Calcium and tip growth in the filamentous fungus Neurosporu crassa Lorelei Bianca Silverman Gavrih A thesis submitted to the faculty of Graduate Studies in partial &IfUment of the requirement for the degree of MASTER OF SCIENCE GRADUATE PROGRAMME Department of Biology York University Toronto, Ontario,Caoada September, 1999
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Calcium and tip growth in the
filamentous fungus Neurosporu crassa
Lorelei Bianca Silverman Gavrih
A thesis submitted to the faculty of Graduate Studies in partial &IfUment of the
requirement for the degree of
MASTER OF SCIENCE
GRADUATE PROGRAMME
Department of Biology
York University
Toronto, Ontario,Caoada
September, 1999
Acquisitions and Acquisitions et Bibliogaphic Servites senrices bibliographiques
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a thesis submitted to the Faculty of Graduate Studies of York University in partial fulfiflment of the requirernents for the degree of
Master of Science
1999 O
Permission has been granted to the LIBRARY OF YORK UNI- VERSITY to lend or selt copies of this thesis, to the NATIONAL LIBRARY OF CANADA to microfilm this thesis and to lend or seIl copies of the film, and to UNIVERSITY MlCROFlLMS to pubfish an abstract of this thesis. The author resewes other publication rights. and neither the thesis nor extensive extracts from it may be printed or other- wise reproduced without the author's written permission.
The tip growth process in Neuropwa WQSW was studied ushg a combined
e1ecrophysiological md c o n f d laser microscopy approach.
1 was interested in whether ion channels and otba transporters are responsible for
the unique electrical properties at the tip, and whether they fhction w the main driving
force for the tip expansion aodlor its regulatioh At est 1 expiorcd whtha ionic fluxes
regulate the tip growth process by voltage clrimping growing hyphal tips. 1 present results
indicating that tip growth is unaffected by changes in transmcmbraae voltage- Therefore,
ionic currents are, not an obligatory requirement of polarized extension of Neurospora
crassa.
However, cytosolic ca2+ does play a key role in tip growth in many organisms.To
confirm the ca2+ role in tip induction in Neurospora, 1 ionophoresed ca2' into the hyphae
and found that ca2' induces subapical initiation of multiple tips near the injection sites.
To directly demonstrate the requirement for ca2+ in h*bal extension, 1 ïnjected #
the hyphae with the ca2+ chelator BAPTA Confocal microscopy, using ratio
fluorescence imaging of ionophoresed ca2+ selective fluorescent dyes Fluo-3 and Fura
Red was used to detemine the subcellular localization of ca2+ and to confirm the
changes of the cytoplasmic fiee ca2+ gradient caused by microinjection of BAPTA
Growing hyphae have a tip-high cytosolic ca2+ gradient. BAPTA ionophoresis rapidly
dissipated the tip high caZf gradient and inhibited growth. Long tem morphological
changes - multibud formation- are probably because l o w e ~ g ca2+ concentration affects
iv
caicineurin control of the conidiation developmentai program-
1 conclude that a tip high Ci2* gradient plays a key role in initiation of tips and
continueci growth in Ne~~oqxmu cnrrsa. The source of ci2+ to maintah the tip-high
gradient is not erraa~eliular Ca2+, but instead some intanai store
First 1 want to thank Dr. Lew fbr ôeing my wond& spervisor, for everything
he tought me, and for bis endless help and encouragement throughout my project in his
laboratoxy 1 leamed that science must be done with passion and a r e and curiosity. the
way he does it. He is such a great professor that things that at first seems to be
ununderstandable becorne so easy der he e~pLained them, tecbiqyes that seems to be
impossible became so cornmon Tbank you for bang a ceneal part of the most important
and interesting years of my We. In addition 1 am gratefbi to D& Heath and White for
invaluable comments and suggestions on my thesis as weii as to my cornmittee members,
Drs. Hood and van Rensburg. 1 would iike to thank Dr. Levina for her extensive help
with ratio imaging and calibration m e as weii for aii precious discussions and
information that she shared with me. Tbanks to Dr. Rethoret and Dr. Hyde for their
assistance with confocal microscopy. Thanks to Yolanda Lew for her wann friendship
and support Many, many thanlrs to Dr. Forer and Sandraa Forer for making me so
welcome in their warm house and interesthg world of music and select culïnary.If you
are reading these acknowledgements, it is also because Dr. Pearhan was the fust to
welcome me and my sister at York Even though we spent a short time in his lab, it was a
great experience and we met such nice people there. I would like especially to thank you
Emina and Moshe David for welcoming us into their great family where we spent
wondefil moments that we will treasure ail our lives.
vi
There are many other people that 1 wodd Like to thank: especiaiiy Ms. Adrienne
Dome for her help and guidance, Mr. Gordon Temple for preparing pictures and slides
and m y collegues Jason, John, Kate, GagaaTbanks to rny M y for loving us so rnuch
as to let us do only what we want and Like and to my twin sister who is the most precious
person in my Me. And finaily t hdcs to Neumspora crassa for lettuig me to explore i ts
disorganized the actin cytoskeleton and changed hyphal diameter and morphology.
However a pH gradient in growing hyphae was not observed using the H' sensitive dye
SNARF- 1.
Sinapis d b a root hairs have a cytosolic pH in the range 7.1-7.5 (Felle and Kepler,
I997), insignificantly more acidic at the tip. Higher plant pollen tube Agupan~hzls
zrrnbeiiattis and fern rhizoid Dryopteris @nus lack a cytoplasmic pH gradient (Parton et
ai., 1997). However, Feijo et al. (1999) reported an acidic domain (using BCECF) at the
extreme apex of Lilium polien tubes, coupled with tip-localized H" influx measured with
H-selective vibrating probe. Therefore, pH gradients are not a consistent feature of tip
growth.
1.5. Calcium and tip growth
1.5.1. caZf ions are required for the growth of Nmopora crusu
Exogenous ca2+ is essential for tip extension in Nmrospoa cmssa, but is not a
major component of the extracellular ionic curent because removal of extemal caZ' with
EGTA stops extension without affectiDg the current (McGillviray and Gow, 1987; but see
below).
Neurospora requires at lest 1 pM w' for growth with normal morphology and
1 O ph4 to attain maximal extension rates. Below 1 jM extracellular ca2', using EGTA
addition to chelate caZ-, extension slowed to a half or a third of the initial original rate
and hyphae formed apical branches or unusually wide bulbous swellings. Hyphal length
was less than half the length of controls, but the mycelial mass was only slightly reduced
(Takeuchi et al., 1988). It appears that the polarized extension has a higher requirement
for ca2' then does biomass increase (Schmid and Harold, 1988). At 2 pM hyphae
continued to extend, appeared morphologically normal, but the flow of transcellular
electric current was consistently reduced suggesting that calcium influx may also
contribute to the electric current that enters the apical region (Takeuchi et ai., 1988).
However, simply chelating ca2+ with EGTA cannot be used to assess the fiaction of
elearical current carried by ca2+. Diminution of the transcellular current is not
necessarily due to changes in ca2+ aux. It may reflect changes in the conductance of
potassium and other ions since it is known that many electrophysiological characteristics
of Neurospora are altered in calcium deficieat media (Slayman, 1965). Surface bound
ca2' may be essentiai in generaîbg hyphd morphofogy by maintainhg the integrity of
the plasma membrane (Slay man, 1965).
ca2+ channel blockers L+a3' and Gi3' had no obvious effect on hypbal extension or
branching. Nifedipine at 100 pM partially inhibited extension and distorted the pattern of
transcelIular electric current, but did not elicit branching (Takeuchi et al., 1988).
However, Corn and Sanders, (1992) reported that ca2* channel antago~sts nifedipine,
ruthenium red and rnethoxyverapamil do not inhibit ca2' Wux; white L,a3' does but it
also depolarises the membrane potential. Thecefore the use of inhibitors to block plasma
membrane ca2' infiux must be regarded with caution because cf side effects or poor
specificity. There is thus no evidence that calcium ions pass across the plasma membrane
by ~a~*channels.
Increasing cytoplasmic ca2+ through treatment for 30 minutes with caZ'
ionophore A23187 induces branching in Nmrospora crassa (Reissing and Kinney,
1983) sugjesting that tip formation may be stimulated by calcium influx. Schmid and
Harold, (1 988) confirmed that the major morphological consequence of A23 187 addition
\vas the rapid appearance of multiple apical branches implying that ca2' gradients may be
required to assure the predornhance of a single hyphal tip. However. A23 187 is not very
specific for ca2' and acts as a cati0n.W exchanger. To establish that the ionophore effect
is due to ca2+ requires that the effect should be dependent on extracellular [ca2'].
Other hyphal organisms also require caZ- for growth The oomycete Achlya
depends on ca2+ for hyphai growth and branching is induced by the addition of ca2'
ionop hore A23 187 (Harold and Harold, 1986). Substantiaf delays in the inhibitory action
of EGTA and ~ a ~ ' suggests that cytoplasmic reservoirs c m supply ca2' needs in the
short term,
in Blasfoc-efla emersonii the transcellular current carried by K' ions requires
no other extracellular ions except ca2* (Van Bmnt et aL, 1982). Removal of ca2+ causes
cells to quickiy fil1 wÏth vacuoles and become visibly abnormai (Stump et al, 1980).
Growth rates in Sbprolegniaferm increase with increasing extemal ca2' up to 50
mM CaCIz and decrease at higher concentration (Jackson and Heath, 1989). In the
absence of externai ca2', growth can occur for a limited time using intemal ca2+, then
stops. Intemal membrane-associated ca2+ locaiized with chlortetracycline can be
modulated by extemal concentration, becoming depleted in hyphae growing in the
absence of ca2' and increasing when extracellular [ca2'] is high The intemal changes
were not as great as extemal ones indicating that the hyphae are capable of regulating
ca2' in the presence of a large concentration gradient. The actin cytoskeleton was altered
in hyphae grown either in high or low [ca27. At ioJ M [ca2-], the hyphae had more
actin in their apical network and peripheral plaques of actin were fùnher fiom the apex
than in more slowly growing hyphae at high (10-' M) or low (-O7) ca2-.
ca2' is also essential for tip growth of pollen tubes. At 20 pM [ca2'] or lower,
growth is reduced, and the pollen tubes tend to burn (Weisenseel and Jaffe, 1976). caZT - uptake into the cytoplasm occurs almost exciusively in the tip region as indicated by the
incorporation of 4S~a2 ' at the tip (Jaffe et al., 1975). Agents that interfere with ca2'
uptake prevent elongation (Weisenseel and M e , 1976; Obermeyer and Weisenseel,
199 1).
Extemai [ca2'] lower than 10 p M inhibits mot ùair ceii extension (Schiefelbein et
al., 1992; Hermmann and Felie, 1995). Maximal growth rates are artained at - 0.3 mM.
1.5 -2. Intracellular ca2'
Intracellular ca2+ probably participates in multiple regulatory ttnctions with
reglation typically occtming when cytosolic [ca2+] levels nses above 0.1 pM (Heath,
1995). As a second messenger, calcium rnay be involved in numerous signa1 transduction
pathways for general cellular activities, including polarized tip growth (Jackson and
Heath, 1993 b), branching (Reissing and b e y , l983), PeniciZZiiunr sponiiation (Roncal
et al., 1 993), cytoplasmic movernent (McKerracher and Heath, 1 986); and other fùnctions
reviewed by Knight et al., (1993) such as chitin synthesis, zoospore motility and cyst
germination, regdation of dimorphism, blue light-induced conidiation circadian rhythms.
infection structure differentiation etc.
In Nawospora crassa. the cytosolic fiee calcium has been measured with ca2*
selective rnicroelectrodes (Miller et al., 1990). The mean value of [ca27+ is 92 f 15 nM.
This low level is probably regulated by ca2' efflux across the plasma membrane by an
K/c~*' antiporter (Stroobant and Scarborough, 1979) that is linked to an electrosenic
ATPase (Miller et al., 1992). ca2' may dso accumulate in intemal stores, possibly
endoplasmic reticulum and mitochondria, fiom where it may be released when necessary-
Vacuolar uptake of ca2' may be responsible for sequeste~g the excess of free ca2' fiom
the cytosol (Cornelius and Nakashima, 1987). Uptake by vacuoles involves active
transport since it is inhibited by vacuolar ATPase inhibitors MN'-dicyctohexyI
carbodiimide (DCCD), N03; and SCN--
In SciproIegniu ferm, a reticulate vacuole system has been proposed as a
significant ca2+ sink in the tip region (Ailaway et al., 1997).
1 -5.3. Intracellular &+ gradients
There is strong evidence in support of the ubiquitous presence of a tip-focused
gradient of cytosoiic fkee ca2' as a general feature of tip growing organisrns.
Tip high gradients of cytoplasmic ca2+ have been observed in the fungus
Neurospora crassa with chlortetracyciine (CTC) (Schmid and Harold, 1988). However,
C~~'-CTC is membrane bound and accumulates in organelles which contain higher
concentration of fkee ca2'; therefore ca2'-CTC fluorescence primarily indicates the
presence of ca2' sequestering organelies. Using a ratiornetric dye technique of acid
loaded calcium sensitive Fluo-3 and calcium insensitive SNARF- 1 Levina et al., ( 1 99 5)
showed that growing hyphae of Neurospora crmsa have a tip-high cytoplasmic free ~ a ' -
gradient which peaked - 3 prn behind the tip (0-07 PM), which is absent in non-growing
hyphae. The gradient was unaffected by ~ d ~ ' (an inhibitor of stretch-activated channels).
A tip high gradient was also observed in the oomycete Saprolegnia f e r a using
either Indo-1 ( G d 1 et al., 1993) or Fluo-3 and SNARF-1 (Hyde and Heath, 1997). The
gradient extends fùrther dong the periphery than the center of the growing hyphae (Hyde
and Heath, 1997); it is very steep within 5 pm of the apex and decays towards a lower
level at about 10-20 pm (Garrill et al., 1993; Hyde and Heath, 1997).
h Fucus sewutus rhizoids Brownlee and Pulsford (1988) ionophoretically
injected Fura-2 to image caZ+ gradients. ca2' was higher at the growing tip in about 50-
60% of ceUs, ranging fiom 105 + 15 nM in the region of the nucleus to 450 2 30 nM at
the extreme apex. Verapamil reduced, but did not abolish the ca2' gradient suggesting
that ca2* influx is at least pactiaiiy responsible for maintenance of ca2' at the tip.
Clarkson et al. (1988) used fluorescence ratio imaging of Fura-2 to measure the
cytoplasmic caZr in tomato (Lycopersicon esculenhm) and oïiseed rape (Braska napus)
root hair ceils but did not consistently see a tip hi& gradient of cytoplasmic ca2*.
Felle and Hepler (1997) used ca2' selective microeiectrodes and pressure injected
dextran-conjugated Fur& ratio imaging in Sinapis alba root kirs to measwe the
cytosolic ca2' concentration. Both methods yield values between 160 and 250 nM for the
basal [ca27 level and of 450 to 710 n M at the tip region. The zone of elevated [ca21
reaches 40 to 60 pm into the cell similar to the region of inward ca2+ net currents
rneasured with an extemal ca2+ selective probe (Felle and Hepler, 1997). The channel
blockers ~ a ~ ' and nifedipine eliminate this flux, stop growth and almost completely
eliminate the cytosolic ca2' gradient (Hert'nma~ and Feue, 1995; Felle and Hepler,
1997). Growth is also inhibited by pressure injected dibromo-BAPTA which causes a
decrease in the [CS] at the tip (Hermmann and Felle, 1995). Non-growing root hairs
may or may not display a ca2* gradient. Thus, a cytosoiic [ca27 tip-high gradient is
essential for tip growth but does not cause growth under al1 cucumstances (Felle and
Hepler, 1997).
Bibikova et al., (1997) used localized photoactivation of the caged calcium
ionophore Br- A23 1 87 to generate an assymetric ca2+ influx across root hair tips of
Arabidopsis tMtiona. Photoaaivsition caused a transient change in the direction of tip
growth toward the bigher [ca2C], foilowed by retum to the orîginai direction within 15
minutes. In poilen tubes of Tr&scantia virginima the ceorientation was permanent
(Bibikova et al., 1997). Tip high ca2+ gradient hnaged by ratio-imaging of microinjected
dextran conjugated Calcium green-2 and Rhodamine was always closely correlated with
the site of active growth, following the direction of growth. Thus, the tip high ca2'
gradient acts as part of the machinery controliing locaiization of secretory vesicle
activity at the apex.
Growing polien tubes of Agcrparrthus umbellahrs exhibited a tip to base gradient
in cytosolic fiee [ca27 imaged using ionophoreticaily hjected Indo-1; the gradient was
not observed in non-growing tubes (Malho et al., 1994). Localized release of ca2'
changed the direction of apical growth towards the site of elevated [ca2']; the gradient of
calcium is one of the factors that directs tip-growth in polien tubes (Maido and
Trewavas, 1996).
In growing pollen tubes of Lifium there is a strict requirement for the presence
of a ca2' gradient (imaged with Fura-2) for tip growth because injection of the ca2'
chelator 5,S3dibromo BAPTA dissipates the tip-high ca2' gradient and inhibits growth
(Miller et al., 1992). Inhibited tubes can reinitiate growth concornitent with re-emergence
of caZ' gradient. The very steep calcium gradient measured in growing pollen tubes with
Fura-2 dextran loaded by pressure injection occurs within the first 10-20 pm proximal to
the tip, reaching 320 n M at the tip and declinhg to a uniform basal ievel of -170 nM
throughout the distal le@ of the tube.
A steep tip-hi& gradient occurs not ody in LiIium fongittomrn but also in
Nicotiana sylvesais and Trukscantia V i r g m i m as rneasured with dextran conjugated
Fura-2 (Pierson et ai, 1996). Pukations in growth rate are wrrelated with tip-locaiized
[ca2'] pulsations (Pierson et al., 1996; Messerli and Robinson, 1997). The gradient
probably derives fkom ca2' entry that is restncted to a s d l area of plasma membrane at
the extreme apex of the tube-
1.6. Fungal ion channels
If ca2' enters the cytoplasm fiom the extracellular medium, then one mode of
entry is via ion channels. Patch clamp of the plasma membrane of Neurospora crassa
revealed two types of channels: spontaneous inward K' channels and stretch activated
inward ca2- channels- They are not preferentially located at the tip (unlike the situation
in Saprolegnia fera, see below), but could be more active at the tip during growth
(Levina et al., 1995). The uniform distribution dong the hypha of the K' channels
suggests a role in K+ uptake to maintain an overall level of positive turgor (Levina et al.,
1995). They play a dispensable roIe in growth via turgor regulation because their
inhibition with TEA (a K' channel blocker) causes only a temporary reduction in growth
rate and reduced sensitivity to hypoosmotic shock (assessed by tip bursting), presumably
due to lower inuacelluiar ~ 7 . ~ d " ininbited stretch activated channels, but only
transiently reduced the rate of tip growth without changing tip morphology. Thus the
channels are not absoiutely essential for tip growth, even though tip high ca2' is
associated with tip growth. There may be ~d~~ insensitive ca2' permeable channels
which transport calcium, whose amplitude might be smaller tban the iimits of resolution
of their recordings. However, Lew (1999) used a self-referencing ion-selective probe to
rneasure ca2' fluxes at the hyphal tip and found that Narrosporu crassa has no net ca2'
flux, the direction of the ca2+ flux beùig almost evenly divided between inward (57.9%
of meamed calcium fluxes) and outward (42.l%), nor does the flux exhibit a tip
localized maximum,
Very and Davies (1 9%) used Iaser microsurgery to expose the plasma membrane
of Nezcrospora crmsu and resolved singleion channef activity by patch clamp. They
detected at least 5 different Channel types: one was a weakly rectifjing channel, probably
anion selective, as suggested by current reversal at the Cl- equilibnum potential, one was
an inward K' channel and another an outwardly rectiQing ca2' permeable conductance,
detected using whole cell level patch clamp measurements. The physiologicai role, if any,
of these channels is unknown.
The plasma membrane of Saproleegnia f e r a contaios two inward ca2'-activated
K' channels and two stretch-activated (SA) ~a~'channels, most abundant at the hyphal
rip (Garrill et al., 1992, 1993). The tip-high gradient of both channels is lost after
dismption of the actin cytoskeleton (Levina et al., 1994). There are two spatially distinct
populations of KT channels: one is found in the absence of SA channels, while the other
is always associated with SA channels; the association is not disrupted by cytochalasin
(Levina et al., 1994). Gd3' inhibited the SA channel activities, completely but reversibly
stop ped hyp ha1 extension, dissipated a tip-high cytosolic ca2' gradient measured with the
fluorescent ca2+ selective dye Indo-1, and inhibited the inward component of tip-
localized net ca2* flux measured with an ion-selective vibrating probe (Gad1 et al.,
1993; Lew, 1999). Because stretch-activated channels are more abundant in the hyphal
tip they appear to generate and maintain a growth-related tip hi@ gradient of cytoplasmic
ca2' by Iocalized uptake of ~ a 2 ' at the hyphal apex, which may be important in
maintainhg the structure of apicai cytoplasm, the movement and fiision of apical vesicle
and thus extension rates and tip shape (Jackson and Heath, 1993; Garrili et al., 1993).
1.7. Objective and rationrie for usuig Newospora crama
The purpose of this project was to investigate the tip growth process in
Neurospora crarsa. Its ease of culturing and relative simplicity makes it an attractive
mode1 organism for studying tip growth. It has a long history as an experimental
organism for research in genetics, biochemistry and electrophysiology. There is also a
library of rnorphological mutants available which may provide additional advantages for
fûrther exploration of how hyp ha1 growth is polarized and regulated.
When 1 started the experiments reported here I found myself codkonted with a
puzzling situation: in Neurospora a tip high ca2+ gradient was found at the apical end of
the growing hypha; however, ca2' channelq rneasured with patch clamp, do not seem to
be essential for hyphal extension (Levina et al., 1995). This raises a number of questions:
Is the calcium essential for hyphal extension and if it is what is its role? I s the influx of
~ a " obligatory for tip growth? in order to answer these questions the fust approach was
to use electrophysiological measurements to assess the importance of ionic fluxes for the
tip growth Initially, 1 had to assess the viability and growth rates of impaled hyphae
because of the technical complexity of the experïments that were to corne. I explored
whether ionic fluxes (iicluding ca23 cegulate tip growth process by using voltage
clamping of growing hyphal tips using longer durations (140 seconds) compared with 30
seconds (Levina al., 1995) and longer periods between clamps to accurately determine
the effect of voltage on growth rates. As the ionic currents were proved not to be an
obligatory requïrement of tip growth in this organism, 1 concentrateci on the role of
cytoplasmic ca2+ on tip growti~ Fim I ionophoresed caZ' into hyphae in order to see its
effects on induction of tip growth process. 1 reinvestigated this issue because previous
reports on increasing cytoplasmic caZf in Neurospora used either ionophores or elevated
extracellular calcium concentration, both indirect and thus diffinilt to interpret. The
present technique uses direct elevation of intemal ca2'. To directly demonsîrate the
requirement for ca2' in tip growth, I hjected the hyphae with the ca2' chelator BAPTA
and monitored both growth and changes in cytoplasmic free ca2' gradients. The
presence, localization and magnitude of the tip high gradient was assessed using ca2'-
selective dyes (Fluo-3 and Fura Red) that are more appropriate than either
chlortetracycline or ratio imaging of Fluo-3 and the H'-sensitive dye SNARF. 1 also used
a different technique to load the hypha than previous reports, obtaining higher level of
fluorescence compared to autofluorescence which allowed more accurate estimation of
cytosolic [c$'].
2. MATERIALS AND METHODS
Neurospora crassa wiid type strain RL2la (Fungal Genetics Stock Center no,
2219, University of Kansas Medical Center, Kansas City, KS) was cultured in 35 mm
growth: mature (more than 200 pm distance m y fkom the tip), young hyphae (at the tip)
and germinating conidia (at the tip) and corn hypbae growing h in solution at the tip
(growing and nongrowing a f k impaiernent). Data are showun as mean standard
deviation, ~ s a m p l e size.
Resting plasma membrane potentiai (mv)
1 Hyphae growing on gel-go substrate Hyphae growhg fia in solution
Mature
-
Young Gennlings
Membrane Potential (rnilliVolts)
Figure 3.2. Relationship between growth rates and plasma membrane potentials
Initial growth rates measured immediately afler recovering fiom impalement and
restarting growth (circles) and between the application of voltage clamphg (squares) are
plotted versus the resting potential recorded at that the. In neither case is there a
relationship between growth rate and resting potential. Data are jittered.
Clamped Potcntial (mil1 iVo1ts)
Figure 3 -3. Relationship between growth rates and clamped potentials.
Upper panel. Growth rates are shown versus clarnped potentials which were
appiied for a duration of -140 seconds. Lower panel. Growth rate difEerence (the growth
rate during the voltage clamp minus the average of the growth rates before and after the
voltage clamp) versus clamped potential. There are fewer data compared with the upper
panel because growth rates before and after the voltage clamp were not aiways available.
There is no relationship between voltage and growth rate. Data are jittered.
driving force for al1 electrogenic transport. &ectively cbnngïng the flux of any ionic
species across the plasma membrane (Figure 2.1). However, growth was unaffecteci: there
was no relationship between growth rate and clamped potential (Figure 3.3 .) implying
that ion transport at the hyphal tip, including @ Mu% is not required for tip growth.
To deterrnine if intracelizdar ca2+ plays a role in tip growth, we injected it
directly into the cytoplasm An example of ca2+ injection is shown in Figure 3.4.
Elevating cytosolic ca2+ induces initiation of branches (nimmarised in Table 3-33, often
multiple: 6 singles, 4 double and 1 triple. The initiation of new branches starts
approximately 7 4 minutes (range 1 to 16) (n=17) after the beginning of ionophoresis.
The branches were located within 43 2 29 pm (range 2- 106 pm) (n=17) fiom the site
of injection. Initiation occurred within 14 2 13 pm (n=17) (range O to 38 prn) of the
growing apex 70% of the hyphae were initiated subapically, the'rest apically. An internai
control is experiments in which calcium could not be ionophoresed into hyphae due to
piugging of the ionophoresis barrei, apparent as a lack of deflection in the potential
recording, white injecting current. In this case, branch induction was uncommon
To determine if elevated ca2' is required for tip growth 1 lowered cytosolic free
[caZ'] using ionophoresis of a ca2' chelator, BAPTA-
Figure 3 -4. Example of a typical a*+ iowphonsis nperiment in Neurospora mard s
hmk
Upper panel The upper trace shows the rrcording of the plasma membrane
potential and caZ' ionophoresis. E1ectrode tests (ET') were performed before and a f k
the impaiement. M e r the impaiement, a -30 mV potential was initidy observeci wbich
slowly hyperpolarized to -134 mV. The aimot injection through the ionophoresis barre1
causes a deflection in the potentiai recorded with the other barreI, The lower trace shows
the injection of m e n t : -2 nA pulses for 9 seconds which cause the deflections shown on
upper trace followed by a 3 seconds pause. During the pause the resting potential retums
to its initial value. When the deflection disappeared, ca2+ was no Longer being
ionophoresed due to plugging of the ionophoresis barrel. Lower panel. Recording of the
growth and the initiation of three branches (arrows) (A -3.3 minutes B 4.9 minutes C
O minutes D 3.7 minutes E 4-8 minutes F 7.7 minutes G 9.6 minutes H 11.6
minutes; zero t h e is the beginning of ca2+ ionophoresis). The fïrst branch appeared 5
minutes after the start of ca2+ ionophoresis (tirne O, C) 50 pm fkom the site of
impalernent, the second &er 8 minutes, 59 ~ i m fiom the site of impalement, and the
third afker 10 minutes 74 fiom the site of impalement. Bar 4 0 p m
I impalement
> C e 3 ln
Resting potencial 50 sec
'1
Ca2'ionop horesis i 5 r m
A-
L
Curcent rnonitor Ie
Table 3 -3. ERect of Ch2+ iowphoresis on branching in Neurospora
Successf'bl injection of calcium was assesseci as described in Figure 3.4. When
ca2+ was clearly being injecteci, 85% of the hyphae induced branches. When the
ionophoretic barre1 was plugged, ody 3 1% of the hyphae had branches. Rarely, (13% of
aU experiments), branches were induced prior to ca2+ injection, probably due to caZ'
leaicage corn the micropipette.
Experiment 1 1 Branch induction
Cases examined
Evidence that caZ' was injected
Yes
No evidence that ca2' was injected
B ranc hing induced prior to pulsing
No
2 13 1 I
9
1
13 4
4 4
3.6. BAPTA cffect on growth and morphology
Ionophoretic injection o f BAPTA either severely inhibited hyphal growth (6 out
of 22 experiments) o r more commody caused complete cessation of growth (16 out of 22
experiments) (Figures 3.5, 3.6) within 3-4 minutes of BAPTA microinjection Dunng
long term recovery (about 20 minutes) after BAPTA injection, hyphae frequently (18 out
of 22) showed changes in hyphal morphology: multiple bud formation (Figure 3 -7. 4 B)
which is similar to the phenotype of hyphae with impaired calcineurin fiinction or wild
type hyphae treated with calcineurin inhibitors Vrokish et al., 1997). This phenotype,
representative for 80% of the hyphae examined d e r BAPTA injection, was occasionally
observed in branches a far distance Erom the impalement site (Figure 3.7. B). In controi
experiments (n=2 l), hyphae injected with KC1 did not show these changes: hyphal
growth was not inhibited by KCI ionophoresis (Figure 3.8) and tip morphology was
normal (Figure 3.7.D).
3.7. Conventional fluorescence microscopy
A variety of fluorescent dyes injected by ionophoresis were quickly distributed
throughout the hyphae due to the diffusion and cytoplasmic streaming. However, the
conjugated dye BSA Fura 2 was more diff~cult to inject compared to nonconjugated
ones (presumably due to its higher molecular weight). In these preliminary experiments
32 out of 56 hyphae recovered fkom impalement, continued to grow, although some
branched more abundantly especialiy with Indo- L ionophoresis. Over time, the dy es
Figure 3 -5. B APTA ionophoretic injedion inhibits hyphd growth: An experimental
example.
Upper panel. Hyphal length versus tirne. Middle panel. Growth rate versus tirne. Lower
panel. Bright field images used for the measmement of growth. (A O seconds B 180
seconds C 325 seconds D 450 seconds E 520 seconds F 580 seconds G 690 seconds
H 8 10 seconds 1 11 10 seconds 1 1690 seconds after impalement). BAPTA injection
(duration 7 minutes) was starteci 450 seconds &a impalement. Bar =IO pm
Figure 3.6. Effect of BAPTA microinjections on hyphal elongation and growth rate:
compiled data.
Hyphae were impaled about 30 pm fiom the hyphal apex. After the hyphae
resumed growth, BAPTA was ionophoresed into the growing hyphae at t h e O for 7-8
minutes. Upper panel. Plot of hyphal length versus t h e . Normally, within approximately
200 seconds of BAPTA ionophoresis, elongation ceased or was markedly reduced.
Lower panel. Growth rates versus the. The symbols and error bars show means + standard errors for length and growth rate respectively compiled for consecutive 200
second time intervals (n=5 to 50). By plotting the data for each hypha as a single iine
rather than only the average values, a distinction can be made among the hyphae that
immediately and irreversibly ce& growing after injection, hyphae that wntinued to
grow for the wbole duration of the experiment at a much slower rate or eventually
stopped growing and hyphae thst recovered growth f i e r inhibition.
growth, destroyed tip zonation of organelles at the tip and dissipateci the intracellular tip
focused ca2+ gradient. Hermmann and Felle (1995) pressure injected dibromo-BAPTA
into the basal region of Sirupis ulba mot hairs. W~thin minutes, it severeiy inhibited tip
growth, eliminated the tip-high [ca2+] gradient and decread the cytosolic [ca27.
The basis for the cessation or inhibition of growth after BAPTA injection would
include both "shuttle b u f f e ~ g " and BAPTA depletion of ca2+, both of which would
affect ca2' regulated cellular processes.
During long term recovery after B U T A injection (-20 minutes), there were
changes in hyphai tip morphology - multiple bud formation - similar to the phenotype
observed in hyphae with impaired calcineUrin hnction or wild type treated with
calcineurin inhibitors (Prokish et al., 1997). This phenotype appeared simultaneously not
only in the main apex, but aiso in branches, some found a relatively far distance away.
This suggests that there is a correlated regulation of main apex and branches, not
unexpected given the correlation between the growth rates of the apex and those of the
branches.
The long-term morphological changes caused by lowering the ca2* concentration
in the tip may be due to modification of calcineurin activity. The increased hyphal width
and budding may be due to defects in ceii waii synthesis andor destabiluation of F-
actin (Halpain et ai., 1998).
Calcineurin is a highly conserved ~a~+/calmodulin-regulated serine/threonine
phosphoprotein phosphatase (Klee and Cohen, 1988). In brain the fùnctional enzyme is a
heterotrimer composed of a catalytic subunit (calcinairin A (CnA) 60 ma), a regdatory
subunit (calcineurin B (CnB) 19 B a ) and calmomilin (Cam). Calmoduiin is a smali,
highly conserved, ubiqyitous protein As a primary 'decodifier' of caz' iaformation, in its
ca2' bound form it acts as a pleiotropic factor which regulates a variety of membrane and
cytoskeletal structurai proteins and enzymes (Cohen and Klee, 1988). The ca2+-
calmodulin complex can alter enqme activity either by directly binding to a target
protein or indirectiy stimulate the target protein through a ca2+ -calmodulin dependent
protein kinase.
Cd3 and calmodulin are both required for the fùil activation of the phosphatase
activity of calcineurin when bound with ca2'and are not interchangeable. The two
proteins recognize distinct binding sites on the calcineurin A subunit (Gao, 1999).
Calmodulin increases the turnover of calcineurin and modulates its response to ca2'
transients while calcineurin B decreases the Km of the enzyme for its substrate,
increasing the affinity of calcineurin for substrate (Stemer and Klee, 1 994).
The ma-1 gene for the catalytic calcineuin subunit is essentiai for apical growth
in Neuropora crassa (Prokish et al., 1997). It is found in high concentration at the
hyphal tip (Kincaid, 1993). Decreased expression causes growth arrest preceded by an
increase in hyphal branching, changes in hyphal morphology and loss of the apparent
apical dominance of the main hypha concomitant with loss of a tip-high ca2' gradient
measured with CTC. Similar responses occur in wild-type hyphe after application of
calcineurin inhibitors, cyclosporïn A and FKSO6 (Prokish et al., 1977).
It seems Likely that both the growth cessation, and long-terq multibud phenotype
caused by ca2+ depldon are due to modification of calmodulin andor caicineurin
dependent processes.
In Neurospora crava ca2'-calrnodulin is known to activate chitin synthase
(Suresh and Subramanyam, 1997), intetacts with actin (Capeiii et al., 1997), tùnctions in
regulation of circadian rhythm (Sakadane and Nakashima, 1996; Suzuki et al., 1996), and
cyclic nucleotide signal transduction (Ortega Perez et ai., 1983). I~ifaibitors of calmodulin
increase the frequency of branching and slow tip growîh (Ortega Perez et al., 1987).
The long term BAPTA effect, aberrant vegetative morphology, resembles
entrance into a "hurrïed" but încomplete conidiation program resulting fiom mis-
scheduled expression of developmentaily regulated genes (Figure 4.3), since spodation
does not normdly occur in submerged culhues (Springer, 1993)- Conidation can be
viewed as an alteration of growth polarity. The first morphological step of
macroconidiation -induced by desiccation, C a , exposure to Light, deprivation of
nutrients -is the transition fiom growth by hyphal tip elongation to growth by repeated
apical budding, in which each apical bud gives rise to the next bud resulting in the
formation of chains of prownidia that resemble beads on a string. The typical time frame
for initiation of conidiation is 2-6 hours, mature conidia are formed after 16 hours. Our
mo rp hological p henotype may correspond to initial preconidial chains whic h can rare1 y
recover to grow by tip elongation and are commody observed 1-2 hours after initiation
of the conidiation developmental program ( S p ~ g e r and Yanofsky, 1989; Springer, 1993;
Vierula, 1996).
In the long-term, ionophotetic injection of the calcium chelator BAPTA causes an unusual tip morphology consisting of multiple buds.
This morphological phenotype is also observed in calcineurin mutants and wiid- type s trains treated with CalcineWin inhibitors.
W e hypoihtsize that the decline in cytoplamiic calcium caused by the injection of the calcium chelator BAVTA results in Iower cakineunn activity which affects gene expression. The result is a morphological phenotype similar to the cafcineu rin mutant.
Figure 4.3. Loag term effects of BAPTA injection into Néurospora craruz
Caicineurin B is required for normal vegetative growth and morphofogy
Nmrospora crama Woethe and Free, 1998). A mutation in the cnb-1 gene which
encodes calcineurin B Sects the ability to repress the entry into conidiation process
causing an abnormal morphology of chahs of swollen, buddmg septated ceils.
Apparently calcineurin activity cepresses the asexual developmental program by
repressing the conidiation specific ccg-1 gene. The production of highly brancheci hypha
with chains of septated cells resembles the formation of conidiophores on aerial hyphae.
4.8. CaIcium and tip growth
As this terrestrial fùngus tives in an environment not very rich in ca2', a
mechanism similar to that of ca2+ "bootstrapping" (Jackson and Heath, 1993b) is
probably occurring. This mechanism was proposed for the maintenance of the gradient in
conditions of low extemal ca2+, but in Our case it may function as the normal one. Unlike
Saprolegnia &rar and pollen tubes, where ca2* fluxes, channels and free calcium
function in a feedback mechanism regulating tip growth, in Neurospora since there is no
net uptake at the tip and because channels are not strictly required for growth, the
mechanisrn must follow a different strategy. In the other organisms the intraceilular tip
high gradient and tip iocalized ca2' influx can be explained by the functioning of caZ'
the channels. in Nmrospora, ca2+ probably enters behind the tip. Vesicles formed via
endoplasmic reticdum/ûolgi body system may accumulate caZ'. These vesicles are then
transported apically. When ciocking at the apical plasma membrane the vesicles would
release their intemal ca2' which will induce vesicle fusion probably via a calmodulin-
mediateci process and may have other fùnctioii~. For example, in Newopru, Capelli et
aL (1997) reported a peptide p47 concentrated at the tip which binds to rcrin and
caimodulin and may play a regdatory role in tip extension by altering the binding of
actin to p47, depending upon calcium concentration
Tip high [ca2'] gradient may be maintaineci simply as a consequence of dilution
due to increased hyphal volume behind the tapered apical region during the contùiuous
advance of the tip. Altematively, the decline in the ca2' gradient f?om the extreme tip to
20 pm proximal may require that a calcium sequestering system be active in this region.
Possible candidates Uiclude: mitochondria, endoplasmic reticulum and calcisomes.
Mitochondria (Heath and Kaminskyj, 1989) and endoplasmic reticulum (Yuan
and Heath, 199 1 a, b) cm act in shoa terni ca2' storage and removal fiom cytoplasm, and
vacuoles as a long term sink (Allaway et al., 1997). In growing fungal hyphae, the
vacuole has the potential to continuakiy enlarge as the hypha extends, increasing its
capacity to store ca2'. They can sequester ca2- and release it when necessary,
functioning as an endogenous buffering system capable of compensating for subaantial
changes in extracellular [ca27 with little change in cytoplasmic [ca2]. caZc from internal
stores are required when the influx behind the tip is reduced or when cytoplasrnic [ca2']
is decreased. The recovery Corn BAPTA injection is compatible with such internal
regdation of cytoplasmic [cazt] .
5. CONCLUSION
The results of my experiments can be readily summarized. 1 presented direct
evidence that :
1). Ionic fluxes at the plasma membrane do not control tip growth.
2). Direzt elevation of cytosolic ca2+ induces tip initiation
3). Direct depletion of cytosolic ca2' inhibits hyphal extension an4 long terrn,
causes the hyphae to shüt to an aberrant morphology due to entq into the conidiation
developmentai pathway.
Taken together these r d t s reveal that tip high gradient is a fùndamental aspect
of tip growth in Neuro~pora crassu and that a minimum level of cytosolic ca2' is
essential for maintenance of tip growth and morphology, possibly regulated by
caicineurin. Because the results show that electrogenic ion transport across the plasma
membrane at the apex is not essentid for the maintenance of tip growth, the r e w e d ca2'
must be supplied fiom some interna1 store. Neither the identity of the internai store
system, nor the regulatov mechanisms controlling ca2' release from these stores are
known. However, the techniques of microinjection we have developed may be ememely
usefül in future research identifjriog and characterizing the regdation of the tip-high ca2'
gradient in growing Narrospora crassa hyphe.
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