RNA Metabolism during Regeneration in Stentor coeruleus1

80 Cytologia 31

RNA Metabolism during Regeneration

in Stentor coeruleus1

Leslie C. Ellwood2 and Ronald R. Cowden3,4

Department of Pathology, J. Hillis Miller Health Center,

University of Florida, Gainesville, Florida, U. S. A.

Received May 9, 1965

Introduction

The ciliate protozoan, Stentor coeruleus, has been a favored organism in which to investigate nucleo-cytoplasmic relationships during intracellular regeneration and reorganization. The morphology of stentors and the changes that occur during regeneration and division have been described in detail and were reviewed in Tartar's (1961) treatise. Studies of either regenerative or replicative morphogenesis have largely been concerned with the

production and ordering of cortical structures. The available experimental evidence, largely based on unpublished observations of Whiteley (cited in Tartar, 1961) and cyto

chemical studies of Weitz (1949) indicate that morphogenesis in stentors involves synthesis of new nucleic acids although Lewis (1962) has reported that no net protein synthesis, but rather interconversion, occurs during regeneration. Whiteley found that stentor regeneration is inhibited by the purine analog, 8-azaguanine, the pyrimidine analog, 2-thiocytosine, and by ribonuclease (RNase). Weisz (1955) had earlier reported that acriflavin, which inactivates nucleic acid metabolism, also inhibits stentor regeneration.

Tartar's (1961, 1963) microsurgical experiments have demonstrated that some function of the macronucleus is essential for regeneration up to the point of formation of the membranellar band primordium, but removal of the macronucleus beyond this point does not hinder the migration and invagination of the cortical structures. Removal of half the macronucleus does not affect the capacity of an individual to regenerate, and even individuals with only a single macronuclear node are capable of regeneration although its onset is retarded by about twenty-four hours. Thus, some quantitative dependence on macronuclear products for regeneration was demonstrated.

Evidence from these lines of investigation suggested that synthesis of new RNA in the macronucleus occurs in the early stages of stentor regeneration. Attempts to demonstrate this by conventional autoradiographic methods were frustrated by the relatively large metabolic pool in these organisms. Consequently, RNA metabolism in stentor regeneration was studied by testing the effects on regeneration of substances known to inhibit RNA synthesis, to destroy RNA, or to interfere with its function in protein synthesis. The antimetabolites employed were actinomycin D, which specifically suppresses DNA dependent RNA synthesis according to Reich et al. (1961), 5-fluorouracil (5-FU) a pyrimidine analogue which interferes with principally RNA synthesis and puromycin which Yarmolinsky and De La Haba (1959) found to specifically inhibit protein synthesis RNase was also used as an agent which might be expected to physiologically destroy RNA. These experiments with RNA antagonists were combined with qualitative and semi-quantitative cytochemical investigations of RNA distribution and levels.

1 This paper is based on a thesis presented in partial fulfillment of the requirements for the degree of Master of Science at the University of Florida, 1964, by L. C. Ellwood.

2 Supported by U . S. Public Health Service Pathology Training Grant 6-294-H-05.3 Supported by U . S. Public Health Service Career Award K3-HD-6176-03.4 Present address: Department of Anatomy

, Louisiana State University Medical School, 1542 Tulane Avenue, New Orleans, Louisiana 70112, U. S. A.

1966 RNA Metabolism during Regeneration in Stentor coeruleus 81

Material and methods

Stentor coeruleus cultures were obtained from a commercial source and maintained in

boiled and filtered pond water containing food organisms fed on a powdered milk solution

as recommended by Tartar (1961). In all experiments, a group of stentors was isolated

from the culture stocks, and all organisms used in a given experiment were treated

together. Regeneration was induced by a brief treatment with 4% urea which induces

shedding of the membranellar band. This was followed by several washes in filtered pond

water. Regeneration following urea damage was comparable in every respect to events

following the more laborious transection techniques.

Antimetabolites were added to pond water medium containing stentors as desired.

Actinomycin D was employed at concentrations between 0.5 to 25.0ƒÁ/ml, 5-FU was used

at 1•~10-3 to 8•~10-3M concentrations, and puromycin dihydrochloride was used at a

concentration of 1•~10-4M. RNase was used in concentrations of 0.05mg/ml to 0.5mg/ml.

The stentors studied cytochemically were fixed in ethanol-acetic acid (3:1). Both whole

mount and serial sections were prepared. For whole mounts, stentors in a minimum of

water were dropped into a small pool of fixative on a slide. Excess liquid was removed

with a capillary pipette after the organisms were firmly attached. The slides were then

transferred into fixative for an additional period, usually about thirty minutes. For

sections stentors were extended before fixation by adding an equal volume of 0.33%

copper acetate to the culture medium as recommended by Kirby (1950). After allowing

the salt to act for eight minutes, the organisms were placed in fixative. Following

fixation, the stentors were washed for ten minutes in 95, 70, 50, and 25 percent ethanol,

and finally water. They were then embedded in 2.5% agar and the cooled agar blocks

were hardened by twenty-four hours' treatment with ten percent neutral formalin. These

were trimmed, dehydrated through 70 and 95 percent ethanol, and absolute tertiary

butanol. They were then infiltrated in a 1:1 slush of tertiary butanol and paraffin, pure

paraffin, and embedded in paraffin. These blocks were sectioned serially at five micra.

Both whole mounts and sections were stained for RNA by the pH 4.0 azure B method

of Flax and Himes (1952) after prior incubation in DNase to remove DNA (Swift 1953).

Semi-quantitative measurements of macronuclear RNA levels were performed using a

commercial version of the microspectrophotometer designed by Pollister and Ris (1947) and

produced by E. Leitz, Inc. of New York. Measurements were performed on sectioned

material and only nodes which completely filled the thickness of the section were selected

for measurement. Three measurements using 590mƒÊ light were made on each of

the measured macronuclear nodes, and the resulting optical density values were averaged.

Ten macronuclear nodes from separate individuals were measured in each group. These

included vegetative stentors; normal regeneration four hours after urea treatment; normal

regeneration eight hours after urea treatment; stentors treated with 2.0ƒÁ/ml actinomycin

D for seventy-two hours; and regenerating stentors which had been subjected to 0.5mg/

ml RNase for twenty-two hours, then removed, treated with urea, and allowed to regenerate

for four hours.

Observations

Normal regeneration of stentors from stocks maintained during the

course of these investigations proceeds as described by Tartar (1961).

After removal of the membranellar band with the urea solution, there is

no apparent activity other than a gradual integration of the former frontal field into the rest of the cortical pattern. At four hours, an easily

discernible, glistening stripe appears at the primordium site, and within another hour, the membranellar cilia have appeared within this band. The

cytologia 31, 1966 6

82 L. C. Ellwood and R. R. Cowden Cytologia 31

entire regeneration process has been divided into seven stages by Tartar

(1961); the first appearance of cilia in the anlage has been defined as stage three. By adjustments in cortical form, the new membranellar band is brought to occupy the normal position and to enclose a new frontal field.

Invagination of a new gullet is completed within eight to nine hours. The

normally beaded macronucleus begins to condense at about the beginning of

gullet invagination and renodulates by the completion of regeneration.

Figs. 1-5. All preparations are 5ƒÊ

sections of stentors which were pre

treated with DNase and stained with

pH 4.0 azure B. Magnification of all

figures is 310•~. 1, normal vegetative

stentor. 2, stentor after 4 hours of

regeneration. 3, stentor after 8 hours

of regeneration. 4, stentor after 72

hours in 2.0ƒÁ/ml actinomycin D. 5,

stentor after 22 hours in 0.5mg/ml

RNase followed by 4 hours of regenera

tion.

As will be noted in Table 1, the microspectrophotometric measurements

of macronuclear levels of RNA indicated that some increase in RNA

concentration could be detected by the fourth hour prior to condensation of

the macronucleus, and that a considerable increase in macronuclear RNA

concentration was evident in the eight hour regenerates after the macronuclei

had renodulated. As may be seen in Fig. 1 through 3, there was some


Table 1. Average extinction values for macronuclei stained for RNA

Table 2. The effect of antimetabolites on regeneration

6*


corresponding increase in levels of cytoplasmic RNA, but this basophilia

was not measurable because of the presence of pigment granules as well as

an increased vesicularity of the cytoplasm. The basophilic material was not

located within the cytoplasmic vesicles, but surrounded them.

Regenerating stentors placed in concentrations of actinomycin D which

effectively suppresses DNA dependent RNA synthesis in mammalian cells

(0.5ƒÁ to 2.0ƒÁ/ml) regenerated on schedule, and did not appear to be

affected by this treatment. When stentors were serially induced to regenerate

in these levels of actinomycin D, they completed two cycles of regeneration

but regeneration was inhibited in the third cycle. Vegetative stentors placed

in 2.0ƒÁ/ml actinomycin D for 72 hours were unable to regenerate; in 90

percent of the cases inhibition was complete, but in 10 percent of the cases

regeneration reached stage three or four where they either remained or the

anlagen were resorbed. Cytochemical studies indicated that stentors treated

for 72 hours with this level of actinomycin D possessed essentially no

macronuclear or cytoplasmic RNA (see Table 1 and Fig. 4).

At higher concentrations actinomycin D suppressed the initial regeneration

in stentors. At a concentration of 15ƒÁ/ml some individuals attained stage

four, but no complete regeneration was noted. At a concentration of 25ƒÁ/

ml, regeneration could be inhibited even if added at the fourth hour, about

one hour prior to the attainment of stage four. Under these conditions,

some individuals reached stage four, but regeneration was never completed.

If this concentration of actinomycin D was added after the sixth hour,

regeneration was neither inhibited nor retarded, but the regenerated

individuals usually died about fifteen hours later.

Concentrations of 1.5ƒÁ/ml of actinomycin D prevents cell multiplication.

In the presence of an abundant food supply, the doubling time of stentors

is about five days. Control animals divided over this period, but the treated

animals did not increase in number.

The experiments with 5-FU gave a similar pattern. At concentrations

of 2•~10-3M, 5-FU did not inhibit the first two regeneration cycles. In

the third cycle no regeneration occurred and most of the organisms were

dead by the sixth hour. At a concentration of 8•~10-3M, some retardation

in the achievement of normal form was noted, and this treatment caused

some distortions in anterior cortical shape even after invagination of the

gullet was complete. A decrease in the size of stentors which did not attainn

normal form was also noted. After thirty-two hours in 8•~10-3M, 5-FU,

the regenerated stentors exhibited extreme cytoplasmic vacuolation and death

ensued before the forty-eighth hour. A concentration of 3•~10-3M did

not produce suppression of a second regeneration cycle, but a longer period

of time was required in the second cycle of regeneration to achieve normal

form.

Stentors treated with urea and placed in 0.5mg/ml RNase failed to


regenerate. Vegetative stentors placed in the same concentration of RNase

failed to regenerate if removed at 24 hours, washed in pond water, treated

with four percent urea, washed repeatedly in pond water, and allowed to

stand in this normal medium . Individuals removed from this RNase

concentration at twenty-two hours, however , were generally capable of

regenerating with only an hour or two's delay . Cytochemical studies

indicated that levels of basophilia in the twenty-two hour RNase treated

material were quite low, but by four hours after urea damage, the levels of

RNA had increased appreciably (Fig. 5), although microspectrophotometric

measurements on the macronuclei indicated that the RNA concentration was

only about a third of that in untreated four hour regenerates (Table 1).

While RNA levels increased during regeneration under these conditions,

levels of basophilia at the end of regeneration were not as high as in controls.

Similarly, basophilia appeared to increase more rapidly in regenerating

individuals than in those simply removed from RNase at the end of twenty

two hours, washed, and returned to pond water.

Puromycin completely inhibited stentor regeneration when used at 1•~

10-4M concentration. A concentration of 5•~10-4 was found to kill the

stentors within three hours. In the lower concentration, vacuolization was

noted near the holdfast at the fourth hour, and this vacuolization gradually

increased throughout the whole animal. Deaths were noted at the eighth

hour with no regeneration having occurred.

The results of various manipulations with antimetabolites on stentor

regeneration are presented in Table 2.

Discussion

The experiments presented in this study have indicated that the agents

known to destroy, prevent synthesis of, or interfere with the function of

RNA will, depending on the mode of action of the individual agent,

eventually suppress regeneration in stentors. The combined cytochemical

and experimental investigation of stentor regeneration has offered some insights

into specific aspects of RNA metabolism in this giant ciliate.

The cytochemical data indicate that both macronuclear and cytoplasmic

levels of RNA increase during the course of regeneration. From Tartar's

(1963) micrurgical experiments, however, it is clear that the essential function

of the macronucleus in regeneration occurs prior to the formation of the

membranellar band primordium. The higher concentration (15ƒÁ/ml-25ƒÁ/ml)

actinomycin D experiments leave little doubt that this function is the

production of new RNA, most probably messenger RNA. Since stable

messenger RNA may be present by six hours and protein synthesis can be

supported if adequate ribosomes are already present, higher levels of

actinomycin D introduced after six hours does not inhibit regeneration but

probably suppresses subsequent RNA synthesis. The fact that the organism


dies at about 15 hours, indicates that a general deprivation of new RNA cannot be tolerated. Since messenger RNA represents a relatively small proportion of the total RNA in any system thus far examined. the cytochemical results probably represent patterns of ribosomal RNA synthesis. They indicate that ribosomal RNA synthesis continues throughout regeneration.

The differential effects of high and low levels of actinomycin D can probably be attributed to the highly polyploid nature of the ciliate macronucleus which was documented for Paramecium by Woodard et al. (1961), and the necessity of blocking either all or a substantial portion of given genetic loci before it significantly affects synthesis of new proteins required in regeneration. Referring once more to the results of Tartar's (1963) micrurgical experiments, it appears that stentors may regenerate if they contain only a single macronuclear node, although the onset of regeneration is retarded. This suggests that a high degree of polyploidy may confer

protection against antimetabolites which act at genetic loci just as this condition protects against radiation damage. An alternative possibility to account for the concentration effect could be that the rate of actinomycin D

penetration may be low as Gross and Cousineau (1963) postulated for sea urchin eggs. The rapid effects of higher concentrations of actinomycin D, however, render this hypothesis less tenable in the case of stentors.

The effects of 5-FU appear to be quite similar to those obtained with actinomycin D. This pyrimidine analog would be expected to strongly affect RNA metabolism as well as other pathways concerned with growth according to Rich et al. (1959). On the other hand, puromycin interferes with the function of transfer RNA, and through this interference it inhibits protein synthesis. Since it acts on the function of RNA rather than its synthesis, an earlier effect on suppression of regeneration could be expected and was observed.

There is still some question about the ability of RNase and DNase to enter living cells and selectively destroy RNA and DNA (Alfert and Das 1912). Nevertheless, the experiments performed with stentor strongly suggest that RNase is capable of performing this role in this organism. Treatments with 0.5mg/ml RNase for twenty-four hours completely destroyed the capacity of stentors to regenerate, although animals treated for shorter times regenerated after a short lag period. Whiteley (unpublished observations reported in Tartar 1961) obtained similar results with RNase and found that the effects of RNase were reversible by addition of RNA to the medium. Longer treatments of vegetative stentors led to progressive decreases in cytoplasmic and macronuclear RNA levels, so the reductions in basophilia noted could not be reasonably explained as post-fixation action by residual enzyme. The rather short lag time noted in animals treated for twenty-two hours with 0.5mg/ml. RNase again suggests that stentors generally operate with a considerable excess of ribosomal RNA beyond the essential level. Although it could be demonstrated that RNA levels were rising by the fourth hour


after their removal from RNase and subsequent treatment with urea, the l

evels were about 1/3 of those obtained in four hour regenerates of untreated

stentors.

Despite many experimental advantages stentors offer such as size , ease of culture, and availability of methods which allow simultaneous induction

of regeneration in large numbers of organisms , they are far from ideal organisms in which to examine immediate metabolic events in intracellular

regeneration because of their large reserve of metabolites . The effects obtained with lower concentrations of actinomycin D and 5-FU are not

unlike the effects of starvation, although the onset is considerably more

acute. Manipulation with antimetabolites which interfere with RNA synthesis

and tracer experiments are more straight-forward in cells with diploid nuclei , or cells with lower levels of reserves.

Summary

1. RNA accumulated during the course of regeneration in both the

macronucleus and cytoplasm of stentors. Levels were higher at eight hours

near the end of regeneration than at four hours.

2. With both actinomycin D and 5-flurouracil, levels which inhibit

RNA synthesis in mammalian cells were initially ineffective in suppressing

regeneration in stentors, but after exposure for relatively long periods, levels

of macronuclear and cytoplasmic RNA was reduced, and regeneration was

inhibited.

3. Very high levels (15-25ƒÁ/ml) of actinomycin D will inhibit regen

eration in stentors if added before achievement of stage four, but if added

at the sixth hour, regeneration is completed. The critical product emanating

from the macronucleus prior to stage four is probably messenger RNA.

4. The results obtained with both actinomycin D and 5-flurouracil

suggest that the high degree of polyploidy of the macronucleus protects the

organism from the effects of antimetabolites which operate on genetic loci.

5. RNase at 0.5mg/ml concentration prevented regeneration, and

appeared to physiologically destroy RNA in the stentors, but organisms

which had almost been depleted of RNA were able to regenerate after a

very short lag period.

Literature cited

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fixation. Acta Histochem. 14: 321-326.

Flax, M. H. and Himes, M. H. 1952. Microspectrophotometric analysis of metachromatic

staining of nucleic acids. Physiological Zoology 25: 297-311.

Gross, P. R. and Cousineau, G. H. 1963. Effects of actinomycin D on macromolecule synthesis

and early development in sea urchin eggs. Biochem. and Biophys. Rec. comm. 10:

321-26.


Kirby, H. 1950. Materials and Methods in the Study of Protozoa. University of California

Press, Los Angeles.

Lewis, B. G. 1962. Protein metabolism in regenerating and non-regenerating Stentor

coeruleus. American Zoologist 2: 424-25.

Pollister, A. W. and Ris, H. 1947. Nucleoprotein determinations in cytological preparations.

Cold Spring Harbor Symp. Quant. Biol. 12: 147-157.

Reich, E., Franklin, R. M. Shatkin, A. J. and Tatum, E. L. 1961. Effect of actinomycin D

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Rich, M. A., Bolaffi, J. L., Knoll, J. E., Cheong, L. and Eidinoff, M. L. 1958. Growth inhibition

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RNA Metabolism during Regeneration in Stentor coeruleus1

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