Vol. 144, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1980, p. 1159-11670021-9193/80/12-1159/09$02.00/0

Oriented Growth of Blastocladiella emersonii in Gradients ofIonophores and Inhibitors

RUTH L. HAROLD' AND FRANKLIN M. HAROLD' 2*

Division ofMolecular and Cellular Biology, National Jewish Hospital and Research Center, Denver,Colorado 80206'; and Department of Biochemistry, Biophysics and Genetics, University of Colorado

Medical School, Denver, Colorado 802622

To investigate whether ion currents help to localize growth and developmentof Blastocladiella emersonii, we grew the organisms in gradients of variousionophores and inhibitors. Gradients were generated by placing into the culturefine glass fibers coated with insoluble inhibitors; in some cases, inhibitors wereadsorbed onto beads of ion-exchange resin. Organisms growing in many of thesegradients exhibited a striking tendency for the thalli to grow toward the fiber.This proved to be misleading; the cells grew not toward the source of theionophore but into the unoccupied zone of inhibition adjacent to the fiber. Fiberscoated with gramicidin-D induced marked effects on the growth of the rhizoids,which were greatly enlarged and grew toward and onto the fiber. None of theother inhibitors produced such effects, except for beads coated with the protonconductors tetrachlorosalicylanilide and compound 1799. The results suggest thatorientation of rhizoid growth results from enhancement of proton flux across theplasma membrane. Growth of the rhizoids was also strongly oriented by gradientsof inorganic phosphate and an amino acid mixture; gradients of glucose, K+, Ca2+,and glutamate were ineffective. We propose that a major physiological functionof the rhizoid is to transport nutrients to the thallus. Finally, we examined theeffects of a series of benzimidazole antitubulins as well as the cytochalasins. Thesedid not orient growth but grossly perturbed the pattern of cellular organization,producing small spherical cells with multiple stunted rhizoids. The findings areinterpreted in terms of the interaction of an endogenous transcellular protoncurrent with elements of the cytoskeleton in the determination of form.

Growth of a cell requires both the productionof specific macromolecules and their proper as-sembly within a framework ultimately con-trolled by the genome. Macromolecule synthesisis now well understood, at least in principle, butthe mechanisms by which growth is localized inspace remain largely mysterious. The presentstudy stems from the proposal, first made byLund (19), that localization is fundamentallyelectrical. In recent years, strong evidence hasbeen obtained that many cells and organismsgenerate extracellular electrical currents thatpossess a definite polarity related to both anat-omy and function. For example, germinatingeggs of the marine alga Pelvetia and germinatingpollen grains of lilies establish a current patternthat predicts and apparently determines thepoint at which outgrowth will occur. The oper-ative ion is probably calcium, which enters thegrowing tip due to the preferential localizationof calcium channels in this region (8, 13, 15; F.M. Harold, Curr. Top. Membr. Transp., inpress). One may therefore expect artificial en-hancement of local calcium entry to stimulatelocalized growth. Indeed, Robinson and Cone

(25) recently documented that a gradient of thecalcium ionophore A23187 polarizes the germi-nation of Pelvetia zygotes, and, in many cases,the effects of applied electrical fields on plantgrowth can be understood in terms of the en-hancement of local calcium fluxes (15, 23).The object of the present study was to deter-

mine whether ion currents play a role in estab-lishing the marked polarization of growth anddevelopment in fungi, particularly in Blastocla-diella emersonii. The life cycle of this aquaticphycomycete (2, 18) begins with motile, non-growing zoospores; when these are inoculatedinto growth medium, they quickly settle down,retract their flagella, and germinate. At thispoint, the basic organization of the organism hasbeen laid down. The cell body is destined togrow into a large coenocytic thallus, containingas many as 100 nuclei. The germ tube elongatesand branches into a filamentous rhizoid thatserves as a holdfast and, we believe, as a trans-port organ (see below). The Blastocladiella or-ganism is thus, in effect, a large and highlydifferentiated single cell (see Fig. 7a). The veg-etative plants can be induced to sporulate at any

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time by replacing the growth medium withbuffered calcium chloride (26). Sporulation isagain a strikingly polarized process that entailsretraction of the cytoplasm from the rhizoids,construction of a baseplate across the thallus,formation of a discharge papilla at the distal endof the thallus, and cleavage of the cytoplasminto zoospores of exquisite architecture (2, 18).Do ion currents help to determine the site and

direction of these biological constructions? Byuse of a vibrating probe (14), we have recentlyfound that the Blastocladiella organism drivesion currents through itself (R. F. Stump, K. R.Robinson, R. L. Harold, and F. M. Harold, Proc.Natl. Acad. Sci. U.S.A., in press). In vegetativecells, positive charges enter the rhizoid and leavefrom the thallus; we do not know what ions carrythe current, but circumstantial evidence pointsto protons. When the organisms enter the spor-ulation pathway, the current pattern reverses;positive current, possibly carried by Ca2", entersthe thallus and leaves from the region of thebaseplate. To learn whether these currents ofprotons and calcium confer direction upon cel-lular processes, we studied the effects of iono-phores, applied both in gradients and in uniformfields, upon growth and development of Blasto-cladiella organisms. Initial observations, de-scribed in an abstract (R. L. Harold, K. R. Ro-binson, and F. M. Harold, Abstr. Annu. Meet.Am. Soc. Microbiol. 1979, I75, p. 107), suggestedthat a calcium current orients growth of thethallus; this conclusion was unwarranted, as willbe shown below. However, we report here thatgrowth of the rhizoid is markedly oriented bygradients of proton-conducting ionophores andcertain nutrients, including Pi and a mixture ofamino acids. The results suggest that the rhizoidserves to transport nutrients to the thallus andthat chemotropic growth of the rhizoid resultsfrom modulation of the proton current throughthe growing cell. Generation of the basic patternof cellular organization appears to be unaffectedby ionophores, but is seriously perturbed byinhibitors of microtubule and microfilament as-sembly.

MATERIALS AND METHODSOrganisms and media. B. emersonii strain L17

was a gift from David Sonneborn and was maintainedin the forn of resistant sporangia as described byLovett (17). Vegetative cells were grown either on thecomplex medium PYG or on the defined medium DM2(26, 28). Zoospores for inoculation were prepared bystandard methods (17, 26, 28) and counted in a he-macytometer just before inoculation. Microscopy andphotography were done with an Olympus IMT in-verted microscope.

Generation of inhibitor gradients. Glass fiberswere drawn and coated with ionophores as described

by Robinson and Cone (25) and then glued into thebottoms of tissue culture dishes (Falcon optilux, 100by 20 mm; General Electric clear silicone glue). Theglue was allowed to cure for 45 min. DM2 medium wasthen added (10 ml) and inoculated with 2 x 105 zoo-spores, which settled to the bottom of the dish. Underour standard incubation conditions, the coated fiberacted as a line source for the inhibitor, and the mediumacted as an infinite sink, thus creating a standingconcentration gradient. Dishes were incubated in thedark at 24°C, with care to avoid movement that woulddisturb the gradient.

Proton-conducting uncouplers which were too sol-uble to be used on fibers were incorporated intoDowex-1 beads, 200 to 400 mesh (75 to 180 ,tm), asfollows. A short column of the resin was washed suc-cessively with a solution of 1 N NaOH, water, andethanol-water (4:1 [vol/vol]); a solution of tetrachlo-rosalicylanilide in ethanol-water (1.5 mg/ml); moreethanol-water; and finally, water. The beads were sus-pended in a solution of poly-L-lysine, 0.2 mg/ml.Dishes were rinsed once with the suspension of beadsand allowed to dry. Dishes containing 20 to 100 beadswere used; again, each bead acted as a point source forthe inhibitor.Chemotropism to nutrients. By a technique of

Stadler (27), small holes were drilled into the bases of35-mm tissue culture dishes with a no. 80 drill bit(diameter, 340 um) in a high-speed drill press. Thecover of the dish was filled with warm DM2 mediumcontaining 1% agar. Before the agar had solidified, thebase of the dish was gently placed on top of the surfaceto form a tight, air-free junction. DM2 medium con-taining a growth-limiting amount of one nutrient wasthen added to the top compartment and inoculatedwith zoospores. The hole acted as a point source forthe limiting nutrient, and the upper compartmentacted as an infinite sink.

Materials. Reagents and inhibitors were purchasedfrom standard suppliers, usually Sigma Chemical Co.Nocodazole and the cytochalasins were purchasedfrom Aldrich Chemical Co. Inhibitors were dissolvedin ethanol or in dimethylsulfoxide; cells in dish culturecan tolerate more than 0.2% of the fonner and 0.1% ofthe latter. We acknowledge with thanks gifts of thefollowing reagents: nigericin and A23187 (R. J. Hosley,Lilly Research Laboratories, Indianapolis, Ind.); io-nomycin (C.-M. Liu, Hoffman-LaRoche, Nutley, N.J.);pactamycin (Sylvia Kerr, University of Colorado Med-ical Center, Denver, and John D. Douros, Develop-mental Therapeutics Program, Chemotherapy, Na-tional Cancer Institute, Bethesda, Md.); carbendazim(H. C. Van der Plas, Landbouwhogeschool, Wagenin-gen, The Netherlands); thiabendazole (L. R. Mandel,Merck Sharpe & Dohme Research Laboratories, Rah-way, N.J.); desformyl gramicidin A and blocked gram-icidin A (Dan Urry, University of Alabama MedicalCenter, Birmingham, Ala.); FCCP and 1799 (P. G.Heytler, E. I. du Pont de Nemours & Co., Wilmington,Del.).

RESULTSGrowth of B. emersonii in gradients of

calcium ionophores. Under the stimulus of

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the discovery that gradients of calcium iono-phores polarize outgrowth of Pelvetia embryos,we began our studies by allowing Blastocla-diella organisms to grow in such gradients pro-duced by the fiber technique. Typical results fora dense culture are shown in Fig. 1. Uncoatedfibers were innocuous (Fig. la), but insertion offibers coated either with A23187 or with thenovel calcium ionophore ionomycin (16) haddramatic effects upon the pattern of growth.Adjacent to the fiber there was a narrow zonedevoid of growth due to the inhibitory effects ofhigh concentrations of the ionophores. Just be-yond this zone the thalli were strongly oriented,growing with their tips toward the fiber; growthof the rhizoids was not affected (Fig. lb).These observations, described in an abstract

(R. L. Harold et al., Abstr. Annu. Meet. Am.Soc. Microbiol. 1979, I75, p. 107), turned out tobe misleading; orientation of thallus growth wasproduced not by the ionophore gradient per sebut by the open space created next to the fiber.This conclusion stems from the following results.(i) A23187 and ionomycin gradients orientedthallus growth in crowded cultures, but not insparse ones. (ii) Orientation in crowded cultureswas elicited by antibiotics that do not transportCa2", notably gramicidin (see below and Fig. 2).(iii) Open space created mechanically was foundto be just as effective as an ionophore gradient(Fig. lc). We are forced to conclude that theorientation of thallus growth is an example ofthe negative autotrophic effect (7). Organisms atthe edge of the zone of inhibition eventuallygrow toward the open space, probably in re-sponse to staling factors produced by theirneighbors (7). We have also noticed that thalliare strongly chemotropic toward oxygen, andthis may be a second factor in directing growthtoward open space. Our experiments thereforeprovide no evidence for localization ofgrowth bycalcium fluxes, although they also do not excludesuch effects.Gradients of gramicidin and of proton

conductors. The need for controls led us togrow B. emersonii in gradients of a variety ofinsoluble ionophores and inhibitors generatedby the fiber technique. The most striking resultswere obtained with gradients of gramicidin D, asshown in Fig. 2. We may disregard the orienta-tion of the thalli and focus on the rhizoids, whichare thickened and enlarged, resembling roots,and are strongly oriented toward the fiber. Letus emphasize that these effects on rhizoid mor-phology and orientation are quite specific. Theywere not observed in gradients of other effectiveinhibitors of growth, including A23187, ionomy-cin, valinomycin (Fig. 3), antimycin, and pacta-mycin. Two gramicidin analogs were also tested;

desformyl gramicidin, with slight ionophore ac-tivity (D. Urry, personal communication) occa-sionally produced slight rhizoid polarization;"blocked gramicidin A," which has no ionophoreactivity, affected neither growth of the cells northe orientation of their rhizoids (data notshown).

Gramicidin is a channel-forming ionophorethat allows the passage of K+, Na+, and H+ (1,24). It is unlikely that Na+ movements are in-volved in the orientation of growth; sodium isapparently not required for growth of B. emer-sonii (28), and its omission did not reduce theeffectiveness of gramicidin gradients (data notshown). Potassium ions are required for growth;orientation of growth in media of very low K+content (0.1 mM) could not be studied becauseunder these conditions the antibiotic becomesexcessively inhibitory to growth. However, thefailure of the powerful K+ ionophore valinomy-cin (1, 24) to orient growth of the rhizoids (Fig.3) suggests that enhanced K+ flux is not respon-sible for the effects of gramicidin D.To test the proposition that the orientation of

rhizoid growth is related to proton movements,we turned to the proton-conducting "uncou-plers" (1). These proved to be too soluble to bestudied by the fiber technique, but in severalcases, useful gradients were produced by beadsof the ion-exchange resin Dowex-1, to which theproton conductors had been adsorbed as de-scribed above. Figure 4a shows orientation ofthe rhizoids toward beads carrying tetrachloro-salicylanilide; compound 1799 was also quiteeffective (data not shown) but carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and theanalogous fluoro-derivative FCCP were still toosoluble. Nigericin elicited some orientation (datanot shown). Beads that had been sham treatedwith ethanol alone were ineffective (Fig. 4b), aswere beads of the cation resin Dowex 50 in theacid form. We conclude that the orientation ofrhizoid growth is probably a reflection of en-hanced proton movement across some mem-brane, presumably the plasma membrane.Chemotropic growth of rhizoids in gra-

dients of nutrients. Chemotropism towardsproton-conducting ionophores presumably re-flects some physiologically useful capacity. Webelieve that the rhizoid, long known to serve asa holdfast, also transports nutrients from themedium to the thallus and that proton conduc-tors mimic the chemotropic effects of certainnutrients.Chemotropism towards water-soluble metab-

olites was investigated by adapting the tech-nique of Stadler (27), in which small holes serveas point sources of the metabolite under study,as described above. Growth of the rhizoids was

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FIG. 1. Growth ofB. emersonii in an ionomycin gradient. (a) Control. A glass fiber was glued to the bottomof the dish, and DM2 medium that was inoculated with zoospores was subsequently added. The organismsgerminated and grew at random. (b) Ionomycin. A glass fiber coated with ionomycin was glued to the bottomof the dish, which was then inoculated as above. Note the zone of inhibition adjacent to the fiber, then aregion of oriented growth, with the thalli growing toward the fiber, and random growth of the organismsfarther out. (c) Open space. A dish was inoculated as above and the zoospores were allowed to germinate.After 1 h, an open zone was created mechanically by scraping with a wooden stick. Note that subsequentgrowth of the thalli was oriented toward this zone. Pictures were taken 16 h after inoculation. Bar, 400 Mm.

FIG. 2. Growth in gradients of gramicidin-D. Procedures were as described in the legend to Fig. 1. (a)Fiber coated with gramicidin-D. Note the zone of inhibition adjacent to the fiber; cells in this region hadstunted thalli but the rhizoids proliferated and grew toward the fiber. Beyond this zone, note oriented growthof the elongated thalli, whereas the rhizoids were normal and randomly oriented. Bar, 200 ,um. (b) Fibercoated with gramicidin-D, at a higher magnification. Note the enlarged and thickened rhizoids growing ontothe fiber. Bar, 40 ,um.

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strongly oriented in gradients of Pi (Fig. 5a) andthe amino acid mixture used in DM2 medium.By contrast, gradients of glucose, K+, Ca2+, andglutamic acid were ineffective (data not shown).No further attempt was made to determinewhich individual amino acids elicited chemo-

FIG. 3. Fiber coated with valinomycin. The anti-biotic inhibited growth of the cells but did not orientthe rhizoids. Bar, 80 pmm.

tropic growth. It is noteworthy that rhizoids oforganisms growing in gradients of Pi and ofamino acids were elongated, thickened and ca-ble-like, quite unlike normal rhizoids but resem-bling those produced by gramicidin. Nutrientgradients did not orient growth of the thalli (Fig.5).Chemotropism towards soluble nutrients may

also underlie a novel mode of growth that wedesignated as "cannibalism." Under certain con-ditions, particular cells send out long, thick,straight rhizoids that appear to penetrate neigh-boring thalli. The crinkled surface and disorga-nized cytoplasm of the thalli and their inabilityto exclude methylene blue leaves no doubt as towho benefits from the union. Such cells areregularly seen in cultures growing in calcium-deficient medium, prepared by omitting thecalcium from DM2 and adding 2,tM ethyleneglycol-bis(,B-aminoethyl ether)-N,N-tetraaceticacid (EGTA) (Fig. 6). We have also occasionallyseen this effect in cells grown in the presence ofthe proton-conducting ionophores tetrachloro-salicylanilide and 1799.

It should be mentioned in passing that growthof the thallus, which is unaffected by nutrientgradients (Fig. 5), nevertheless exhibits strikingtropisms of its own. Thalli are strongly chemo-tropic towards oxygen, as shown particularly bythe effects of occasional air bubbles; rhizoids arealso chemotropic towards oxygen, but less mark-edly than= the thalli. Second, as mentioned

FIG. 4. Orientation of rhizoids in a gradient of tetrachlorosalicylanilide. Pictures were taken after 16 h;bar, 80 pm. (a) Tetrachlorosalicylanilide was adsorbed onto beads of Dowex-1 resin, 200 to 400 mesh, whichwere attached to the culture dish with polylysine. Note enlarged rhizoids growing toward and onto the bead.(b) Control, with a resin bead sham-treated with ethanol. The cells were larger than in (a) becausetetrachlorosalicylanilide inhibited growth; rhizoids were normal and not oriented.

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it~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.MFIG. 5. Chemotropic growth ofrhizoids in a gradient ofPi. Pictures were taken after 16 h; bar, 250 um. The

black zone is a hole in the bottom of the culture dish, approximately 450tm in diameter, as described in thetext. (a) Cells growing in a gradient of PA. The lower dish contained DM2, and the upper dish (in which thecells were growing) contained DM2 with only 5 M Pi. (b) Control, no gradient; both upper and lower dishescontained DM2. Note that the cells in (a) obtained enough Pi to attain almost normal size; cells farther awayfrom the hole (not shown) were much smaller.

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FUNGAL GROWTH IN IONOPHORE GRADIENTS 1165

sidered above either inhibit growth or modulateits direction, but have no obvious effects on thepattern of cellular organization. By contrast,both antitubulins and cytochalasins have char-acteristic effects upon the general structure ofBlastocladiella organisms.

Fig. 7b illustrates the effects of a fiber coatedwith nocodazole, an insoluble benzimidazole de-rivative thought to inhibit microtubule assemblyin a manner analogous to that of colchicine (3,

,.<.>/̂.<Jw s 5, 10). Cels growing adjacent to the fiber werenot only smaller but grossly misshapen; in place

**/ ^ ' ~ w of the ovoid thallus and single bushy rhizoid onefinds "ticks," which are round little cells withstubby rhizoids all over. Farther from the fiber,the cells were larger and roughly spherical orslightly elongate, with multiple stunted rhizoids.The morphology became progressively morenormal with distance from the fiber; nocodazoledid not orient the growth of either the thallus orrhizoid.Other benzimidazole inhibitors of microtubule

assembly (3, 5, 10) were either too soluble or too/ | rP] >weak to be applied in gradients, but their effects

upon morphology in uniform fields were muchlike those described for nocodazole. Sphericalcells and multiple stunted rhizoids were pro-

_......

duced by the following compounds when addedeither together with the zoospores (0 min) or togermlig (90 mm after inoculation): carbenda-

*~~~~,~~zirn, 8 AM; thiabendazole, 20A!M; and griseoful-vm, 100 ,uM. It is noteworthy that none of theseinhibitors affected the morphology or motility ofthe zoospores nor did they prevent their germi-nation upon addition of 50 mM KCI.

__ We have also examined the effects of cyto-chalasins A through E. When added to germlingsin dish culture all elicited gross morphological

_ > ;.effects similar to those of antitubulins; thalliwere globose or misshapen, and rhizoids ap-peared branched or thomy and generally muchthickened. Cytochalasin A was the most potentof the series and too powerful for study in gra-dients; less than 0.5 ,tg/ml was sufficient to to-tally block growth. Cytochalasins D and E were

FIG. 6. Cannibalism in B. emersonii. Cells were effective at 2 ,Ag/ml, and B and C were effectivegrown for 20 h in Ca2 -deficient medium (DM2 minus at 10 ,ug/ml. When added to zoospores, cyto-calcium, supplemented with 2 pLM EGTA). Note the chalasins A and B blocked formation of the germcable-like rhizoids linking large cells to their smaller tube.neighbor. Bar, 50 tmn.

FIG. 7. Effect of nocodazole on morphology of B. DISCUSSIONemersonii. (a) Normal cells in DM2. (b) Nocodazole,44uM, added 1.5 h after inoculation of the zoospores. The shape of a growing cell of B. emersoniiPictures were taken after 16 h; bar, 100 gun. appears to be subject to two kinds of forces. One

set determines the basic organization of the cellabove, thalli repel each other and rarely cross into the thallus and rhizoid and later controlseach other's paths; rhizoids suffer no such inhi- the formation of such elements as the baseplatebitions. and discharge papilla. To be sure, the pattern isInhibitors of cytoskeleton assembly per- not immutable; occasionally one sees multiple

turb cellular organization. The reagents con- rhizoids or multiple papillae, and these aberra-

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tions are evidently a function of the environ-ment. Nevertheless, it appears that the organismpassing through its life cycle executes a set ofinstructions that was fixed from the time ofgermination (if not before). Superimposed onthe basic pattern of organization, a variety ofmodulations can be seen that are clearly envi-romnental in origin. These include the tendencyof organisms in crowded culture to assume agreatly elongated form, with the thallus perpen-dicular to the substratum; the timing of sporu-lation; and the various tropisms of both thallusand rhizoid. These two kinds of morphogeneticprocesses may, but need not, have the samephysical basis. Our findings lead us to suspectthat they are, in fact, quite distinct.The basic observations concern chemotropic

growth of the rhizoid in gradients of nutrients(Pi and amino acids) and of proton-conductingionophores. In both of these circumstances andalso in cells growing cannibalistically, the rhi-zoids grew up the gradient and were morpholog-ically quite unlike those of cells grown in DM2medium (or in PYG). To rationalize the obser-vations, we propose that there is normally aproton current through the rhizoid, with protonsentering the filaments as suggested by the elec-trical measurements (Stump et al., in press).From what is known of the proton circulation inother fungi (4, 8) we expect protons to be activelyexpelled by proton-translocating ATPases andto leak back into the cell by passive channels;preferential localization of pumps in the thallusor of leaks in the rhizoidal filaments would thengenerate a current of protons across the cell(Stump et al., in press). We postulate furtherthat this proton current exerts a directional ef-fect upon the growth of the rhizoid and thatboth nutrients and proton conductors exert theirchemotropic effects by modulating the protonflux across the plasma membrane, presumablyat the tip. It is well known that nutrient trans-port into fungal cells often occurs by symportwith protons (4, 8), and one can thus imagine avery direct connection between the presence oftransportable nutrients and enhanced protonflux across the membrane. Alternatively, andperhaps more likely, recognition of such nutri-ents as Pi by specialized receptor sites at the tipof the rhizoid may regulate the passage of pro-tons through local channels. The proton-con-ducting ionophores bypass the physiologicalchannels, providing alternate routes for protonsto cross the rhizoid plasma membrane.This hypothesis begs the question, what is the

physiological function of the (putative) protoncurrent? We suspect that it is connected withthe translocation of nutrients through the rhi-zoid into the thallus, although we have no evi-

dence to support this suggestion. A second func-tion, by hypothesis, must be to guide the growthof the rhizoid. This could happen if the region ofmaximal proton entry were destined to becomethe region of maximal growth, i.e., the growingtip. Localization of outgrowth in a Pelvetia em-bryo by a calcium current (13, 15, 22, 25) illus-trates this relationship, but it does not appear tofit our observations with Blastocladiella orga-nisms. These are better accommodated by thehypothesis that the growing tip is fixed by otherfactors, but the tip grows in such a way as tomaximize proton flux across its surface. Just howa proton flux could be sensed, confer directionupon the growing rhizoid, and produce the char-acteristic root-like morphology, are questionsthat we do not wish to consider at this time.There seems to be some link to nutrient depri-vation since starving cells have thickened rhi-zoids, albeit they are never as striking as thoseseen in cells growing in an appropriate gradient.

If there is merit to the proposal that tropismsof the rhizoid result from modulation of a protoncurrent through this organelle, it may be morewidely applicable in the plant world. Weisenseeland Jaffe (30) showed that germinating lily pol-len expels protons from the grain and generatesa transcellular ion current. Barley roots take upprotons into the growing zone just behind thetip (29) and growth of the root may possibly beguided by this current. Indeed, dinitrophenol (aclassical uncoupler and proton conductor) isknown to polarize the outgrowth of Fucus em-bryos (12).Our experiments implicate proton movements

in tropisms of the rhizoid but not in those of thethallus. In addition to proton conductors wetried a range of other ionophores, including theK+ ionophore valinomycin and the calcium ion-ophores A23187 and ionomycin (16), withoutsuccess. The failure of calcium ionophores tomodulate growth patterns or tropisms was sur-prising since calcium movements have been sostrongly implicated in the localization of growthin plant and animal systems (13, 15, 25). Wecannot conclude that calcium currents do notplay such a role in Blastocladiella organisms,only that our experiments to date have turnedup no evidence to support it.

Finally, we would point out that none of theionophores, including those for protons, seemedto alter the basic pattern of cellular organizationinto the thallus and rhizoid (possible exceptionsare CCCP, FCCP, and nigericin at relativelyhigh concentrations); antimycin and pactamycinalso had no effect at this level. The pattern is,however, seriously perturbed in cells grown inthe presence of either cytochalasins or benzimi-dazole inhibitors of microtubule assembly. Such

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cells are smaller than normal ones, are sphericalor irregularly shaped, and bear multiple stuntedrhizoids. We plan to explore the genesis of thesedistortions in the future, but it seems appropri-ate here to call attention to the parallel betweenour observations and those made with plant andalgal cells grown in presence of colchicine. Sev-eral laboratories have reported that such cellsare spherical and possess cell walls whose micro-fibrillar organization is abnormal (9, 11, 21).Taken in conjunction with ultrastructural data,these observations have given rise to the pro-posal (9, 20) that the deposition of cellulosemicrofibrils is controlled by a network of corticalmicrotubules located just beneath the plasmamembrane. One may well imagine the locationof cellulose synthase complexes (6) to be deter-mined in this manner. Perhaps a similar rela-tionship holds for the deposition of chitin fibrilsin the cell walls of fungi. Since the shape offungal cells is ultimately expressed in a rigid cellwall, the study of fungal morphogenesis maycome to turn on the interactions among ioncurrents, cytoskeleton, and wall assembly.

ACKNOWLEDGMENTS

We are deeply indebted to Kenneth Robinson (Departmentof Physiology, University of Connecticut Health Center, Far-mington) for introducing us to the fiber technique for gener-ating gradients, to Mayer Goren of this Department for helpwith the resin beads, and to Robert Kennedy (Department ofPhysiology, University of Colorado Medical Center) for drill-ing fine and unobstructed holes. This work could not havebeen done but for the generous gifts of inhibitors listed in thetext.The research was supported in part by research grant PCM

78-15123 from the National Science Foundation.

LITERATURE CITED

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2. Cantino, E. C., and G. L. Mills. 1976. Form and functionin Chytridiomycete spores, with emphasis upon Blas-tocladiella emersonii, p. 501-556. In D. J. Weber andW. M. Hess (ed.), The fungal spores form and function.John Wiley and Sons, Inc., New York.

3. Davidse, L. C., and W. Flach. 1978. Interaction of thia-bendazole with fungal tubulin. Biochim. Biophys. Acta543:82-90.

4. Eddy, A. A. 1978. Proton-dependent solute transport inmicroorganisms. Curr. Top. Membr. Transp. 10:279-360.

5. Friedman, P. A., and E. G. Platzer. 1978. Interaction ofanthelmintic benzimidazoles and benzimidazole deriv-atives with bovine brain tubulin. Biochim. Biophys.Acta 544:605-614.

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