The role of the nucleus in the formation and maintenance of the contractile vacuole in Amoeba proteus

Experimental Cell Research 62 (1970) 326-330

THE ROLE OF THE NUCLEUS IN THE FORMATION AND MAINTENANCE OF THE CONTRACTILE VACUOLE IN AMOEBA PROTEUS C. J. FLICKINGER

and R. A. COSS

Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo. 80302, USA

SUMMARY The role of the nucleus in the formation and maintenance of the contractile vacuole in amebae was investigated using microsurgery. Amebae were cut in two, forming nucleated and enucleated portions that either contained or lacked a contractile vacuole. The total numbers of cells and the numbers containing a contractile vacuole in these different categories were determined at intervals after the operation. In both nucleates and enucleates that initially contained a contractile vacuole, the contractile vacuole was maintained for as long as the cell survived. Enucleated fragments that lacked a contractile vacuole did not regenerate one. In contrast, nucleated portions initially lacking a contractile vacuole regenerated a contractile vacuole, usually within 1 h. The results indicate that the nucleus is required for the formation of a new contractile vacuole but that the nucleus is not necessary for the maintenance of the contractile vacuole.

Different cytoplasmic organelles depend upon the nucleus to varying degrees.Mitochondria and chloroplasts are partially independent of the nucleus. They contain their own DNA, exhibit traits that are inherited independently of the nuclear genes,and reproduce by growth and division [8, 9, 14, 151.In contrast, Golgi complexes are highly dependent upon the nucleus. They decline in size and number following enucleation or actinomycin treatment in amebae [5, 61 and Acetabularia [31]. In amebae, Golgi complexes are rapidly regenerated following the introduction of a new nucleus into enucleated cells [7J The endoplasmic reticulum in amebae is dependent upon the nucleus for maintenance of its normal form, but not for its survival. It persists in enucleated amebae but undergoes extensive changes in configuration, forming cisternae that are longer and narrower than Exptl Cell Res 62

normal and that appear progressively to encircle parts of the cytoplasm [5]. The contractile vacuole of protozoa is another highly organized systemof cytoplasmic membranes. Fine structure studies [4, 10, 13, 19, 22,251 have shown that it consists of a central membrane-bounded vacuole and, in some species, a series of radially arranged canals. A multitude of small tubules or vesicles is clustered in the cytoplasm around the main vacuole membrane. In amebae, mitochondria are concentrated in a layer surrounding the vesicles [IO, 19, 221. The dependence of the contractile vacuole upon the nucleus may readily be studied in amebae. Cells can be cut into nucleated and enucleated halves, with either part containing the contractile vacuole. The study of enucleated cells over a period of time permits the determination of whether the contractile

Nucleus and contractile vacuole

vacuole can survive in the absence of the nucleus. In addition, by observation of both nucleated and enucleated fragments initially lacking a contractile vacuole, it becomespossible to determine whether the contractile vacuole can be regenerated, and whether the presence of the nucleus is required for regeneration. The results of the present study indicate that the nucleus is required for the formation of a new contractile vacuole, but that the presence of the nucleus is not necessary for the maintenance of the contractile vacuole.

321

vacuole with respect to the nucleus was known. The four types of fragments; N+, N-, E+ and E-, were maintained separately under identical conditions in Syracuse watch glasses at 21°C without food organisms for 4 to 8 days. The total number of cells and the number containing a contractile vacuole were determined daily by observation with a stereomicro-

scopeat x 40-80.

RESULTS

Nucleated (N) and enucleated (E) fragments initially either possessed(+) or lacked (-) a contractile vacuole. The following total numbers of cells were formed by micromanipulation and were observed at daily intervals for 4 to 8 days: N+, 125; N-, 139; E+, 136; E-, 118. Within the pairs of N+ and E-, MATERIALS AND METHODS and N - and E +, the numbers of cells differ Stock cultures of Amoeba proteus were maintained in Prescottmedium[23]with dailyfeedingsof washed slightly becausein a few instances one member of the pair lysed during the cutting proTetrahymena. The amebae were not fed on the day preceding an experiment to avoid the presence of cess.Table 1 shows a sample experiment. The many large food vacuoles that complicate the obtotal numbers of cells in the four different servation of the nucleus and contractile vacuole. Individual amebae were placed in small droplets classesare compared with the numbers conof medium on the surface of a glass slide coated with taining a contractile vacuole at daily intervals about 2 mm of 2 % agar as for nuclear transplantation by the technique of Jeon [12]. The medium was for 1 week after operation. In other experiwithdrawn with a braking pipette, flattening the cells ments, the proportion of cells surviving at on the agar and permitting the visualization of the nucleus and contractile vacuole. The amebae were cut different times varied, but the results with into two approximately equal parts with a glass respect to the presenceor absenceof contracneedle controlled by a deFonbrune micromanipulator. The operation was visualized at a magnification tile vacuoles were identical to the experiment of x 180 under a compound microscope. shown. In both nucleated and enucleated porThe location of the contractile vacuole, whether in the nucleated part or the enucleated part, was de- tions containing a contractile vacuole (N+ termined in each case at the time the cell was cut. Less and E +), the contractile vacuole was mainthan 5 % of the cells appeared to contain more than one contractile vacuole and were not used in these tained as long as the cell survived. Contractile experiments. All of the cells on any given slide were vacuoles in enucleated cells (E +) not only cut so as to yield the same kind of progeny; i.e., either (1) a nucleated portion containing a contractile persisted but also continued to undergo cyclic vacuole (N C) and an enucleated part lacking a confilling and emptying. All of the nucleated tractile vacuole (E-), or (2) a nucleated fragment lacking a contractile vacuole (N -) plus an enucleate portions initially lacking a contractile vacuole containing a contractile vacuole (E+). Since the (N-) regenerated one by the day after the nucleus and contractile vacuole were frequently found in proximity to one another, it was sometimes neces- operation. In no case did an enucleated cell sary to gently separate them with the needle before without a contractile vacuole (E-) reform cutting in order to produce N-, E + pairs of approximately equal size. the organelle. After the operation, the amebae were transferred Since nucleated cells initially lacking a conto a Syracuse watch glass using a braking pipette. The enucleated halves were readily identified and tractile vacuole (N -) regenerated one in less separated from the nucleated parts on the basis of the than a day, additional experiments were contendency of the enucleates to become rounded, their failure to adhere to the substrate and their lack of ducted to determine how rapidly a contractile motility. Since the contractile vacuole was identified vacuole is formed. Amebae were cut into at the time of cutting, the location of the contractile Exptl Cell Res 62

328 C. J. Flickinger & R. A. Goss Table 1. The presence of a contractile vacuole in amebafragments E+

N-

N+ Days after operation

Total

With CV

Total

With CV

0 :

34 34 32

34 34 32

42 42

0 42

3 4 5 6 7

ii 22 20 17

31 24 22 20 17

;i 27 23 21 17

:: 27 23 21 17

E-

Total

With CV

Total

With CV

E 31 31 26 22 14 5

36 33 31 31 26 22 14 5

:i 24 22 18 15 12 8

0 0 0 0 0 0 0 0

N + , nucleate initially containing a contractile vacuole. N - , nucleate initially lacking a contractile vacuole. E + , enucleate initially containing a contractile vacuole. E - , enucleate initially lacking a contractile vacuole. CV, contractile vacuole. All cells were maintained in Prescott medium [23] at 21 “C without food organisms..

nucleated and enucleated parts with the contractile vacuole included in the enucleate portion (N- and E +). The nucleated fragments (N -) were observed at 5 min intervals for 1 h and then at 15 min intervals for an additional hour or until all cells had formed a contractile vacuole. The percent of cells containing a contractile vacuole at intervals after the operation is shown in fig. 1. In this experiment a contractile vacuole appeared in some cells within 10 min, and all the cells formed a contractile vacuole by 1 h after the operation. 100 90 60 70 60 50 40 30 20 IO 10

i?o

30

40

50

60

Fig. 1. Abscissa: time after operation (min); ordinate:

% cells containing a contractile vacuole. The regeneration of a contractile vacuole in nucleated ameba fragments initially lacking a contractile vacuole (N -). Total number of cells = 23. ExptI Cell Res 62

DISCUSSION The results indicate that the nucleus is required for the formation of a new contractile vacuole following its removal by microsurgery. A contractile vacuole was regenerated within one hour in nucleated fragments (N -) but did not reappear in enucleated cells (E -). The nucleus does not appear to be necessary for the maintenance of the contractile vacuole once it is formed, however, because contractile vacuoles persisted in enucleates (E +) as long as the cells survived. Contractile vacuoles in enucleates(E +) continued to undergo cyclic filling and emptying, but their rate of activity has not been compared quantitatively with that of normal amebae. Comandon & deFonbrune [3] also found that the contractile vacuole persisted in enucleated amebae. They observed that its rate of emptying remained normal, at least for 3 days, but that the maximal volume attained by the vacuole began to decline 2 days after enucleation. The contractile vacuole also persists in enucleated Stentor with a normal rate of pulsation ([I, 241,cited in [29]). More detailed studies of the physiology of contractile vacuoles, however, might reveal abnormali-

Nucleus and contractile vacuole

ties in their function in enucleated cells. Furthermore, it is possible that although contractile vacuoles persist in enucleates, they may possessstructural abnormalities that lie beneath the resolution of the light microscope and would be detectable by electron microscopy. Previous experiments [18, 201 have also shown that contractile vacuoles in nucleated amebae can be regenerated following their removal. In early work on amebae, however, the regeneration of a contractile vacuole in enucleated cells was reported [ll], cited in [29]; [20]. The basis for this difference from the present study is not clear, but one possibility is suggestedby a problem encountered in the early phases of this study. In our initial experiments, we attempted to cut amebae with a braking pipette and then to separate them into groups of nucleates and enucleates with and without contractile vacuoles. It proved very difficult to distinguish between the classes of cells, especially to ascertain whether a contractile vacuole was initially present or absent in a fragment, which often had assumeda rounded shape. Consequently, the results upon following the cells for several days were equivocal. It was to avoid this possibility of initial errors in classification of the fragments that we used the more tedious but more precise technique of cutting the cells while they were flattened on the surface of an agar-coated slide. This method permitted the direct visualization of the nucleus and contractile vacuole and enabled us to determine the location of both when the ameba was cut in two. The regeneration of a contractile vacuole in enucleated Stentor has also been reported [2, 26, 27, 281, cited in [29]). Perhaps there is a species difference between amebae and stentors in the ability of enucleates to form a contractile vacuole. Alternatively, the apparent formation of a new contractile vacuole

329

in enucleate stentors may actually be due to the enlargement of a persisting part of the original contractile vacuole included in the enucleate fragment, as has previously been suggested [29]. The origin of the new contractile vacuole is at present unknown. Much of the early literature on the origin of contractile vacuoles was clouded by the controversy over whether the contractile vacuole was a stable structure or disappeared and reformed once during each cycle of filling and emptying (see [30]). In addition, the relationship between the surrounding granules, now known to be mitochondria, and the contractile vacuole itself was debated (e.g., [16, 17, 18, 20, 21, 301). The system described in this report is a favorable one in which to investigate the formation of the contractile vacuole, because nucleated cells initially lacking a contractile vacuole are readily obtained and the organelle develops rapidly and in a reproducible fashion. Electron microscope studies of the origin of the contractile vacuole are in progress. The authors wish to acknowledge the technical assistance of Mrs Heather Taylor. This study was supported by a research grant from the American Cancer Society (E-500) and by Program Project HD-02282, Health Sciences Advancement Award FR-02084,and TrainingGrant5TOl-HDOO17204 from the National Institutes of Health. Dr Flickinger is the recipient of a Research Career Development Award from the National Institute of General Medical Sciences.

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Received March 9, 1970