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

395 Wellington Street 395, rue W d l m OtiawaON K 1 A W OttawaOfU K 1 A W Canada Canads

The author has granted a non- exclusive licence ailowing the National Library of Canada to reproduce, loan, distribute or seil copies of this thesis in microform, paper or electronic formats.

The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fiom it may be printed or otherwise reproduced without the author's permission.

L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distriiuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique.

L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.

CALcIUMANDTIPGROWTHIN'rHE FILAMENTOUS FUNGUS Neurospora cmsa

by Lorelei Bianca Silverman Gavrila

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

internai universe:

mugh cndoplasmic reticulum plasma mKloth endoplasmic

vii

IntraceMar pH gradients .................................................................................. 17

calcium and tip gr0 wth. ..........................~...............................~~~~...~~..~.~.~~..~...... 19

1.5.1. Ca2+ions are requmd forîhegrowîhofNe~~oprm~~assa .................. 19

1 - 5 2 Inîraceltular Ca2+ ........................................................................... .... ...... 22

1 . 5 3 . Intraddar Ca2+ gradients .................................................................... -23

Fungal ion channeIs ......................................................................................... -26

1.7. Objective and ratiode for using Neuropspora wusa ........... ..... .................. 2 8

2 . MA'I'ElUALS AND METKODS ................................................................................ -30

. 2.1 Culturing Ne2nosporu Cr- ........................................................................... -30

2.2. Growth rates measwements .............................................................................. 3 1

2.3. Electrophysiology .. .. .............................. i ............................................................ 31

2.3.1. Micropipette Mrication and membrane potemial recordings ................ -31

2.3 .2 . Voltage clamping .................................................................................... -34

. . . 2.4. Calcium q ectton .................................................................................. .. 37 ..........

. . . 2.5. BAPTA mjectron ......................................................... .'. .................................... -37

2.6. Conventional fluorescence microscopy ........................................................ -38

2.7. Laser scaaning confocal fluorescence microscopy ............................................ -39

. * 2.7.1. Calcium fluorescent mdtcators... .................... ...-.. ...... 39

. . . . ......................................................................... 2.7.2. Microqeaion protocoL -40

............................................................................... 2.7.3. Confocal microscopy 42

.................................................................................... 2.7.4. Autofluorescence -42

.................................................................................... 2.7.5. Image processing -45

ix

......................... 2.7.6. In vitro ratiomettic calibration ofFluo-3 and Fura Red -46

3 . RESULTS ..........................................................................*......................................... 50

.................................................................... 3.1. Growth rate measurements ....... .. 50

3 .2 . Branch and main hyphal tip growth rates are d a t e c i .................................... 50

............................................................ 3 .3 . Plasma membrane p o t d and growth- 53

3 .4 . Voltage clamping- ............................................................................................... 53

3 .5 . Calcium ionophoresis .......................................... ....-......................................... 57

3.6. BAPTA effect on growth and morphology ........................................................ 60

............................................................. 3 .7 . Conventionai fluorescence miaosapy -60

................ 3.8. Laser Scannning fluorescence rnicroscopy ratio imaging of calcium 65

. . . .................................................................................. 3 3.1 . Dye distribution.. -65

.......................... 3 . 8 .2 . Calcium gradient in growing and nongrowing hyphae -68

2.e ............................................ 3 .8.3. BAPTA effect on Ca gradients -68

4- DISCUSSION .............................................................................................................. 73

4.1. Assesrnent of growth rates after impalement ............. : ....................................... -73

4.2. Plasma membrane potentials .............................................................................. -74

............................................................................................... 4.3 . Voltage clamping -76

4.4. Calcium injection ............................ .. ............................................................. -77

......................................................................................... 4.5. Imagllig calcium -78

2+ ................................................................................. 4.6. Ca gradient 80

............................................................. 4.7. BAPTA dissipates the calcium gradient 84

4.8. Calcium and tip growth ................................................................ -90

X

5 . CONCLUSION ........................................................................................................ -92

6 . REFERENCES ........................................................................................................ ..93

LIST OF TABLES

Table 2-1-

Table 2.2,

Table 3.1.

Table 3 .2 .

Table 3 .3 .

Table 4.1.

Summary of voltage ciamp durations for the second protocoL ...................... 35

Relative fluomcence intensity of signai for autofluorescent and dyes

loaded growing and nongrowing hypha e. ....................................................... 44

Cornparison of growth rates for impaled and unimpald Narrcqora crassa

hyphae ............. .. ............................................................................... 1

Plasma membrane potentials recorded fÎom different stages of hypbal

gr0 wth, ............................................................................................................ 54

Effkct of ca2+ ionophoresis in Netcrospora crassa hyphal tip ......................... 59

Calcium concentraiion in dBerent tip-growing organisms ..................... -82

LIST OF FIGURES

Figure 2.1 . Voltage clamping of membrane potentids ..................................................... -36

Figure 2.2. Relative fluorescent iatensities for autofluorescent and d yes injected

.................................................................... hyph ...................................... .. 43

Figure 2.3. Imaging uniform layers of buffer solution .................................................... 47

xi

Figure 2.4. In vitro talibration came of Fiuo-3 and Fura Red ratio of emksion

2 intensities versus [Ca *] &ee ........................................................... 49

Figure 3.1. Relationship between brrnoh tip and main apex growth rates .................... 5 2

Figure 3.2. Relationship betwctn growth rate vasus plasma membrane poteiitkls ........ 55

Figure 3.3. Relationship betwan growth rates and clamped potentials ....................... 56

Figure 3 .4 . Example of a îypical Ca2+ ionophoresis experiment in Neutoqporu crassz's

...*...................*.................................. ................ h y p h .............. .... .. 58

Figure 3 .5 . BAPTA ioeophontic injection inhibits hyphal growth: An experimental

.......................................................................................................... m p l e -61

Figure 3.6. Effect of BAPTA microinjections on hyphai elongation and growth

........................................................................................ rate: compileci data 62

Figure 3.7. Long tam effects of BAPTA injection in N w o p r a crusa

........................................................................................................... hyp hae -63

Figure 3.8. Control for B APTA microinjection: KCl ionophoresis into growing

............................................................................................................ hyp hae -64 . .

Figure 3 .9 . Homogenous cytoplasmic distribution of Fluo-3 and Fura Red fluorescent

....................................................... dyes within Neurospora crassa hyp hae -66

Figure 3.10. Dye sequestration in Neurospora crara hypha imaged by coafocal

........................................................................... microscopy -67

Figure 3.1 1 . Ewmple of a calcium distribution in growing Neurospora crussa hypha

................................... ionophoretically injected with Fluo-3 and Fura Red -69

Figure 3.12. Calcium distribution at growing and nongrowing hyphal apices ................. 70

Figure 3.13. Changes in the qathl distrr'brition and the mapitude of [mi gradient

............................................................................. iffa B APTA inj ectioa -72

Figure 4.1. Sutnmary of the main electmgenic transporters potmtdiy involved in

tip gcowth reguiation ................................................................. -75

........................................................ Figure 4.2. Shuttte di€fÛsion rnechanisrn -85

Figure 4.3. Long tam effects of BAPTA injection inîo Neuropora craçsa .............. -89

ABBREVIATIONS

AS31 87- cation ionophore

ATP- adenosine triphosphate

BAPTA- 1,2 bis(ortho1aminophe~}ethane-Ns~,ET acetate

BCECF- 2'7'-bis-(2-CIUbOxya~r~5<and6)-carboxyfluoftSCeiP

BS A bovine serum albumen

Cam- calmoduiin

CCD- charge wupled device

CTC-c hlortdracycline

DCCD N'N" dicyclohexylcarûodiimide

dibromo BAPTA- (see BAPTA above)

EGTA- ethyleneglywl-bi@-aminoethyL~N,N,W ,N' tetraacetîc acid

F-actin- filamentous actin

Fluo-3 - 2, 7dicloro-6-hydroxy-3 -0xo-9-xa11thenyl4' -methyl-2,Z7-(ethy1enedioxy)dianilhe

N,NyN',Ny tetracetic acid f

& -dissociation constant

MOP S 3 ' (N-morpholino) pro panesulfonic acid

PIPES- 1,4 piperazinediethanedonic acid

SA-stretch activatecl

SNARF- 1 -seminaphthorhodafluor

TEA- tetraethylamonium

V, trammembrane potentiai

1. INTRODUCTION

1.1. Ecological perspective

In terrestrial ewsystems h g i have evolved as primary agents of decay, an

essentid step in the recycling of carbon, nitmgen and other inorganic and organic

molecules (Griffin, 1981). Nearly aii h g i are aerobes and ail of them are heterotrophs

obtaining nutrients as parasites, necmtrophs (fùngi that kit1 the tissudorganism they

invade) or saproîrophs. Fungi can not enguiffood because they have hard chitinous walls.

Instead, they secrete powerfiil digestive enzymes into the immediate environment that

break food d o m into smaller molecules; these dissolved nutrients are then absorbed into

the fingus through the tùngai wail and membrane (Manseth, 1995).

Because they are nonmotde, h g i have evolved special patterns of growth and

morphology to adapt rapidly for successfùl survivd and proliferation optimizing their

ability to invade new territones and obtain nutrients. Fungal spores, often air dispersed,

fia germinate ïnto hyphae, the dominant vegetative tùagal stm&ure, a tube of constant

diameter consistently shaped with a well defined polarity (GamII et al., 2992; Jackson

and Heath, 1993b). As the hypha extends, starGng a few hundred microns behind the

hyphal tip, it usually becomes segmented at regular intervals by incomplete cross walls

(septa) with a variety of pore structures dowing for continuity and free movement of

cytoplasm and migration of organelles and nuclei (Griffin, 1981). Within the mycelium

(defined as the total hyphae fiorn an individual tùngus), hyphae ofien are interconnected

by anastomoses through which cytoplasm can flow fiom one part of a mycelium into

another (Zalokar, 1959). The mycelium structure has a high area to volume ratio that is

ideal for the efficient absorption of nutrients. Hyphae at the pwing edge of the colony

continue to elongate via a process d e d tip growth and branch at different intervals.

Metaboiïsm, volume increases, tubular extension rates, and regdation of the

direction of growth mus be finely coordinated during hyphal extension. In this

introduction to the process of tip growth, I wiU foas on Neuropora crussa, the

ascomycete fiingus 1 am using to aramine the control of tip growth. Neuro~para crussa

utilizes tip growth to fonn a characteristic radidy spreading colony. its molecular

genetics are very well characterized (Perkins, 1992) and a variety of morphological

mutants, possibly due to lesions in the tip growth process have been identifieci (Vierula,

1996; Perkbs et ai., 1982). There is aiready a considerable body of knowledge regarding

the structure and physiology of the hyphal apex in Neurospra crama. Pertinent

information f?om other tip-growing organisms will also be presented.

L.2. Tip growth process

Most prominent in the fùngal kingdom, tip growth is also found in widely

divergent celi types: prokaryotic actinomycetes, protists including algae and oomycetes,

pollen tubes and root hairs of higher pIants.

Tip growth is characterized by a precisely regulated dynamic equilibrium between

the synthesis and localized extension of c d wall and plasma membrane and the

application of an expansive force derived fiom turgor pressure and the cytoskeleton Tip

growth involves the coordination of cell wall synthesis and apical deposition, via

polarized transport and exocytosis of wali vesicle contents at the tip, forward migration of

the entire cytoplasm with respect to the lateral ceil wall and plasma membrane in order to

maintain its apical position and movement of individual organelles within this migrating

cytoplasm to maintain a characteristic distribution (Heath, 1990)- The critical events of

this complex process are localued within the kst 5-10 pm of the tip (Jackson and Heath,

1993 b).

1.2.1. Cytological polarity

The first 50-100 pm of the growing end of the Nwospora crassa hypha, ending

at the first septum, diflers substantiaily tiom the rest-of hypha, being non-vacuolated

(Zalokar, 1959).

The extreme tip (0-5 pm) of the hypha is filled predominantly with vesicles -100

nrn in diameter (Seiler et al., 1997; Collinge and Trincï, 1974). Mitochondria and

endoplasmic reticulum begin to appear a few Pm behind the tip, increasing in number

distally. Mitochondria are abundant and situated slightly closer to the tip han

endoplasmic reticulum (Collinge and Trinci, 1974). Smooth endoplasmic reticulum is

sparse and more or less evenly dispersed throughout the diameter of the hypha, while the

more distal regions of the hyphal apex appear to be particularly well endowed with rough

endoplasmic reticulum and an abundance of free ribosomes (Collinge and Trinci, 1974).

Nuctei are first observed about 10-15 pm behind the tip. They are very dense in the

young region, but aiways absent at the ememe apex (first 0-1 0 pm) (Collinge and Trinci,

1974).

A similar organelle distribution was obsewed for the oomycete Saprokgnia ferar

(Heath and Kaminskyj, 1989). The most apical 1-2 pm zone is filled with Golgi body

derivecl exocytotic wall vesicles (Heatb and Kaminskyj, 1989; Yuan and Heath, 199 La).

Mitochondna are rare in the most apical 5 pn, attab theù maximum abundance in the 5-

10 ptn zone (Heath and Kaminskyj, 1989; Aiiaway et ai, 1997) and rernain abundant for

at l e s t the fist 20 p m Golgi bodies are absent £kom the 6rst 0-5 pxn and occur at

approxirnately equal fiequencies throughout the rest of the first 20 Fm. Nuclei do not

occur at the ememe apex, they first appear in the 20-20 Pm zone (Heath and Kaminskyj,

1989; Aliaway et ai., 1997). Vacuolation begins at about the level of the most apical

nuclei (-10 Pm fiom the tip) and graduaiiy increases so that at 400 pm behind the tip,

the hyp ha is filled by large vacuoles (Yuan and Heath, 199 1 a, b).

In Nerrrospora crassa histochemical staining revealed welI-defied zones dong

the hyphae. A variety of enzymatic activities such as peroxidase, alkaline and acidic

phosphatase and P-galactosidase were observed only in the 10-50 pn zone. RNA staining

occurred in a 10- 100 pm zone. The mitochondrial enzymes succinate dehydrogenase and

cytochrome osidase were found fiom 100 to 150 pm with lesser activity in a 50-100 pm

zone. Glycogen \vas distributed in a spatial pattern similar to that of mitochondrial

enzymes. This means that the 50-150 pm zone is where polysaccharides are hydrolyzed

into sugar to supply metabolic energy required for protein, membrane and ce11 wall

synthesis (Zalokar, 1959).

1.2.2. Ce11 wall polarity

There is aiso a "cell wall polarity" determined by the requirernents of localized

ce11 wall synthesis. The importance of the ceii wall in d e t e m g füngal morphology is

supported by the tact that enzymatic removal of the ceii wall nom hyphal filaments

results in spherical protoplasts (e.g. Levina et al., 1995), hyphal growth resuming only

after the wall is aiiowed to regeaerate. Using longitudinal and senal transverse

sectioning, Trinci and Coiiinge (1975) found four waü zones dong Neurospora crassa

hyphae: apical extensible; subapical not extensible; a zone of formation of a secondary

wall, extemal to the prirnary waü; and a mature zone where wall lysis may occur during

starvation to provide nutrients to the celi. Wall thickness remains unchanged from the tip

to about 50 Pm behind ( 4 5 nm thick) and then starts to increase, becoming constant

after reaching a thickness of -275 m. A very short region at the very tip of the hypha

must be active in laying down new ce11 wall. Here a special concentration of enzymes

necessary to build new celi wall constituents is required. Chitin is the most important

ultrastructural component of the fùngal ce11 wall. Chitin synthesis occurs at the apex

(Ruiz-Herrera, 1992). Chitin synthase is cornpartmentalized in membranous vesicle

(chitosomes) abundant in the vicinity of the hyphal tip (Sietsrna et al., 1996) and becomes

activated after vesicle hsion with the plasma membrane (Gooday, 1983).

Mahadevan and Tatum (1967) obtained four fiactions of the ce11 wall: fraction 1

glucan-peptide-galactosamine complex (glucose, galactosarnine, glucuronic acid,

glucuronolactone, arnino acids glycine, alanine, leucine, isoleucine, valine, aspartic acid

(Mahadevan and Tatum, (1965)); fiaction [I f?ee sugars and amino sugars (manose,

manitol, glucosamine (Mahadevan and Tatum, 1967)); fiaction III laminarin-like P 1-3 P 1 -

6 glucan; fiaction IV chitin. Hyphae stained with antisemm to fraction 1 showed

maximum fluorescence in the apical a d o r subapical regions: in both cases, fluorescence

showed a sharp decrease with distance behind the hebapical regions. Hyphae stained with

antisem &mt M o n III showed niiitly fluore~ce~~t t@, with fluorescence

increasing with distance fiom the tip. Hjphae stained with antiscrum to naaion N show

fluorescence only et the sites of hyphai hctwes (Hadey d Kay, 1976).

1.2.3. Vesicle distribution polnnty

Enzymes and prrairpors for biosynthesis of new cell waii polymers and plasma

membrane are produced in the endoplasmic r e t i d u m ail dong the hypha, packaged into

special vesicles in Golgi bodies or their equivalents, transportecl vectorially through

cytoplasmic streaming to the apex via actin microfilaments or microtubules, exocytosed

and incorporated at the extreme apex during tip growth or branch formation (Gooday,

1983; Wessels, 1986)-

The vesicles and other components concentrated at the tips are organized into an

assembly visible in phase contrast microscopy caiied the apical body or Spitzenkorper.

In Neuro.!pora crczssa, s m d sateiiite Spintzenk6rpers, which- arise behind the apex,

migrate toward the apical pole where they merge with the main Spitzenk6rper (Lopez-

Franco et ai., 1994). Hyphe ofNeurospora c r a w have a tendency to meander, yet they

maintain an overall direction of growth- Growth directionality (Le. change of direction in

the establishment of a new axis) was closely correlated with a sustaïneci shifi of the

Spitzenkorper position away from the existing axis. It is likely that the Spitzenkorper

functioning as a vesicle supply center (Riquelme et al., 1998) dictates hyphal

morphology. It is certainiy not univerdy required, since many tip-growkg organisms

do not have a Spitzenk6rper and may control vesicle supply via alternative mechanisms

(Heath and van Rensburg, 1996 and Regdado, 1998).

The vesicles are usudly more or less uniformiy distributed throughout the cross

sectional area of the h y p h Vesicles are generated fiom Golgi bodies throughout the

cytoplasm of the penphed growth zone (Trinci, 1973). They may concentrate in the

extension zone of a hypha not because they are mainly generated io the cytoplasm within

this region (this would imply a very fast rate of synthesis of membrane and wall

precursors), but due to the transport of vesicles at a constant rate into the tapered region

of the tip, where the volume of groundplasm per unit hyphal length is decreasing at a

progressive rate (Collinge and Trinci, 1974). About 38 000 vesick per minute must fise

at the hyphal apex to maintain normal extension rates at 25' C.

Neurosporu crama has a pulsed hyphal elongation with fiequent pulses (1 3-14

per minute) which hypothetically correlates with the overalI rate of vesicle discharge

(Lo pez-Franco et al., 1994).

1 2.4. Cytoskeletal polarity

In fùnsi, vesicle exocytosis coincides with the actin-rich apex (Heath, 2990).

Picton and Steer (1982) suggested that a tension-bearing apical cap of F-actin associated

with the plasma membrane regulates tip growth in plant and fungal cell. The extensibility

of actin would regulate the extensibility of the tip in response to the turgor, a gradient in

yield being responsible for generation of the tip shape.

In Neurospora mmsa, F-a& is mainly concentrated at the growing hyphal tips

(Tinsley et aL, 1998) where it forms a unifonn cap (Barja et ai, 199 1). Subapically, actin

is visuaiited predominantiy as dots consistently sphencal in shape, but varying in nurnber

and size in different hyphae. F-sain was suggested to be attached to the subapical plasma

membrane and the ceii wall by an integrin-regdatory system (Heath, 1994; Bruno et ai.,

1996). Degousee et al. (unpubiished) used indirect immunofluorescence and found that F-

actin, spectrin, and a possible integrin homologue are more concentrated at the tip of

N m s p o r a crassa Spectrh is the dominant component at the tip where it forms a very

strong cap and a steep gradient and may be the dominant wmponent of tip morphology

generation. lntegrins may be involved in plasma membrane-ce11 wall adhesion consistent

with punctuate staining at the tip and dong the hyphae well behind the extrerne tip

Oegousee et al., unpubiished).

Proper actin and microtubule organization is required for tip initiation and

extension in Neir~oqora crassa. At lower concentrations the ant i-act in dmgs

cytochalasin A and B cause an increase in branching; at higher concentrations they cause

abnormal swollen, irregular hyphae and eventually inhibit hyphai growth (Allen et al.,

1980). Cytochalasin A and B also prevent conidiai germ tube emergence (Barja et al.,

1993).

Microtubules, some >50 p m in length, are associated with ce11 cortex and located

in aii regions of the hyphae. They extend parailel to the a i s of the hypha fiom distal

regions of the hyphae to the apex (Bruno et ai., 1996). The mti P tubulin îungicide

benomyl and nocodazole, both suppress liiear growth of Neurospora crassa hyphae at

p M concentrations, though the growth is relatively insensitive to nocodazole (incomplete

inhibition at 20 PM) ( l h t et al., (1988)).

Organelle movement in Neurospora craca is also a microtubule-dependent

process. Treatment with 10 PM nocodazole for 30 minutes caused a complete

disappearance of microtubules and reversibly blocked directed Vansport of al1 organelles,

whereas the anti-a& agent cytochalasin D was without efFect at concentration up to 20

PM (Steinberg and Schliwa, 1993; Seiler et ai., 1997).

Kinesin is a molecular motor that may function to translocate organelles along

microtubules. A kinesin mutant Iacks a Spitzenkorper and exhibits highly branched

hyphae of gnarled appearance (Seiler et al., 1997), perhaps by alteration of production

and transport of secretory vesicles to sites of ceU wall formation. Interestingly,

organeliar movement is unaffected Thus, microtubules do appear to play a role in hyphal

extension.

An actin distribution similar to that of Neirrospora massa is found in growing

hyphae of the oomycete Saprdegnia f e r a The tapered extensible portion of hyphal tip,

where the cell wall is plastic, contains a high concentration of filamentous actin (F-actin)

organized into a finely fibrilar apical cap, which differs in organization from the actin in

subapical inextensible regions of the hypha where a peripheral array of coarser filaments

(cables) and plaques is observed. The actin inhibitor cytochalasin E disrupts the normal

pattern of peripheral cytoplasmic actin populations in the hyphal tip and alters tip

morphology. Initially, there is a transient growth acceleration, followed by rounding of

hyp ha! apices, then swellhg and cessation of growth (Jackson and Heath, 1990; Garrill

et al., 1993; Levina et al-, 1994; Gupta and Heath, 1997). In addition to its role in tip

morp hogenesis (Heath, IWO; Jackson and Heath, 1990; 1993 a) F-actin which permeates

the cytoplasrn may be important in cytoplasrn and organelle movement (Jackson and

Heath, 1993 a), vectonal ce11 wdl vesicle transport and exocytosis (Heath and Kaminskyj,

1989; Heath, 1990) and the bulk movement of cytoplasm in BasidrdroboIus m a p s

(McKerracher and Heath, 1 987).

In pollen tubes, actin microfilaments accumulate in the apical region of the

growuig tube where bey fom a dense matrix that may resist turgor pressure of the

growing tube. Actin microfilaments found as cables dong the shank of the tube may be

involved in the transport and directional movement of secretory vesicles to apical

docking sites essential for celi elongation (reviewed by Taylor and Hepler, 1997).

1.3. Ionic currents and tip growth

There is a wide spread belief that ionic currents are not only a manifestation of

cellular polarity, but may be involved in generating and maintainmg this polarity during

tip growth (Kropf et al., 1984; JafFe and Nuccitelli, 1977). The assymetric spatial

distribution and/or activity of electrogenic ion transport proteins (porters and pumps) in

the plasma membrane causes a flow of electncal charge through the cytoplasrn and in the

extracelhlar space. Ion transport is an essentiai component of ceIl homeostasis,

maintainhg ionic balance, cytoplasmic pK turgor pressure and the uptake of ions and

other metabolites d u ~ g gr~wth by coupling solute transport to electrochemical

gradients.

1 -3.1- Electrical fields

A wide range of animal and plant cens have been shown to migrate or become

p olarized or aligned in electrical fields. Exogenous electric fields may polarize mycelial

fungi either by electrophoresing the protek and vesicles in the cytoplasm or by

influencing the distribution of key morphogenic proteins in the plasma membrane (Jaffe,

2977; J&e et ai., 1974). The high capaçitance of bioiogical membranes would favor the

latter possibility since intracellular components would b e electrically insufated fkom the

field, while membrane associated proteins may carry charged groups protuding into the

extracytoplasmic space which would be exposed directly to and mobilized by the

exogenous electric field (Robinson and Jaffe, 1975: McGillviray and Gow, 1986). if

these proteins are invoived in the generation of ceU polarity the endogenous and

exogenous fields could bring about polarity by influencing their topographical

distribution.

Various fungi redirect growth towards the anode or cathode (gdvanotropism)

when exposed tu an exogenous voltage field (Gow, 1994). Neurospora crassa and Achlya

grow and form branches toward the anode, rhizoid growth in A[lomyces is strongly

directed towards the anode, whiie Aspergilllrs and Mircar mzrcedo exhibit tropism towards

the cathode (McGillivray and Gow, 1986) as do hyphae of AlZomyces macrogynus (De

Silva et al., 1992). Germination of Neuroqora crassa conidia is also highly polarized in

electrical fields, occunhg on the anodic side of the spores and the tubes continue to

grow towards the anode. The electrical field also stimulates branching in Natrospora

crussa (McGillviray and Gow, 1986).

Pollen tubes also possess gdvanotropism, growing towards the cathode (Malho et

al., 1994). It has been proposed thaî they may orient their growth in response to the small

potential gradient down the stigma @¶alho et al-, 1994).

The existence of an intracellular cwrent was first demonstrated in Nkurospora

crassa using intemal electrodes (Slayman and Slayman, 1962). They observed a gradient

of the membrane potential between the apex and distal region of hypha that would cause

electric current flow into the tip- The membrane potential at the tip was 100 mV more

positive thm the potential recorded one centimetre back; most of the voltage &op occurs

within the first millimetre. Lntraceiiular currents were also observed, and extensively

characterised in Achlya (Kropf; 1986).

1 .3 .3. Extracellular currents

The existence of an extraceMar current was Iater established using vibrating

probe electrodes. Growing hyphae of Neurospora crassa, like many other tip-growing

O rganisms (germinating algal eggs, plant roots, lily pollen grains, water molds) drive

endogenous electric currents of about 0.2 pA/cmZ (McGillviray and Gow, 1987) through

themselves such that positive charges enter into the apical region, flow along the hyphal

length and exit distally nom the tnink. To complete the current loop, charge flows

through the extracellula. medium from tmnk to tip.

The generation of a transce1luIa.r current is a very early event in the establishment

of ce11 polarity, l o d k h g the site of growth and differentiation in rnany cases the

appearance of Localized inward electricd m e n t precedes the emergence of a tip and

accurately predicts the site of the future branch as in hyphae of Achlya bisexuuIis (Kropf

et al., 1983), the rhiioid of Pelvetia zygotes (Nuccitteli, 1978) and the gem tube of iily

pollen grains (Weisenseel et ai, 1975).

There are a number of electrogenic transporters that may contribute to the

extracellular current. The fùngal plasma membrane KATPase is an electrogenic proton

translocating pump (Slayman and Gradman, 1975; Scarborough, 1976) which generates a

large electrochemical proton gradient that drives autrient uptake into the hypha As in

Achlya (Kropf et ai., 1984; Kropf, 1986), protons may be expelled distdy fkom the

Newospura hyphal mrok by the H'-ATP~s~ and a significant fiaction of the proton

expelled may return into the apical region of the hyphae via specific transport proteins

such as H'lglucose cotransporters (Slayman and Slayman, 1974) or by nHT/amino acid

symporters as in Achlya wopf et al., 1984; Gow et ai., 1984; Kropf 1986; Schreurs and

Harold, 1988). Other symporters, for example nH'/~hosphate (Versaw and Metzenberj,

1995) and n H K ' (Rodnguez Navarro et al., 1986), may also contribute to K reentry.

Some of the ions that rnay carry the current have been identified by ion

substitution experiments. In Neuroqorra tram, the only ions or substances whose

omission had significant effects on the ioaic current were potassium, phosphate, calcium

and glucose. Both K+ and phosphate contribute to the flow of positive charges into the

apex. However, the absence of K' and phosphate also causes thinner hyphae and reduced

growth rates. Phosphate caused the pater attenuation of both current and extension rate

(McGillviray and Gow, 1987), by 4W. If phosphate contributes to inward current, it

would have to be undergohg e f l n at the tip to explain the aîtenuation of positive

current entry. Phosphate is essential for growth and may be required for current

generation without actualiy contributhg directly to the ionic current.

The rernoval of giucose reduced the magnitude of the inward current without

greatly affecting hypbd extension although hyphal diameter decreased by half- The

contribution of glucose to the inward current of Neurospora crma might be due to the

activity of a H+/glucose symport (McGiilviray and Gow, 1987). However, this is unlikely

since the H'/glucose symport (glucose transport system U) would be repressed at the

concentrations of glucose used in these expenments (Slayman and Slayrnan, 1974;

Scarborough, 1976). Since both the apical inward and the outward current greatly

diminished when glucose was removed, the requirement for glucose may reflect the

necessity for glucose metabolism to provide ATP for proton pumping. The dependence of

the current on metabolism was confrrmed by the inhibitory effects of cyanide (0 and

a i d e (Na). Increasing the pH of the medium (equivalent to reducing the fiee proton

concentration) drarnatically reduced the inward current deasity, suggesting that protons

carry most of the current (McGillviray and Gow, 1987)-

In Achlya increasing the pH caused the inward current to drop and then to shift to

outward concomitant with growth cessation (Kropf et al., 1984). When pH was returned

to 6.5, the inward current quickly reappeared at the tip and growth resurned. The

dependence of the inward current upon the extemal pH suggests that protons carry the

charge influx in Achiya as well as in Neirrospora. The majority of ionic current is not

carried by in other organisms. In the marine algae Fucus and Pelveria, Cl* is the major

source of current e m y (Nucciteili and J&e, 1976), as is ca2'. Thus, ca2' i d u x and Cl-

efflux create the inward ament in Pelvetia eggs (Robinson and JafEe, 1975; Nuccitelli

and Jaffe, 1976). In lily polien germ tubes, Ky influx dominates the inward current and

H' the outward current (Weisenseel and M e , 1976). The inward current is camed by

protons in barley roots (Weisenseel, 1979). Ion-sekive probe measurements revealed an

inwardly directed net ca2+ aiment at the tip of growing root hairs of Sirrcyis alba L.

(Hermmann and Feiie, 1995). and Arabidopsts thaliana (Schiefelbein et al., 1 992).

Ln growing ceIIs of the water mold Blasrociadiella emersonii, a positive current of

a 1 w c m 2 enters the rhizoid and leaves from the thallus, protons carry the current, which

reverses during sporulation when part of the current is probably carried by ca2' (Sturnp et

al-, 1980).

TransceIiular ion current flowing along the hyphal Iength may be a part of the

pol arizing mec hanism that establishes an intracellular voltage field which could direct

vesicles to the apex or induce both cytoplasmic and ceIi membrane asymmetry by

redistri buting charged macromolecules and organelles within the hyp ha by self-

electrophoresis or electroosmosis (JafEe et al., 1974; Jaffe 1977; Kropf, 1986). Most

proteins bear a net charge on their surfàce and may be subject to an electrophoretic force

established by ionic currents at the tip. Theoretically the endogenous fields in

A~eurospora (0.5 V/cm, Slayman and Slayrnan, 1962) and Achlya (0.2 Vkm or greater

immediately behind the tip) are of suscient magnitude to transport anionic cellular

constituents and localize them at the growing tip by self electrophoresis. That

nongrowing hyp hae failed to circulate current would support this specuiation (Kropf,

1986).

1.3 -4. Ionic currents are not essential for tip growth

In a few cases, nongrowiug Neurospora hyphae were found with a normal inward

current at the apex (hyphal extension dtimatety resurned) and growing hyphae with an

outward current (McGiiiviray and Gow, 1988; Talceuchi et al., 1988). These current

patterns, although rare, indicate that there is no tight correlation between the intensity of

transcellular electric current and the rate of hyphal extension

In Achlya, the emergence of a new branch sometimes caused the inward current

at the main apex to diminish or to reverse transiently corn inward to outward without

affecthg the growth rate of the original tip (Schreurs and Harold, 1988). If a self

electro p horetic mec hanism was operating, vesicles would be expected to move away

from the growing apex under these condition. Thus, self electrophoresis cm not always

provide an adequate explanation for growth localization.

Gow et al. (1984) suggested that growth of Achfya was invariably associated with

proton influx even when hyphae were branching. The attenuation and reversal of the

direction of electrical current was thought to be due to transitory fluxes of some other

cation, with a greater and oppositely charged efflux that masked the continual apical

influx (Kropf et al., 1984; Gow et al., 1994). If transcellular ion currents do play an

indispensable role in localization and maintenance of hyphal polarity, then it wouid seem

likely that it is the flow of H? or some other specifk ion that is important and not the

total flow of electricai charge.

Discontinuities in the correlation between tip growth and the pattern of flux of

endogenous electrical currents suggested that ion gradients e-g. protons or calcium are

more likely to be important to tip growth than cytoplasmic eiectrid fields across the

cytoplasm (Gow, 1984)-

1-4- Intracellular pH gradients

Growing hyphae of Neuropru do generate an external pH gradient: the

medium surroundhg the apex is slightly more alkaline than the bulk phase imply hg a net

K f l u x at the apex (Takeuchi et al., 1988)- In Achba as weI the extemai pH is siightiy

alkaline at the tip in growing, but not in nongrowing hyphae. The apical extemai

alkalinity may be due to the tra~l~cellular proton current (Gow et al., 1984; Kropf et al.,

1984). However, apical allcaLinity in Achlya may also be due to ammonia production

(Schreurs and Harold, 1988) and not wdepletion by H'/ amino acid symport activity.

Proton influx at the growing hyphal apex wouid be expected to cause a iocalized

acidification of the cytoplasm at the site of proton current entry compared with the

subapical cytoplasm zone of outward current. Another dominant source of cytoplasmic

protons is acids produced during metabolism.

Changes in external pH have only a small transient effect on cytoplasmic pH,

which was reported as 7.2-7.4 by Sanders and Slayman (1 982) and 7.57 by Parton et al.,

(1997). Intraceildu pH must be controlied within reiatively narrow Limits for the fùngus

to survive. Metabolism alone is capable of controlling p b since p h is unaffected

when the w-ATPase is ïnhibited by orthovanadate (Sanders and Slayman, 1982).

Acidification or alkalinkation of the cytoplasm in Neuroqpora slowed down, but did not

abolish hyphal growth Modification of extemai pH causes the hyphal tips to exhibit an

undulating growth pattefll and reduced growîh rates (Parton et aL, 1997)-

The presence or absence of a cytoplasmic pH gradient at the hyphal apex is

controversial. Parton et al. (1997) observed none using pH sensitive fluorescent dye

(SNARF-1). However, Robson et ai. (1996) reported a pH gradient at the extending

hyphai tips that is up to 1.4 pH units more aikaline than more distd regions also using a

pH sensitive dye, BCECF-AM . Both the magnitude and the length of the pH gradient

were strongty correiated with the rate of hyphai extension, and eradication of the gradient

arrested growth. As hyphai extension rate increased, both the pH at the tip and the length

of the pH gradient increased, suggesting that in fiingi hyphal extension is cntically

dependent upon the presence of an aikaline pH gradient at the tip.

In Saprolegnia fera, Bachewich and Heath (1997) found that cytoplasmic

acidi fication reduced hyp ha1 growth, aitered organelle morphology and posit ioning,

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

tissue culture dishes on solid substrate (2% w/v gellan gum, ICN Biochemicals. Cleveland,

OH) containing VogeI's minimai rnedÏum (Vogel, 1956) supplemented with 2% sucrose.

The conidial inoculum was placed near the edge of the dish which was placed in the dark

for about 5-7 hours at 28 OC or over night (14 hours) at room temperature. The culture

was then covered with a buffer solution (Levina et al., 1995) containing 10 mM KI, 1

m . CaC12, 1 mM Mg&, 10 mM PIPES (1,4 piperazïnediethanesulfonic acid), pH

adjusted to 5.8 with KOH and with the osmoiality adjusted with sucrose to 260

mosmolBcg- a balancing point, Lying between 0.125 M and 0.25 M sucrose, in which

normal extension of the hyphal apex takes place wîthout significant change in the tip

morpholog (Robertson & Rizvi, 1968). Mycelia were also grown on 3.5 x I cm

scratched strips of dialysis membrane overlyins solid substrate (2% agar w/v) containing

VogeI's minimal medium plus 2% sucrose. In this case, the conidial inoculum was placed

in the center of the Petri dish. Strips of dialysis tubing bearïng hyphae cut from the

periphery of the culture were attached to the bqttom of tissue culture dish with tape and

immediately covered with buffer solution.

2-2. Growth rates measurements

Growth rates were determined fiom the cideo monitor screen or fkom video

thermal prùits using caiiirations with a stage micrometer. Unless otherwise noted, a

CCD carnera (KP-MW Hitachi Denshi Ltd., Japan) was used to take the images. The

rneasurernent resolution was about 0.2-0.4 pm. Growth rates were calculated for hyphae

growing kee in solution within the mycelid mat or at the edge of the colony.

Mea~ements were also made immediately afler recovery h m the impalement, when the

hyphae resumed growth and und the hyphae grew out of the video screen area (typically

-14 minutes later). The slow growth rates for hyphae attached to dialysis tubing (3.4 & 0.4

pm min-' (n=6)) or gellan gum (7.1 + 4.4 pm min'' ( ~ 8 ) ) before impalement, but 2.2 5 t

prn min-' (n=6) d e r Unpalement) combineci with the fact that Unpalement of hyphae

atrached to substrate caused vacuolation and growth cessation precluded their use.

Therefore, irnpalements were performed using hyphae floating fiee in solution, (that is

orowing into the solution) which recover normal growth d e r impalement. Y

2.3- Electrophysiology

2.3.1. Micropipette fabrication and membrane potential recordings

Double-barrel micropipettes used to impaie the hyphae were constructed by

placing two borosilicate (KG-33) glas capillaries with intemal filaments (1 mm outer

diameter, 0.58 mm inner diameter, Friedrich and Dimmock Inc., Millville, NJ) within a

nichromium heating filament. The capillaries were heateâ, then twisted 360' and puiled

on a modifieci vertical puller (model P-30, Sutter Instruments hc., Novato, CA) to yield

micropipette tips with aperture of 0.05 pm, overall tip width of 0.2 pm, and a resistance

of 10-20 Mohm when b a c w e d with 3 M KCl (Lew, 1991; 1996). The pipettes were

backfilled by syringe with a KCl solution just pnor to use. in generai, the higher the

concentration of KCl in the micropipette, the Lower the resistance but the larger the

dfisional leak of KCL into the ceIl (Pumes, 198 1)- KCL at 3 M caused vacuolation of

thin hyphae growing on substrate, while 200 mM or 1 M KCl were not very efficient

because of the hi& resistance of the micropipettes tips. The best results were obtained

using larger hyphae floating fhe in solution, and 3 M KCL

In preliminary experirnents, the micropipettes tended to plug about 9 minutes

after impalement. To avoid plugging, we tried using micropipettes with a larger aperture,

(which unfortunately damaged the cells), increased KCI concentrations and Tnton X- 100

(0.1% to 5%) added to the pipette solution (Kropf 1986). Contrary to the results of

Kropf (1 986) in Achlya, Triton X-200 in the micropipette tip caused the resting potential

to depolarize to -89 mV (3M KCI + 5% Triton X-100), -86 mV (3M KCI + O S % Triton

X-100) and -1 12 mV (3M KCl + O. 1% Triton X-100) compared with the normal resting

potential of about -130 mV.

The double-barreled micropipettes were comected with a chlorided Ag wire to

two intracellular electrometers (model IE-25 1, Warner Instruments, Sarasota Springs, FI).

The impalements were c o b e d by injecting current through one micropipette and

o b s e ~ n g a voltage deflection in the other micropipette (there was no crosstalk observed

between the barrels, tested before and after each impalement). Vokage and currents were

monitored on a Tektronfx 22 1 1 digital storage oscilioscope.

The reference electrode was constnacted tiom a chloridecl Ag wire inserted inside

a glass capillaq tube (1 -5 mm inner diameter, 1.8 mm outer diameter) fiame-bent into an

L shape, fiiied with 2% agar in 3 M KCL and seaied at the straight end with a silicon-based

seaiant, leavhg the wire protmding- An adjustable holder held the reference electrode and

the L-shape tip was placed in the buffet solution that covered the mycelia, thereby

comecting the solution to the electricai circuit ground.

Hyphae growing fiee in solution were held during the impalement with a action

micropipette held by a stage-mounted holder and c o ~ e c t e d via tubing to a 60 mi syringe.

Suction on the pipette was created via the syringe, but was applied only when required

because many tirnes the mechanical support alone was sufficient to prevent the movement

of the hypha durhg impalement.

Hyphae were impaied within -26 pm of the hyphal apex with the double barre1

micropipettes. Only hyphae without cytoplasmic leakage f i e r impalements were used for

further experiments. The impalements were performed using a water immersion objective

lens (40X, NA 0.75) on a Nikon Optiphot microscope equipped with two Leitz

micromanipulators. The set up was mounted on top of a vibration isolation table and was

shielded by a Faraday cage.

Resting plasma membrane potentials were recorded for mature and young hyphae,

and germinating conidia aod for growing and nongrowing tips. The apparent resting

potentiai is the difference between the potentials measured when the microelectrode is

inside and outside the cell (the amplifier being zeroed with the pipette outside the cell)

(Purves, 1981).

2.3 -2. Voltage ciamping

Mer hyphae recovered fiom irnpalements and restarted gowth, voltage

cIamping experiments were performed using an operational amplifier configured for

voltage clamping (Lew, 1991). By using voltage clamp, one can experimentaily modiQ

the rate and direction of electrogenic ion transport through the membrane (Figure 2.1 .).

The simplest way to present this is to describe the effect of voltage clamping on the ratio

of outward (f44 and inward (ri) flux of the ion species n:

where 2 and ho are the ion concentrations inside and outçide respectively, z,, is the

valence of the ion, B =F/RT where F is the Faraday's constant, R is the rnolar gas

constant and T is the absolute temperature, Vm is the clarnped potential and Vn is the

Nernst equilibrium potential for the ion n. The flux ratio is exponentially dependent on the

clarnped potential and the direction of the net flux reverses at the Nernst potential (Weiss,

Dunng voltage clamps, the clamping current was monitored to assure that it did

not change due to pluggiog of the micropipette tip. The apical plasma membrane was

clamped in the range nom -200 mV to +50 mV. The a-e duntion of each clamp was

140 seconds (Table 2.1.). In initial arperimeats, voltage chnping was applied for each

hypha according to the foilowing protocol: -200 mV, -100 mV, +50 mV, O mV, -50 mV

for about 4 minutes at each voltage (Data not shown). In sub~e~uent experiments, the

protocol was changed to dow the hypha to grow at nad restirig potential betwan the

application of voltage clampingins In this case the membrane potential was clamped at mher

a negative (-150 mV, -100 rnV, -200 mV) or positive (+50 mV or +25 mV) d u e during

an experiment. For both protocols, growth rates were recordeci every 1 or 2 minutes. The

duration of the expriment was limaed because the hyphae grow out of the video screen

area and it was impossible to move the stage while the hypha is impaled.

Table 2.1. Summary of voltage clamp durations for the second protocol

Clamped Voltage

Duration of voltage clamp (range)

Mean @ SD) duration ofvoltage clamp

Number of experiments

CF H+ K+

Figure 2.1. Voltage clamping of membrane potentials

Voltage clamping holds the intracellular electlical potentiai at a specified value.

Clarnping the membrane at either a positive or a negative value can rnodifL the flux and

direction of electrogenic ion transport through the membrane. If the inaux or etnux o f

certain ions is an absolute requirement for hyphal growth, then changing the ionic flux

would affect (either inhibiting or stimulating) hyphal growth.

ca2+ H+ K+

A 4 4

A +ve potential inside

-ve potentiai inside v

4

t

ca2+ H+ K+

7

4

f

t v

2.4. Cakium Injection

Ca '' was injected into the hyphal tips using current injection (ionophoresis). The

injection protocol used puises of t1.7 nA current for 9 seconds foiiowed by a 3 seconds

pause for a total period of 2-3 minutes. One barrel of the double barrel micropippete was

mled with 3M KCl to record the membrane potential and the cation ejecting barre1 was

filled with different ratios of KCI and CaClz (50 mM: 25 mM; 3 M: 30mM; 50 mM:0.5

mM; 500 mM: 5 mM; O mM: 25 mM) for ionophoresis. The best results (least plugging)

were obtained with 3 M KCI: 30 mM CaClt. Growth rates were recorded as well as the

initiation of branches for about 20 minutes-

2.5. BAPTA Injection

B APTA [ 1-2 bis (ortho-arninop henoxy) ethane-N, N, N', NT- tetrapotassium acetate]

is a highly selective calcium chelating reagent (&=160 nM at pH4.0) which can be used

ro control ca2' concentrations either inside or outside of cells (Haugland, 1996). After the

hypha resumed growth, BAPTA was injected into the hypha by applying pulses of -2 nA

for 7 seconds, followed by a 4 seconds pause for duration of 8 minutes. The ionophoresis

barrel was filled with 50 mM BAPTA + 100 mM KCI while the other barre1 was filIed

with 3M KCI. During microinjection of the hypha the recording of the membrane potential

was used as an indicator of successfûl ionophoresis. The control for negative current used

to deliver BAPTA was KCI ionophoresis (data were selected Eom voltage clamping

expenments to match the conditions in BAPTA expenments).

2.6, Conventionai Fluorescence Microscopy

ca2' selective fluorescent dyes were used to wnfirm that BAPTA injection into

hyphae effectively decreased fiee [ca27i- Because of technical difnculties reported by

Knight et al. (1993) using fluorescent dyes in funW preliminary experiments were

performed to examine the e E i of injecting different fluorescent dyes on hyphai

cytology, especially dye sequestration The dye distriiution and cornpartmentdiration

were assessed subjectively. The viability of the dye-loaded hyphae was assessed by the

general appearauce of the hyphae, presence of growth and cytoplasmic streaming, and

the extent of vacuolation. Six fluorescent dyes were ionophoreticaiiy injected into hyphae:

Fluo-3 (pentaammonium salt or pentapotassium salt TEF Labs, Austin, TX or Molecular

Probes Inc., Eugene, OR, respectively), BSA-Fura 2, Calcium Green- 1 and Indo- 1

(potassium sait, TEF Labs, Austin, TX), 6-carboxy fluorescein and Lucifer yeilow

(potassium salt, Molecular Probes Inc., Eugene, OR).

The tip of one barre1 of the micropipette was filled with the dye solution (0.5 to 5

mM), then bacffilled with 3M KCl to provide electrical conductance. The injection of

dyes was computer controlled using pulses of -2 nA for 9 seconds followed by a 6 second

pause for a duration of about 6 minutes. The dyes were loaded to a level which provided

an adequate signal for visudization without pemirbing the normal activity of the hyphae.

This Ievel was judged visually. The excitation light source was a Nikon high pressure

mercury lamp (100 W) (Mode! HB-1010 MF, Nikon, lapan). For all dyes a Fluo-3 filter

block (excitation 485 nm, dichroic 505 nm, emissicn 530 nm, Omega Inc., Brattieboro,

VT) was used. The fluorescence images were observeci on an image intensifier camera

Growth rates and the level ofbranching were recorde&

2.7. Laser Scanning Confocal Fluorescence Microscopy

Intraceiiular fkee ca2+ distribution at the growing hyphal apex was examined using

confocai fluorescence rnicroscopy which allows high-resolution opticai sectionhg through

the hyphae because ody light £?om the confocal plane (0.5-0.7 pn) is coilected, while out-

of-focus light fiom the excited fluorophore is rejected (D-i'berto et al., 1994).

2.7.1. Calcium fluorescent indicators

The ca2' gradient in growing and nongrowing hyphae was measured by injecting

into hyphae two calcium indicators Fluo-3 and Fura Red and using ratio imaging. Ratioing

the fluorescence intensities measured at two different wavelengths, results in the

canceIlation of artifaçtual variations in the fluorescence signal (caused by nonuniform

indicator distribution within the hypha or variation in loading etficiency of different

hyphae) that might otherwise be interpreted as changes in ca2' concentration.

The single emission dye Fluo-3 (pentapotassium salt ce11 impermeant, &= 325

nM) is excited by visible light (maximum excitation at 506 nm, emission 525 nm)

(Haugland, 1996). Essentiaüy nonfluorescent unless bound to ca2', Fluo-3 green

fluorescence increases 40 to 100 fold upon binding ca2'. Because the spectral maxima

remain unchanged upon binding to ca2' tbis dye alone can not be used for ratiometric

rneasurements; howewer, simultaneous loading of hyphae with FIuo-3 and Fura Red,

which exbibits reciprocal change in fluorescence intensity upon bhding ca2+, were used

to obtain ratiometric measurements of intracellular ca2' (Lipp and Niggh, 1993; Read et

al., 1992).

Fura Red (& 4 4 0 nM, Haugland, 1996) is a visible dual-excitation wavelength

ratiometric dye (excitation m ~ u m 474 nm, emission 656 am). The CC free and the

ca2' bound forms of the indicator have distinct spectra, with the maxima located at

dîfïerent wavelengths (the spectra show shifts in excitation wavelength with an

isobesbectic point). The fluorescence emission of Fura Red decreases at higher ca2-.

Initially, a 1 :2 ratio of the two fluorescent calcium indicators Fluo-3 (33.33 PM)

and Fura Red (66.66 pM) was used (Lipp and Niggli, 1993); but because the signals were

Iow, especially Fura Red, the ratio was modified to 1:3 and the concentrations in the

micropipette were increased to 0.33 mM Fluo-3 and 0.99 mM Fura Red.

2 - 7 2 hkroinjection Protocol

The hyphae were Mpaled -55 Fm behind the apex using two hydraulic

micromanipulators (mode1 MO -108 and mode1 MO-203, Narishige, Japan). hpalements

were performed this far fiom the apex to avoid possible confusion between naturally

occumng ca2' gradients and anincialy elevated ca2' levels at the ïmpdement site. The

microeiectrode was not removed f?om the cell &er the microinjection. The dyes were

injected ionophoretically using an electrometer (model IE-20 1, Wamer Instrument Co.,

FL) and a duo 773 Electrometer (World Precision Instruments, FL) to apply -2.5 to -IO

nA currents for duration of 1 second to several minutes, Potdals were monitored on a

oscilioscope (model 221 1, Telaronk, ON). As ca2' ions bound by a dye molecule are not

avaiIabIe for celIular metabolism, the intraceMar dye concentrations must be sufficient to

provide adequate fluorescence signal for detection, but low enough so as not to interfere

with intraceMar physiology (Hyde, 1998; Dfiberto et al., 1994). Fluorescence intensity

was judged visualiy and by applying a histogram after laser d g 8

In BAPTA experiments, after hyphae recovered fkom the impalement and resumed

growth, the hyphal length was recorded, the hypha was ionophoretically injected with

Fluo-3 and Fura Red, the growth and fluorescence measured, then the hypha was injected

with BAPTA and the growth and fluorescence measured again. One barre1 of the

micropipette was filled with the two dyes at the tip and the other with 50 mM BAPTA +

1 00 mM KCI; both barrels were backfilled with 3M KCl. Fluorescence images and bright

field images of the hyphae (taken wÏth a TV carnera mode1 WV 1550, Panasonic) were

printed on a thermal video printer (Mode1 P40U Mitsubis6 Japan).

2.7.3. Confocal Microscopy

Ratio fluorescence imaging was performed on a BioRad MRC-600 confocal

apparatus (Bio-Rad, Mississauga, ON, Canada) attacheci to a N~kon Optiphot 2

microscope (Nikon, Japan) equipped with a krypton argon mixed gas laser and with two

filter blocks K1 (dual excitation, 488 nm and 568 nm; double dichroic 568 nm and 488 nm

reflection and 500-540 nm and 600-660 nm transmission; and emission 522 nm) and

SNARF (522 nm excitation, 600 nrn dichroic and 640 nm emission).

The FIuo-3/Fura Red mixture was excited with 10% laser intensity (neutral density

filter 1) at 488 nm because these dyes have overlapping excitation spectra. Emitted

fluorescence was detected simuitaneously with two photomultiplier tubes at 522 nm for

Fluo-3 and 640 am for Fura Red.

Dye bleaching was negligible during recording. No kinetic correction of the

individual fluorescence transients was necessary since Lipp and Niggli (1 993) found no

significant kinetic differences between the cal'-binding properties of Fura Red and Fluo-3

when measuring rapid ca2' release signals in citrate free solution.

2.7.4. Autofluorescence

The autofluorescence was deterrnined pnor to injection by imaging-im area of the - .

hyphal tip using the same settings as when the dye fluorescence was measured.

Autofluorescent sipals Eom the hypha could not be subtracted from the dye signal since

Autofluorescence

Dye fluorescence

Fura Red red fluorescence Fluo-3 green fluorescence

Figure 2.2. Relative fluorescence intensities for auto fluorescent and dyes injected hypha

recorded in the same conditions, with the same settings and without contrast

enhancement. Fluorescent intensity measurements were perfonned using a box placed in

the centrai area of the hypha.

Table 2.2. Rdative &or- btmsïties of s~lfofiuomsccnce and dye-loadcd growing

and nongr- hyphae masurrd in the sunt d region of the hypha Average

fluorescent intcnsitics for the two dyes am shown fw lpowme md nongrowing h y p h in

the le& cohimns. Autofh~orescence (as a percent) for hyphat in which both measuremenfs

were done am shown in the ri@ wiumns. In .II cases, the setthgs (phhole 2/3 open,

channe1 ga ï~8 .2 , black level=5) were tht same*

Signal

Autofluorescence 1 I F 5 Nongrowing 1

Autofluorescence

Growing

Fluorescence

Nongrowing

4.4 5 1.4

n=19

52.2 21 -6

n=13

Fluorescence

Growing

Fura Red

Fuca Red

50.0 2 32.5

n=13

Autafluorescence:Dye fluorescence

YO

the hypha is constantly elongating and changing in shape. Fortunately, the

autofluorescence detected from these hyphae is very even and much weaker than the dye

fluorescence, about 11% of Fluo-3 aad 6% of Fura Red signals (Figure 2.2 and Table

2.2.); therefore, the interference 6om autofluorescence was negligiite. Photon counting

gain and offset were set up using autofluorescent hyphae.

2.7.5. Image processing

Fluorescence images were detected and recorded in the fast photon counting mode

using a 40X water immersion objective (NA 0.75)- The image acquisition was controlIed

using COMOS software. The number of scans used to coiiect the data was selected to be

the fewest (usually 10) needed to reveal a good image.

The fluorescence images of hyphae were separated into Fluo-3 and Fura Red files

using the SOM program and were converted to Fluo-3/Fura Red ratio images by dividing

the n u 0 3 by Fura Red images pixel by pixel using the image-piocessing software Scion

image (a PC port of the popular Macintosh NM-image) running on a Compaq personal

cornputer. The image ratios are independent of the amount of dye measured, but

proportional to ion concentration (Read et al., 1992). 20 pixels wide transects along the

rnidline length of the hyphae were used to masure the fluorescence ratio intensities at the

hyphal apices. The apical end of the tpmect was positioned as close to the hyphal tip as

possible, avoiding the edges and extended back as far as possible (the limitation was the

loss of media1 section focus). The fluorescence emission ratio intemity profiles along the

transect (itensity versus distance) were then expressed as free calcium concentration

[ca2Yi in n M using an in viao caibration cuve (section 2.7.6.) and ploted vernis distance

to quanti@ the steepness and magnitude of the [Ca2+]i gradients at the tip.

2.7.6. In vitro ratiometric caltiration of Fluo-3 and Fura Red

In vitro calibration was performed ushg a calcium calibration buffer kit with

magnesium #2 (Molecuiar Probes, Eugene, Oregon, USA) that contain eleven dilutions of

calcium buffers in deionized water (resistance 17.9 Ohm) and simulate the intraceliular

magnesium pg2+ may compete with ca2' for binding to the dyes, thus reduciug the fkee

dye available for the complexation with ca23, ionic strengtb, pH and viscosity. Because

cells contain very low levels of fiee ca2+, ca2+ solutions were buffered with the calcium

chelator EGTA to precisely calibrate the ca2' dye indicators. EGTA -ethylenegiycol bis

( p aminoethy1ether)-N,N,N,N' , W tetraacetic acid- has a much greater selectivity for ca2*

oïer M~'' compared with other chelators and therefore can be used to control ca2' in the

presence of physiological concentrations of M~~ (Haughland, 1996).

The opposite [ca27;-sensitive responses of the dyes were calibrated in vitro by

rario imaging uniform layers of solution (Figure 2.3.). First an in vitro calibration of

concentration of the dye mixture injected into hyphae was performed by imaging layers of

solution containing different Fluo-3 : Fura Red concentrations in dH2O (1 pM : 3 pM, 1 O

ph.1: 30 pM, 20 pM: 60 CLU, 30 pM: 90 pM, 40 pM: 120 pM, 50 pM: 150 pM and 100

FM: 300 M. The closest to the mean fluorescence intensity found for injected hyphae

\vas at 10 ph4 Fluo-3: 30 pM Fura Red; therefore, the final total dye concentration

cover

microscope slide

solution

Figure 2.3 - Imaging unifonri layers of buffer solution

20 pl droplets of each calibration buffer solution (40 pM final concentration of

Fluo-3: Fura Red) were placed on the X intersection scratched with a diamond knife

on a glass slide and covered with a cover glass on which a colored point was marked

on the upper side- 50 pi distilled water was placed on the top of the cover glass to

assure a complete film between the coverslip and the water immersion objective. The

distance nIled with buffer solution required 2.7 full turns of the fine focus as

deterrnined by focusing on the edge of the cut and on the imer surface of the coverslip

below the colored mark Focushg first on the edge of the cutting and then up 1 -8 turns

assured that the focal plane was in the solution.

in the hyphae was estimateci at about 40 pM. The concentration of dyes, the optical

arrangement of the microscope and laser detection settÏngs were the same as for hyphal

imaging. The biack level was adjusted for &O- Images were corrested for the

background, but not for fluorescence crossover for the two dyes-

The fluorescence at the two emission waveiength of each solution was measured

and divided Fluo-3 by Fura Red. The ratio of emission intensities was plotted versus free

[ca27. Under these conditions a 100 times increase in Fluo-3 to Fura Red ratio was

obtained over the calcium concentration ranging fiom O to 39.8 p M at which point Fluo-3

fluorescence approaches saturation.

The resulting sigrnoidal in vitro calibration curve presented in Figure 2.4. was fit

using nonhnear regression to the phenomenological equation to yield the & for the

fluorescent dye indicators:

ratio= N(I + 10- )

where A is the maximum ratio, B is the apparent dissociation constant (Log &) of the two

fluorescent indicator dyes, and pCa is -log[ca2'].

Data £tom al1 experiments were analyzed using SYSTAT statisticai package

software (SYSTAT Inc., Evanston, IL, USA) or were transferred to the plotting prograrn

SYGRAPH.

-7 -6

log 1CaZl

Figure 2.4. In vitro calibration curve of Fluo-3 / Fura Red ratio of emission intensities

(0) versus [ca27 fkee.

Solution containing fiee ca2*(0 to 39.8 FM), 40 FM final concentration of dyes

MOPS buffered (pH 7.2), 1 rnM ~ g ~ ' , high K' (1 00 mM KCl) were placed on a slide and

imaged on the confocai microscope. The same instrument setthgs were used as those for

hyp hae imaging experiments. The emission fluorescent Uitensities of Fluo-3 (A) and Fura

Red (O) in increasing concentrations of fiee ca2* are also shown divided by 10. The

estimated I(d for the Fluo-3 and Fura Red ratio was 930 nM-

3. RESULTS

3.1. Growth rate mcrsurements

Mer impalements, the hyphe usuaiiy stopped growing and sometimes bdged at

the tip, then recommenced growth within 7-8 minutes (or within 3 4 minutes when using

larger hyphae). hiring recovery, the new tip was formed at the very apex of the ceil or

emerged slightly to the side of the initial tip. If tip swehg occured, the swoiien region

was still visible when hypha had recoverd and had resumed growth. Evenhially, the

recovered hypha showed a normal morphology.

The growth rate for impaied hyphae was 1.1 _+ 1 -3 p d m i n ( ~ 5 2 ) immediately

after growth restarted. The growth rate continued to increase until the hyphae grew out of

the video screen area ( w i h -14 minutes). By then, the growth rate for impaled hyphae

was 7.0 2 3.4 pmlmin (n=52), significantly fower than for unimpaied hyphae at the edge

of the colony (1 3.1 t 5.4 pmhin, n=14) (t-test, p<10-3) (Table 3.1).

3.2. Branch and main hyphd tip growth rata are correlateci

Growth rates for the main hyphal tip and branches behind the main apex

measured during voltage clarnping experiments were compared and found to be strongly

conelated (Figure 3.1 .).

Table 3.1. Cornparison of growth rates for impded and unimpaîed Newoqora aarsa

hyphae. For the irnpaled hyphae the initiai growth rates w m rnemmed immediately after

recovery nom impalement when the hypha resumed growth The nnal growth rates is the

last growth rate recordad, typically -14 minutes Mer, when the hypha grew out of the

vide0 screen area. Initial and finai growth rates are si@candy lower than for unimpaled

hyphae (t-test, p < l ~ - ~ ) . Hyphal growth rates within the colony are not sîatisticaîiy

different f?om those taken at the edge of the colony (t-tee p=O.OSI). Data are shown as

mean + SD, (sample size). Data for impaled hyphae were taken fiom the voltage

clamping experiments.

Unimpaled hyphae Impaled hyphae

Wit hin the colony At the colony edge Initial growth rate Final growth rate

O 2 4 6 8 10 12 14 16 18

Main Hyphal Tip Growth Rate (pm min-')

Figure 3.1. Relatiooship between branch tip and main apex growth r a t s showhg a strong

correlation (linear regression: branch growth rate= 0.79 1 +O. 59 1 *main 2 =0.484, pc 1 O'*,

n=103). The central iine is the best fit fiom regression aaalysis, the outer lines are 95%

confidence intervals.

3.3. Plasma membrane potentirl rad growth

The plasma membrane potentid can be generated by a combination of passive

ionic diffusion, electrogenïc CO-transport and ion pumping. In Nèurosporn, an ATP-

dependent electrogenic pump contributes significantly to the negative inside electrical

potentid across the plasma membrane (Slayman, 1965). H'/gIucose and other H'

cotransporters as w e l as ion channels will also affect the potential-

The potentials of various types of hyphae were measwed to determine if there

were any dflerences that could be due to di£Ferences in ion transport (Table 3.2). Plasma

membrane potentials for the mature hyphae, that is, large hyphae located within the

myceliai mat, are more negative than for the growing hyphal apices at the edge of the

hyphal colony (t-test, p=0.009) which are signïfïcantly more negative than potentials at

apices of young ged ings (t-test, p<10-3) and conidial g e m tubes (t-test, p < l ~ J ) . The

plasma membrane potentials are significantly less negative for non-growing tips in

solution compared to growing ones (t-test, p=0.007). However there was no significant

correlation between potential and growth rate O;igure 3.2.).

3.4. Voltage clamping

To diectly explore the effect of the voltage on the hyphal elongation rates,

the apical plasma membrane was clamped in the range fiom -200 mV to +5O mV for

durations of about 140 seconds for each voltage clampkg in different experiments

(Table 2.1). Holding the intraceliular electrical potential at a specified vaiue modifies the

Table 3.2. Plasma membrane potentd (mV) rccorded fkom différent stages of hypbai

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.

" -600 -400 -200 O 200 400 600 800 1000 1200 1400 1600

Time (seconds)

Figure 3.7. Long term effects of BAPTA injection in Neurospora crassa hyphe.

The muiti bud phenotype first began to appear 20-25 minutes after BAPTA ionophoresis.

A and B show two examples 48 and 42 minutes d e r BAPTA ionophoresis, respectively.

C is redrawn from Prokish et al. (1997) to show the similar morphological phenotype

after treatment with a calcineurin inhibitor. D shows a control phenotype, d e r KCl

ionophoresis. Bar= 20 Pm.

Figure 3.8. Control for BAPTA microinjection: KCl ionophoresis into growing hyphae.

Upper panel. Hyphal length versus the. The inaease in le@ was not affécted b y KCl

ionophoresis (begun at time O). Lower panel Growth rates versus tirne. Continuai

increase in growth rate after recovery fkom impalement was unaffected by KCI

ionophoresis. Both individual experhents ( n 4 l ; thin lines) and mean 2 standard error

compiled fiom consecutive 200 second time interval (circles; n=lI to 35) are shown.

The data were selected fiom voltage clamp experiments based on two criteria: relatively

long growth periods afker ionophoresis and long duration of ionophoresis (average, 340

smnds; range: 60-90 seconds) to match the conditions in BAPTA microinjection

experiments (ionophoresis for 420- 480 seconds, currents of about -2 nA). K'

ionophoresis (3.8 +/- 2.2 a n=23; range: 0.9- 8.6 aA) and Cl' ionophoresis (-8.2 +/-5.2

nA; n=11 range: -3 to -21 nA). Experiments include voltage clamps from-200 mV to

+SO mV.

gradually became sequestered, but in the short terni (20 minutes), the technique definitely

permits the study of cytoplasmic @+ in living hyphae (data not shown).

3 -8. Laser scanning fluorescence microrcopy

3.8.1. Dye distribution

Impalement often caused temporary cessation of hyphal growth, but the hyphae

usuaiiy recovered within 2 to 5 minutes. Mer growth resumed, the hyphae were injected

with Fluo-3 and Fura Red and continueci to grow at a slower rate (6.3 2 4.3 ) r d min,

n=28) wmpared to unimpaled hyphae (t test, p=û.OOL), but withio the normal impaied

hyphae range. Hyphae which did not resume growth (non-growing hyphae) were also

injected with the dyes-

Following dye injection, the dye distribution remained diffuse (Figure 3.9.) and

the fluorescence could be monitored for 10-20 minutes for the rnajority o f hyphae. Both

dyes had a similar intracellula. distribution, al1 subcellular stnictural elements

accumulating or excluding the two indicators to the same degree (147 out of 150

experiments). Rarely, fine punctate, partïculate or reticulate dye sequestration variable in

size and shape was o b s e d . The dye sequestration was a dynamic process, that is the

fluorescent pattern changeci over time (Figure 3.1 0.).

Figure 3.9. Homogenous cytoplasrnic distribution of Fluo-3 and Fura Red fluorescent

dyes within Neurospora massu hyphe.

Left panels. Series of confocal images fiom a siagie growing hypha injected with the

fluorescent dyes Fluo-3 (left) and Fura Red (right). For 26 minutes after dye

io no p horesis, uniforni homogenous fluorescence was O bserved using laser scanning

confocal microscopy. Thus the dyes were located in the cytoplasm and did not partition

into organeiles; and therefore, "report" cytosolic [CaZ7. Central panel. Correspondhg

bright field images of the same h y p k Bar = 10 pm Time of image acqisition represent

minutes fiom the injection of dyes A O minutes, B 9.2 minutes, C 26 minutes. Right

panel. Ratio images of the confocal images in the left panel.

Time of image acquisition

2.4 minutes

4.8 minutes

Figure 3.10. Dye sequestration in Neurospora massa hyphae imaged by confocai

microscopy. A, B, C, D are a he-series of confocal Mages of a hypha loaded with

Fluo-3 and Fura Red. T h e is the minutes after dye ionophoresis. B p l O pm.

3.8 -2. Calcium gradient in growing and nongrowing hyphae

Fluorescence ratiometnc d y s i s of dye-loaded growing hyphae revealed a tip

high [ca2f1i gradient which extends over the e s t 10 p fiom the tip that was less

pronounceci in non-growing hyphae. Figure 3.11. displays an experimental example for a

growing hypha, compiled data are shown in Figure 3.12. Overall, there were variation in

the magnitude and extent of the cdcnim gradient for individuai hyphae which iacluded

growing hyphae without apically elevated ca2-. Ciear tip-high ca2+ gradients were

observed in -76 % (n=24) of growing hyphae. The intracellular fiee ca2+ concentration

was 25 1 2 14 nM (mean 5 SD, d o ) 0-5 pn h m the apex and 193 2 7 (n=39) n M

20- 25 prn behind the apex For nongrowing hyphae [ca27 was 203 _t 8 n M (n=40) 0-5

pm fiom the apex and 170 _+ 16 n M (n=39) 20-25 behind the apex.

3.8 -3. B APTA effect on ca2' gradients

These experiments were technically very dinicult to perform because of the multiple

ionophoretic treatments that had to be done within a brief period of time. Some

experiments (9 out of 3 1) were discarded due to l o s of focus. Other experiments were

discarded due to poor media1 sections îhrough the apex, dye overloading (resulting in

saturat ing signais), underloading, and rare1 y, dye sequestratio~~ In the remainder, B APT A

injection effectively dissipates the [ca2qi gradient and consistently caused a decline in

f?ee calcium level (21 out of 22 experiments) that could be visualised immediately &et-

BAPTA injection throughout most of the hypha; the effect was more marked at the apex,

Figure 3.1 1. Example of a calcium distribution in a growiag Neurosporu crassa hypha

ionophoreticall y microinjected with Fluo-3 and Fura Red.

Confocal microscopy fluorescence images of a growing hypha (A Fura Red; B.

Fluo-3) are shown with a bright field image of the same hypha (C). D. FIuo-3: Fura Red

ratiometric pseudocolor image of [ca2' Ii indicating the presence of a typical tip high

calcium gradient for a growiog hypha E. Comsponding transect profile of the ratio

values dong the length of the hypha, converted to [ca2C]. Bars= 10 Pm.

10 20 3 0

Distance from the tip (pm)

Distance from the tip (pm)

Figure 3.12. Calcium distribution at growing and nongrowing hyphal apices.

The mean fiee calcium concentrations are shown versus the distance from the tip

for growing (circles, n=24) and nongrowing (squares, n=24) hyphae. High [ca27i in the

tip region gives rise to a tip to base gradient which extends over a relatively short region

(10 p) to a basai level. Growing hyphae had a pronounced tip bigh [ca2'] compared to

nongrowiag hyphae and higher fîee calcium levels throughout the first 25 p. The thick

lines are exponential best fits to the averaged data Thin h e s are hear best fits 2 99%

confidence intervals, demonstrating the highly statistically significant difference between

the ca2' gradients in growing and nongrowing hyphae.

reducing the magnitude of the gradient. An example is shown in Figure 3.13. The

dissipation of the gradient occurs simultaneously with the inhibition of growth as

illustrateci in the bright field images. Three types of growth responses were found:

frequently, in 26 out of 3 1 experiments the hyphae stopped growing, 4 hyphae

restarted growth and 5 out of 3 1 hyphae did not stop pwing. In 2 out of 2 1 experiments

the tip high ca2+ gradient was dissipateci to the basal levd (25 pm behind the apex); in 5

out of 21 to an intermediate level between the tip and basal level and in 9 out of 21 at a

level lower than the basal one. For hyphae that initiaily stopped growth after BAPTA

ionophoresis, than restarted growth, in 3 of the 4 cases the level of calcium increased

when the hyphae restarted growth after the initial reduction caused by BAPTA injection.

500

425

" 350 f - 1 275 -

" 9 9 zoo

12s

50 O 10 20 30 -0 10 20 30

Dirtaica fmm the tip ()rd a Diiuoca fmm th. tig km)

B U T A injection d

Time (minutes)

Figure 3.13. Changes in the spatial distribution and the magnÏtude of [ca2'ji gradient

after BAPTA injection.

A Fluorescence ratiometnc image and quaotitation of hypha injecteci with Fluo-3

and Fura Red 2.5 minutes before starting BAPTA injection shows regions of elevated

caZC Iocalized at the hyphal apex which are absent 1 minute &er ionophoretic injection

of BAPTA (E3). The disappearance o f the tip gradient was concomitant with growth

cessation within 2 minutes (lower panel).

4. DISCUSSION

4.1. Assessment of growth mes after impalement

Al1 the techniques used in these expenments were based on the impalement of the

hyphae, which proved to be a very suitable experimental approach in Neuruspora crassa

The hyphae resume growth after the irnpalemeat, permitting a varïety of precise readily

monitored experimentai changes to the intracellular medium in growuig hyphae. The

growth rates for impaled hyphe after r e c o v e ~ g îkom impalement are lower than for

unimpaled hyphae; however, they continue to increase towards those for unimpaled ones.

There were two dEculties with waiting for a retum to the initial growth rate. One was

the inability to keep the tip in the field of view f i e r it grew out of the screen The other

was the ever-increasing distance between the growing tip and the micropipette tip. Better

results were obtained with bigger hyphae which proved to recover faster and eventually

grew at a higher rate-

The growth rates for apex were correlated with those of the subapical branches,

suggesting a synchronised regulation of tip growth. The hyphal unit, rather than the apex

alone affects growth rate, supported by an early observation that hyphal growth rate is

proportional to the length of the hypha (Smith, 1924).

4.2. Plasma membrane potentials

The plasma membrane potential of mature hyphae was -30 mV more negative

than at the tips of growing hyphae. Popatova et al. (1988) reported similar values of

membrane potentiai in the apical ceiis (-120 to -145 mV) which were aiways depolarized

10-30 mV cornpared to the hypha 200-500 p m proximal nom the apex. In Achlya the

membrane potential near the tip was at least 10 mV more positive than the potential

recorded 500 pm back (Kropf et ai., 1984). The depolarized apical potentiai may be due

to the presence of H'lglucose cotransport at the apex (Slayman and Slayman, 1974)

andor H'K' (Blatt and Slaymaq 1987; Rdnguez Navarro et ai., 1986), and

H'/phosphate cotransport (Versaw and Metzenberg, 1995) consistent with the normal

apical location of H' influx. It may also be due to lower W A T P ~ S ~ activity in the apical

region of the hypha (Figure 4.1). The H ' A T P ~ s ~ is an integral membrane protein (Auer et

al., 1998) which generates -80% of membrane potential in N m s p o r a (Slayman, 1965;

Slayman et al., 1973). The ATP- dependent ion pump extrudes H' across the plasma

membrane at a rate of 5-20 pA/cm2, consuming 30 % of cellular ATP turnover (Slayman

et al., 1973). Its activity would compete with the metabolic needs of biosynthesis and

intraceliular movement required for tip growth-

Using immunolocalization, Obermeyer et al., (1992) found that w-~TPase is

abundant in the plasma membrane of poiien grains, but is absent or sparsely distributed in

the plasma membrane of poiien tubes. However, in Neurospora, Degousee et al.

(unpublished) found H'ATP~S~ to be Localized at the hyphai apex

H* Glucose H+ caZ' H Arnim acid

Figure 4.1 . Summary of the main electrogenic transporters potentiall y involved in

tip growth regdation.

UnWre a previous report that non-growing apices were more hyperpolarïzed than

growing (-1 15 t 45 mV (FI 7) compared to -64 f: 56 mV (II*), p=0.02) (Lewt 1 WS), 1

found that the plasma membrane potentiai for nongrowhg tips (-109 f: 49 mV, n-1) was

significantly less negative than for growing ones (-132 2 23 mV, n=107) p=û.007. The

cause of this difference may be the larger sample size. It is unlikely to be the distance

between the apex and the impalement site, because I found no relationship between the

potentiai and the site of impalement within 60 fiom the tip.

4.3. Voltage clamping

This work extends the previous observations of Levina et al. (1995) who clamped

the membrane potential at the hyphal apex in the range fiom -300 mV to +L 50 mV and

found that the hyphal elongation rates were unaffected. However, these previous

experiments used very brief (30 to 120, mostly 30 seconds) voltage clamps, and a much

longer voltage clamping might be required to affect the growth rate; therefore I extended

the duration of the clamp in the experiments reported here.

Voltage clamping of the apical plasma membrane in the range fiom -250 to +50

mV, for an average duration of 240 seconds will severely affect voltage dependent ion

£luxes. However, growth was unaf5ected indicatiag that electrogenic ion transport across

the plasma membrane at the apex is not essential for the maintenance of tip growth

In an initial set of expenments, the voltage clamping protocol did not include

intervals beîween the clamps (which allows a cornparison between 'normal' and voltage

clamp growth rates), making possible to clamp for longer duration (average 240 seconds)

and allow a cornparison among gcowth rates at dinecent positive and negaîive voltage.

Although the growth rates were slower, they were Ulliiffected by voltage clamping fkom

-250 to + 50 rnV. In the second protocol, 1 applied either a positive or a negative clamp

before and after letting the hypha grow at its normal membrane potential, but this

protocol limiteci the duration of voltage clamping to -120 seconds. However, the results

of both protomls and the results of Levina et al. (1995) are in agreement: voltage clamp,

even for long duration is without effect on growth rates. This approach to m o d e the

activity of channeis and porters is more appropriate than using inhibitors or blockers

whose effect on tramporters in Neuropra is poorly cbaracterized, o r only assumed.

4.4. Calcium injection

In order to confirm a ca2' role in tip induction 1 injected ca2' into hyphae.

Increasing cytoplasmic [ca27 induced singie or multiple branches near the injection site.

Under normal conditions, the first branch occws 52 2 30 p u fiom the tip, the second at

17 1 2 127 yrn (Levina et al., 1995). Branches occumng aRer ca2' ionophoresis are closer

to the tip (-14 Pm fiom the tip) and therefore are considered induced. Indirect evidence

that caZ' entry induces apical branches was obtained using the ca2' ionophore A23 187,

but ca2' dependence was not clearly demonstrated (Reissing and Kimey, 1985).

üV irradiation of growing hyphae of SaproIegnia f e r a increases cytoplasmic

[ca2*] followed by the formation of one or more branches within about 4 minutes toward

the subapical side of the irradiation site (Grinberg and Heath, 1997). It is not known if

both phenomenon are unrelated responses to UV damage. In Sqrolegnia a gradient of

ca2' forms as the bmch develops but not prior to bud appearance indicating either an

unknown initiator or s u b d e t d l e [ca29 concentratioas are needed to initiate the tip,

much less than those required for continueci tip extension (Hyde and Heath, 1997)- Either

an unknown initiator or undetectable ca2' elevation could induce the formation of radial

arrays of F-actin which does occur during branch initiation (Bachewich and Heath,

1997).

The direct elevation of fiee calcium level ushg microinjection probably creates

regions with conditions favorable for branch initiation, especiaily the activation of branch

initiation factors, and the accumulation of the precursors for membrane and ceIl wall

synthesis. The accumulation of vesicles may cause the formation of a Spitzenkorper. caZ+

may also reorient the cytoskeleton causing re-arrangement and local expansion of

cytoplasm, membrane and cell wd. The effect is Wrely to be relatively locaiized because

ca2' has a low mobility in the cytoplasm (JaSe et al., 1975). Diffusion of ca2' in

cytoplasm is much slower than in fiee solution probably due to the activity of ca2'-

binding proteins, ca2' sequestration, and the long diffusion. pathway caused by the

cytoskeletal network.

4.5. Imaging calcium

1 used ratio fluorescence imaging on a confocai microscope to determine the

subcellular localization and dynamic changes of the cytoplasmic fiee ca2' and to confirm

the effect of BAPTA injection on the cytoplasmic fkee ca2+ gradient. By ionophoresing

the fluorescent dyes Fluo-3 and Fun Red into the hyphae we were able to use a ratio

imaging technique and avoid the drawbacks of non-ratiometnc single dye quatltitation

PrevÎous reports examinhg Caf' in Neurospora crassa used CTC (Dicker and

Turian, 1990; Prokish et al., 1997; Schmid and Harold, 1988) which is sensitive only to

high fiee [ca27, or image-ratioing of acid loaded ca2' sensitive Fluo-3 and ca2-

insensàive, H+ sensitive SNARF-I dyes (Levina et al., 1995). The present method of

ionophoresis gave much better loading efEciencies, providing a good level of

fluorescence versus autoffuorescence, and both dyes are caZC specific.

After ionophoresis, the dye distribution remains difise and couid be imaged

accurately for -20 minutes. In contrast to a previous report (Knight et al., 1993) we did

not have serious problems associated witb dye sequestration Few hyphae injected with

Fluo-3 and Fura Red showed punctate, particdate or reticulate fluorescence ap pearance

probably due to sequestration into some organellar system(s) as a response to cell

damage. We speculate that excessively high dye probe concentration may trigger a toxic

response by the cell, resulting in sequestration. Indeed, 1 found-it to be relatively easy to

overload the cell using ionophoresis, a technique that Knight et al. (1993) also used.

Sequestration could involve an anion transport mechanism that may serve to detoxie the

fungal cytoplasrn by removing naturally occurring unwanted anions (Cole et al, 1 997). I t

may reduce the cytosolic dye concentration to levels which prevent precise [ca2-]i

measurement. Furthermore, dye fluorescence fiom organelles can contùse measurements

of cytoplasrnic [ca27i.

In addition to the absence of sequestration, both dyes also had a similar

distribution; that is, al1 subcellular structural elements accumulated or excluded the two

indicators to the same degree. It was extremely rare to observe difEerent distribution of

the dyes, and only a long time after injection of dyes hto the hyphe. As long as the two

indicators cudîstributed uniformly the differences or changes in the local concentrations

of the dyes mixtures will have no influence on the calculateci intraceilular ca2'

concentration-

4.6. ca2' gradient

Fluorescence ratiomeuic analy sis of dye-loaded gro wing hyp hae reveals a tip

high [ca2'-Ji gradient in 76% of the analyzed hypha which extends over a relatively short

region within the first -25 f b m the tip to a uniform basal level- The calcium

concentration was estimateci at 25 1 2 14 n M 0-5 pm fiom the tip of growing and 203 2 8

n M for nongrowing hypha; and 193 2 7 for growing and 170 t 16 for nongrowing hypha

20-25 pm behind the tip. Thus, the growing hyp hae had a pronounced tip high ca2'

gadient compared to nongrowing hyphae and higher fiee calcium levels throughout the

first 25 W. The values reported are somewhat hi-er than those rneasured with ca2'

selective microelectrodes -90 nM (Miller et al., 1990) in mature hyphae within the

myceIial colony. When Fluo-3 was ratioed against SNARF-1, Levina et al- (1995)

reported a ca2+ peak - 3 pm b e h d the tip (tmnsects were not used, Limiting resolution).

The low level of fluorescence relative to autofluorescence precluded the estimation of

[ca2-] 25 pm behind the tip because the concentration of calcium in this region was

below the level of accurate measuremexit. However, the dope of the gradient was

established at about 10 fold. The gradient was only present in growiog hyphae indicating

an obligatory role in tip growth Wevina et al., 1995).

The presence of a slight ca2' gradient in noagrowing hyphae compareci with the

results of Levina et aL(1995) may be explained by the difference between the definitions

of nongrowing hyphae in these two experirnents, Hyde and Heath, (1997) define

nongrowing hyphae of Saproiegnnia fera as ones which transiently ceased growth for

unidentined reasons and typicaily occurred in colonies. I âid not find Nëurospora crmm

hyphae which stopped growing *out reason and then restarted growth (this does not

mean that the phenornenon does not exïst). In our case, nongrowing means a hypha

which normally grew before the impalement and did not resume growth aftmards. Such

hyphae may maintain a part of the tip high ca2+gradient present when it was growing. In

the other cases of hyphae which were not growing (Levina et al., 1995: Hyde and Heath,

1997) one can not be sure when these hyphae recovered fkom dye loading or fiom the

cutting of the dialysisis tubuig at the edge of the colony which occurred 1 hour before,

and when they stopped growing, giving time for the hypha to. lose the gradient present

while they were growing.

There is a large variance among the calcium concentration reported for tip-

growing organisms due to different techniques and dyes used, not to mention the variety

of organisms, but al1 report values in the nM range (Table 4.1).

Table 4.1. Calcium concentration in d i n i tip growing O-

Tip and Organism I techniques

0-5 pm fiom

the tip

251 +14 n M

Ratio ~h10-3/F&a Present data

Red ionophoresis fkom the tip

203 5 8 n M

925 15 n M seledive

microelectrode

Ratio Fhio-3

SNARF-1 acid

loaded

Miller et al,

1990

Peak at 3 pm

70 nM

ten fold lower Levina et al.,

1995

Garrill et ai,

1993

At the

extreme apex

76 nM

Acid loaded

RatioFIuo-3/

SNARF-1 acid

loaded

Hyde and

Heath, 1997

450 + 30 DM

extreme apex

ionophoresis Fura Fucars I Brownlee and

Pulsford,

1998

Lycopericon and

ionophoresis Brasda root hair

Clarkson et

al,, 1998

Felle and

Fura-2 dcxtran

pressure injection

ionophoresis

at the

extreme tip

320 n M the

highest

nrst 10-20 p

to îhe tip

170 n M

pressure injection

750-3000 n M

pulses at the

extreme apex

200 n M 2.5

p behind the

tip

Furaldextran

pressure injection

tubes

Cilium

long~uonrm,

Nicotiana

si~vesfnsfns.

Traâèscantia

virgrhicma pollen

tubes

Pierson et al,

1994

Malho et al.,

1994

Miller et aL,

1992

Pierson et al.,

1996

Wymer et al.,

1997

4.7. BAPTA dissipate the calcium gradient

Once the [ca27 distribution was resolved, the next step was to demonstrate the

effect of BAPTA on the ca2+ gradient and tip growtb. The reîationship between the tip

high intracellular gradient and the process of tip elongation was established through

microinjection of BAPTA

Dissipation of the [Ch2+& gradient can be achieved experimentally with BAPTA

injection into hyphae, under which conditions the hyphae stop growing or, rarely, grow

at lower growth rates. A reduction in [ca27i occurred and could be visualized right after

BAPTA injection throughout most of the hypha, but the eEect was more m k e d at the

apex, reducing the magnitude of the gradient. Rapid dissipation of the [cazf1 gradient by

BAPTA injection hto hyphae ocairred simultaneously with the inhibition of growth.

Therefore, BAPTA inhibition of growth is probably due to the disniption of ca2' role in

directing polarized growth. In hyphae which resumed growth (13% of the hyphae

inhibited by B APTA) a caZi gradient was normally reestablished .(75% of the hyp hae).

BAPTA was proposed (Speksnijder et al., 1989) to dissipate the tip high ca2'

gradient by "shuttle buffering" of Ca '+(Figure 4.2). If the buffer has a caZf dissociation

constant between the high and low ca2- concentrations of the gradient, then it will

preferentially bind free ca2+ in regions of high [ca27 and quickly dif£Ùse to regions of

low [ca2C] where the ion will be relwed, generating a eee buffer that is able to cliffuse

back to high [ca2c] regions and repeat the cycle. The dissociation constant of BAPTA

(- 160 nM) is in the required range for effective "shuttle buffering".

Growing %

Ionophorctic injection of the calcium chelator, BAPTA, dissipates the t i p high calcium gradient,

BAPTA - BAPTA

.a2++

BARA- c a 2 + BAPTA- Ca

Figure 4.2. Shuttle dmsion mechanism

Similar BAPTA effects are reporteci for pollen tubes and root hairs. Pierson et al

(1 994) injected BAPTA buffers in Lilium pollen tubes. BAPTA reversibly inhibited

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.

6. REFERENCES

AUaway W., M o r d AE., Heath IB. and Hardham A X (1997) Vacuolar

reticulurn in oomycete hyphai tips: An additional component of the ca2+ regdatory

system. Fungal Genet. Biol. 22: 209-220

Allen E.D., Aiuto R and Sussman A S . (1980) E f f i of cytochalasins on

Newoqora crassu L Growth and ultrastnictwee Protoplasma 102: 63-75

Auer M., Scarborough G-A and Kuhlbrandt W. (1998) Tiiree-dimensional map of

the plasma membrane H'ATPase in the open configuration Nature 392: 840-843

Bachewich C. and Heath I.B. (1997) Radial F-actin arrays precede new hypha

fo m a t ion in Saprolgnïa: irnp lications for establishing polar growth and regulating tip

morphogenesis. J. CeU Sci. 1 1 1 : 2005-20 16

Bachewich C.L. and Heath I.B. (1997) The cytoplasmic pH influences hyphal tip

growt h and cytoskeieton-related organization. Fungal Genet. Biol. 2 1 : 76-9 1

Barja F., Nguyen Thi B.-N. and Turian G. (1991) Loçalization of a d n and

characterization of its isoforms in the hyphae of Neurospora crassa. FEMS Microbiol.

Lett. 77: 19-24

Baja F., Chappuis, M.L. and Turian G. (1993) Differential effects of

anticytoskeletal compounds on the localization and chernical patterns of actin in

germinating conidia of Neurospora crussa. FEMS Microbiol. Le* 107: 26 1-266

Bibikova T.N., Zhigilei A, and Gilroy S. (1997) R w t hair growth in Arubidopsis

thaliana is directed by calcium and endogenous polarity- Planta 203: 495-505

Blatt M-R and Slayman CL- (1987) Role of active potassium transport in the

regulation of pH by nonanimal cells. Proc. NatL Acad. Sci USA 84: 2737-2741

Brownlee C. and Pulsford AL. (1988) ViJualisation of cytoplasmic ca2' gradient

in Fums serratus rhizoids: correlation with ceIl structure and polarity. 1. Celi Sci- 91:

249-256

Bruno KS-, Aramayo R, Minke P.F., Metzenberg RL, and Plamann M, (1 996)

Loss of growth polarity and mis1ocalization of septa in Neuropra mutant altered in the

regulatory subunit of CAMP-dependent protein kinase. Ernbo J. 15: 5772-5782

Capeiii N., Baja F., Van Tuinen D., Momat J., Turian G. and Ortega Perez R

(1997) Purifkation of a 47-Kda calmoduh-binding polypeptide as an actin-binding

protein fiom Neurospora c r m m FEMS Microbiol. Lett. 14: 21 5-220.

Clarkson D.T., Brodee, C., Ayling, S-M. (1988) Cytoplasmic calcium

rneasurements in intact higher plant ceils: results from fluorescence ratio imaging of

Fura-2. J. Ce11 Sci, 9 1 : 71-80

Cohen P. and Klee C.B. (1988) Calmodulin: Molecular Aspects of cellular

Regulation.vol. 5 . Elsevier, Amsterdam

Cole L., Hyde G. J. and Asford AE. (1 997) Uptake and compartmentalisation of

fluorescent probes by Pisolifhus tinctorius hyphae: evidence for an anion transport

mechanism at the tonoplast but not for fluid-phase endocytosis. Protoplasma 199: 18-29

Collinge AJ. and Trinci A P J . (1974) Hyphal tips of wild-type and spreading

colonial mutants of Narrospora massa- Arcb Microbiol. 99: 353-368

Cornelius G. and Nakashima K (1987) Vacuoles play a decisive role in calcium

homeostasis in Neurospoa crassa- J. Gee Microbiol. 133 : 2341 -2347

Corzo A and Sanders D. (1992) Inhibition of Ca2' uptake in Neurcspora cr-

by La3+: a mechanistic study. J. Gen MigobioL 138: 1791-1795

Degousee N. & al (unpublished)

De Silva L-R, Youatt J., Gooday G.W. and Gow N-AR (1992) Inwardly diuected

ionic currents of AZZomyces macrogymrs and other water moulds indicate sites of proton-

driven nutrient transport but are incidental to tip g r o d Mycol. Res- 96: 925-93 1

Dicker J.W. and Turian G- (1990) Calcium deficiencies and apical branching in

wiid type and 'fiost' and 'spray' morphological mutants of Neurospora cr-. J. Gen.

Microbiol, 136: 1413-1420

Dilibreto P A , Wang XF. and Herman B. (1994) Confocai imaging of ca2+ in

cells. Meth. in Ce11 Biol, 40: 243-262

Feijo J.A., Sainhas J., Hackett G. R, Kunkel J.G. and Hepler P.IC (1999)

Growing pollen tubes poses a constitutive alkaline band in the clear zone and a growth-

dependent acidic tip. J.Cell Biol. 144: 483-496

Felle H.H. and Hepler P.K. (1997) The cytosolic ca2+ concentration gradient of

Sinapis aCba root hairs as reveaied by ca2+ -selective rnicroelectrode tests and Fura-

Dextran Ratio Imaging. Plant Physiol. 114: 3945

Gao Z.H. and Zhong G. (1999) Calcineurin B and calmodulin-binding identified

with phage-displayed peptide libraries. Gene 228: 5 1-59

Garriil A-, Lew RR and Heath 1. B. (1992) Stretch aaivated ca2+ and ca2'-

activated K+ charnels in the hyphal tip plasma membrane of the oornycete SaproIegna

fermc. J. Cell Sci. 10 1 : 721 -730

G a r d , A, Jadwn S.L., Lew RR and Heath *.B. (1993) Ion charnel activif~ and

tip growth, tip-locatized -ch-activateci channels generate an essential Ci2+ gradient Ui

the oomycete SiqproIegnia fer= Eur- J- Ce1 Biol, 60: 3 58-365

Gooday G.W. (1983) The hyphal tip. In Fungal diEerentiation, A contemporary

synthesis. J.E. Smith editor pp. 3 15-356, Marcel Dekker, New York

Gow N-AR (1984) Traashyphal e1ectrica.i currents in fiingi. J. Gen. Microbiol.

130: 33 13-33 18

Gow N.AR, Kropf D.L., Harold FM. (1984) Growing hyphae of Achlya

bisemalis generate a longitudinal pH gradient in the surrounding medium. J. Gen,

Microbiol. 130: 2967-2974

Gow N.AR (1994) Growth and guidance of the fiingal hypha. Microbiol. 140:

3 193-3205

Grifin, D.H. (198 1) Fungal Physiology. John Wiley & Sons, Inc., New York

Grinberg A and Heath I.B. (1997) Direct evidence for ca2+ regdation of hyphal

branch induction- Fungal Genet. Biol. 22: 127-1 39

Gupta G.D. and k a t h I.B. (1997) Actin disruption by latrunculin B causes

turgor-related changes in tip growth of SqroIegnia f e r a hyp hae. Fungal Genet. Biol. 2 1,

64-75

Halpain S., Hipolito A and Saffer L. (1998) Calcineurin destabilizes F actin.

Regulation of F-actin stability in dendritic spines by glutamate recepton and calcineuria.

J. Neurosci. 18, 9835-9844

Harold RL. and Harold F.M. (1986) Ionophores and cytochalasins modulate

branc hing in AchCya bisexuuiis. J. Gen. Microbiol, 1 3 3 : 2 13 -2 19

Haugland RP. (1996) EEaadbook of Fluorescent Robes and Research Chemicals,

M o l d a r Probes Inc. Eugene, OR USA

Heath LB. and Kaminskyj S-G-W. (1989) The organization of tip-growth-related

organeiies and microtubules reveaied by quantitative adysis of the fieeze-substituted

oomycete hyphae. J. Cell Sci- 93: 41-52

Heath 1-B. (1987) Preservation of a labile codcai array of actin filaments in

growing hyphal tips of fùngus mroiegnia ferm Eur. I. CeN. Biol. 44: 10-16

Heath I.B. (1990) The roles of actin in tip growth of fùngi- Int- Rev. Cytol. 123:

95-127

Heath I.B. (1994) The cytoskeleton In: Growing Fungus. Gow N-AR and Gadd.

G.M. (eds) Chaprnan and Ha& London pp. 91-134

Heath I.B. (1995) Integration and regulation of hyphal tip growth, Cm. J. Bot. 73

(suppl. 1) S 13 1-139

Hermann A and Felle HH. (1 995) Tip growth in root haïr cells of Sinapis alba

L: significance of intemal and extemal ca2' and p H New PhytoL 129: 523-533

Hunsley D. and Kay D. (1976) Wall structure of Neurospora hyphal apex:

immunofluorescent locaiization of waii surface antigens. J. Gen. Microbiol. 96: 233-

248

Hyde G.J. and Heath I.B. (1997) ca2+ gradients in hyphae and branches o f

Saeegn ia ferm. Fungal Genet. Biol. 2 1 : 238-25 1

Hyde GJ. (1998) Calcium imaghg: a primer for mycologists. Fungal Genet.

Biol. 24, 14-23

Jackson S.L. and Heath IB . (1989) Effects of exogenous calcium ions on tip

growth, intraceiiular C2? concentration and actin arrays in hyphae of fimgus &prokgnia

fera . Exp. M y d 13: 1-12

Jackson S L and Heath IB. (1990) Evidençc that actin reinforces the extensib!e

hyphal apex of the oomycete &pro/egnia fertu. Protoplasma 157: 144-153

Jackson S.L. and Heath IB. (1993a) The dynamic behavior of cytoplumic F-

actin in growing hyphae Protoplasma 173 : 23-34

Jackson S.L. and Heath I.B. (1 993b) Roles of calcium ions in hyphai tip growth-

Microbiol. Rev. 57: 367-382

Jaffe L.F., Robinson KR and Nucciteiü R (1974) Local cation entry and self

elecrophoresis as an intracellular localization mechanism, Ana N. Y. Acad. Sci. 238:

3 72-3 89

JafKe L-A, Mesenseel M-K and JafFe L.F. (1975) Calcium accumulation within

the growing tips of pollen tubes. J. Cell Biol. 67: 488492

Jaffe L.F. and Nucciteili R (1977) Electrical control of development. AMU. Rev.

Biophys. Bioeng. 6: 445-476

JaEe L.F. (1977) Electrophoresis dong ceU membrane. Nature 265: 600-602

Kincaid R (1993) Calmodulin-dependent protein phosphatases from

microorganism to man. in: Shenolikar S., Nairn AC. (eds) Advances in Second

Messengers and Phosphoprotein Research Vol. 20, Raven Press, New York, pp. 1-23

Klee C.B. and Cohen P. (1 988) The cahodulùi-regulated protein phosphatase. In:

Cohen P., Klee C.B. (eds) Calmodulin: Molecular Aspects of Cellular Regulation. vol.5

Elsevier, Amsterdam, pp. 225-248

Knight H, Trewavas AJ. and R d ND. (1993) Confoul rnicroscopy of lMng

fimgal hyphae microinjected with Ca2+ -sctlsitivt fluorescent &es. MycoL Res. 97

:1505-1515

Kothe G. 0. and Free S.J. (1998) Calcincurin Subu& B is required for normal

vegetaîke gr& in Neraoqma cr- Fungai Genet, BioL 23 : 248-258

Kropf D L , Lupa MD.A, CaidwtU J.H. and Harold F U (1983) Celi poknty:

Endogenous ion current p r d e and prcdid b d g in the water mold Achlycrycr

Science 220: 1385-1387

Kropf DL., Caidweii J.H., Gow N.AR and M d F M (1984) TransccUular

ion currents in the water mold Ac-. Amino acid proton symport as a mechanism of

current entry. J. C e U Biol. 99: 486-496

Kropf D.L. (1986) Electrophysio~ogical properties o f A c h b hyphae: Ionic

currents studied by intraceUular patentid recording. J. Cd Biol. 102: 1209-12 16

Levina N.N., Lew RR and Heath I.B. (1994) Cytoskeletal regulation of ion

channel distribution in the tip-growing orgdm SproZegnia ferm. J. Ceil Sci 107: 127-

134

Levina, N.N., Lew, RR, Hyde, G. J. and Heath, I.B. (1995) The role oKa2+ and

plasma membrane ion channels in hyphal tip growth of Neurospoora crcmwa J. Cell Sci.

108,3405-3417

Lew, RR (1991) Electrogenic traasport properties of growing Arabihpsis root

hairs. Plant Physiol. 97: 1527-1 534

Lew RR (1996) Pressure regulation of the electncal properties of growhg

Arabidopsis thalianu L. root hairs- Plant Physiol. 1 12: 1089-1 100

Lew RR (1998) Mapping fuqgal ion chnnel location Fungai Genet. BioL 24:

69-76

Lew RR (1999) Comprvlfive anaiysis of calcium and proton fluxes magnitude

and location in the growing hyphal O-m &pro-a fer= and Narroqcwa CI-

Eur. J.Ceii Biol, In press,

Lipp P. and Niggli E. (1993) Ratiomeaic confiml ca2' measunments wiîh

visible wavelength indicators in isolated cardiac myocytes. C d Calcium 14: 359-372

Lapez-Franco R, Bartnicki-GarCia S., and Bracker CE. (1994) Puisecl growth of

fûngal hyphal tips. Pmc. NatL Acad. Sct USA 91: 12228012232

Mahadevan P R and Teaim EL. (1965) Relationship of the major constituents of

the Nèurospora Cr- cd-waU wild type anci colonial morphology. J. Bacterd 90:

1073-1081

Mahadevan P.R and Tatum EL. (1967) Localization of stnicturd polymers in

the ceii waii of Nëwospora c~ar54- J. Cell Biol. 35: 295-302

Malho R, Read N.D., Pais US. and Trewavas AJ. (1994) Role of cytosolic fkee

calcium in the reorientation of pollen tube g r 0 6 Plant J. 5: 33 1-341

Malho R and Trewavas AJ. (1996) Localized apical increases of cytosolic fkee

calcium control pollen tube orientation. Plant Cell8: 1935-1949

Mameth J.D. (1995) Botany. An Introduction to Plant Biology, second edition

S aunders College Publishing

McGiLiviray A M and Gow N.AR (1986) Applied elearical fields polarize the

growth of mycelid h g i J. Gen MicrobioL 132: 2515-2525

McGiiiviray AM. and Gow NAR (1987) The transhypbal electrical ament of

Nnrrosgora crossa is carried principaüy by protons. J.Oen MicrobioL 133: 2875-2881

Mc Kerracher L.J. and Heath IB. (1986) Polanzcd cytopiasmic movement and

inhibition of saltations i n d u d by calcium-mediaid e&as of microbeam in fimgal

hyphal. Ceil Md. Cytoskel. 6: 136145

McKerracher L.J. and Heath I.B. (1987) Cytoplasmic migration and intraceMar

organelie movements dunng tip growth oftùngal iryphae. Exp, Mycol 1 1: 79-100

Messerli M and Robinson KR (1997) Tip locaked Ca2+ puises are coincident

with peak pulsatile growth in pollen tubes of Lihm dongi/hroyllllf. J. Ceil Sci. 12694278

Miller AJ., Vogg G. and Sanders D. (1990) Cytosolic calcium homeostasis in

fungi: Roles of plasma membrane transport and intraceîiuîar sequestration of calcium

Proc. Natl, Acad. Sci. USA 87: 9348-9352

Miller D.D., Callaham D.A, Gross D.J. and Hepler P K (1992) Free ca2+

gradient in growing pollen tubes of Liizum. Keii Sci 10 1 : 7-12

Nuccitelli R and JafEe LE. (1976) The ionic wmponents of the m e n t pattern

generated by developing fùcoid eggs. Dw. Biol. 49: 5 18-53 1

Nucciteili R (1978) Ooplasmic segregation and secretion in the Peivetia egg is

accompanied by a membrane generated electrical airrent. Dev. Biol. 62: 13 -33

Oberrneyer G. and Weisenseel UH. (1991) Calcium channel blocker and

calmodulin antagonists a f k t the gradient of free calcium ion in My pollen tubes. Eur. J.

Celi Biol. 56: 3 19-327

Obermeyer G., Lutzelshwab M, Hamaun HG. and Weïsenseei UH. (1992)

Imrnunoiocaiization of H'ATPBSCS in the piasma membrane of pollen graias ad pollen

tubes of LiIium C e - . Protoplasma 171 : 5563

Ortega Perez R, Van Tuinen D., Marme D. and Turian G- (1983) Calmodulin-

stimuiated cyclic nucleotide phosphoditstcrase fiom Nmaspra QQEFP Biocbim

Biophys. Acta 758: 84-87

Ortega P e r a R and Turian G- (1987) Cytomorphologid deffects produced by

anti-caimodulin agents in outgmwing genn tubes and elongatïng hyphe of Neur03jpora

CTLISSLI. Cytobios. 49: 137-145

Parton RM., Fischer S., Milho R, Papasouliotis O., Jelitto TC., Leonard T. and

Read M.D. (1997) Pronound cytopiasmic pH gradients are not required for tip growth

in plant and fimgal ceiis. J. Ceil Sci. 1 10: 1 187-1 198

P e r m D.D., M o r d A, Newmeyer D. and Bjorkman, M. (1982) Chromosomal

loci of Neuro~pora crussa. Microbiol. Rev. 46: 426-570

Perkins D.D. (1992) Neurcpru: The organism behind the molecular revolution

Genetics 130: 687-70 1

Picton J.M. and Steer MW. (1982) A model for the mechanism of tip extension in

pollen tubes. J. Theor. Biol. 98: 15-20

Pierson E.S ., Miller DD., Callahm D.A, Shipley A M, Rivers B .A, Cred M.

and Hepler P.K (1994) Pollen tube growth is coupled to the e~traceilular calcium ion

flux and the intraceilular calcium gndient, effect of BAPTA-type buffers and hypertonie

media. Plant Ceil 6: 18 15- 1828

Pierson ES, Miller DD., Cailaham D A , van Aken J., Hackctt G. and HcpIer

P-K (19%) Tip localized calcium entcy fiuduates diiriog pollen tube growth. Dev. BioL

174: 160-173

Potapova T.V., Aslanidi KB., BelzersLayya T.A and Levina N.N- (1988)

TransceUd~ ionic aunats studied by imrieclhilu m a i rrcordings in Neuroqara

massa hyphae. FEBS Leît 241: 173-176

Prokish H, Yarden O., Dieminger M, Tropdu8 M. and Bartheiiness LB. (1997)

Impairement of calcineurin funaion in Neraoqmu nasia reveais its essentiai role in

hyphal growth, morphology and majntc?nance of the apical Ca2+ gradient, MOL Gm

Genet. 256: 104-1 14

Purves RD. (1981) Microelectrode Mahods for IntracelMar Recording and

Ionophoresis Academic Press

Read ND. Man W.T.G., Knight UR, Knight K. Knight UR, Malho R,

Russell A, Shacklock P.S. and Trewavas AJ. (1992) Imaging and measurement of

cytosolic fiee calcium in plant and fimgal cells. J. Microscopy 166: 57-86

Regalado C.M. (1998) Roles of calcium gradients in hyphal tip growth: a

mathematical model. Microbiol. 144: 277 1-2782

Reissig, J.L. and Kinney S.G. (1983) Calcium as a branching signai in

Nmrosporra crasH- J. Bacieriol- 154, 3: 1397-1402

Riqyelme M., Reynaga-Pena CG-, Gierz G. and Bartnicki-Garcia Sr (1998) What

determines growth direction in fimgal hyphae. Fungal Genet. BioL 24: 10 1-109

Robertson NF. and Rizvi S R I L (1968) Some observattions on the water relations

of the hypha ofhreurarpora crcrssp AM. Bot. 32: 279-29 1

Robinson KR and JdEe LE. (1979) Poiarized fucoid eggs drive a calcium

current t h u g û th«~~~eEves. Science 187: 70-72

Robson GD., Prebble E., Rickers A, Hoskïng S., Denning D.W-, Trinci A F J .

and Robertson W. (19%) Pol- growth of fùngai h y p k is defhed by an alkaline pH

gradient- Fungai Genet, Biol, 20: 289-298

Rodriguez-Nawun, A, B k UR and Slayman C L (1986) A potassium-proton

symport in Neurospora cmss~. J. Gen Physiol. 87: 649-674

R o n d T., Ugalde U.O. and hsîoza A (1993) Calcium-induad conidiation in

- . PeniEilium cyclp ïm: Caidum Triggers cytosolic -on at the hyphal tip. J.

BactenoL 175: 879-886

Ruiz Herrera J. (1992) Fungal Ce11 Waii: Structure, Synthesis and Assembly.

CRC Press Inc. Boca Raton

Sadakane Y. and Nakashima H. (1996) Light-induced phase shifting of the

circadian conidiation rhythm is inhibited by caimodulin antagonists in Neurospora crassa

J. Biol. Rhythms 1 1 : 234-240

Sanders D., Hansen U-P. and Slayman CL. (1981) Role of the plasma membrane

proton pump in pH regulation in non-animal ceiis. Proc. NatL Acad. Sci. USA 78:

5903-5907

Sanders D. and Slayman CL. (1982) Prodominant role of oxidative metabolism

net proton transport in the eykasrotic microorganism hire~~ospora. J. Gen Physiol. 80:

3 77-402

Scarborough G.A. (1976) The Néuropra plasma membrane ATP-ase is an

electrogenic pump. Proc. Natl. Acad. Sci. USA 73: 1485-1488

Schiefelbein S.W., Shipley A and Rowse P. (1992) Calcium influx at the tip of

growing roof-hairs alls ofbabk&~sis Mr'unu. Pianta 187: 455459

Schmid J. and Huold F M (1988) Duai roies for dcium ions in apical growth of

Neurospora crussa J- Gm. MicmbioL 134: 2623-263 1

Schraus W.JA and Huold F M (1988) TransceUular proton current in AcMjm

bisexualis hyphae: Relationsbip to polarkd growth, ProcProc Nd. A d . S c i USA 85:

1534-1538

Seiler, S. Nargang FE., Steinberg G. and Schliwa M. (1997) Kinesin is essential

for celi morphogrnesis and polacized seaetion in Nelaospora massa. EMBO J. 16: 3025-

3034

Sietsma J.H., Beth Diu A, Ziv V., Sjollema K.. A and Yarden 0. (1996) The

iocalization of cbitin synthase in membranws vesicle (chitosomes) in N m q w r u

crassa. Microbiol. 142: 1591-1 596

Slayman C.L. and Slayman C. W. (1 962) Measurement of membrane potentials in

Neurospora crassa Science 136: 876

Slayman C.L. (1965) Eleztricaî properties of Neutopru c r a . Effects of

extemal cations on intracellular potential. J. Gen PhysioL 49: 69-92

Slayman CL., Long W-S. Lu C-Y. H (1973) The reIationship between ATP and

an electrogenic pump in the plasma membrane of Neurcpra crmsu. J. Membrane Biol.

14: 305-338

Slayman C.L. and Slayman C.W. (1974) Depolarization of the plasma membrane

of Nrnospora massa during active transport of glucose. Proc. Natl. Acad. Sci. USA 7 1 :

1935-1939

Slayman CL. a d Gradmm D. (1975) Electrogmic tnnsport in die plrcima

membrane of Neumpom Biophys. J. 15: %8-971

Smith J.H (1924) On the carly growth rate of the individual fûngus hypha New

PhytoL 23 : 65-79 cited in Zaiokar (1959)

Speksnijder JE., Miiler AL., Weisearrel MH., Chen T-H. and Jafn LI. (1989)

Calcium buffa injections block fucoid eggs developmeaî by hcilitating calcium

difision Proc. N d Ad. Sci USA 86: 6607-661 1

Springer ML. and Yano$Ly C. (1989) A morphoIogicai and gmnic anaiysis of

conidiophore demiopment in N i - CI~SSO. Gcnes Dev. 3: 559-571

Springer ML. (1993) Genetic control o f fimgal différentiation: the three pathways

of Neurospra cr=- Bioessays 15,6: 365-374

Steinberg G. and Schliwa, H, (1993) OrganeHe movement in the wiid type and

wd-less & sg, os-1 mutants of Neurospura ausa are mediated by qtopiasmic

microtubule. J. Cell Sci- 106555464

Stemmer P.M. and Klee C.B. (1994) Dual calcium ion regdation of caicinewin

by calmoduh and caicineufia B. Biochem 33,22: 6859-6866

Stroobant P. and Scarborough G A (1979) Active transport of calcium in

Neurospora plasma membrane. Proc. Nd. Acad. Sci. USA 76: 3 102-3 106

Shimp RF., Robinson KR, Harold RL. and Harold F M (1980) Endogenous

electrical curents in the water mold Blasruccadieila emersonii during -growth and

sporuiation Proc. NatL Acad. Sci, USA 77 no 11: 6673-6677

Suresh K and Subramanyam C. (1997) A putative role for calmodullli in

activation of Neurospoo crmsa chitin synthase. FEMS Microbiol. Lett. 150: 95-1 00

Suzukï S-, Km&i S. .ad Nakashime H. (1996) Mutants wuth sadh i t y

to a calmodulin antago* &kct the Eirad*ii dock in lhuqpam arrrcrr Genet 143:

11 75-1 180

TaLeuchi Y., Schmid J-, Caidweil J H d Harold F.M. (1988) Transceiiui~ ion

curretits and extension of Neuroqwa mario hyphae. J. Memb. Bi01 10 1 : 3341

Taylor LP. and Hepler P X (1997) Poiien gaminrtion and tuôe g r o d Anou.

Rev. Plant. PhysioL Plant MOL BioL 48: 461-91

That TC, Rossier C., Barja F., Turian G. and Ross VP, (1988) Induction of

multiple gam tubes in N'èurapra WOSSQ by antitubuiin agents. Eur. J. Cdl BioL 46:

68-79

Tinsley JW, Lee LK, MinLe P.F. and Plamann M. (1998) Analysis of actin and

actin related protein 3 (ARP3) gene expression following induction of hyphal tip

formation and apolar growth in NeuropwaQ MOL Gen Genet- 259: 60 1-609

Trinci AP. and Collinge AJ. (1975) Hyphal wail growth in Neuropra c r ~ ~

and Geofrichum canddum. J, Gen MicrobioL 91: 355-361

Trinci APJ- (1973) Growth of wild type and spreading colonial mutants of

Newospora crassa in batch culture and on agar medium Arch. Mikrobiol. 9 1 : 1 13- 126

Van Brunt J., Caldwell J.H. and Harold F.M. (1982) Ciradation of potassium

across the plasma membrane of Blaç0s:ladieida emersonii: K? channel. J. Bacteriol. 150:

1449-1461

Versaw W.K. and Metzenbeq RL. (1995) Represiile cation-phosphate

symporters in Neuropra massa Roc. NatLAdSci. USA 92: 38843887

V a y A-A, DNies IM(1988) Lrta mimsurgery parnits fimgai p h m a

membrane single ion channt1 nsoiution at the hgphl tip. Appiied Environ MiaobioL

64,4: 1569-1572

Vieda, P.S. (1996) The genetics of morphogaresigaresis in Nerrrqmm CTL~SSII In

Chin, S-W., Moore, D. (eds) Pastems in Fungai Development Cambridge University

Press- Cambridge pp 87-104

Vogei, KJ. (1956) A cornrenient growth medium for Neurospora ~ e d i u m N).

MIcrobiol. Gen. Buii. 13: 4246

Weisenseel MN, NucciteIli R and Jaf& L.F. (1975) Large electïid aurents

transverse growing pollen tubes. J. Ceil. BioL 66: 556-567

Weisenseel MN. and Jae LE. (1976) The major growth current througb lily

polien tubs enters as H+ and leaves as H*. Planta 133: 1-7

Weisenseel UK, Dom A and JafFe L.F. (1979) Natural currents transverse

growing roots of barley (Hord' vulgwe L). Plant Physiol. 64: 5 12-5 18

Weiss T.F. (1996) Cellular Biophysics Vol 1 Transpo~ p.531 The MIT Press,

Cambridge, Massachusetts.

Wessels J.G.H. (1986) Ceii wail synthesis in apical hyphal growth. Int. Rev.

Cytol. 104: 37-79

Wymer CL., Bibikova T.N. and GiJroy S. (1997) Cytoplasmic fia calcium

distributions during the development of root hairs of Ardi(Iopsis th; r l i~ l l l~ Plant 1. 12:

427-43 9

Yuan S. and Heath I.B. (1991a) A cornparison of fluorescent membrane probes in

hyphal tips ofSaproIegniaferm. Exp. MycoL 15: 103-115

Y- S. and Heath [.B. (1991b) Chlortetracyche stliMng p.nrms of growing

hyphal tips of oomycetc SapKOtcgnfc fm. Exp. MycoL 15: 91-102

Zalokar M (1959) Growth ami dinerentiation of N-ru cr~rro hyphae. Am

J, Bot. 46: 602-610