Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3
Correspondence
Roger R. Lew
planters{at}yorku.ca
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ABSTRACT |
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Both authors contributed equally to the work.
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INTRODUCTION |
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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.
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METHODS |
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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 kryptonargon 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 2550 µM. The fluorescence was detected by confocal microscopy using a Bio-Rad MRC-600 apparatus equipped with a kryptonargon 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 56 h, at which time the germlings had grown about 100200 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
).
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RESULTS |
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DISCUSSION |
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Ca2+ requirement for growth in fungi
Fungal growth requires extracellular calcium. Ca2+ concentrations greater than 10100 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 412 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+] 30160 nM higher than the basal [Ca2+] of about 220 nM 1020 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
).
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ACKNOWLEDGEMENTS |
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Received 14 February 2003;
revised 9 April 2003;
accepted 8 May 2003.
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