Inhibition of movement in slime mold plasmodia by specific carbohydrates

INHIBITION OF MOVEMENT IN SLIME MOLD PLASMODIA BY SPECIFIC CARBOHYDRATES JOHN R.

DENBO and

DONALD

M.

MILLER

Department

Department

of Biology, Grinnell College, Grinnell, IA 50112, U.S.A. and of Physiology, Southern Illinois University. Carbondale, IL 62901. U.S.A. (Rewired

I Augusr1977)

Abstract--i. Oriented migrating plasmodia of the acellular slime mold P. polycrphntum Schw. respond to small (0.125 M) substratum con~ntrations of common monosaccharides by a significant reduction in migration rate. 2. This effect was noted with D-galactose and o-mannose, and to a lesser extent with o-glucose and o-fructose. 3. Inhibition of movement was specific for the carbohydrates tested and not related to their general

osmotic properties. 4. Although no correlation was observed between a monosaccharide’s and its at&y to support growth, monosaccharides reported as powerful attractants of the pjasmodium INTRODUCTION

The plasmodial stage of the acellular slime mold Physarum polycephalum Schw. exhibits a vigorous and rhythmical protoplasmic streaming, which Kamiya (1959) termed “shuttle-how”, and a directed ameboid type of movement term “migration”. Although much information is available concerning the control of shuttle-flow streaming (see reviews by Kamiya, 1959; Jahn & Bovee, 1969; Komnick et al., 1973) very little information exists concerning the control of plasmodial migration. Traditional studies of plasmodial migration have focused on factors controlling the direction of movement or “taxis” of the plasmodium. Jiinsson (1883) and Clifford (1897) observed that plasmodia would migrate up a water gradient (hydrotropism) and against a current of running water (rheotropism). Watanabe rt al. (1937) demonstrated that migrating plasmodia would orient toward the cathode in a d.c. electric field (galvanotaxis) but Anderson (195I) proved that the movement toward the cathode actually resulted from anodal inhibition rather than cathodal stimulation. Coman (1940) found that certain acids, bases and salts had attractant or repellent effects on piasmodia, and Carlile (1970) extended these studies demonstrating an apparent positive chemotaxis to certain simple sugars and other nutrients capable of supporting plasmodial growth. Recently interest has also developed in factors capable of controlling the rate of plasmodial movement. Plasmodia which have been unidirectionally oriented on 2”/;, non-nutrient agar substrata were shown by Miller & Anderson (1966) to migrate at a constant rate with pauses in migration at 6-8 hr intervals. Such migratory pauses had previously been observed by Guttes & Guttes (1963) during the synchronous mitosis in the plasmodium. Denbo & Miller (1976) demonstrated that migration rate was also controlled by the temperature, osmolarity, pH and concentrations of specific ions in the substratum. These studies indicated that the action of many plasmodial repellents might be explained by their ability to inhibit the rate of plasmodial migration in the direction 269

found

to inhibit

ability to inhibit migration

migration

have previous&

been

of the source of the repellent, rather than stimulating migration away from the repellent source. In the present study we have examined several common which have been previously monosaccharides reported to be plasmodial attractants for their ability to specifically alter the rate of plasmodial migration. MATERIALS

Ejirt

AND METHODS

of cc~mnmn tnonosuccharides on migration rate

A strain of Ph~saru~ po~.ycepha~u~ Schw. was obtained from Carolina Biological Supply Co.. (Burlington, N.C. 27215) and propagated in the plasmodial stage by the method of Camp (1936) as modified by Miller & Anderson (1966). Prior to the migration studies plasmodia were starved by random migration across 2% non-nutrient agar substrata for 12-15 hr. Such a procedure was found to lengthen the interval between mitotic pauses in migration to about 16 hr and was found to be necessary in order to obtain highly reproducible migration rates. Following the 12-15 hr period of starvation, plasmodia were oriented between strips of Parafilm on 20/1 non-nutrient agar substrata (pH = 9.0, temperature 22°C) as described by Denbo & Miller (1976). The agar substrata contained varying concentrations of one of the following sugars: sucrose; u-glucoie; D-mannose; o-fructose; and u-galactose. Groups of ten plasmodia were migrated on agar&bstrata containing 0.025 M. 0.05 M. 0.10 M. and 0.20 M concentrations of the above-mentioned sugars. Each plasmodium was ailowed to migrate for 6 hr on the above substrata, after which time an average migration rate was calculated. At I-hr intervals the distance (S) (in cm) each plasmodium had migrated was measured to verify that migration rate had remained constant throughout the experiment and that no mitotic pauses in migration had occurred. The data gathered were analyzed by a one-way analysis of variance (ANOVA) with comparisons between various “within group” means being made by the Tukey method as described by Glass & Stanley (1970).

M~croplasmodia werr grown in pure culture by the method of Daniel & Baldwin (1964). Cultures grown in Daniel’s semidefined growth medium (SDGM). in which D-glucose served as the major carbon source, served as positive growth controls. while cultures grown in SDGM


m-Ij”--MOLAR

200 CONCENTRATION

OF

SUGAR

Fig. I. The effect of different carbohydrates on the mlgration rate of plasmodia. Plasmodia were oriented on ?‘I,, non-nutrient agar substrata (pH 9.0) containing different concentrations of various carbohydrates. Each point rcpresents the mean migration rate of a group of IO plasmodia. Statistically significant differences between mean migration rates of the different groups of plasmodia were determined by the Tukey method (P = 0.01) following oneway analysis of variance. All migratton studies were conducted in the dark at 23 24’C (See text for results).

without glucose served as negatlvc growlh controls. C arhohydrates whose ability to support grouth were to he tested. replaced glucose at exactly its concentration in SDGM. i.e. 0.9”,,. Growth studies were initiated h> the mnoculation of eight replicate cultures with an amount of microplasmodia equivalent to 0.01 mg protein/ml from 3-day old stock cultures. Aliquots were removed from each of the replicates at 24-hr Intervals and analyzed for total protein by the method of Lowry rt ul. (1951). During the growth studies all cultures were incubated in the dark at 21 C. RkSIILTS

Plasmodia migrating on 0.025 M concentrations of all monosaccharides tested maintained migration rates significantly lower (P = 0.01) than plasmodia migrating on an equivalent concentration of sucrose (Fig. 1). I>-galactose and u-mannose produced the greatest inhibition compared to 0.025 M sucrose. while n-fructose and I,-glucose produced smaller degrees of inhibition. Comparisons between means revealed that the difference in migration rate between the groups migrating on I,-glucose and I,-fructose was not significant (P = 0.01 ). while all other hetwerngroup comparisons were significant (P = 0.111). At 0.05 M concentrations of I)-galactosc and t>-mannose plasmodia migrated significantly slower (P = 0.01) than those migrating on equivalent sucrose concentrations. However. the difference in migration

Table

I, Summar)

Concn 0.025 M 0.050 M 0.100 M

0.‘00 M

of mean migration various concentrations Sucrose 1.23 cm ht3.20 7.51 1 2’

On the 0. IO M substrata o111~ 1~-nx1111mxc rnl~~htd migration over and above that produced hq an ~x~~I*~ alent sucrose concentration (Tabk I t At 0.10 M cotlcentrations all monosaccharides dcpresscd migration to the same extent as 0.3) M sucrose. and therL> UC’IC no significant differences hetwcen group mc‘an~ *Attempts to establish the threshold conccntl-;itlon\ of the above sugars necessary to produce an inhlhltion of migration rate stgnificantl! greater than an equivalent concentration of sucrose revealed that tor I)-glucose and I)-fructose the threshold ~a\ 111 the I z t 0 ’ M range. and for r)-galactose and I,-~llanr1ose.

Iv;,5

t

i

IO

i

and

I

IO

‘Xl

\‘i~ilcL’ll-

Figure 2 illustrates the ability 01 \arloub monosaccharides tested in the migration studies to support the growth of microplasmodia. The highest growth rate and peak protein concentration occurred in media containing I+glucose as the major carbon source. These cultures attained a peak protein calcentration of 5.4 mg/ml at 72 hr incubation. I,-Mannose supported the next highest rate of growth with a probable peak of 4.0 mg/ml at about X0 hr. WLialactose and I+fructose yielded similar growth curves. both exhibiting a 72 hr peak protein concentration of 2.X mg/ml. II-Fructose sustained a maximum protein concentration 24 hr longer than the I)-galactose cultures. Sucrose yielded a small initial peak of 2.3 mg/ml at 50 hr. after which time the protein concentration dropped rapidl) to the levels m control flasks containing no major carbon source. At this time cnsphcrulation began to occur.

The data on microplasmodial growth obtained in this study are not in complete agreement with the results of a previous study by Carlile (1970). (‘arlile’s study indicated virtually no growth in media in which I)-fructose was the major carbon source, whereas the present studies indicated moderately good growth on I)-fructose. A much earlier study by Daniel & Baldwin (1964) also found moderately good growth in I)-fructose. C‘arlile’s study also indicated much better growth on L)-mannose than was found in the present study. The present study does agree with (‘arlile’s (1970) observation that sucrose does not support microplasmodial growth. The small 50 hr peak 5cen in

rate> of group\ of plasmodia of different carbohydrates ~;alactosc

‘.X1 2.x I 2.37 I.54

ht3wecn

trations.

2.1x

2.15 2.38 1.64

Mannohe

mugrating

f- ructosc

2.52 I .90

XX 2.130

I .x3

2.36

I.13

I .35

on

Inhibition of movement in slime mold plasmodia

0.0

30.0

60.0

90.0

120.0

150.0

HOURS

Fig. 2. The effect of common carbohydrates on the growth of microplasmodia of Physarum polycephulum. Each point

represents the mean of eight replicate cultures. Microplasmodia were incubated in the dark at 22°C.

Fig. 2 represents glucose carry-over into the experimental cultures from the 3-day stock cultures. Daniel & Baldwin (1964) also observed that no microplasmodial growth occurred on sucrose. EfSPct of carbohydrates

on migration

rate

Each of the four monosaccharides tested in this study inhibited plasmodial migration rate to a greater extent than predicted on the basis of their osmotic activities alone. This specific inhibition was most apparent at dilute concentrations, i.e. 0.025 M and 0.05 M, and was completely masked at higher concentrations by the more general phenomenon of osmotic inhibi~on previously reported by Denbo & MiIIer (1976). D-Mannose and D-galactose produced the greatest amount of inhibition, with D-fructose and D-glucose producing lesser amounts. Comparing Fig. 1 with Fig. 2 it is interesting to note the apparent lack of correlation between the ability of a monosaccharide to be utilized to support growth and its ability to specifically inhibit migration rate. For example, ranking the sugars by ability to support growth yields D-glucose > D-mannose > o-galactose > o-fructose, while ranking by ability to inhibit migration yields u-mannose and D-galactose S D-fructose and D-ghCOSe. Carlile (1970) hypothesized that certain monosaccharides which supported plasmo~al growth also served as plasmodial attractants. D-glucose, D-mannose, and D-galactose were listed as examples of such attractants. It is interesting that this study has also shown that these sugars alter the rate of plasmodial migration. Since plasmodial taxis was not a variable studied in the present experiments it is not possible to determine from the data at hand whether changes in plasmodial migration rate were related to changes in direction of migration or whether both result from a more fundamental process. However, it would be interesting to extend the present studies to a wider spectrum of piasmodial attractants, determining if they too always produce changes in migration rate. Furthermore, the concentrations of the sugars used

271

in the present studies were approximately the same as those used by Carlile (1970) to demonstrate the presence of positive chemotaxis. In the study by Carlile, D-fructose was reported to be unable to support plasmodial growth and was hlso not found to be a plasmodial attractant. However, this may not be an exception to the hypothesis that plasmodial attractants also inhibit plasmodial migration rate, since it is clear in the present study that o-fructose does support the growth of the strain of P. ~o~ycepha~~~ utilized in this experiment. At the present time, experiments are continuing in an effort to determine whether or not the strain of P. po!ycephaium used in these experiments is attracted by D-fructose. The mechanism by which certain monosaccharides inhibit movement in the plasmodium remains to be elucidated. Miller (1972) suggested that certain carbohydrates might interact with receptors on the plasma membrane of the plasmodium (activating adenyl cyclase) leading to an elevation of the internal cyclic 3’:Sadenosine monophosphate (CAMP) concentration and that this in turn would alter migration rate. AIthough there is abundant evidence for a specific role for CAMP in controlling the movement (and morphogenesis) of the cellular slime molds, little information is available concerning the role of this nucleotide in the physiology of the acellular slime mold. In E. coli Adler (1975) discovered that the ability of these organisms to respond to specific attractant molecules (some of which were monosaccharides) was mediated by specific membrane-associated receptor proteins. Furthermore, these proteins were associated with membrane systems for the transport of these sugars from the external environment. Also. while the ability to transport the sugar was essential for positive chemotaxis, the ability to metabolize the sugar was not, since non-metabolizable analogs had the same attracting action as the normal substrates. Nothing is known about the specificity of monosaccharide transport in P. polycephalum but the present study does make clear the lack of correlation between the relative ability of a monosaccharide to inhibit migration and its relative ability to be metabolized in a way leading to net plasmodial growth. Ueda et al. (1975) measured changes in potential differences across the external surface of the plasmodium in response to varying concentrations of certain salts, amino acids and simple sugars shown to be capable of orienting the motive force of streaming of a piasm~ium in a Kamiya chamber. He .reported that substances capable of orienting the polarity of the motive force of streaming (i.e. D-galactose, D-glucose, o-mannose) exhibited specific concentration-dependent threshold effects on perturbing the potential difference from one portion of the surface of the external plasma membrane to another. Furthermore, these membrane perturbations occurred at exactly the same threshold concentratibn necessary to orient the polarity of the motive force. Interestingly, the threshold concentrations for these monosaccharides were in the same concentration range used by Carlile (1970) in his studies on chemotaxis and were also in the 0.025 to 0.05 M range in which inhibition of migration rate was observed in the present study.

32

JOHN R. DI+HO

and DONAI 1) M. MII I IX

The studies of Ueda t’f crl. (1975) also suggest why substances serving as plasmodial attractants might be expected to inhibit migration rate. In the Kamiya chamber the polarity of the motive force of streaming results from the net transport of protoplasm from one side of the chamber to the other. Jahn (1964) dctermined that the volume of protoplasm transported in any one time interval was proportional to the flow during that same interval. The amount of protoplasm participating in the flow at any one time would be determined by the gel- sol equilibrium within the plasInodiLiln. Therefore, any factor altering the gelLso1 ~~luiiibrium of the pl~~snl~~di~in~would be expected to shift the polarity of the motive force as measured by the Kamiya chamber. Mast & Presser (1932) observed that the maximum rate of movement in amebar was associated with the maintenance of an “optimum” sol gel ratio in the cytoplasm. Environmental factors such as changes in certain ion concentrations (including hydrogen ion) shifted the ratio away from the optimum and reduced or totally inhibited the rate of movement. Denbo & Miller (1976) have demonstrated that a similar situation also exists in plasmodia of P. i)t’i~,c.l,phcrlzcllr and explained the alteration of mjgration rate produced by changes in pH. metal cation concentrations and substrata with high osmotic pressures. If positive chemotaxis to certain monosaccharides was mediated at one point by processes leading to the alteration of the sol -gel ratio of the pkdsmodium away from its physiologically “optimum” point. then thts would obligatorily cause a decrease in plasmodial migration rate. The nature of the sol- gel system of the plasmodium remains unclear. Tremendous amounts of evidence reviewed by Komnick ut al. (1973) indicate the presence of the actomysinoid contractile gel system. On the other hand. Sheen c~fcl/. (1969) used histochemical staining techniques to demonstrate the presence of a mu~opolys~~cch~lride-like material making large contributions to the ge1 system of the cortical ectoplasm of the plasmodium. Furthermore. this material appeared to be of the same composition as that of the slime coat of the plasmodium. Since that time McCormic et ul. (1970) isolated and partially characterized the material of the slime coat and found a major constituent to be galactosamine. If galactosamine and mannosamine polysaccharides are also important constituents of a “second” plasmodial gel systern. then factors regulating the synthesis and degradation of this polysaccharide might be important regulators of the equilibrium of this system. Such an hypothesis might partially explain why I>-galactose and I,-mannose inhibited migration more effectively than n-glucose and it-fructose. REFERENCES AI)LI.R J. (lY75) Chemotaxls III bacteria. In Primitiw Sew,sor!’ mtl (‘ol?r,,llr,ric,otin,l S!,.UUJI,Y (Edi ted bq <‘AKLILI M. J.). Academic Press. New York. ANLIEHSON J. D. (19.51) Galvanotaxis of slime mold. .I. ~CVI.

PhK5kl.

3s. I 16.