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
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Summary |
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Key words: Fungal tip growth, IP3 receptor, Ca2+ gradient, Phospholipase C, Ca2+ channels, Planar lipid membrane
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Introduction |
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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, 1999), root hairs
(Felle and Hepler, 1997
) and
pollen tubes (Pierson et al.,
1996
), but not in the fungus Neurospora crassa
(Lew, 1999
;
Silverman-Gavrila and Lew,
2000
). Therefore, plasma membrane stretch-activated
Ca2+ channels, identified as the mechanism of Ca2+ entry
in S. ferax (Garrill et al.,
1993
), or another type of Ca2+ channel
(Very and Davies, 2000
) 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,
2000
).
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, 2001) (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.
|
In animal cells, the major site for IP3 receptors is the ER
(Berridge and Irvine, 1989). 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, 1985
),
and from vacuoles and tonoplast vesicles
(Alexandre et al., 1990
;
Allen et al., 1995
). The plant
IP3 receptor was characterized in detail by Biswas
(Biswas et al., 1995
). In
fungi, IP3 activates Ca2+ release from vacuoles of
Candida albicans (Calvert and
Sanders, 1995
), Saccharomyces cerevisiae
(Belde et al., 1993
) and N.
crassa (Cornelius et al.,
1989
). 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., 1990
). There
are reports of IP3-sensitive Ca2+ stores other than
vacuoles in cauliflower inflorescences
(Muir and Sanders, 1997
) and
in growing pollen tubes: in the nuclear-rough ER region
(Franklin-Tong et al., 1996
;
Malho, 1998a
) and from
vesicles or ER (Zheng and Yang,
2000
). 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., 2000),
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, 1991
). In
C. albicans, there is an increase in IP3 during germ tube
formation of the yeast to hyphal morphological transition
(Gadd and Foster, 1997
).
Ins(1,4,5)P3 kinase activity is higher during germ tube
formation compared with the yeast morph. Prior et al.
(Prior et al., 1993
;
Prior et al., 1994
) and
Lakin-Thomas (Lakin-Thomas,
1993a
; Lakin-Thomas,
1993b
) 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., 1998
) 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.,
1995
), including Li+ inhibition of inositol metabolism
and hyphal growth (Hanson,
1991
). Besides fungal organisms, there is evidence of
IP3-mediated morphogenesis in tip-growing pollen tubes
(Malho, 1998a
;
Kost et al., 1999
).
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,
2001). 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.
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Materials and Methods |
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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, 1976) 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,
1981), ER (16/29%) (Borgeson
and Bowman, 1983
) and plasma membrane (29/39%)
(Bowman et al., 1981
)
interfaces. The actual vacuolar membrane density has been reported to vary
from 1.06 to 1.30 g cm-3
(Vaughn and Davis, 1981
),
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, 1981
), with
EGTA present (Bowman and Bowman,
1982
), 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., 1982), 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, 1992)] 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, 1978).
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., 1995).
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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Results |
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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,
1993). 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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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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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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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.,
2001) and the tip-localized cytoplasmic Ca2+ activity
was found to be in the nanomolar range (400-600 nM)
(Silverman-Gavrila and Lew,
2000
; Silverman-Gavrila and
Lew, 2001
). 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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|
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%).
|
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., 1997).
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.,
2001
). 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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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,
2001). 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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Discussion |
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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,
2001). 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,
2001
). 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,
2001
). 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.,
2001
; Diver et al.,
2001
). In higher plants, capacitative Ca2+ entry has
been postulated to play a role in pollen tube growth
(Malho, 1998b
). 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, 1999
), modifying the
electrical driving force for Ca2+ entry by voltage clamp has no
effect on hyphal growth (Silverman-Gavrila
and Lew, 2001
), 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., 1993)], in
N. crassa [dantrolene (Cornelius
et al., 1989
)], in red beet root [heparin and TMB-8
(Brosnan and Sanders, 1990
;
Alexandre et al., 1990
)], and
C. albicans [partial inhibition by heparin
(Calvert and Sanders, 1995
)].
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.,
1998
). 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,
1987
). 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, 1959
).
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., 2001).
Ca2+ release by type 3 is much less affected by ATP compared with
type 1 (Thrower et al., 2001
),
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.,
1998). 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., 2001
), 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., 1985), the
oomycetes S. ferax (Yuan and
Heath, 1991
) and N. crassa
(Schmid and Harold, 1988
). In
N. crassa the CTC gradient is very similar to the steep gradient of
apical vesicles (Collinge and Trinci,
1974
) (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.,
2001
).
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, 2000) 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, 1999
).
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