The function of slime from Physarumflavicomum in the control of cell division HENRYR. HENNEY, JR., A N D MORTAZAASGARI
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Depnrtment ofBiology, University of Horrston, Hor~ston,Texas 77004 Accepted July 24, 1975 in H E N N E YH, . R., JR., and M. ASGARI.1975. T h e function of slime fromPl~ysrrr~rt~~Jklvicotn~rrn the control of cell division. Can. J . Microbiol. 21: 1866-1876. A haploid cell of the myxomycete Physorrrn~fln\~icotnumundergoes cytokinesis, producing a large population ofcells. However, after syngamy, cytokinesis no longeroccurs but karyokinesis does and subsequent growth results in the formation of a diploid syncytial plasmodium. Slime, which is produced by the plasmodium but not the haploid cells, was aseptically isolated and purified, and tested for its effect as a cytokinetic regulator. Slime (a viscous, high molecular weight, acidic glycoprotein) affected cytokinesis of the haploid myxamoebae growing in pure culture in soluble media, and the effect was concentration dependent. In simple media, a slime concentration of about 6 x lo-' p g protein per cell suppressed cytokinesisabout 50%. unequally inhibited the synthesis of protein, RNA, and DNA, but stimulated respiration. T h e biological activity of slime was not species specific and it also affected the bacterium BrrciN~rssrrbtilis by inhibiting cytokinesis, stimulating oxygen uptake, and producing an aberrant cell morphology. Slime was inactivated by heat, fragmentation, and incubation with dithiothreitol, mercaptoethanol, and the proteolytic enzyme papain (EC 3.4.22.2). The inhibitory effect of slime on cell division of haploid cells could not be achieved using mucin or various polyanions. The possible role of slime in the production of the diploid syncytium is discussed. H E N N E YH. . R.. JR., et M. ASGARI.1975. T h e function of slime from Pl~yscrrrrt~~flapnr~icon~rrtn in the control of cell division. Can. J. Microbiol. 21: 1866-1876. Une cellule haplolde d u rnyxomycetePl~ysrrrrrt~lflr~prr~icotnrrm produit, par suite de cytokineses, une grande population de cellules. Toutefois, apres la syngamie, la caryocinkse remplace la cytokinese et la croissance subsequente conduit h la formation d'un plasmode syncytial diplo'ide. La matikre visqueuse qui est produite par le plasmode, mais non par les cellules haploides, fut isolee e t purifiee aseptiquement, et testte pour son effet en tant que regulateur de cytokinese. La matiere visqueuse (une glycoproteine acide d e poids moleculaire eleve) affecte la cytokinese des myxamibes haploldes croissant en culture pure, en milieux solubles, et cet effet varie avec la concentration. Dans les milieux simples, une concentration en substance visqueuse d'environ wg de proteine par cellule riduit d'environ 50% la cytokinese, inhibe inegalement la 6 x synthese de proteine, de RNA et de DNA, mais stimule la respiration. L'activite biologique de la substance visqueuse n'est pas spicifique aux especes; elle affecte aussi la bacterie Bncillrrs slrbtilis en inhibant la cytokinese, en stimulant I'absorption d'oxygene et en produisant une morphologie cellulaire aberrante. La substance visqueuse est rendue inactive par la chaleur, la fragmentation et I'incubation en presence de dithiothreitol, de mercaptoethanol, e t de I'enzyme proteolitique papaine (EC 3.4.22.2). L'effet inhibiteur de la substance visqueuse s u r la division cellulaire des cellules haploides ne peut t t r e obtenu par I'emploi d e mucine ou de polyanions divers. Le r6le possible d e la matikre visqueuse dans la production de syncytes diploi'des est discute. [Traduit par le journal]
Introduction Evidence for the presence within cells of specific regulators of cell division has been presented in recent years. Egyiid and SzentGyorgyi (1966a, 19666) and Szent-Gyorgyi et al. (1967) found that a-ketoaldehydes inhibited cell division in concentrations that did not inhibit respiration. Using bacteria1 cultures they found that the a-ketoaldehydes altered division by 'Received M a y 28, 1975.
specifically interfering with protein synthesis. The inhibitor reacted with SH groupsof proteins, and protein synthesis was reactivated by thiol compounds. Because of the presence of a naturally occurring reductase for the a-ketoaldehydes in cells, the inhibition was reversible under conditions where proliferation was desirable. Also, recent work has dealt with specific endogenous mitotic inhibitors called chalones (Forscher and Houck 1973; Houck and
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HENNEY AND ASGARI: CONTROL O F CELL DIVISION
Hennings 1973). Chalones possess cell specificity and reversibility but lack species specificity. A wide variety of animal cells which appear to be under chalone control include epidermis, liver, melanocyte, fibroblast, kidney, granulocyte, and lymphocyte. The chalones appear to be macromolecules (protein or glycoprotein) of molecular weights about 30 000 to 50 000 (although the range of values reported is from 2000 to I00 000). Although chalones have not been completely purified, they do not appear to be excessively toxic and chalones inhibit tumor cells as well as controlling growth of normal tissue (Forscher and Houck 1973; Houck and Hennings 1973). The myxomycetes are eucaryotes that are ideally suited to research of this type because of their unique life cycle (Henney and Henney 1968b; Gray and Alexopoulos 1968; Cheung et a/. 1974). The life cycle is characterized by a number of stages which undergo differentiation. Physarum flavicomum is a heterothallic myxomycete (Henney and Henney 19686) whose diploid plasmodium (Henney and Henney 1 9 6 8 ~ ;Henney and Lynch 1969) and haploid myxamoebae swarm-cells (Henney et a/. 1974; Henney and Asgari 1975) can be grown in pure culture in partially and completely defined media under controlled laboratory conditions. The heterothallic, haploid cells can be maintained indefinitely in pure culture as long as opposite mating types are kept separate. These cells undergo cytokinesis and large populations of myxamoebae-swarm cells are produced. However, when opposite mating types are mixed, syngamy occurs and two myxamoebae or two swarm cells form a diploid zygote (Henney and Henney 19686). After zygote formation, cytokinesis no longer occurs but karyokinesis does, and subsequent growth results in the formation of a large multinucleate mass of protoplasm (the plasmodium) (Henney and Henney 19686; Gray and Alexopoulos 1968; Cheung et a/. 1974). Since cell division occurs in the haploid amoebae but not in the diploid plasmodium, it is reasonable to assume that the plasmodium might produce and contain a regulator (inhibitor) of cell division. Although growing diploid plasmodia of P. J¶avicomum contain and secrete slime, haploid myxamoebae-swarm cells d o not produce slime. It appears that the biosynthesis of slime is initiated at the time of formation of the acellular diploid phase. Slime is composed of protein
1867
consisting of at least 17 different amino acids, while the other component is a polysaccharide consisting of the hexose galactose (Simon and Henney 1970). Since there is no general agreement for naming such a polymer (Gottschalk 1972), we have loosely referred to it as a glycoprotein (Simon and Henney 1970). Correlated with the presence of slime in the growing plasmodium is the inability of the cell t o undergo cytokinesis. However, at one stage of the life cycle, individual cellular units (sclerotia) are produced by the differentiation and cytokinesis of the starving acellular plasmodium. Slime is abundantly eliminated from the differentiating plasmodium at the early stage of sclerotial formation (Cheung er a/. 1974). In ultrastructural studies slime has not been detected in sclerotia, which are the only diploid cellular stage of the life cycle (Cheung er ul. 1974). For these reasons we deduced that plasmodial slime could play a role as an endogenous regulator of cell division. In this study we demonstrate that plasmodial slime does affect cytokinesis, respiration, and macromolecular biosynthesis of haploid cells of P. J¶avicomum. In addition, slime also inhibits cell division and alters respiration and the morphology of the bacterium Bacillirs subtilis.
Materials and Methods Gro~vttrof Organisnis Pl~ysnrrtti~ flavicornitnr variety 1 plasmodia (American Type Culture Collection (ATCC) 26496) and haploid cells were isolated (Henney and Henney 19680, 19686; Henney e t 01. 1974) and maintained in semidefined (SD and BTC) and defined media as previously described (Henney and Henney 19680; Henney and Lynch 1969; Henney e t al. 1974; Henney and Asgari 1975). Growth was determined by increases in protein content and by direct cell counts using a hemacytometer (Neubauer), as previously described (Henney and Henney 19680; Henney et 01. 1974). Oxygen uptake was measured using a Gilson respirometer. Concanavalin A, chondroitin sulfate, bovine mucin, and dextran sulfate were obtained from Sigma; heparin was from Nutritional Biochemical Corporation. Growth of Bacillrrs subtilis (ATCC 6051) in 3.1% trypticase soy broth (Baltimore Biological Laboratories) was measured by monitoring optical density a t 660 nm. Aseptic Isolation of Slime Procedures for the isolation and chemical analyses of slime produced by P. flaoicomr~rn plasmodia grown for 6 days in S D medium (but containing a final concentration of only 1.25 pg hematinlml) have been published (Simon and Henney 1970). However, in this study, aseptic conditions were maintained by collecting slime, a n d precipitating it with 3 volumes of 95% alcohol,
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CAN. J. MICROBIO L. VOL. 21, 1975
in sterile 250-ml Nalgene centrifuge bottles. The slime precipitate was quickly washed free of residual ethanol with 118 volume sterile distilled water and then dissolved in 118 volume sterile distilled water by incubation at 4 "C for 48 h. Acidified acetone precipitation (Simon and Henney 1970) was omitted in this work since it increased fragmentation of slime. The sterility of the slime was confirmed by incubation in five microbiological test media previously described (Henney and Henney 1968b), as well a s S D and BTC media. Slime viscosity was measured at 25 "C using a Cannon viscometer. Biological Activity of Slittre The effect of slime on growth of cultures was tested by adding slime to test cultures, and an equal volume of sterile distilled water to the control cultures. These cultures, therefore, contained equivalent concentrations of medium and inoculum. Some experiments were performed by adding slime plus an equal volume of twice-concentrated medium to the test cultures; the control cultures were unaltered so that they contained the same total volume and concentration of medium plus inoculum. Samples were then removed from these cultures, at specified intervals, for growth analyses.
Slime Modification In some experiments, slime, at a final concentration of 200 pg protein/ml, was incubated on a rotary shaker (160 rpm) for 24 h a t 36 "C with 5 m M 0-mercaptoethanol (ME), or 5 m M dithiothreitol (DTT), or 1 mg/ml papain (EC 3.4.22.2) (Worthington). Slime was reisolated by alcohol precipitation and dissolved in glassdistilled water. Electron Microscopy Samples were fixed for 3 h in 2.5% glutaraldehyde buffered with 0.15 M sodium cacodylate, pH 7.2, and washed three times with the same buffer. Samples were dehydrated through a graded series of acetone, lyophilized, and coated with a thin layer of gold. Observations were made with a Cambridge S4-10 Stereoscan electron microscope (SEM) at 10 kV and recorded o n Polaroid type 55 film.
Results The effect of slime on the growth and oxygen uptake of haploid cells of P. jlauicon7um in S D medium (Henney et al. 1974) is shown in Fig. I . Cells in S D media containing 3.2 x l o p 4 pg Biosyt~tlresisof Macrot?rolec~~les pg slime Haploid cells of P. fi'avicot~i~itn were incubated in BTC slime protein per cell and 4.8 x medium (with and without slime) containing either protein per cell attained a cell yield at 5 days of 5 pCi uniformly labelled 14C-protein hydrolysate (Sch- only 66% and 59%, respectively, of control warz/Mann, Orangeburg, New York), or 5 pCi [2-14C]- cultures without slime (Fig. 1A). However, uracil, or 10 pCi [2-14C]thymine (New England Nuclear, Boston). Samples were removed at 18-h intervals, added oxygen uptake was stimulated 21% and 62%, respectively, over that of control cultures to an equal volume of chilled 10% (w/v) trichloroacetic acid (TCA), and incubated for 30min at 4°C. The without slime (Fig. 1 B). precipitates were collected by centrifugation at 6000 x g Figure 2 shows the effect of slime on growth for 10 min and then washed twice with chilled 5% and oxygen uptake of haploid cells in the simpler TCA. The precipitates were dissolved in Hyamine BTC medium (yeast extract of SD medium rehydroxide, added t o cocktail A, and radioactivities determined using a liquid scintillation spectrometer as placed by biotin and thiamine; Henney et al. previously described (Lynch and Henney 1973). 1974). At slime protein concentrations of pg per cell, pg per cell and 8 x 4 x Electrophoresis oxygen uptake was stimulated 22% and 47%, Polyacrylamide-gel disk electrophoresis was performed on slime by use of 3.74, and 5% acrylamide respectively, over that of control cultures (Fig. 2A), but cell yields were only 82x and 41 % separating gels and a buffer pH of 9.5. The chemical formulation for this process was that outlined by Canalco of the corresponding control cultures (Fig. 2B). (1965 Data Sheet, Canal Industries, Bethesda, Maryland). In the BTC medium (Henney et al. 1974) and The slime was detected in the gels by staining with 0.5% defined medium M4 (Henney and Asgari 1975), alcian blue (glycoprotein stain), or, in some cases, 0.2% pg coomassie blue (protein stain). All gels were measured slime protein concentrations near 6 x and the R, values were calculated by dividing the distance per cell produced a growth inhibition of about the slime traveled from the top of the gel (cathode) 50%. A 50% inhibition of cell division in the by the distance the tracking dye traveled. complex S D medium required 5 to 10 times higher slime-protein concentrations per cell Isoelectric Focusing than in the simpler media. Isoelectric focusing (LKB, Rockville, Maryland) was The effect of various concentrations of slime performed, with a pH range of 3.5 to 10 and 3 to 6 using a 14, ampholyte concentration and a stabilizer &50% on haploid cells growing in BTC medium is gradient of sucrose, t o determine the purity of the slime illustrated in Fig. 3. The slime protein concentraand the isoelectric point (PI). The samples used in each tions were 3, 7, 9, and 12 x pg per cell, experiment contained. 10 to 25 mg slime protein. Slime which produced a stimulation of oxygen uptake fractions after isoelectric focusing were dialyzed for 96 h of 10, 15, 21, and 40%, respectively, compared using six 100-volume changes of glass-distilled water.
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HENNEY AND ASGARI: CONTROL O F CELL DIVISION
MINUTES
FIG.I . The effect of plasmodia1 slime on (A) the cell division, and (B) oxygen uptake of haploid cells of P.flavicornum in SD medium. Cells were grown at 25 "C on a rotary shaker (165 rpm). Test flasks contained slime and a n equal volume of twice-concentrated medium. Test and control flasks contained equivalent concentrations of medium and inoculum. Oxygen consumption was measured with a Gilson respirometer at 25 "C. Culture with no slime, a; culture with 3.2 x pg slime protein per cell, ;culture with 4.8 x pg slime protein per cell, 0.
with controls (Fig. 3A), but cell yields of only 69, 34, 15, and 12%, respectively, of control cultures (Fig. 3B). Slime-inhibited cells were viable after transfer to fresh media. Figure 4 illustrates the similar appearance of normal myxamoebae and myxamoebae inhibited by slime as determined with the scanning electron microscope. There is no evidence for the presence of a slime coat on the cell surface of inhibited cells. The influence of slime on the incorporation of radioactive precursors into protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) of haploid cells of P. flavicomum is summarized in Table 1. The presence of slime decreased incorporation of radioactivity into those fractions by 54, 66, and 43%, respectively,
when compared with control cultures. These calculations were on a basis of counts per minute per cell yield and do not merely reflect the decreased cell yield obtained in the presence of slime (Table 1). The slime used in this study was dissolved in sterile glass-distilled water after isolation and alcohol precipitation, since it was determined that dissolving it in the basal salts used in our media (Henney and Lynch 1969), potassium phosphate buffer (0.02 M, pH 7.5), or tris(hydroxymethy1)aminomethane buffer (0.02 M, pH 7.5) did not improve its biological activity (i.e., ability to inhibit growth of haploid cells). Also, sterile slime stored at room temperature, 4 "C, or in the freezer, exhibited the same biological activity. However, as illustrated in Fig.
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CAN. J. MICROBIOL.
FIG. 2. The effect of plasmodial slime on the (A) oxygen uptake, and (B) cell division of haploid cells of P. flaoicomutn grown in BTC medium. The cells were grown at 25 "C on a rotary shaker (165 rpm). Oxygen consumption was measured at 25 "C with a Gilson respirometer. Control flasks contained a volume of water equivalent to the volume of slime used in test flasks. Culture with 4 x pg slime protein per cell, 0 , and control culture, 0;culture with 8 x pg slime protein per cell, 0 , and control culture, 1.
VOL. 21,
1975
5A, after incubation at temperatures above 40 "C the biological activity was decreased. The greatest loss of activity occurred between 80 and 100 "C (Fig. 5A) and slime incubated in a boiling water bath for 15 min was devoid of biological activity. Slime also lost its growth inhibitory properties when incubated at 51 "C for various periods of time (Fig. 5B). The greatest loss of biological activity occurred after incubation for 80 to 100 min at 51 "C (Fig. 5B). Increased handling, such as repeated alcohol precipitations, caused fragmentation of slime and reduced its biological activity. This accounts for the variability in activity we encountered in some of our initial experiments. Other treatments also reduced the biological activity of slime. For example, exposure of slime to DTT, ME, or the proteolytic enzyme papain, eliminated its inhibitory effect on the growth of haploid cells of P. Jlavicomzim. In addition, the viscosity of slime was reduced by those treatments. The relative viscosity of native slime, at a concentration of 10 pg protein per millilitre, was estimated to be 1.15 centipoise. The relative viscosities after slime was exposed to DTT, ME, papain, or boiling were, respectively, 74, 80, 72, and 73% of that of native slime. Disk electrophoresis of biologically active slime using 5% and 3.7z acrylamide separating gels resulted in the slime not entering the separating gels (Fig. 6A). Bovine mucin also - 14
-. -A
6-
A
*
B
/ -12
A
u
,i - 1 0
I
.
E
-8
-
N
-
2-
-
""
-4 -2 9
30 MIHUTtS
60
70 0
I
'
4
7
10
0A1S
FIG.3. The effect of various concentrations of plasmodial slime on the (A) respiration, and (B) cell division of haploid cells of P.flaoicomum. The cells were grown in BTC medium a t 25 "C on a rotary shaker (165 rpm). The oxygen uptake was measured at 25 "C with a Gilson respirometer. Dilutions of medium in test flasks, because of the addition of slime, were corrected by the addition of a n equal volume of twice-concentrated BTC medium. Control ( 0 ) ; cultures with 3 x l o w 5( O ) , 7 x lo-' ( I ) , 9 x (A), a n d 1.2 x (A) pg slime protein per cell, respectively.
1871
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H E N N E Y AND ASGARI: CONTROL O F C E L L DIVISION
FIG.4. Scanning electron microscopy of myxamoebae of Pliysur~~niflavicomum. ( A ) normal cells, x 3000; (B) slime-inhibited cells, x 6800.
failed to enter the gels. However, after exposure of slime to DTT, ME, or papain, the slime penetrated the 5% acrylamide separating gels, and after staining with alcian blue only one band was noted with an RJ of about 0.17
(Fig. 6B, C , E). This band also stained weakly with coomassie blue (Fig. 6D). Isoelectric focusing of slime for about 12 h revealed the presence of two closely positioned slime fractions which were at the acidic end of
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CAN. J. MICROBIOL. VOL. 21, 1975
TABLE 1
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Influence of slime on incorporation of radioactive precursors into macromolecules
Precursor
Radioactive fraction
U-I4C-protein hydrolysate U-14C-protein hydrolysate
Protein Protein
-
RN A RN A
-
[2-'4C]uracil [2-14C]uracil [2-I4C]thymine [2-14C]thymine
Slimea
Total cpm per cultureb
+
825 410 246 459
825 410 381 515
%
incorporation 100.0 46.2 100.0 33.6
+ +
DN.4 DNA
Cpm + Cell yield per culturebsc cell yield
100.0 57.1
+
Indicates absence; indicates presence of 5 x lo-' ug slime plrotein per cell. bAfler 54 h incubation in BTC medium.
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Depnrtment ofBiology, University of Horrston, Hor~ston,Texas 77004 Accepted July 24, 1975 in H E N N E YH, . R., JR., and M. ASGARI.1975. T h e function of slime fromPl~ysrrr~rt~~Jklvicotn~rrn the control of cell division. Can. J . Microbiol. 21: 1866-1876. A haploid cell of the myxomycete Physorrrn~fln\~icotnumundergoes cytokinesis, producing a large population ofcells. However, after syngamy, cytokinesis no longeroccurs but karyokinesis does and subsequent growth results in the formation of a diploid syncytial plasmodium. Slime, which is produced by the plasmodium but not the haploid cells, was aseptically isolated and purified, and tested for its effect as a cytokinetic regulator. Slime (a viscous, high molecular weight, acidic glycoprotein) affected cytokinesis of the haploid myxamoebae growing in pure culture in soluble media, and the effect was concentration dependent. In simple media, a slime concentration of about 6 x lo-' p g protein per cell suppressed cytokinesisabout 50%. unequally inhibited the synthesis of protein, RNA, and DNA, but stimulated respiration. T h e biological activity of slime was not species specific and it also affected the bacterium BrrciN~rssrrbtilis by inhibiting cytokinesis, stimulating oxygen uptake, and producing an aberrant cell morphology. Slime was inactivated by heat, fragmentation, and incubation with dithiothreitol, mercaptoethanol, and the proteolytic enzyme papain (EC 3.4.22.2). The inhibitory effect of slime on cell division of haploid cells could not be achieved using mucin or various polyanions. The possible role of slime in the production of the diploid syncytium is discussed. H E N N E YH. . R.. JR., et M. ASGARI.1975. T h e function of slime from Pl~yscrrrrt~~flapnr~icon~rrtn in the control of cell division. Can. J. Microbiol. 21: 1866-1876. Une cellule haplolde d u rnyxomycetePl~ysrrrrrt~lflr~prr~icotnrrm produit, par suite de cytokineses, une grande population de cellules. Toutefois, apres la syngamie, la caryocinkse remplace la cytokinese et la croissance subsequente conduit h la formation d'un plasmode syncytial diplo'ide. La matikre visqueuse qui est produite par le plasmode, mais non par les cellules haploides, fut isolee e t purifiee aseptiquement, et testte pour son effet en tant que regulateur de cytokinese. La matiere visqueuse (une glycoproteine acide d e poids moleculaire eleve) affecte la cytokinese des myxamibes haploldes croissant en culture pure, en milieux solubles, et cet effet varie avec la concentration. Dans les milieux simples, une concentration en substance visqueuse d'environ wg de proteine par cellule riduit d'environ 50% la cytokinese, inhibe inegalement la 6 x synthese de proteine, de RNA et de DNA, mais stimule la respiration. L'activite biologique de la substance visqueuse n'est pas spicifique aux especes; elle affecte aussi la bacterie Bncillrrs slrbtilis en inhibant la cytokinese, en stimulant I'absorption d'oxygene et en produisant une morphologie cellulaire aberrante. La substance visqueuse est rendue inactive par la chaleur, la fragmentation et I'incubation en presence de dithiothreitol, de mercaptoethanol, e t de I'enzyme proteolitique papaine (EC 3.4.22.2). L'effet inhibiteur de la substance visqueuse s u r la division cellulaire des cellules haploides ne peut t t r e obtenu par I'emploi d e mucine ou de polyanions divers. Le r6le possible d e la matikre visqueuse dans la production de syncytes diploi'des est discute. [Traduit par le journal]
Introduction Evidence for the presence within cells of specific regulators of cell division has been presented in recent years. Egyiid and SzentGyorgyi (1966a, 19666) and Szent-Gyorgyi et al. (1967) found that a-ketoaldehydes inhibited cell division in concentrations that did not inhibit respiration. Using bacteria1 cultures they found that the a-ketoaldehydes altered division by 'Received M a y 28, 1975.
specifically interfering with protein synthesis. The inhibitor reacted with SH groupsof proteins, and protein synthesis was reactivated by thiol compounds. Because of the presence of a naturally occurring reductase for the a-ketoaldehydes in cells, the inhibition was reversible under conditions where proliferation was desirable. Also, recent work has dealt with specific endogenous mitotic inhibitors called chalones (Forscher and Houck 1973; Houck and
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HENNEY AND ASGARI: CONTROL O F CELL DIVISION
Hennings 1973). Chalones possess cell specificity and reversibility but lack species specificity. A wide variety of animal cells which appear to be under chalone control include epidermis, liver, melanocyte, fibroblast, kidney, granulocyte, and lymphocyte. The chalones appear to be macromolecules (protein or glycoprotein) of molecular weights about 30 000 to 50 000 (although the range of values reported is from 2000 to I00 000). Although chalones have not been completely purified, they do not appear to be excessively toxic and chalones inhibit tumor cells as well as controlling growth of normal tissue (Forscher and Houck 1973; Houck and Hennings 1973). The myxomycetes are eucaryotes that are ideally suited to research of this type because of their unique life cycle (Henney and Henney 1968b; Gray and Alexopoulos 1968; Cheung et a/. 1974). The life cycle is characterized by a number of stages which undergo differentiation. Physarum flavicomum is a heterothallic myxomycete (Henney and Henney 19686) whose diploid plasmodium (Henney and Henney 1 9 6 8 ~ ;Henney and Lynch 1969) and haploid myxamoebae swarm-cells (Henney et a/. 1974; Henney and Asgari 1975) can be grown in pure culture in partially and completely defined media under controlled laboratory conditions. The heterothallic, haploid cells can be maintained indefinitely in pure culture as long as opposite mating types are kept separate. These cells undergo cytokinesis and large populations of myxamoebae-swarm cells are produced. However, when opposite mating types are mixed, syngamy occurs and two myxamoebae or two swarm cells form a diploid zygote (Henney and Henney 19686). After zygote formation, cytokinesis no longer occurs but karyokinesis does, and subsequent growth results in the formation of a large multinucleate mass of protoplasm (the plasmodium) (Henney and Henney 19686; Gray and Alexopoulos 1968; Cheung et a/. 1974). Since cell division occurs in the haploid amoebae but not in the diploid plasmodium, it is reasonable to assume that the plasmodium might produce and contain a regulator (inhibitor) of cell division. Although growing diploid plasmodia of P. J¶avicomum contain and secrete slime, haploid myxamoebae-swarm cells d o not produce slime. It appears that the biosynthesis of slime is initiated at the time of formation of the acellular diploid phase. Slime is composed of protein
1867
consisting of at least 17 different amino acids, while the other component is a polysaccharide consisting of the hexose galactose (Simon and Henney 1970). Since there is no general agreement for naming such a polymer (Gottschalk 1972), we have loosely referred to it as a glycoprotein (Simon and Henney 1970). Correlated with the presence of slime in the growing plasmodium is the inability of the cell t o undergo cytokinesis. However, at one stage of the life cycle, individual cellular units (sclerotia) are produced by the differentiation and cytokinesis of the starving acellular plasmodium. Slime is abundantly eliminated from the differentiating plasmodium at the early stage of sclerotial formation (Cheung er a/. 1974). In ultrastructural studies slime has not been detected in sclerotia, which are the only diploid cellular stage of the life cycle (Cheung er ul. 1974). For these reasons we deduced that plasmodial slime could play a role as an endogenous regulator of cell division. In this study we demonstrate that plasmodial slime does affect cytokinesis, respiration, and macromolecular biosynthesis of haploid cells of P. J¶avicomum. In addition, slime also inhibits cell division and alters respiration and the morphology of the bacterium Bacillirs subtilis.
Materials and Methods Gro~vttrof Organisnis Pl~ysnrrtti~ flavicornitnr variety 1 plasmodia (American Type Culture Collection (ATCC) 26496) and haploid cells were isolated (Henney and Henney 19680, 19686; Henney e t 01. 1974) and maintained in semidefined (SD and BTC) and defined media as previously described (Henney and Henney 19680; Henney and Lynch 1969; Henney e t al. 1974; Henney and Asgari 1975). Growth was determined by increases in protein content and by direct cell counts using a hemacytometer (Neubauer), as previously described (Henney and Henney 19680; Henney et 01. 1974). Oxygen uptake was measured using a Gilson respirometer. Concanavalin A, chondroitin sulfate, bovine mucin, and dextran sulfate were obtained from Sigma; heparin was from Nutritional Biochemical Corporation. Growth of Bacillrrs subtilis (ATCC 6051) in 3.1% trypticase soy broth (Baltimore Biological Laboratories) was measured by monitoring optical density a t 660 nm. Aseptic Isolation of Slime Procedures for the isolation and chemical analyses of slime produced by P. flaoicomr~rn plasmodia grown for 6 days in S D medium (but containing a final concentration of only 1.25 pg hematinlml) have been published (Simon and Henney 1970). However, in this study, aseptic conditions were maintained by collecting slime, a n d precipitating it with 3 volumes of 95% alcohol,
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CAN. J. MICROBIO L. VOL. 21, 1975
in sterile 250-ml Nalgene centrifuge bottles. The slime precipitate was quickly washed free of residual ethanol with 118 volume sterile distilled water and then dissolved in 118 volume sterile distilled water by incubation at 4 "C for 48 h. Acidified acetone precipitation (Simon and Henney 1970) was omitted in this work since it increased fragmentation of slime. The sterility of the slime was confirmed by incubation in five microbiological test media previously described (Henney and Henney 1968b), as well a s S D and BTC media. Slime viscosity was measured at 25 "C using a Cannon viscometer. Biological Activity of Slittre The effect of slime on growth of cultures was tested by adding slime to test cultures, and an equal volume of sterile distilled water to the control cultures. These cultures, therefore, contained equivalent concentrations of medium and inoculum. Some experiments were performed by adding slime plus an equal volume of twice-concentrated medium to the test cultures; the control cultures were unaltered so that they contained the same total volume and concentration of medium plus inoculum. Samples were then removed from these cultures, at specified intervals, for growth analyses.
Slime Modification In some experiments, slime, at a final concentration of 200 pg protein/ml, was incubated on a rotary shaker (160 rpm) for 24 h a t 36 "C with 5 m M 0-mercaptoethanol (ME), or 5 m M dithiothreitol (DTT), or 1 mg/ml papain (EC 3.4.22.2) (Worthington). Slime was reisolated by alcohol precipitation and dissolved in glassdistilled water. Electron Microscopy Samples were fixed for 3 h in 2.5% glutaraldehyde buffered with 0.15 M sodium cacodylate, pH 7.2, and washed three times with the same buffer. Samples were dehydrated through a graded series of acetone, lyophilized, and coated with a thin layer of gold. Observations were made with a Cambridge S4-10 Stereoscan electron microscope (SEM) at 10 kV and recorded o n Polaroid type 55 film.
Results The effect of slime on the growth and oxygen uptake of haploid cells of P. jlauicon7um in S D medium (Henney et al. 1974) is shown in Fig. I . Cells in S D media containing 3.2 x l o p 4 pg Biosyt~tlresisof Macrot?rolec~~les pg slime Haploid cells of P. fi'avicot~i~itn were incubated in BTC slime protein per cell and 4.8 x medium (with and without slime) containing either protein per cell attained a cell yield at 5 days of 5 pCi uniformly labelled 14C-protein hydrolysate (Sch- only 66% and 59%, respectively, of control warz/Mann, Orangeburg, New York), or 5 pCi [2-14C]- cultures without slime (Fig. 1A). However, uracil, or 10 pCi [2-14C]thymine (New England Nuclear, Boston). Samples were removed at 18-h intervals, added oxygen uptake was stimulated 21% and 62%, respectively, over that of control cultures to an equal volume of chilled 10% (w/v) trichloroacetic acid (TCA), and incubated for 30min at 4°C. The without slime (Fig. 1 B). precipitates were collected by centrifugation at 6000 x g Figure 2 shows the effect of slime on growth for 10 min and then washed twice with chilled 5% and oxygen uptake of haploid cells in the simpler TCA. The precipitates were dissolved in Hyamine BTC medium (yeast extract of SD medium rehydroxide, added t o cocktail A, and radioactivities determined using a liquid scintillation spectrometer as placed by biotin and thiamine; Henney et al. previously described (Lynch and Henney 1973). 1974). At slime protein concentrations of pg per cell, pg per cell and 8 x 4 x Electrophoresis oxygen uptake was stimulated 22% and 47%, Polyacrylamide-gel disk electrophoresis was performed on slime by use of 3.74, and 5% acrylamide respectively, over that of control cultures (Fig. 2A), but cell yields were only 82x and 41 % separating gels and a buffer pH of 9.5. The chemical formulation for this process was that outlined by Canalco of the corresponding control cultures (Fig. 2B). (1965 Data Sheet, Canal Industries, Bethesda, Maryland). In the BTC medium (Henney et al. 1974) and The slime was detected in the gels by staining with 0.5% defined medium M4 (Henney and Asgari 1975), alcian blue (glycoprotein stain), or, in some cases, 0.2% pg coomassie blue (protein stain). All gels were measured slime protein concentrations near 6 x and the R, values were calculated by dividing the distance per cell produced a growth inhibition of about the slime traveled from the top of the gel (cathode) 50%. A 50% inhibition of cell division in the by the distance the tracking dye traveled. complex S D medium required 5 to 10 times higher slime-protein concentrations per cell Isoelectric Focusing than in the simpler media. Isoelectric focusing (LKB, Rockville, Maryland) was The effect of various concentrations of slime performed, with a pH range of 3.5 to 10 and 3 to 6 using a 14, ampholyte concentration and a stabilizer &50% on haploid cells growing in BTC medium is gradient of sucrose, t o determine the purity of the slime illustrated in Fig. 3. The slime protein concentraand the isoelectric point (PI). The samples used in each tions were 3, 7, 9, and 12 x pg per cell, experiment contained. 10 to 25 mg slime protein. Slime which produced a stimulation of oxygen uptake fractions after isoelectric focusing were dialyzed for 96 h of 10, 15, 21, and 40%, respectively, compared using six 100-volume changes of glass-distilled water.
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HENNEY AND ASGARI: CONTROL O F CELL DIVISION
MINUTES
FIG.I . The effect of plasmodia1 slime on (A) the cell division, and (B) oxygen uptake of haploid cells of P.flavicornum in SD medium. Cells were grown at 25 "C on a rotary shaker (165 rpm). Test flasks contained slime and a n equal volume of twice-concentrated medium. Test and control flasks contained equivalent concentrations of medium and inoculum. Oxygen consumption was measured with a Gilson respirometer at 25 "C. Culture with no slime, a; culture with 3.2 x pg slime protein per cell, ;culture with 4.8 x pg slime protein per cell, 0.
with controls (Fig. 3A), but cell yields of only 69, 34, 15, and 12%, respectively, of control cultures (Fig. 3B). Slime-inhibited cells were viable after transfer to fresh media. Figure 4 illustrates the similar appearance of normal myxamoebae and myxamoebae inhibited by slime as determined with the scanning electron microscope. There is no evidence for the presence of a slime coat on the cell surface of inhibited cells. The influence of slime on the incorporation of radioactive precursors into protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) of haploid cells of P. flavicomum is summarized in Table 1. The presence of slime decreased incorporation of radioactivity into those fractions by 54, 66, and 43%, respectively,
when compared with control cultures. These calculations were on a basis of counts per minute per cell yield and do not merely reflect the decreased cell yield obtained in the presence of slime (Table 1). The slime used in this study was dissolved in sterile glass-distilled water after isolation and alcohol precipitation, since it was determined that dissolving it in the basal salts used in our media (Henney and Lynch 1969), potassium phosphate buffer (0.02 M, pH 7.5), or tris(hydroxymethy1)aminomethane buffer (0.02 M, pH 7.5) did not improve its biological activity (i.e., ability to inhibit growth of haploid cells). Also, sterile slime stored at room temperature, 4 "C, or in the freezer, exhibited the same biological activity. However, as illustrated in Fig.
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CAN. J. MICROBIOL.
FIG. 2. The effect of plasmodial slime on the (A) oxygen uptake, and (B) cell division of haploid cells of P. flaoicomutn grown in BTC medium. The cells were grown at 25 "C on a rotary shaker (165 rpm). Oxygen consumption was measured at 25 "C with a Gilson respirometer. Control flasks contained a volume of water equivalent to the volume of slime used in test flasks. Culture with 4 x pg slime protein per cell, 0 , and control culture, 0;culture with 8 x pg slime protein per cell, 0 , and control culture, 1.
VOL. 21,
1975
5A, after incubation at temperatures above 40 "C the biological activity was decreased. The greatest loss of activity occurred between 80 and 100 "C (Fig. 5A) and slime incubated in a boiling water bath for 15 min was devoid of biological activity. Slime also lost its growth inhibitory properties when incubated at 51 "C for various periods of time (Fig. 5B). The greatest loss of biological activity occurred after incubation for 80 to 100 min at 51 "C (Fig. 5B). Increased handling, such as repeated alcohol precipitations, caused fragmentation of slime and reduced its biological activity. This accounts for the variability in activity we encountered in some of our initial experiments. Other treatments also reduced the biological activity of slime. For example, exposure of slime to DTT, ME, or the proteolytic enzyme papain, eliminated its inhibitory effect on the growth of haploid cells of P. Jlavicomzim. In addition, the viscosity of slime was reduced by those treatments. The relative viscosity of native slime, at a concentration of 10 pg protein per millilitre, was estimated to be 1.15 centipoise. The relative viscosities after slime was exposed to DTT, ME, papain, or boiling were, respectively, 74, 80, 72, and 73% of that of native slime. Disk electrophoresis of biologically active slime using 5% and 3.7z acrylamide separating gels resulted in the slime not entering the separating gels (Fig. 6A). Bovine mucin also - 14
-. -A
6-
A
*
B
/ -12
A
u
,i - 1 0
I
.
E
-8
-
N
-
2-
-
""
-4 -2 9
30 MIHUTtS
60
70 0
I
'
4
7
10
0A1S
FIG.3. The effect of various concentrations of plasmodial slime on the (A) respiration, and (B) cell division of haploid cells of P.flaoicomum. The cells were grown in BTC medium a t 25 "C on a rotary shaker (165 rpm). The oxygen uptake was measured at 25 "C with a Gilson respirometer. Dilutions of medium in test flasks, because of the addition of slime, were corrected by the addition of a n equal volume of twice-concentrated BTC medium. Control ( 0 ) ; cultures with 3 x l o w 5( O ) , 7 x lo-' ( I ) , 9 x (A), a n d 1.2 x (A) pg slime protein per cell, respectively.
1871
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H E N N E Y AND ASGARI: CONTROL O F C E L L DIVISION
FIG.4. Scanning electron microscopy of myxamoebae of Pliysur~~niflavicomum. ( A ) normal cells, x 3000; (B) slime-inhibited cells, x 6800.
failed to enter the gels. However, after exposure of slime to DTT, ME, or papain, the slime penetrated the 5% acrylamide separating gels, and after staining with alcian blue only one band was noted with an RJ of about 0.17
(Fig. 6B, C , E). This band also stained weakly with coomassie blue (Fig. 6D). Isoelectric focusing of slime for about 12 h revealed the presence of two closely positioned slime fractions which were at the acidic end of
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CAN. J. MICROBIOL. VOL. 21, 1975
TABLE 1
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Influence of slime on incorporation of radioactive precursors into macromolecules
Precursor
Radioactive fraction
U-I4C-protein hydrolysate U-14C-protein hydrolysate
Protein Protein
-
RN A RN A
-
[2-'4C]uracil [2-14C]uracil [2-I4C]thymine [2-14C]thymine
Slimea
Total cpm per cultureb
+
825 410 246 459
825 410 381 515
%
incorporation 100.0 46.2 100.0 33.6
+ +
DN.4 DNA
Cpm + Cell yield per culturebsc cell yield
100.0 57.1
+
Indicates absence; indicates presence of 5 x lo-' ug slime plrotein per cell. bAfler 54 h incubation in BTC medium.
