnitude and location along growing hyphae of Saprolegnia ferax and Neurospora crassa. Eur J Cell Biol 78, 892–902.[Medline]

Messerli, M. A. & Robinson, K. R. (1997). Tip localized Ca2+ pulses are coincident with peak pulsatile growth rates in pollen tubes of Lilium longiflorum. J Cell Sci 110, 1269–1278.[Abstract/Free Full Text]

Messerli, M. A., Danuser, G. & Robinson, K. R. (1999). Pulsatile influxes of H+, K+ and Ca2+ lag growth pulses of Lilium longiflorum pollen tubes. J Cell Sci 112, 1497–1509.[Abstract/Free Full Text]

Messerli, M. A., Creton, R., Jaffe, L. F. & Robinson, K. R. (2000). Periodic increases in elongation rate precede increases in cytosolic Ca2+ during pollen tube growth. Dev Biol 222, 84–98.[CrossRef][Medline]

Nakatani, K., Chen, C. & Koutalos, Y. (2002). Calcium diffusion coefficient in rod photoreceptor outer segments. Biophys J 82, 728–739.[Abstract/Free Full Text]

Ortega-Perez, R., Irminger-Finger, I., Arrighi, J. F., Capelli, N., van Tuinen, D. & Turian, G. (1994). Identification and partial purification of calmodulin-binding microtubule-associated proteins from Neurospora crassa. Eur J Biochem 226, 303–310.[Abstract]

Osherov, N. & May, G. S. (2001). The molecular mechanism of conidial germination. FEMS Microbiol Lett 199, 153–160.[CrossRef][Medline]

Pierson, E. S., Miller, D. D., Calahan, D. A., Shipley, A. M., Rivers, B. A., Cresti, M. & Hepler, P. K. (1994). Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. Plant Cell 6, 1815–1828.[Abstract/Free Full Text]

Pierson, E. S., Miller, D. D., Callahan, D. A., van Aken, J., Hackett, G. & Hepler, P. K. (1996). Tip-localized calcium entry fluctuates during pollen tube growth. Dev Biol 174, 160–173.[CrossRef][Medline]

Prokisch, H., Yarden, O., Dieminger, M., Tropschug, M. & Barthelmess, I. B. (1997). Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient. Mol Gen Genet 256, 104–114.[CrossRef][Medline]

Pu, R. & Robinson, K. R. (1998). Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa. J Cell Sci 111, 3197–3207.[Abstract/Free Full Text]

Ramsdale, M. & Lakin-Thomas, P. L. (2000). sn-1,2-Diacylglycerol levels in the fungus Neurospora crassa display circadian rhythmicity. J Biol Chem 275, 27541–27550.[Abstract/Free Full Text]

Rasband, W. S. & Bright, D. (1995). NIH Image: a public domain image processing program for the Macintosh. Microbeam Anal Soc J 4, 137–149.

Robson, G. D., Wiebe, M. G. & Trinci, A. P. J. (1991). Low calcium concentrations induce increased branching in Fusarium graminearum. Mycol Res 95, 561–565.

Schiefelbein, J. W., Shipley, A. & Rowse, P. (1992). Calcium influx at the tip of growing root hairs of Arabidopsis thaliana. Planta 197, 455–459.

Schmid, J. & Harold, F. M. (1988). Dual roles for calcium ions in apical growth of Neurospora crassa. J Gen Microbiol 134, 2623–2631.[Medline]

Silverman-Gavrila, L. B. & Lew, R. R. (2000). Calcium and tip growth in Neurospora crassa. Protoplasma 213, 203–217.

Silverman-Gavrila, L. B. & Lew, R. R. (2001). Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungus Neurospora crassa. Eur J Cell Biol 80, 379–390.[Medline]

Silverman-Gavrila, L. B. & Lew, R. R. (2002). An IP3-activated Ca2+ channel regulates fungal tip growth. J Cell Sci 115, 5013–5025.[CrossRef][Medline]

Smith, G. D., Keizer, J. E., Stern, M. D., Lederer, W. J. & Cheng, H. (1998). A simple numerical model of calcium spark formation and detection in cardiac myocytes. Biophys J 75, 15–32.[Abstract/Free Full Text]

Suresh, K. & Subramanyam, C. (1997). A putative role for calmodulin in the activation of Neurospora crassa chitin synthase. FEMS Microbiol Lett 150, 95–100.[CrossRef][Medline]

Tanaka, Y., Hata, S., Ishiro, H., Ishii, K. & Nakayama, K. (1994). Quick stretch increases the production of inositol 1,4,5-trisphosphate (IP3) in porcine coronary artery. Life Sci 55, 227–235.[CrossRef][Medline]

Tellez-Inon, M. T., Ulloa, R. M., Glikin, G. C. & Torres, H. N. (1985). Characterization of Neurospora crassa cyclic AMP phosphodiesterase activated by calmodulin. Biochem J 232, 425–430.[Medline]

Torralba, S. & Heath, I. B. (2001). Cytoskeletal and Ca2+ regulation of hyphal tip growth and initiation. Curr Top Dev Biol 51, 135–187.[Medline]

Torralba, S., Heath, I. B. & Ottensmeyer, F. P. (2001). Ca2+ shuttling in vesicles during tip growth in Neurospora crassa. Fungal Genet Biol 33, 181–193.[CrossRef][Medline]

Turner, B. C., Perkins, D. D. & Fairfield, A. (2001). Neurospora from natural populations: a global study. Fungal Genet Biol 32, 67–92.[CrossRef][Medline]

Vogel, H. J. (1956). A convenient growth medium for Neurospora. Microb Genet Bull 13, 42–46.

Wang, J. H. (1953). Tracer-diffusion in liquids. IV. Self-diffusion of calcium ion and chloride ion in aqueous calcium chloride solutions. J Am Chem Soc 75, 1769–1770.

Wilkinson, L. (1988). SYSTAT: The System for Statistics. Evanston, IL: SYSTAT.

Wymer, C. L., Bibikova, T. N. & Gilroy, S. (1997). Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana. Plant J 12, 427–439.[CrossRef][Medline]

Yin, H. L. & Janmey, P. A. (2003). Phosphoinositide regulation of the actin cytoskeleton. Annu Rev Physiol 65, 761–789.[CrossRef][Medline]

Received 14 February 2003; revised 9 April 2003; accepted 8 May 2003.



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